BLACPMA0902

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

Achyrocline satureioides
Volumen 9, Nmero 2, Marzo de 2010

Editoriales | Editorials
BARANDA (Chile) Hait, tierra de esperanza.
CESPEDES y ALARCON (Chile) La ciencia chilena se pone de pie.
Artculos | Articles
DELAZAR, et al. (Reino Unido) Ornithogalum cuspidatum Bertol. bulbs, a source of free radical scavengers and phytosterols.
RETTA et al. (Argentina) Diferenciacin de las especies Achyrocline satureioides, A. flaccida y Gnaphalium gaudichaudianum por sus perfiles cromatogrficos.
GARCA-BORES et al. (Mxico) Photoprotective activity and general toxicity of Yucca periculosa stilbenes.
MARTNEZ et al. (Argentina) Los remedios naturales en la prevencin y cuidado de la salud oral de los tobas del Chaco Central (Argentina).
ARANCIBIA et al. (Argentina) Aromatic plants from Patagonia: chemical composition and antimicrobial activity of the essential oil of Senecio mustersii and S. subpanduratus.
CESPEDES et al. (Chile) Anti-inflammatory Activity of Aristotelia chilensis Mol. (Stuntz) (Elaeocarpaceae).
DAMIAN BADILLO et al. et al. (Mxico) In vitro antioomycete activity of Artemisia ludoviciana extracts against Phytophthora spp.
FAZIO et al. (Venezuela) Antitumour and anti-inflammatory activities in a hydroethanolic extract of Lindackeria paludosa, a South American shrub.

2010 The Authors
2010 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 9 (2), i
BLACPMA ISSN 0717 7917
Comit Editorial | Editorial Board

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

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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 (2), 84
Editorial
www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 9 (2) 2010 | 84

Hait, tierra de esperanza
[Haiti, land of hope]
Benito BARANDA.
1,*

1. Amrica Solidaria (www.americasolidaria.org)
Enviado: 2010-02-06

El terremoto lo ha devastado todo, se ha llevado
vidas, historias, cantos, colores, familias, negocios,
casas, barrios, escuelas, hospitales, oficinas de
gobierno, iglesias (inclusive a cado la catedral con su
arzobispo muerto), y aun en puerto prncipe se respira
polvo y se siente ya ese olor a cuerpos
descompuestos. Todo lo avanzado con dedicacin y
sacrificio en los ltimos aos, el esfuerzo diario de
miles de haitianos y haitianas que se dedicaron a
trabajar por otros con generosidad, hoy se derrumba,
se oscurece y nos provoca una gran tristeza y
nosotros mismos sentimos un cuota de desesperanza.
Sin embargo en medio de las nubes negras que asolan
esas hermosas y ricas tierras, en ese pueblo de 'tierras
altas' donde viven personas trabajadoras y golpeadas
por siglos con la angustia y el abandono, hoy que ya
se acerca la temporada de las lluvias, luego las
tormentas y huracanes, aun hay esperanza. Esa
esperanza la escriben a diario lps mismos haitianos y
haitianas, la proclaman aun en sus cantos en cada
esquina de puerto prncipe, la manifiestan ya en el
resurgir del comercio callejero, en los deseos
irrefrenables de volver a la escuela (aunque en la
ciudad se calcula que el 80% de ellas est
inutilizable), y la demuestran en sus deseos de
trabajar y levantarse nuevamente, de reconstruir con
sus manos la ciudad y de alzarlas tambin para alabar
y ayudar a otros.
En ese territorio los jvenes profesionales
voluntarios y voluntarias de Amrica Solidaria estn
dejando nuevamente parte de sus vidas, en aos
pasados 70 de ellos y ellas donaron su tiempo, cario
y profesin a personas con nombre, a familias y
comunidades, hoy nuevamente retoman el servicio y
ya se alistan 10 para sumarse a otros 3 que estn all,
se espera que en el 2010 se llegue a tener
simultneamente 40 voluntarios colaborando con la
reconstruccin y desarrollo de Hait, fortaleciendo
sus instituciones y colaborando con la consolidacin
de la red solidaria que los mismos haitianos han
creado.
Creemos y seguiremos convencidos que es posible
superar las injusticias y la pobreza en Amrica, en
especial en los pases ms pobres y con mayores
grados de exclusin social, y especficamente en
nuestro subcontinente latinoamericano, juntos
lograremos en las prximas dcadas reescribir la
historia y que mejor que recomenzarla en el primer
pas libre de nuestro continente (y seguramente en el
primero de personas procedentes de frica que
lograron dicha libertad).
La pobreza, la miseria, las grandes desigualdades
y la falta de justicia y ausencia de libertad, es posible
superarla si nos ponemos de acuerdo, nos entregamos
y sacrificamos, si somos capaces de soar juntos un
continente diferente, con espacios de libertad y
desarrollo para todas y todos, con oportunidades de
crecimiento para cada ser humano que nace en
nuestras tierras, con solidaridad y sin individualismos
perniciosos sino ms bien con la promocin de la
iniciativa personal y colectiva tendiente al bien
comn. Para ello es necesario poner nuestra
inteligencia y voluntad al servicio de los dems, no
de nosotros mismos ni de nuestros propios deseos de
poder o de acumulacin de bienes, sino ms que
nunca se requiere que construyamos lo posible para
la felicidad de muchos.
Nuestro rico y hermoso continente, las personas
que en el hemos nacido y vivimos, queremos algo
diferente, y esperamos que las grandes naciones de
la tierra lo entiendan con claridad y nos ayuden a
lograrlo, queremos que cada nios y nia nacida aqu
goce de la libertad y de las oportunidades para
desarrollarse en plenitud, con felicidad, amor e
igualdad.

2010 The Authors
2010 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 9 (2), 85-86
Editorial

La ciencia chilena se pone de pie
[Chilean science gets up]
Carlos L CESPEDES
1
, Julio ALARCON
2

1
Laboratorio de Bioqumica Vegetal y Fitoqumica-Ecolgica. ;
2
Laboratorio de Sntesis y Biotransformacin de Productos Naturales.
Departamento de Ciencias Bsicas, Universidad del Bo-Bo, Av. Andrs Bello s/n, Casilla 447, CP 3780000, Chilln, Chile
Enviado: 2010-03-17

Para la comunidad cientfica de Chile, el
comienzo de este ao 2010 ha sido muy duro, durante
la madrugada del 27 de Febrero 2010, ocurri un
fuerte terremoto grado 8.8 que sacudi las regiones
quinta, sexta, sptima, octava, novena, y regin
metropolitana, dejando a muchas ciudades, pueblos
costeros y del interior casi en ruinas. Muchos
edificios de las Universidades de Santiago
(Universidad de Santiago de Chile, USACH),
Valparaso (Catlica de Valparaso), Talca
(Universidad de Talca), Chillan (Universidad del
Bo-Bo), Concepcin (Universidad de Concepcin)
y Temuco (Universidad Catlica de Temuco,
Universidad de la Frontera) resultaron con graves
prdidas, destrozos de infraestructura, material de
trabajo (vidrio, reactivos y solventes), y de valiosos
equipos. As, la comunidad cientfica chilena lamenta
la terrible perdida del edificio de la Facultad de
Ciencias Qumicas de la Universidad de Concepcin,
el que resulto completamente destruido tanto por el
terremoto como por el casi inmediato incendio de
prcticamente todo el edificio. Lo ms lamentable es
la perdida de mucha investigacin, algo que muy
probablemente tomara algunos aos en recuperarse.
La comunidad cientfica en Chile, espera que la
agencia estatal para la ciencia y la tecnologa
(Comisin Nacional de Investigacin Cientfica y
Tecnolgica/CONICYT) de las garantas y el apoyo
para recuperar equipos y materiales, algo imperioso y
fundamental para poder comenzar y/o reiniciar las
investigaciones, y as poder volver a obtener las
evidencias cientficas que permitan recomenzar con
el chequeo de hiptesis, y obtencin de datos que
permitan responder en parte a las numerosas
preguntas cientficas que se haban generado en las
investigaciones que estaban en curso.
La regin centro-sur incluye a las Universidades:
Catlica de Valparaso (UCV), de Valparaso (UV),
de Santiago de Chile (USACH), de Talca
(UTALCA), del Bo-Bo (UBB: en sus dos Campus
Chillan y Concepcin), de Concepcin (UDEC, en
sus tres Campus: Concepcin, Chillan y Los
ngeles), Catlica de la Santsima Concepcin
(UCSC), Catlica de Temuco (UCT), de la Frontera
de Temuco (UFRO) y la Austral de Valdivia (UAV).
Todas estas universidades con algn tipo de daos,
pero las que resultaron con ms daos fueron las
UDEC, UBB y UTALCA.
Figura 1: Imgenes de la devastacin causada por el terremoto en
las instalaciones universitarias de la Regin del Bo-Bo.

.
Cspedes y Alarcn La ciencia chilena se pone de pie


Figura 2. Productividad Cientfica nacional. (Fuente: CRUCH, Consejo de rectores de Chile).

Todas estas universidades poseen en conjunto una
productividad cientfica significativa, sumando entre
toda el 33% nacional, datos que no hacen sino
evidenciar el potencial de la investigacin cientfica
de las regiones, que con fuerza contribuyen al
desarrollo cientfico y tecnolgico de Chile
Finalmente, este esfuerzo de trabajo
mancomunado y solidario requiere del apoyo de las
autoridades (Intendentes, Gobernadores, Alcaldes,
CORFO, CONICYT, Ministerio de Educacin-Div.
Educ. Superior), para que unidos no solo nos
pongamos de pie (de lo cual estamos ms que seguros
de lograrlo), si no que llevemos a nuestros
investigadores, profesores, y estudiantes de pre y
posgrado a un nivel superior al que se tena antes de
este terrible terremoto y tsunami. Esta experiencia es
el mejor ejemplo de que se debe apoyar
incondicionalmente a la ciencia bsica, pilar
fundamental en los avances del conocimiento de la
naturaleza. Los conocimientos adquiridos a travs de
la investigacin cientfica, no siempre tienen o
poseen una aplicacin mediata y/o inmediata, sino
que muchas veces se aplican a largo plazo, como por
ejemplo las investigaciones en sismologa, y es en
este minuto cuando resulta relevante el conocimiento
adquirido por nuestros colegas de la Universidad de
Chile, expertos en sismologa, quienes prcticamente
hacen investigaciones cientficas en este campo del
conocimiento con un mnimo de apoyo.

La universidad est daada pero no est en el
suelo ni devastada
Rector Sergio Lavanchy, Universidad de Concepcin, Diario El
Sur, 9 de Marzo 2010.

2010 The Authors
2010 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 9 (2), 87 - 92
Articulo Original | Original Article



Analyses of phytosterols and free radical scavengers in the bulbs of
Ornithogalum cuspidatum Bertol.
[Analisis de fitosteroles y captadores de radicales libres en bulbos de Ornithogalum cuspidatum Bertol.]
Abbas DELAZAR
1
Ehsan NAZIFI
1,2
Ali MOVAFEGHI
2
Hossein NAZEMIYEH
1
Salar HEMMATI
1
Lutfun NAHAR
3

Satyajit D. SARKER
4,*

1School of Pharmacy and Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2Department of Plant Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
3Drug Discovery and Design Research Division, Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, City
Campus South, MA Building, Wulfruna Street, Wolverhampton WV1 1LY, England, UK
4Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, MM Building, Molineux Street, Wolverhampton WV1
1SB, England, UK
Abstract
The gas chromatography-mass spectrometry (GC-MS) analyses of the methanol (MeOH) extract of the bulbs of Ornithogalum cuspidatum led to
the identification of thirteen phytosterols, namely cholesterol, 3,5-didehydro-stigmastan-6,22-diene, 4,4-dimethyl-5-cholest-7-en-3-one, 4-methyl-
cholesterol, 5-cholestene-3,7-diol, campesterol, cholest-4-ene-3,6-dione, stigmast-4-en-3-one, stigmasta-3,5-dien-7-one, stigmasterol, 5-ergostenol, -
sitosterol, -sitosterol The free radical scavenging activity of the MeOH extract, and its solid-phase extraction fractions were assessed by the 2,2-diphenyl-1-
picryl hydrazyl (DPPH) assay, and the 40% MeOH-water fraction showed the highest degree of free radical scavenging property (RC50 8.87 x 10
-2
mg/mL),
compared to that of the positive controls Trolox and quercetin 3.07 x 10
-3
and 2.78 x 10
-4
mg/mL, respectively. The 40% MeOH-water fraction also had the
highest level of total phenolics content.

Keywords: Ornithogalum cuspidatum; Liliaceae; phytosterol; GC-MS; antioxidant.

Resumen
El anlisis GC-MS del extracto metanlico (MeOH) de los bulbos de Ornithogalum cuspidatum llevo a la identificacin de trece fitosteroles, a
saber colesterol, 3,5-didehidro-stigmastan-6,22-dieno, 4,4-dimetil-5-colest-7-en-3-ona, 4-metil-colesterol, 5-colesten-3,7-diol, campesterol, colest-4-en-
3,6-diona, estigmast-4-en-3-ona, estigmasta-3,5-dien-7-ona, estigmasterol,
5
-ergostenol, -sitosterol, -sitosterol. La actividad captadora de radicals libres
del extracto MeOH, y sus fracciones tras extraccin en fase slida se evaluaron mediante el ensayo del DPPH y la fraccin 40% MeOH-H2O exhibi la
capacidad mas alta (RC50 8.87 x 10
-2
mg/mL), comparada con los controles positivos Trolox y quercetina (3.07 x 10
-3
and 2.78 x 10
-4
mg/mL,
respectivamente). La fraccin 40% MeOH-H2O contiene el nivel mas alto de fenoles totales.

Palabras Clave: Ornithogalum cuspidatum; Liliaceae; fitosterol; GC-MS; antioxidante.

Recibido | Received: January 28, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: December 2, 2009.
Publicado en Lnea | Published Online: March 19, 2010.
Declaracin de intereses | Declaration of interests: Authors have no competing interests.
Financiacin | Funding: none declared
This article must be cited as: Abbas Delazar, Ehsan Nazifi, Ali Movafeghi, Hossein Nazemiyeh, Salar Hemmati, Lutfun Nahar, Satyajit D. Sarker. 2010. Ornithogalum
cuspidatum Bertol. Bulbs, a source of free radical scavengers and phytosterols Bol Latinoam Caribe Plant Med Aromat 9(2):87 92. {EPub March 19, 2010}.

*Contactos | Contacts:. E-mail: s.sarker@wlv.ac.uk ; Tel: +44 (0)1902 322578 ; Fax: +44 (0)1902 322496.

Delazar et al. Ornithogalum cuspidatum Bertol. bulbs, a source of free radical scavengers and phytosterols


INTRODUCTION
The genus Ornithogalum (Liliaceae)
encompasses ca. 150 perennial bulbous species,
which are mostly distributed in the temperate
climates of Europe, Asia, and Africa (Bryan, 1989;
Du Plessis et al., 1989; Ghannamy et al., 1987). The
bulbs of certain Ornithogalum species are known to
contain a variety of steroidal glycosides (Kubo et al.,
1992; Kuroda et al., 2002, 2004; 2006). While the
physiological roles of these steroidal glycosides in
plants are yet to be fully understood, they are known
to possess antimicrobial, mould inhibiting, and insect
deterrent properties. Thus it is assumed that steroidal
glycosides may play a role in plants defence
(Morrisey and Osbourn, 1999; Gus-Mayer et al.,
1994). These structurally diverse compounds have
also been observed to kill protozoans and molluscs,
impair the digestion of protein and the uptake of
vitamins and minerals in the gut, cause
hypoglycemia, and to act as antifungal, antiviral and
antioxidant agents. Reports on studies on steroidal
glycosides for their membrane-permeabilizing,
immunostimulant, hypocholesterolemic and
anticarcinogenic properties have established that
these compounds affect growth, feed intake and
reproduction in animals (Francis et al., 2002). To our
knowledge, with exception of a few previous studies
(Gahreman, 1997; Nazifi et al., 2008; Delazar et al.,
2009) on some species of Ornithogalum, there has
been no report on any systematic studies on the
steroidal components of the bulbs of Ornithogalum
cuspidutum, a native perennial to Iran, Iraq and
Turkey. We now report on identification of thirteen
phytosterols from the bulbs of O. cuspidatum using
GC-MS as well as the free radical scavenging activity
of the MeOH extract and its solid-phase extraction
fractions.
MATERIALS AND METHODS
Plant Material
The bulbs of Ornithogalum cuspidatum Bert. were
collected from Maraghe in the North-West of Iran
during April-May 2006. A voucher specimen (TUM-
ADE 0284) representing this collection has been
retained in the herbarium of the Faculty of Pharmacy,
Tabriz University of Medical science, Iran.
Solid-phase extraction
A portion (2 g) of the MeOH extract was
subjected to solid-phase extraction on a C
18
Sep-Pak
cartridge (10 g) using a step gradient of H
2
O:MeOH
and DCM to obtain seven fractions 1-5, H
2
O:MeOH
= 80:20 (0.212 g), 60:40 (0.115 g), 40:60 (0.180 g),
20:80 (0.041 g), 00:100 (0.027 g) and fraction 6,
100% DCM (0.030 g), respectively. All fractions
were concentrated using a rotary evaporator at a
maximum temperature of 45 C.
Liebermann-Burchard test for steroids
The MeOH extract and all solid-phase fractions
were examined for the presence of steroids using the
Liebermann-Burchard test. Acetic anhydride (2 mL)
was added to the extract (100 mg), the mixture was
thoroughly stirred, heated for 2 min on a water bath
and allowed to stand at r.t. Sulphuric acid (2 mL) was
gently added to 0.7 mL of the supernatant acetic
anhydride layer. The blue to green color of the upper
layer indicated the presence of phytosterols in the
MeOH extract and its fractions.
Preparation of trimethylsilyl (TMS) ether
derivatives
The steroidal compounds present in the positive
fractions were converted to their trimethylsilyl
derivatives using trimethylsilyl chloride. Each
fraction was mixed with trimethylsilyl chloride (100
L) in glass sealed tubes using an ultrasonic bath for
2 min and then vortexing briefly. The tubes were
incubated at 60 C for 45 min. Thereafter, the solvent
was evaporated under a stream of nitrogen and the
TMS ether derivatives were dissolved in 0.2 mL of n-
hexane, the tubes were sonicated in an ultrasonic bath
for 2 min, vortexed and centrifuged for 3 min. The n-
hexane layer was transferred to another tube,
avoiding any solid particles, and analyzed by the GC-
MS. After derivatization, the tubes were stored at -20
C for subsequent analyses within 3 days (Paresh and
Normen, 1998).
Gas Chromatography-Mass Spectrometry
The GC-MS analyses were carried out in a
Shimadzu GC-MS-QP 5050A gas chromatograph
fitted with a DB1 (methyl phenyl sylonane, 60 m
0.25 mm i.d.) capillary column. Carrier gas, helium
with a flow rate of 0.6 mL/min; column temperature,
5 min in 180 C, 180-260 C at 3C/min, 5 min in
260 C, 260-280 C at 0.2 C/min, and finally 5 min


in 280 C; injector temperature, 280 C detector
temperature, 290 C, Volume injected, 1 L of TMS
ether derivatives in n-hexane (2%); Split ratio, 1:8.
The MS operating parameters were as follows:
ionization potential, 70 eV; ion source temperature;
290 C; quadrupole 100 C, Solvent delay for 100%
MeOH fraction 32.0 min and for DCM fraction 42.0
min, scan speed 2000 amu/s and scan range 30-600
amu, EV voltage 3000 volts.
Identification of the compounds
The identification of phytosterols was based on
direct comparison of the retention times and mass
spectral data with those for the TMS derivatives of
standards and by computer matching with the Wiley
229, Nist 107, Nist 21 Library, as well as by
comparison of the fragmentation patterns of the mass
spectra with those reported in the literature (Adams,
2004; Paresh and Normen, 1998; Massada, 1978).
Pure chemical standards of phytosterols used in
this work were cholesterol, stigmast-4-en-3-one,
stigmasta-3,5-dien-7-one, stigmasterol, 5-
ergostenol, -sitosterol, and -sitosterol (Sigma-
Aldrich, UK).
Antioxidant Assay
2,2-Diphenyl-1-picrylhydrazyl (DPPH), molecular
formula C
18
H
12
N
5
O
6
, was obtained from Fluka
Chemie AG, Bucks. Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) was obtained
from Sigma-Aldrich, UK. The method used by Takao
et al.

(1994) was adopted with suitable modifications
(Kumarasamy et al., 2007). DPPH (8 mg) was
dissolved in MeOH (100 mL) to obtain a
concentration of 80 g/mL.
Qualitative Aanalysis: Test samples were applied
on a silica gel TLC plate and sprayed with DPPH
solution using an atomiser. It was allowed to develop
for 30 min. The colour changes (purple on white)
were noted.
Quantitative Analysis: 2,2-Diphenyl-1-
picrylhydrazyl (DPPH), molecular formula
C
18
H
12
N
5
O
6
, was obtained from Fluka Chemie AG,
Bucks. Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) was obtained
from Sigma-Aldrich, UK. The method used by Takao
et al.

(1994) was adopted with suitable modifications
(Kumarasamy et al., 2007). DPPH (8 mg) was
dissolved in MeOH (100 mL) to obtain a
concentration of 80 g/mL.
Serial dilutions were carried out with the stock
solutions (10 mg/mL) of the plant extracts/fractions
to obtain concentrations of 5x10
-1
, 5x10
-2
, 5x10
-3
,
5x10
-4
, 5x10
-5
, 5x10
-6
mg/mL. Diluted solutions (2
mL each) were mixed with DPPH (2 mL) and
allowed to stand for 30 min for any reaction to occur.
The UV absorbance was recorded at 517 nm. The
experiment was performed in duplicate and the
average absorption was noted for each concentration.
The same procedure was followed for the positive
controls Trolox and quercetin. The RC
50
value,
which is the concentration of the test material that
reduces 50% of the free radical concentration, was
calculated as mg/mL.
Total Phenolic Contents (TPC)
The TPCs of the MeOH extract and its fractions
were determined by the modified Folin-Ciocalteu
assay (Jung et al., 2008). Briefly, the reaction
mixtures contained 250 L of each sample at various
concentrations (0.4-2 mg/mL) and 750 L of Folin-
Ciocalteu reagent, and were kept at ambient
conditions for 5 min, followed by the addition of 2
mL of 7.5% Na2CO3. The final mixture was diluted
to 7 mL of total volume with deionized H2O. The
reaction mixtures were kept in the dark at ambient
conditions for 1 h to complete the reaction. Then, the
absorbance was measured at 765 nm. All experiments
were conducted using gallic acid (Sigma-Aldrich,
UK) as a calibration standard, and the results were
recorded as mg of gallic acid equivalent per 100 g of
dried extract or fraction.
RESULTS AND DISCUSSION
The DPPH antioxidant assay is based on the
principle that 2,2-diphenyl-1-picryl-hydrazyl
(DPPH), a stable free radical, is able to decolorize in
the presence of free radical scavengers (antioxidants).
The odd electron in the DPPH radical is responsible
for the absorbance at 517 nm, and also for visible
deep purple color (Kumarasamy et al., 2007). When
DPPH accepts an electron donated by a free radical
scavenger, the DPPH is decolorized, and the extent of
desulfurization can be quantitatively measured from
the changes in absorbance. In the TLC-based
qualitative antioxidant assay using the DPPH spray,
the MeOH extract, solid-phase fractions of the
MeOH extract of the bulbs of O. cuspidatum
displayed weak to moderate free radical scavenging
activity indicated by the presence of a faint
yellow/white spot on a purple background on the


Table 1. RC
50
values of the MeOH extract and its fractions in the DPPH assay, and their total phenolics content (TPC)
Extract and fractions RC
50
(mg/mL) TPC (mg gallic acid equivalents /100 g)
MeOH Extract 3.74 x 10
-1
13.5
Fraction of 20% MeOH in water 3.85 x 10
-1
15.9
-2
70.5
-1
49.0
-1
36.4
-1
19.2
Fraction of DCM 15.1 x 10
-1
8.1
Trolox 3.07 x 10
3
-
Quercetin 2.78 x 10
4
-
Table 2. Phytosterols present in the fractions of the MeOH extract of the bulbs of O. cuspidatum.
Compounds Rt
a
% M Formula Mass spectral data
b

Solid-phase fraction 100% MeOH
Cholesterol 90.6 0.57 386 C
27
H
46
O 73 (87.3), 129 (100), 213 (9.6), 329 (60.7), 353 (24.5), 368 (46.4),
458(21)
5-Ergostenol 94.9 0.99 400 C
28
H
48
O 43 (100), 55 (60.2), 213 (24.6), 289 (23.2), 315 (31.7), 382 (23.3), 400
(38.9)
5-Cholestene-3,7-diol 96.5 0.56 402 C
27
H
46
O
2
43 (84), 57 (73.2), 73 (80.6), 129 (44.7), 147 (43.6), 455 (100), 544
(11.8), 546 (1.8)
4,4-Dimethyl-5-cholest-7-
en-3-one
99.0 0.65 412 C
29
H
48
O

41(45.1), 55 (100), 69 (50.4), 87 (57.4), 119 (25.3), 397 (3.3), 412 (25.7)
Campesterol 104.0 9.35 400 C
28
H
48
O 55 (45.8), 69 (24.8), 73 (75.4), 129 (100), 255 (15), 382 (50.2), 457 (6.3),
472 (20.1)
-Sitosterol 106.7 1.00 414 C
29
H
50
O 43 (100), 55 (66.9), 107 (37.2), 255 (19.9), 329 (31.2), 396 (21.2), 414
(33.7)
Stigmasterol 108.3 8.11 412 C
29
H
48
O 55 (62.7), 69 (41), 83 (100), 129 (41), 255 (21.9), 394 (16.1), 484 (13.3)
-Sitosterol 116.5 10.73 414 C
29
H
50
O 43 (90.1), 57 (55.6), 95 (42.6), 129 (100), 255 (16.9), 357 (46.6), 396
50.8), 486 (21.1)
Stigmast-4-en-3-one 123.8 0.21 412 C
29
H
48
O 43 (41.9), 81 (23.2), 95 (21.5), 124 (100), 229 (29.9), 370 (7.4), 412
(13.9)
Solid-phase fraction 100% DCM
3,5-Didehydro-stigmastan-
6,22-diene
79.9 1.47 394 C
29
H
46
43(100), 57 (64.3), 81 (53.3), 105 (29.4), 135 (81.2), 158 (23.9), 379 (4),
394 (50.6), 455 (3.9)
4-Methyl-cholesterol 103.7 6.47 400 C
28
H
48
O 43(100), 55 (50.4), 73 (69.4), 107 (35.9), 129 (96.3), 255 (16.7), 367
(23.5), 382 (46.9), 457 (6.7), 472 (20.6)
-Sitosterol 106.6 1.41 414 C
29
H
50
O 43 (100), 57 (68.4), 107 (40), 255 (15.9), 329 (27.1), 396 (17.2), 414
(36.2)
Stigmasterol 107.9 4.00 412 C
29
H
48
O 55 (10.3), 69 (45.8), 83 (100), 129 (37.5), 255 (19.6), 394 (16.1), 484
(11.3)
Cholest-4-ene-3,6-dione 110.2 0.55 398 C
27
H
42
O
2
43 (100), 57 (40.9), 81 (34.1), 124 (47), 133 (57.5), 385 (22.9), 398
(11.8)
-Sitosterol 116.4 13.95 414 C
29
H
50
O 43 (92.1), 57 (56.7), 95 (42.1), 129 (100), 255 (15.9), 357 (51.4), 396
(44.7), 486 (20.7)
Stigmasta-3,5-dien-7-one 117.8 1.81 410 C
29
H
46
O 43 (82.8), 55 (60.5), 75 (95.5), 107 (31.8), 174 (100), 187 (18.1), 269
(10.6), 410 (22.1), 413 (18.5), 488 (14)
(a) Rt in minutes corresponding to the TMS derivatives of O. cuspidatum sterols.
(b) m/z values correspond to the non-derivatised sterols. Mass spectral data reported for phytosterols was identified after comparison
with GC-MS of TMS derivates of standards or correlation with fragmentation patterns of standards.



TLC plates. The DPPH scavenging capacity of the
extracts was compared with known antioxidants,
Trolox and quercetin. The RC
50
(the concentration
of the extract/compound at which it reduces 50% of
the DPPH absorbance at 517 nm) values of the
MeOH extract, fractions and positive controls are
presented in Table 1. The total phenolics content
(TPC) of the MeOH extract and its fractions was
determined by the modified Folin-Ciocalteu assay
(Jung et al., 2008). The results demonstrated a clear
correlation between the TPC values and the free
radical scavenging potency of the extract and the
fractions (Table 1). Among the fractions, the 40%
MeOH-water fraction showed the highest level of
free radical scavenging activity (RC50 = 8.87 x 10
-2

mg/mL), and it had the highest TPC value (70.5 mg
gallic acid equivalents per 100 g). Thus, the free
radical scavenging activity of the MeOH extract of O.
cuspidatum was predominantly because of the
phenolics compounds.
As the Liebermann-Burchard showed the
presence of steroidal compounds in the MeOH
extract and its 100% MeOH and DCM solid-phase
fractions, they were analyzed by the GC-MS. The
results of the GC-MS analyses leading to the
identification of steroidal compounds from the 100%
MeOH and the 100% DCM fractions of the MeOH
extract of O. cuspidatum are summarized in Table 2.
Thirteen steroidal compounds were identified in the
MeOH extract of O. cuspidatum, meanwhile nine and
seven steroidal compounds were identified,
respectively, from the 100% MeOH and the 100%
DCM fractions. It was noted that the 100% MeOH
and the 100% DCM fractions were composed of at
least 32.2 and 29.7% steroidal compounds,
respectively. -Sitosterol, stigmasterol and -
sitosterol were present in both fractions. -Sitosterol,
campesterol and stigmaterol were the most abundant
steroids in the 100% MeOH fraction, 10.73%, 9.35%
and 8.11%, respectively. In the 100% DCM fraction,
-sitosterol, 4--methyl cholesterol and stigmasterol
were the main components (13.95%, 6.47% and
4.00%, respectively).
The present study established that the bulbs
of O. cuspidatum are a rich source of phytosterols as
well as free radical scavengers. Phytosterols possess
cholesterol-lowering properties (Ostland et al., 2003),
and protective effects on development of coronary
heart disease (Brufau et al., 2008; Fassbender et al.,
2006). The mechanism of phytosterols action is based
on its ability to reduce cholesterol absorption (Brufau
et al., 2008). The evidence suggests that -sitosterols
improve urinary symptoms and flow measures in men
with of benign prostatic hyperplasia (Wilt et al.,
2000; Gerber, 2002). Epidemiological data suggest
that the phytosterol content of the diet is associated
with a reduction in common cancers including
cancers of the colon, breast, and prostate. In addition,
phytosterols have effects that directly inhibit tumor
growth, including the slowing of cell cycle
progression, the induction of apoptosis, and the
inhibition of tumor metastasis (Bradford and Awad,
2007). Thus, the bulbs of O. cuspidatum might be
used in the formulation of food supplements as one of
the active ingredients.
CONCLUSIONS
As phytosterols posses various human health
protecting properties (Ostland et al., 2003), the bulbs
of O. cuspidatum might have some uses as food
additives. In addition, the moderate level of free
radical scavenging property of the fractions because
of phenolics compounds could also be useful in food
industry.
REFERENCES
Adams RP. 2004. Identification of Essential Oil
Component by Gas chromatography/ Quadrupole
Mass spectroscopy. Allured Publishing Corporation,
Illinois, U.S.A.
Bradford PG, Awad AB . 2007. Phytosterols as anticancer
compounds. Mol Nutr Food Res 51:161-70.
Brufau G, Canela MA, Rafecas M. 2008. Phytosterols:
physiologic and metabolic aspects related to
cholesterol-lowering properties. Nutr Res 28:217-225.
Bryan JE. 1989. In: Bulbs. Timber Press, Portland, vol 2,
pp 298.
Delazar A, Nazifi E, Movafeghi A, Nahar L, Nazemiyeh
H, Moghadam SB, Asnaashari S, Sarker SD. 2009.
GC-MS analysis of Ornithogalum procerum. DARU .
in press. .
Du Plessis N, Duncan G. 1989. Bulbous Plants of Southern
Africa. Tafelberg Publ. Ltd., Cape Town, RSA, pp.
192.
Fassbender K, Ltjohann D, Dik MG, Bremmer M, Knig
J, Walter S, Liu Y, Letimbre M, von Bergmann K,
Jonker C. 2006. Moderately elevated plant sterol
levels are associated with reduced cardiovascular risk-
-the LASA study. Atherosclerosis 196:283-288.
Gerber GS. 2002. Phytotherapy for benign prostatic
hyperplasia. Curr Urol Rep 3, 285-291.
Francis G, Kerem Z, Harinder MPS, Becker K. 2002. The
biological action of saponins in animal systems: a
review. Br J Nutr 88, 587605.


Ghahreman A. 1997. Flora of Iran. Published by Research
Institute of Forests and Rangelands . RIFR. , Tehran,
Vol. 16, pp. 165.
Ghannamy U, Kopp B, Robien W, Kubelka W. 1987.
Cardenolides from Ornithogalum boucheanum. Planta
Med 53:172178r
Gus-Mayer S, Brunner H, Schneider-Poetsch HA, Rudiger
W. 1994. Avenacosidase from oat: purification,
sequence analysis and biochemical characterization of
a new member of the BGA family of beta-
glucosidases. Plant Mol Biol 26:909921.
Jung HA, Jung YJ, Yoon NY, Jeong DM, Bae HJ, Kim
DW, Na DH, Choi JS. 2008. Inhibitory effects of
Nelumbo nucifera leaves on rat lens aldose reductase,
advanced glycation endproducts formation, and
oxidative stress. Food Chem Toxicol 46:38183826.
Kubo S, Mimaki Y, Sashida Y, Nikaido T, Ohmoto T .
1992. New cholestane bisdesmosides from the bulbs
of Ornithogalum thyrsoides. Bull Chem Soc Jpn 65,
11201124.
Kuroda M, Mimaki Y, Yokosuka A, Hasegawa F, Sashida
Y. 2002. Cholestane glycosides from the bulbs of
Ornithogalum thyrsoides and their cytotoxic activity
against HL-60 leukemia cells. J Nat Prod 65:1417
1423.
Kuroda M, Mimaki Y, Ori K, Sakagami H, Sashida Y.
2004. Steroidal glycosides from the bulbs of
Ornithogalum thyrsoides. J Nat Prod 67:16901696.
Kuroda M, Ori K, Mimaki Y . 2006. Ornithosaponins A-
D, four new polyoxygenated steroidal glycosides from
the bulbs of Ornithogalum thyrsoides. Steroids
71:199-205.
Kumarasamy Y, Byres M, Cox PJ, Jaspars M, Nahar L,
Sarker SD. 2007. Screening seeds of some Scottish
plants for free-radical scavenging activity. Phytother
Res. 21:615-621.
Massada Y. 1976. Analysis of Essential Oil by Gas
Chromatography and Mass Spectrometry. John Wiley
and Sons, New York, U.S.A.
Morrissey JP, Osbourn AE. 1999. Fungal resistance to
plant antibiotics as a mechanism of pathogenesis.
Microbiol. Mol Biol Rev 63:708724.
Nazifi E, Delazar A, Movafeghi A, Hemmati S,
Nazemiyeh H, Nahar L, Sarker SD. 2008. GC-MS
analysis of the dichloromethane extract of the bulbs of
Ornithogalum cuspidatum Bert. Family: Liliaceae.
from Iran. Rec Nat Prod 2, 94-99.
Ostlund RE, Racette, SB, and Stenson WF. 2003.
Inhibition of cholesterol absorption by phytosterol-
replete wheat germ compared with phytosterol-
depleted wheat germ. Am J Clin Nutr 77:13851589.
Paresh CD, Normen L. 1998. Capillary column gas
liquid chromatographic separation of 5-unsaturated
and saturated phytosterols. J Chromatogr A 816, 177
184.
Takao T, Watanabe N, Yagi I, Sakata K. . 1994. A simple
screening method for antioxidants and isolation of
several antioxidants produced by marine bacteria from
fish and shellfish. Biosci Biotechnol Biochem 58,
1780-1783.
Wilt T, Ishani A, MacDonald R, Stark G, Mulrow C, Lau
J. 2000. Beta-sitosterols for benign prostatic
hyperplasia. Cochrane Database Systematic Review 2:
CD001043.

2010 The Authors

Artculo Original | Original Article


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Diferenciacin de las especies Achyrocline satureioides, A. flaccida y
Gnaphalium gaudichaudianum por sus perfiles cromatogrficos
[Differentiation of the species Achyrocline satureioides, A. flaccida and Gnaphalium gaudichaudianum by their
chromatographic profiles]
Daiana RETTA
1
; Roco FERNANDEZ PENUTO
1
; Mara CORREA
1
; Martha GATTUSO
2
;
Susana GATTUSO
2
; Arnaldo BANDONI
1

1
Ctedra de Farmacognosia-IQUIMEFA. Facultad de Farmacia y Bioqumica. Universidad de Buenos Aires-CONICET. Junn
956, 2 piso. C1113AAD Buenos Aires, Argentina.
2
Ctedra de Farmacobotnica, Facultad de Ciencias Bioqumicas y Farmacuticas,
Universidad Nacional de Rosario Suipacha 531, S2002LRK, Rosario, Argentina.
Abstract
The analysis of the chromatographic patterns of samples of inflorescences of Achyrocline satureioides, A. flaccida and Gnaphalium gaudichaudianum
of Argentina by thin layer and gas chromatography was performed. The profiles by thin layer chromatography of A. satureioides and A. flaccida were similar,
except that in A. satureioides, there is a violet zone (Rf 0.45) in predominance to the orange zone (Rf 0.4) found in samples of A. flaccida. In A. flaccida the
orange zone is major than the violet zone, mentioned before. Referring to G. gaudichaudianum, two different patterns were found, which are completely
different than those obtained with the samples of Achyrocline. One group shows two intense orange-magenta zones (Rf 0.5 y 0.1) that are absent or there are
found in lower intensity in the second group. The profiles by gas chromatography of the volatile fraction show common peaks corresponding to alpha pinene,
limonene, 1,8-cineole, alpha copaene and beta caryophyllene. There were some quantitative differences among profiles (especially with respect to alpha
copaene), but overall they were not useful for the discrimination between species. TLC was useful for the correct identification of these species, very similar
between themselves.
Keywords: Achyrocline satureioides; Achyrocline flaccida; Marcela; Gnaphalium gaudichaudianum; TLC; GC.
Resumen
Se realiz el estudio de los perfiles cromatogrficos por capa fina y cromatografia gaseosa de muestras de inflorescencias de Achyrocline satureioides,
A. flaccida y Gnaphalium gaudichaudianum de Argentina. Los perfiles por cromatografa en capa fina de A. satureioides y A. flaccida son semejantes,
excepto que para A. satureioides se observa una banda de color violeta (Rf 0.45) que predomina respecto de la banda naranja difusa (Rf 0.4) en muestras de A.
flaccida. En A. flaccida se observa predominancia de la banda naranja por sobre la violeta, mencionadas antes. Respecto a G. gaudichaudianum, se observan
dos perfiles distintos, que a su vez son completamente distintos a los de las muestras de Achyrocline. Un grupo presenta dos bandas color naranja-fucsia
intenso (Rf 0.5 y 0.1) mientras que en otras muestras, stas no se encuentran o son de menor intensidad. Los perfiles obtenidos por cromatografa de gases de
las fracciones voltiles de las tres especies presentan picos mayoritarios comunes identificados como alfa pineno, limoneno, 1,8-cineol, alfa copaeno y beta
cariofileno. Si bien se observaron ciertas diferencias cuantitativas entre algunos compuestos (especialmentec alfa copaeno), los perfiles no permitieron
discriminar entre las especies. La tcnica por TLC facilita la correcta identificacin de estas especies, en forma sencilla y rpida.
Palabras Clave: Achyrocline satureioides; Achyrocline flaccida; Marcela; Gnaphalium gaudichaudianum; CCF; CG.

Recibido | Received: October, 15, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: January 4, 2010.
Publicado en Lnea | Published Online 25 March 2010
Declaracin de intereses | Declaration of interests: authors have no competing interests.
Financiacin | Funding: This work was financed by UBACYT B014 and PICTR -0284
This article must be cited as: Daiana Retta; Roco Fernandez Penuto; Cecilia Correa; Martha Gattuso; Susana Gattuso; Arnaldo Bandoni. 2010. Diferenciacin de las especies
Achyrocline satureioides, A. flaccida y Gnaphalium gaudichaudianum por sus perfiles cromatogrficos. Bol Latinoam Caribe Plant Med Aromat 9(2):93 99. {EPub 25 March
2010 }.
*Contactos | Contacts: e-mail: dretta@ffyb.uba.ar
Retta et al. Diferenciacin cromatografica de A. satureioides, A. flaccida y G. gaudichaudianum

www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol.9 (2) 2010 | 94

INTRODUCCION
El gnero Achyrocline (Asteraceae)
comprende unas 15-20 especies y ocurre
principalmente en Amrica del Sur y Central, con
extensin a frica tropical y zonas montaosas de la
Isla de Madagascar (Giangualani, 1976; Nesom,
1990). En Argentina habitan ocho especies,
distribuidas principalmente en el norte y centro del
pas (Zuloaga y Morrone, 1999). Es bien conocida la
dificultad que presenta la correcta identificacin de
stas especies, dado que poseen caracteres
morfolgicos muy semejantes entre s (Giangualani,
1976).
La especie ms utilizada, debido a la amplia
distribucin en nuestro pas, y de mayor inters
comercial es Achyrocline satureioides (Lam.) DC. Es
una planta herbcea, perenne, tomentosa, que se
conoce con los nombres vulgares de marcela,
marcelita, marcela blanca. Sus partes areas e
inflorescencias son tradicionalmente usadas como
digestivas, antiinflamatorias, antiespasmdicas,
antidiabticas y antiasmticas (Ratera y Ratera, 1980;
Toursarkissian, 1980). En Argentina no solamente es
usada en medicina tradicional y en la formulacin de
fitoterpicos, sino tambin en la elaboracin de
productos alimenticios, dado que posee un intenso
aroma que recuerda al levstico y su sabor es amargo.
En Uruguay se comercializan adems, productos
cosmticos, debido a sus propiedades antioxidantes y
antiinflamatorias.
La demanda actual, en la regin, est cubierta
por la colecta de material silvestre, lo que lleva
muchas veces a que sta se encuentre mezclada con
otras especies similares, como Achyrocline flaccida
(Weinm.) DC. con la cual comparte parcialmente su
rea de distribucin y es tambin conocida como
marcela o marcela amarilla. Sus inflorescencias
son principalmente empleadas como digestivas,
antiespasmdicas, febrfugas, tnico, antihelmnticas
(Hieronymus, 1882; Parodi, 1886). Otra especie
contaminante o adulterante es Gnaphalium
gaudichaudianum DC. (Asteraceae), conocida como
vira vira o marcelita. Es tambin empleada en
trastornos digestivos y su morfologa es similar a las
dos especies de Achyrocline antes mencionadas
(Martnez Crovetto, 1981).
Existen estudios botnicos comparativos de
identificacin de dichas especies (Amat 1988; Gattuso
et al., 1998, 2008; Petenatti et al., 2004) donde se
describen los caracteres diacrticos de diferenciacin,
pero dado que stos resultan sutiles, suele presentar
cierta dificultad la identificacin de las mismas.
Esto plante la necesidad de encontrar una
tcnica analtica sencilla, rpida, accesible que permita
obtener mayores evidencias para la identificacin de
estos materiales, en particular cuando se presentan en
polvo o triturados.
El anlisis por cromatografa en capa fina
(TLC) de la especie A. satureioides se encuentra
descripta en la Farmacopea Brasilera (2003), faltando
el anlisis cromatogrfico de A. flaccida y G.
gaudichaudianum.
Por lo expuesto, se propuso la bsqueda de
una tcnica por TLC, que permita determinar los
perfiles cromatogrficos que caractericen dichas
especies en Argentina.
MATERIALES Y METODOS
Material Vegetal
Se analizaron muestras de Achyrocline
satureioides (Lam.) DC., Achyrocline flaccida
(Weinm.) DC., Gnaphalium gaudichaudianun DC.
(Asteraceae) y Gnaphalium aff. gaudichaudianun de
distintas regiones de Argentina (Tabla 1). La
recoleccin e identificacin botnica del material,
inflorescencias en todos los casos, estuvo a cargo de
las Doctoras Martha Gattuso y Susana Gattuso. Los
ejemplares de herbario fueron depositados en la
ctedra de Farmacobotnica, de la Facultad de
Ciencias Bioqumicas y Farmaceticas de la
Universidad Nacional del Rosario.
Extraccin
Extraccin: 2 g de material vegetal se
extrajeron por maceracin en hexano, por 24 h. Se
filtr y se llev a sequedad en evaporador rotatorio a
presin reducida a temperatura no mayor a 40 C y se
reconstituy con 2 ml de hexano.
Cromatografa en capa fina
Se emplearon cromatoplacas de slica gel
Merck como fase estacionaria. Como fase mvil se
emple una mezcla de Tolueno: Acetato de etilo:
cido actico (9:1: III). La siembra se realiz en
banda de 1 cm. La distancia de desarrollo fue de 10
cm. El revelado se realiz por aspersin con el
reactivo de anisaldehdo sulfrico y posterior
aplicacin de calor, en estufa a 105 C. La observacin
se realiz a la luz natural.


Cromatografa de Gases (GC-FID-MS)
Los aceites esenciales fueron extrados de 100
gramos de las inflorescencias desecadas a temperatura
ambiente de cada especie, por hidrodestilacin durante
3 horas usando una trampa tipo Clevenger. El producto
obtenido se sec con sulfato de sodio anhidro y
almacen a 2 C hasta su anlisis.
Para el anlisis de los aceites esenciales se
utiliz un GC-FID-MS Perkin Elmer Clarus 500,
equipado con un inyector automtico (relacin de split:
1:100) conectado por un divisor de flujos a dos
columnas: a) polietilenglicol PM ca. 20,000 y b) 5%
fenil-95% metil silicona, ambas de 60 m x 0.25 mm
con 25 m de espesor de fase. La columna polar se
conect a un detector FID, mientras que la no polar se
conect a un FID y a un detector de masa cuadrupolar
(70 eV), mediante un sistema de venteo (MSVent).
La fase mvil fue Helio, flujo constante de 1.87
mL/min. La temperatura fue programada de acuerdo al
siguiente gradiente: 90-225C a 3C/min, luego
isotrmico por 15 min. El inyector y ambos FID se
usaron a 255C y 275C, respectivamente. El volumen
de inyeccin fue 0.2 L de una solucin etanlica al
10%. La temperatura de la lnea de transferencia y la
de la fuente inica fueron 180C y 150C
respectivamente; el rango de masas buscado fue 40-
300 Da (10 scan/segundo).
La identificacin de los constituyentes se
realiz por comparacin de sus ndices de retencin
(relativos al de los n-alcanos C
8
-C
20
) obtenidos en
ambas columnas, con los de compuestos de referencia.
Adems, cada espectro de masa obtenido fue
comparado con los de las bases de datos electrnicas
tradicionales (Adams, 2007, Wiley/NBS, 2008) y con
los desarrollados en nuestro laboratorio a partir de
patrones o aceites esenciales de composicin qumica
conocida. El anlisis cuantitativo se realiz usando el
mtodo de porcentaje de reas, sin considerar alguna
correccin por factores de respuesta. Se consider para
cada constituyente la respuesta ms baja entre las
obtenidas en las dos columnas usadas.
Tabla 1: Datos de recoleccin e identificacin del material
vegetal.
Muestra Especie Lugar de recoleccin
S1/1554 A. satureioides Dpto. Punilla- Crdoba
S2/1631 A. satureioides Ro Espinillo- Crdoba
S3/1892 A. satureioides Villa Ventana- Buenos Aires
S4/1627 A. satureioides Icho Cruz- Crdoba
S5/1626 A. satureioides Los Reartes- Crdoba
S6/1629 A. satureioides
Santa Rosa de Calamuchita-
Crdoba
S7/1894 A. satureioides Dique La Florida- San Luis
S8/1895 A. satureioides
Yacanto de Calamuchita-
Crdoba
S9/1890 A. satureioides Coronel Rosales- Buenos Aires
F1/1546 A. flaccida Dpto. Coln- Entre Ros
F2/1660 A. flaccida Santo Tom- Corrientes
F3/1662 A. flaccida Jardn Amrica- Misiones
F4/1664 A. flaccida
Ruta 127, lmite Entre Ros y
Corrientes
F5/1587 A. flaccida Pronunciamiento- Entre Ros
F6/1663 A. flaccida Cua Pir- Misiones
F7/1559 A. flaccida Dpto. San Javier- Misiones
F8/1661 A. flaccida San Jos- Misiones
G1/1632 G. aff. gaudichaudianum Los Reartes- Crdoba
G2/1628 G. gaudichaudianum
Crdoba
G3/1633 G. aff. gaudichaudianum Los Reartes- Crdoba
G4/1625 G. aff. gaudichaudianum
Villa General Belgrano-
Crdoba
G5/1936 G. gaudichaudianum
Crdoba
G6/1893 G. gaudichaudianum El Trapiche- San Luis
G7/1896 G. gaudichaudianum Departamento Capital- San Luis
G8/1902 G. gaudichaudianum El Libertador- Corrientes
G9/1937 G. gaudichaudianum Ro Espinillo- Crdoba



Figura1: Cromatografa en capa fina de muestras de A. satureioides (el crculo indica la banda de inters).

S1 S2 S3 S4 S5 S6 S7 S8 S9

Figura2: Cromatografa en capa fina de muestras de A. flaccida (el crculo indica la banda de inters).

F1 F2 F3 F4 F5 F6 F7 F8

Figura 3: Cromatografa en placa fina de muestras de G. gaudichaudianum (el crculo indica la banda de inters).

G1 G2 G3 G4 G5 G6 G7 G8 G9


Figura 4: Perfiles por cromatografa gaseosa de la fracciones voltiles de (A) A. satureioides, (B) A. flaccida y (C) G. gaudichaudianum.

(A) A. satureioides

(B) A. flaccida

(C) G. gaudichaudianum



RESULTADOS
Cromatografa en capa fina
En las figuras 1, 2 y 3 se muestran los perfiles
cromatogrficos obtenidos de cada especie.
De los perfiles de las muestras de A.
satureioides y A. flaccida se observ que los mismos
son semejantes. Pero existe una diferencia que podra
discriminar ambas especies: en A. satureioides se
observa una banda de color violeta de Rf aproximado
0.45 que predomina y se resuelve bien, respecto de la
banda naranja difusa, que puede estar presente o no, de
Rf cercano a 0.4. En A. flaccida se observa
predominancia de la banda naranja (Rf 0.4) por sobre
la violeta mencionada para A. satureioides, que
prcticamente no es detectada.
Ambas especies presentan algunas bandas en
comn: una banda pardusca de Rf 0.65, debajo de esta
se encuentra una banda anaranjada y por debajo una
banda rosada de menor importancia.
Respecto a las muestras de G.
gaudichaudianum, se observan dos perfiles distintos,
que a su vez son completamente distintos a los de las
muestras de Achyrocline. Un grupo de muestras
presenta dos bandas color naranja-fucsia intenso, en Rf
0.5 y 0.1, mientras que en otras muestras, stas no se
encuentran o son de menor intensidad. En todas las
muestras se observa una banda naranja en Rf 0.15,
0.35 y 0.9. Existe tambin una banda rosada en Rf 0.6.
Cromatografia de Gases (GC-FID-MS)
Los perfiles obtenidos por cromatografa de
gases de las fracciones voltiles de las tres especies se
presentan en la figura 4. Los picos mayoritarios
comunes fueron identificados como alfa pineno,
limoneno, 1,8-cineol, alfa copaeno y beta cariofileno a
8.4, 10.5, 10.7, 23.0 y 24.9 minutos, respectivamente.
Si bien se observaron ciertas diferencias cuantitativas
entre algunos de los perfiles obtenidos (por ejemplo el
correspondiente a alfa copaeno), no se pudo encontrar
una diferencia estadsticamente significativa por el
anlisis de varias muestras de cada especie.
DISCUSIN Y CONCLUSIONES
Si bien la identificacin primaria del material
vegetal se realiza por medio de estudios botnicos, en
el caso de la muestras de marcelas esto resulta
bastante difcil, dado que las caractersticas que las
diferencias son sutiles y requieren de un estudio
bastante minucioso y preciso del material, dando lugar
a posibles errores en la identificacin, en particular si
esta es realizada por personal no entrenado. Por otro
lado, el empleo de tcnicas cromatogrficas, como
TLC, resulta ms sencillo, est ampliamente
difundido, es de fcil acceso, econmico y
reproducible.
Si bien, ambos estudios son complementarios,
hasta el momento solo se encontraba descripto el
anlisis por TLC para la especie A. satureioides
(Farmacopea Brasilera, 2003), pero no se indicaba
diferencias respecto a A. flaccida. De hecho, con el
sistema que indica la Farmacopea Brasilera para
marcela, se obtienen perfiles similares tanto para A.
satureioides como para A. flaccida, con lo cual no
permitira discriminarlas entre s. Adems, en dicha
norma se emplea Celulosa como fase estacionaria, con
la que se suele obtener menor resolucin de las bandas
presentes en las muestras que con Slica gel.
Como parte de la bsqueda y optimizacin de
un sistema cromatogrfico que caracterizara estas
especies, se probaron distintos sistemas y distintos
tipos de extractos. Partiendo de extractos realizados
con etanol al 80% y sistemas cromatogrficos
especficos para compuestos de mayor polaridad, no se
evidenciaron diferencias significativas en los perfiles
de las especies de Achyrocline, por lo tanto se decidi
partir de un extracto de menor polaridad. La tcnica
que aqu se propone permite diferenciar las tres
especies y junto con la identificacin botnica permite
obtener resultados ms slidos, aportando mayores
evidencias, que contribuyen a minimizar la ocurrencia
de errores en la identificacin del material.
Por otro lado, dado que el extracto que se
emplea para la siembra es hexnico, se cree que
muchas, aunque no todas las bandas detectadas,
pueden corresponder a los componentes voltiles
presentes en el aceite esencial de dichas especies. El
estudio de aceites esenciales por cromatografa
gaseosa de muestras de A. satureioides y A. flaccida
provenientes de Argentina ya ha sido reportado, no
encontrndose diferencias significativas entre dichas
composiciones (Labuckas, D. et al., 1999; Retta et al.,
2009a, 2009b).
Como prueba de esto se adjuntan los perfiles
obtenidos por cromatografa de gases de las fracciones
voltiles de las tres especies (figura 4), donde se
observan solamente diferencias cuantitativas entre las
especies, pero que se invalidan totalmente al evaluar
distintas poblaciones.


Este trabajo resulta un claro ejemplo de lo
importante y til que sigue siendo el empleo de la
cromatografa en capa fina como criterio de
identificacin de material vegetal. Respecto a las
muestras de G. gaudichaudianum, se han observado
dos perfiles diferentes. Si bien algunas de las muestras
fueron clasificadas como afines a dicha especie, las
diferencias qumicas observadas no se encuentran
correlacionadas con diferencias en la identificacin.
Ambos perfiles se obtuvieron tanto para muestras
identificadas como G. gaudichaudianum como para
aquellas que fueron tentativamente identificadas como
G. aff. gaudichaudianum. Lo cual indica la
coexistencia de ambas composiciones qumicas en
muestras de esta especie. A fin de profundizar en
dichas diferencias, el anlisis de los componentes
voltiles de esta especie se encuentra bajo estudio por
nuestro grupo de trabajo.
Para concluir podemos decir que esta tcnica
resulta de utilidad para discriminar estas especies tan
similares entre s, en particular cuando se trata de
muestras que se presentan en polvo o trituradas.
AGRADECIMIENTOS
Este trabajo fue subsidiado por los Proyectos
UBACYT B014 y PICTR -0284.
REFERENCIAS
Adams RP. 2007. Identification of Essential Oils
Components by Gas Chromatography / Quadrupole
Mass Spectroscopy. Allured Publ. Corp., Carol Stream,
IL.
Amat A. 1988. Uso de los caracteres histofoliares para
identificar las especies argentinas del gnero
Achyrocline DC. (Asteraceae). Acta Farmacutica
Bonaerense 7: 75-83.
Farmacopeia Brasileira 4 ed., vol.1, 2003. Ateneu, Rio de
Janeiro.
Gattuso S, Gattuso M. 1998. Caracteres anatmicos y
exomorfolgicos distintivos de Achyrocline
satureioides (Lam.) DC. (Asteraceae-Inuleae). Acta
Farmacutica Bonaerense 17: 255-61.
Gattuso M, Cortadi A, Rodrguez MV, Mc Cargo J, Retta D,
Bandoni A, Ferraro

G, Gattuso S.

2008. Caracteres
florales de A. satureioides, A. flaccida y G.
gaudichaudianum. Bol Latinoam Caribe Plant Med
Aromat 7: 247-256.
Giangualani R. 1976. Las especies argentinas del gnero
Achyrocline (Compositae). Darwiniana 20: 549-576.
Hieronymus J. 1882. Plantae Diaforicae, Florae
Argentinae. Ed. Boletn de la Academia Nacional de
Ciencias en Crdoba, Buenos Aires, Argentina, Tomo
4, pp. 344.
Labuckas D, Maestri D, Grosso N, Zygadlo J. 1999.
Essential oils of Achyrocline satureioides, Achyrocline
alata and Achyrocline tomentosa. Planta Med 65: 184-
186.
Martnez Crovetto R. 1981. Plantas utilizadas en la
medicina popular del NO de Corrientes. Ministerio de
Cultura y Educacin, Fundacin Miguel Lillo,
Tucumn, Argentina, pp.106, 111.
Nesom G. 1990. Taxonomy of Achyrocline (Asteraceae:
Inuleae) in Mexico and Central America. Phytologia
68: 181-185.
Parodi D. 1886. Plantas usuales del Paraguay, de Corrientes
y de Misiones. Ed. Coni, Buenos Aires, Argentina.
Petenatti E, Nievas C, Petenatti M, Del Vitto L. 2004.
Medicamentos Herbarios en el Centro-oeste Argentino,
IV. Marcelas y Vira-vira en Muestras Comerciales.
Acta Farmacutica Bonaerense 23: 484-491.
Ratera E, Ratera M. 1980. Plantas de la Flora Argentina
Empleadas en Medicina Popular. Ed. Hemisferio Sur.
Buenos Aires. Argentina, pp. 62-64.
Retta D, Di Leo Lira P, van Baren C, Elechosa M, Jurez M,
Molina A, Bandoni A. 2009. Composicin del aceite
esencial de las partes areas de Achyrocline
satureioides provenientes de ocho poblaciones del
centro de Argentina. En Resmenes del Primer
Congreso Internacional de Farmacobotnica de Chile y
Tercera Reunin BLACPMA, Chilln, Chile.
Retta D, Gattuso M, Gattuso S, Di Leo Lira P, van Baren C,
Ferraro G, Bandoni A. 2009. Essential oil composition
of Achyrocline flaccida (Weinm.) DC. (Asteraceae)
from different locations of Argentina. Biochem
Systemat Ecol 36: 877-881.
Toursarkissian M. 1980. Plantas medicinales de la
Argentina. Hemisferio Sur S. A. Buenos Aires, pp. 25-
32.
Wiley/NBS. 2008. The Wiley/NBS Registry of Mass
Spectral Data. Wiley & Sons, Inc., New York, NY.
Zuloaga F y Morrone O. (eds) 1999. Catlogo de las plantas
vasculares de la Repblica Argentina II. Missouri
Botanical Garden Press.

2010 The Authors




Este es un articulo de Acceso Libre bajo los trminos de una licencia Creative Commons Atribucion-No Comercial-No trabajos derivados 3.0 Internacional (http://creativecommons.org/licenses/by-nc-
Photoprotective activity of Yucca periculosa polyphenols
[Actividad fotoprotectora de los polifenoles de Yucca periculosa]
Ana Mara GARCA-BORES
1,4-
, Christian BELLO
1
, Yukiko CAMPOS
1
, Jos del Carmen BENITEZ
2
, Saul FLORES
3
,
Margarita CANALES
1
, Tzasn HERNNDEZ
1
, Jos Guillermo AVILA ACEVEDO
1

1
Laboratorio de Fitoqumica, UBIPRO,

Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autnoma de Mxico,
Tlalnepantla 54090, Edo. de Mxico, Mxico;
2
Laboratorio 1, UMF, Facultad de Estudios Superiores-Iztacala, Universidad Nacional
Autnoma de Mxico, Tlalnepantla 54090, Edo. de Mxico, Mxico;
3
Laboratorio de Recursos Naturales, UBIPRO, Facultad de Estudios
Superiores-Iztacala, Universidad Nacional Autnoma de Mxico, Tlalnepantla 54090, Edo. de Mxico, Mxico
4
Posgrado en Ciencias
Biolgicas, Universidad Nacional Autnoma de Mxico
Abstract
The aim of this work was to investigate the potential utility of the methanolic extract of the bark of Yucca periculosa, as well as trans-3,3',5,5'-
tetrahydroxy-4'-methoxystilbene (MS), resveratrol, and naringenin for their potential as photochemopreventive agents. All substances have photoprotective
effect against UV-B induced cell death in Escherichia coli, with MS and resveratrol showing the highest photoprotective properties. The sun protection factor
(SPF) of the substances was evaluated by a guinea pig bioassay and a histopathological skin study. All substances prevented skin damage induced by UV and
have an SPF higher than the octyl methoxycinnamate (OMC) a commercial sunscreen. The results showed the trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene
isolated from Y. periculosa may afford substantial protection against the damages caused by UV exposure.

Keywords: Photoprotective activity; polyphenols; trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene; Yucca periculosa.
Resumen
El objetivo de este trabajo fue investigar el efecto fotoprotector del extracto metanlico de la corteza de Yucca periculosa, y de las sustancias
aisladas del extracto: el trans-3,3',5,5'-tetrahidroxi-4'-metoxiestilbeno (MS), el resveratrol y la naringenina. Todas las susbstancias tuvieron un efecto
fotoprotector al evitar la muerte cellular de Escherichia coli inducida por la radiacin UV, siendo el MS y el resveratrol los que poseen mayores propiedades
fotoprotectoras. El factor de proteccin solar (FPS) de las sustancias se evalu en cobayos y mediante un estudio histopatolgico de la piel irradiada con UV.
Se determin que todas las sustancias previenen del dao histolgico en la piel inducido por la UV, adems poseen un FPS mayor que el octil
metoxicinnamato OMC, un filtro solar comercial. Los resultados muestran que el MS de Y. periculosa presenta un mayor efecto fotoprotector.

Palabras Clave: Actividad fotoprotectora; polifenoles; trans-3,3',5,5'-tetrahidroxi-4'-metoxiestilbeno; Yucca periculosa.

Recibido | Received: July,17, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: December 21, 2009.
Publicado en Lnea | Published Online 25 March 2010
Financiacin | Funding: This work was partially financed by grant 52485 CONACYT, IN213309 PAPIIT-DGAPA-UNAM and an internal grant from FES-Iztacala PAPCA-
UNAM
This article must be cited as: Ana Mara Garca-Bores, Christian Bello, Yukiko Campos, Jos del Carmen Benitez, Saul Flores, Margarita Canales, Tzasn Hernndez, Jos
Guillermo Avila. Photoprotective activity of Yucca periculosa polyphenols. Bol Latinoam Caribe Plant Med Aromat 9(2):100 108. {EPub 25 March 2010 }.
*Contactos | Contacts: E-mail: boresana@yahoo.com; Tel.: + 52-5-623-11-36; Fax + 52-5-623-12-25;

Garca-Bores et al. Photoprotective activity of Yucca periculosa stilbenes


INTRODUCTION
Yucca periculosa Baker is a plant of the Agavaceae
family, which is native to the states of Oaxaca, Puebla,
Tlaxacala and Veracruz in Mxico. The bark of this
plant was used in traditional medicine as anti-
inflammatory and to treat pain caused by ear infections
(Aguilar et al. 1994). In previous studies in our
laboratory found that the methanolic extract contains
polyphenolic compounds with antioxidant properties:
resveratrol and trans-3,3',5,5'-tetrahydroxy-4'-
methoxystilbene (MS) (Torres et al. 2003). The MS
showed strong radical scavenging and even stronger
antiplatelet activity than did resveratrol (Piacente et al.
2004). We also isolated from Yucca periculosa a
flavanone, naringenin. Naringenin possess some
antioxidant activity, though its activity is poorer in
comparison with many other phenols (Erlund, 2004).
Botanical antioxidants have attracted considerable
attention because of their skin photoprotective effects.
This has generated a great interest in using topical
antioxidants for the prevention of photocarcinogenesis
and photoaging. Exposure of the skin to ultraviolet
(UV) radiation from the sun, particularly to its UV-B
component (280-320 nm), can result in erythema,
edema, hyperplasia, hyperpigmentation, sunburn cells,
immunosuppression, photoaging, and skin cancer
(Afaq and Mukhtar, 2006). Recent changes in lifestyle
and the decrease in the earths ozone layer have led to
a significant increase in the amount of UV-B radiation
that people receive, leading to a surge in the incidence
of skin cancer and photoaging. As these trends are
likely to continue in the foreseeable future, the adverse
effect of UV-B has become a major human health
concern (Baliga and Katiyar, 2006; Nichols and
Katiyar, 2009).
A growing awareness of the risks associated with
skin exposure to UV-B radiation over the past decades
has led to increased used of sunscreen products.
Sunscreens provide protection from UV-B radiation by
producing a protective layer on the skin in which UV
light is absorbed by organic compounds (Rodil and
Moeder, 2008). Sunscreens are useful, but their
protective properties are not adequate enough to
prevent the risk of UV-induced skin cancer, due to
their inadequate use, incomplete spectral protection,
and toxicity (Baliga and Katiyar, 2006). Therefore, the
development of novel strategies to reduce the
occurrence of skin cancer and delay the process of
photoaging is a highly desirable goal. One approach to
reduce the occurrence of skin cancer and photoaging is
through photochemoprevention, which is defined as
the use of agents capable of ameliorating the adverse
effects of UV-B on the skin (Afaq and Mukhtar, 2006;
Baliga and Katiyar, 2006; Nichols and Katiyar, 2009).
In recent years, considerable interest has been focused
on identifying naturally occurring botanicals for the
prevention of photocarcinogenesis. A wide variety of
botanicals, mostly flavonoids and other phenolic
substances, have been reported to possess substantial
anticarcinogenic and antimutagenic activities, due to
their antioxidant, anti-inflammatory and sunscreens
properties (Afaq and Mukhtar, 2006; Baliga and
Katiyar, 2006; Adhami et al. 2008) or promote the
repair of molecules like DNA adducts (Nichols and
Katiyar, 2009).
One of the most studied compounds is resveratrol.
It was showed to possess the potential to ameliorate
the damage caused by UVB exposure (acute,
semichronic and chronic) to SKH-1 mice. It appears
that the protective effects of resveratrol are mediated
via its antioxidant potential and its ability to modulate
cell cycle and apoptosis signaling pathways (Reagan-
Shaw et al. 2008), although has not been determined
its sunscreen potential.
Most of the natural polyphenols can absorb UV
radiation. Therefore, when applied topically, they can
prevent penetration of the radiation into the skin like a
sunscreen. This ability of natural polyphenols as
sunscreens can reduce the damage induced by UV in
the skin (Nichols and Katiyar, 2009). The present
study was designed to estimate the potential utility of
topically applied polyphenolic compounds isolated
from Y. periculosa like photochemopreventive agents.
With this aim, we evaluated the photoprotective effect
of the methanolic extract, stilbenes and naringenin of
Yucca periculosa against UV-B induced cell death and
UV-B induced skin damage in guinea pigs as well as
to obtain the sun protection factor.
Plant material
Yucca periculosa Baker (Agavaceae) bark was
collected in September 2004 in Zapotitln de las
Salinas, Puebla, Mxico and it was identified by Dra.
Edith Lopez Villafranco of the IZTA Herbarium at the
Factultad de Estudios Superiores Iztacala, UNAM. A
voucher specimen was deposited at the IZTA
herbarium (Voucher n IZTA 27516).


Extraction procedure
Dried and ground barks of Y. periculosa (1,000 g)
were extracted at room temperature with methanol.
The MeOH extract was filtered and concentrated under
vacuum. The remaining residue from the methanolic
extract was redissolved in MeOH, and hexane was
added to it in a separatory funnel. After solvent-
solvent extraction, the fat-free methanolic extract was
removed from the hexane portion. The methanol layer
was evaporated under reduced pressure, and the
extract was kept in the dark at 4 C until it was tested.
The MeOH extract yield was 24.1% w/w, and the
extract yield of the hexane portion was 0.8%. Ten
grams of the MeOH extract was used in the bioassays,
and 230 g was submitted to silica gel column
chromatography (G60, Merck) as the solid phase.
Elution was carried out with CHCl
3
:MeOH mixtures.
Fractions of CHCl
3
:MeOH (95:5, 9:1 and 8:2) gave the
following natural products: naringenin (0.718 g) (1),
resveratrol (0.348 g) (2), and trans-3, 3', 5, 5'-
tetrahydroxy-4'-methoxystilbene (MS) (2.143 g) (3),
respectively. The pure substances were analyzed and
characterized by their Rf, UV and
1
H NMR
spectroscopic data (Fig. 1). Identification of the
compounds was conducted by both spectroscopic
analyses and direct comparisons with authentic
samples (Wenker and Gottlieb, 1977; Oleszek et al.
2001; Torres et al. 2003).
Protective effect against UV-B induced cell death
A strain of Escherichia coli (ATCC 25922)
was grown in a heart and brain infusion broth (Bioxon-
112) until the culture reached a concentration of 10
7

cells/mL (O.D. 0.3 read at 550 nm). The bacteria were
centrifuged for 10 min at 5000 rev/min, suspended in
Ringer PBS (pH 7.0), and transferred into quartz
cuvettes (Pye Unicam B53875 A, thickness 1 mm,
capacity 4 mL). Each photoprotective substance was
dissolved in MeOH (2 mg/mL) and put in a quartz
cuvette. A cuvette containing bacteria was placed
behind the cuvette containing the photoprotective
substance, forming one experimental unit. The
experimental units were irradiated with a UV-B lamp
(312 nm, Spectroline EB-280C), with an irradiation
dose of 0.60 J/cm
2
(Avila et al. 2005). The number of
surviving bacteria was detected in accordance with the
dilution method at different time periods. The
substances employed were the Y. periculosa MeOH
extract, naringenin, resveratrol and MS. The positive
control was octyl methoxycinnamate (OMC) (ISP
VAN DIK) and the negative control was MeOH. Tests
were repeated in at least three independent
experiments and the assays were performed in
triplicate.
The mortality rate (K) was calculated by linear
regression analysis with Microsoft Excel.

Fig. 1. Structures of naringenin 1, resveratrol 2, and trans-3, 3', 5,
5'-tetrahydroxy-4'-methoxystilbene (MS) 3.

1

2

3
Photoprotective activity against UV-B induced skin
damage
Adult female guinea pigs of the Hartley strain
weighing 300-350 g were used in this study. The
animals were selected at random for each group. The
animals were reared on laboratory chow, fed ad
libitum, and had free access to water at all times. The
room was maintained at 22 2 C with natural
daylight. All animal experiments in this study were
approved by the Institutional Biosecurity and Animal
Ethics Committee.
O
O
OH
HO
HO
OH
OH
HO
HO
OH
OMe
OH
HO


The dorsal skin of the guinea pigs to be irradiated
was shaved with an electric clipper (Oster Mod 274-
01), followed by application of hair removal cream
(Velvinette-Wella) a day before exposure of the
animals to UV-B radiation. The skin was rinsed under
warm tap water and dried. After 24 h, the dorsal skin
was treated with 2 mg/cm
2
of the photoprotective
substances OMC, Y. periculosa MeOH extract,
naringenin, resveratrol, MS, or vehicle (setting gel-Stil
Net) or was left untreated. Five animals at a time
from each group were then wrapped with 7.5 cm wide
tape containing six exposure windows (three windows
each side of the spinal line) 2.0 cm
2
in area. After 15
min, the animals were placed under a bank of 5 UV-B
lamps (312 nm, Spectroline EB-280C) with an
irradiation of 0.60 J/cm
2
. All irradiance measurements
were performed using a calibrated radiometer
(Spectroline DM-300HA) at the same distance from
the lamps as used during cutaneous exposures.
Irradiation times of the six exposure windows on each
animal were set to bracket the suspected SPF of the
substance being tested. The exposure windows were
covered at the end of each time point. After irradiation,
the tape was removed from the animals (Bissett et al.
1991; Avila et al.2005).
Sun protection factor
Sunburn erythema is the most conspicuous and
well-recognized acute cutaneous response to UV
irradiation, and it is the most widely used endpoint in
dermatological photobiology. The molecules
responsible for light absorption (chromophores) that
initiate sunburn inflammation have not been precisely
identified. However, the action spectrum of erythema
is consistent with the hypothesis that UV interactions
with DNA are of major importance. Indirect oxidative
damage might also occur secondarily to endogenous
photosensitization reactions (Matsumura and
Ananthaswamy, 2004). A widely accepted method for
sunscreen efficacy measurement is SPF, which is
defined as the ratio of the dose of UVR (290-400 nm)
required to produce 1 MED (Minimal Erythema Dose)
on sunscreen-protected skin (after application of 2
mg/cm
2
of product) over the dose required to produce
1 MED on unprotected skin (Bissett et al. 1991).
Visual assessment of skin reaction (perceptible
unambiguous erythema) was performed 16-24 h after
UV-B exposure by three trained observers in the same
room under the same lighting conditions. For each
animal, the MED on unprotected skin and that on skin
protected by the substances were recorded. A
statistical analysis was performed on all the collected
data. The non-parametric methods for Kruskal-Wallis
and Mann-Whitney U-tests were used to determine the
level of significance against the vehicle in each of the
experimental SPF determinations. P-values less than
0.05 were considered statistically significant.
Histopathological study
After 24 h of irradiation, the animals were
sacrificed using sodium pentobarbital. The UV-B
exposed portion of cutaneous tissue was quickly
removed and fixed in 10% buffered formalin,
embedded in paraffin, and sectioned at 6 m. Sections
were stained with hematoxylin and eosin dyes (H&E
stain). Five slides were checked for each of the five
animals, and photomicrographs were obtained using a
Nikon Labophot-2 microscope with a Nikon Coolpix
digital camera.

Table 1. Sun protection factor (SPF) in guinea pigs

Compounds
(2 mg/cm
2
)
SPF Exposition time without
erythema (min)
Without protection - 20 2.0
OMC
*

2.0 0.1 40 4.5
MeOH extract
*+

3.4 0.5 68 9.5
Naringenin
*+

3.6 0.6 72 10.1
Resveratrol
*

5.0 0.7 100 12.3
MS
*^

5.6 0.5 112 8.5
OMC: octyl methoxycinnamate; MS: trans-3, 3, 5, 5-
tetrahydroxy-4-methoxystilbene. *p< 0.05 statistical significance
compared with the group without protection, + p< 0.05 statistical
significance compared with the group with OMC, p< 0.05
statistical significance compared with the groups with resveratrol.



Figure 2. Protective effect against UV-B induced cell death of E. coli.
A

B

A) Without protection (K= 0.87, R
2
=0.93). B With protection, OMC (+) (K= 0.13, R
2
=0.99); MeOH extract (^) (K= 0.12, R
2
=0.99); 1:
Naringenin (o) (K= 0.25, R
2
=0.99); 2: Resveratrol (-) (K= 0.09, R
2
=0.93); 3: MS, trans-3, 3, 5, 5-tetrahydroxy-4-methoxystilbene () (K=
0.07, R
2
=0.85). Each group represents the mean of three independent experiments.
-2
-1
0
1
2
3
4
5
6
7
8
9
0 2 4 6 8 10 12
Time (minutes)
L
o
g

#

s
u
r
v
i
v
o
r
s
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40 50 60 70 80 90 100
Time (minutes)
L
o
g

#

s
u
r
v
i
v
o
r
s
OMC MeOH extract 1 2 3


Figure. 3. Histology of skin of UVB-irradiated mice treated with polyphenols of Yucca periculosa.
Sub-minimal erythema dose (S-MED) 20 Minimal erythema dose (MED). 20
a) Normal skin

b) Skin without protection MED= 20 min,

c) OMC S-MED (20 min) and MED (40 min)

d) MeOH extract S-MED (~40min) and MED (~70 min),

e) 1: Naringenin S-MED (~40 min) and MED (~ 70 min)



Sub-minimal erythema dose (S-MED) 20 Minimal erythema dose (MED). 20
f) 2: Resveratrol S-MED (~80 min) and MED (~100 min)
,

g) 3: MS S-MED (~80 min) and MED (~120 min)

D dermis; DK dyskeratosis; EP epidermis; PE perivascular edema; PI perivascular infiltration; SbC Sunburn cell; SC stratum corneum;
SP spongiosis; TEP Thickening epidermis..
Photoprotective activity
The protective effect against UV-B induced cell
death was evaluated using E. coli as a cell model. The
results showed that the bacteria population (~10
7
)
without protection reached cell death at 10 min, with a
mortality rate of 0.87 (Fig 2A). Naringenin possesses
pronounced photoprotective activity when compared
to the negative control, though the results show that it
was less active than OMC as a positive control (cell
death at 35 min). The MeOH extract and resveratrol
both protected their respective bacteria populations in
a similar manner and with OMC did not reach cell
death until 60 min. The MS protected the bacteria
more efficiently than the positive control did; the
bacteria population protected by this compound did
not reach cell death until 90 min of irradiation with
UV-B (Fig 2B).
The constant mortality K is a parameter that
indicates the range of inactivation of E. coli. The data
in Fig. 2 show the photoprotective effect of the
phenols of Y. periculosa tested. In the present work, all
substances (OMC, methanolic extract, naringenin,
resveratrol and MS) protected the bacterial population
from the lethal effects of UVR. All substances
presented K values lower than experiments without
protection (Fig 2B). In the experiments with
protection, the K ranged between 0.07 and 0.25. MS
showed a strong photoprotective effect against UV-B
induced cell death; the K (0.07) was 12.5-fold below
the K without protection (0.87). The bacterial decay
depends mainly on the dose of radiation that induces
damage to DNA. E. coli was inactivated when exposed
to UV. The effectiveness of UV light in the biological
inactivation primarily results from the fact that DNA
molecules absorb UV photons between 200 and 320
nm, with peak absorption at 265 nm. In case of lethal
damage, DNA replication is blocked by DNA
alterations, mainly cyclobutane pyrimidine dimer
(CPD) and the pyrimidine (64) pyrimidinone (6
4PP), which ultimately results in reproductive cell
death. The exposure of a bacterial culture to UV-B
produces the rapid decline in population due to
damage to the DNA (Oguma et al. 2001; Taghipour,
2004).
The SPF values of the tested substances in this
study were determined on guinea pigs (Table 1). The
negative control (guinea pigs with vehicle) showed
perceptible erythema at 20 2 min; this time was
considered as the MED. All the substances of Y.
periculosa were more active than OMC (SPF 2.0


0.1), because a significant difference was observed in
comparison with controls (p<0.05). The SPFs obtained
for the MeOH extract and for naringenin were 3.4
0.5 and 3.6 0.6, respectively. Resveratrol and MS
were the compounds with the highest photoprotective
activity (p<0.05). Their SPFs were 5.0 0.7 and 5.6
0.5, respectively. These compounds also retarded the
appearance of erythema at about ~100 min. The
methanolic extract and the three isolated compounds
have maximum absorptions in the UV-B region of the
electromagnetic spectrum and are, therefore,
potentially photoprotective substances. These
substances absorb in the UV-B region as follows:
naringenin (
max
288 nm), resveratrol (
max
305 nm)
and MS (
max
316 nm). In addition, resveratrol and MS
have molar extinction coefficients higher than the
OMC (c 37 800 M
-1
cm
-1
and c 45 300 M
-1
cm
-1
,

respectively). These results explain the protective
properties of the methanolic extract and the
compounds isolated in both models against UV-B
induced cell death and skin damage.
Sunscreens have long been used to protect against
the acute effects of UVR. OMC is a widely used UV-B
filter in various cosmetic formulations. It is known that
all organic sunscreen agents may induce adverse
effects such as irritation, allergic contact reaction,
photoallergy, or phototoxicity. Kullavanijaya and Lim
(2005) reported photosensitization and/or
photoallergic reactions induced by this compound.
Previous studies have shown that when exposed to
sunlight, this UV-B filter will change from octyl-p-
methoxy-trans-cinnamate (E-OMC) to octyl-p-
methoxy-cis-cinnamate (Z-OMC). The study showed a
hypsochromic shift and reduction of the molar
extinction coefficient (trans:
max
310 nm, c 24 000M
-
1
cm
-1
; cis:
max
301 nm, c 12600M
-1
cm
-1
) (Tarras-
Wahlberg et al. 1999).
A histological evaluation was also performed on
the guinea pig skin exposed to UVR, and both the
unprotected skin and the skin protected by each of the
substances. The histological changes after 20 minutes
of UV irradiation in guinea pig skin compared with
normal skin (Fig. 3a) included thickening of stratum
corneum and epidermis, intra-/intercellular and
perivascular edema, perivascular infiltration,
dyskeratosis, and spongiosis, as shown in Fig. 3b. The
guinea pigs treated with a topical, sub-minimal
erythema dose of the methanolic extract, the isolated
compounds or OMC did not show these UVB-induced
inflammatory changes, as shown in the left panels of
Figs. 3c to 3g. The histopathological study of the skin
samples exposed at MED with protection showed that
a topical application of each of the experimental
treatments had a different effect on the skin, which
could be an indication that the protection was also
linked to the modulation of cellular processes. The
appearance of erythema in animals treated with
resveratrol and MS occurred at ~ 100 minutes, while
those animals treated with naringenin or the methanol
extract had an appearance of erythema ~ 70 minutes.
Finally, those animals treated with OMC had an
appearance of erythema ~ 40 minutes of exposure to
RUV. Many agents, like ultraviolet light filters, affect
the transmission of ultraviolet light to human skin. In
addition, there are agents, such as antioxidants, that
can modulate the effects of ultraviolet light on the
skin. Most of the naturally occurring
chemopreventitive polyphenolics exert multifaceted
action, and any clinical applications using these
substances should be based on the precise
understanding of the physiologically relevant action
mechanisms. Treatment of UVB-irradiated HaCaT
cells with naringenin enhances the removal of CPD
from the genome, as observed by both direct
quantitation of CPD in genomic DNA and immuno-
localization of the damage within the nuclei.
Naringenin could protect skin from UVB-induced
damage and carcinogenesis via an inhibition of
excessive apoptosis and accelerated elimination of
UVB-induced promutagenic and precarcinogenic CPD
lesions (El-Mahady et al. 2008). Resveratrol imparts
protection from short-term UV-B exposure-mediated
cutaneous damages in SKH-1 hairless mice (Afaq et
al. 2003; Reagan-Shaw et al. 2008). MS has anti-
inflammatory and antiplatelet properties and prevents
the carbonylation of blood proteins (Wenzig et al.
2008). It is necessary to study the molecular
mechanisms of MS-mediated protection of UV-B
damage of skin. In the case of the methanolic extract,
the effect on the modulation of cellular processes
could be diverse, because it is a mixture of
compounds. It is also made up of other polyphenolic
compounds that are polar in nature and that may
interact with the cellular components of skin.
CONCLUSION
The increase in skin cancer morbidity and mortality
is alarming and expensive, in both human and
economic terms. New strategies are needed to combat
this disease. The development of promising
chemopreventitive agents is a demanding process that
requires continuous dedication and funding for the


development of agents from start to finish. The
research of natural products with chemopreventitive
properties has focused on the antioxidant, anti-
inflammatory and antimutagenic activities of the
compounds. In addition, this study shows that the
polyphenolic compounds isolated from Y. periculosa,
in particular, MS, are able to absorb UVR, reducing
the transmission of this type of radiation to the skin
and this is the first report of MS as a photoprotective
agent. These compounds thus provide photoprotection
due to their antioxidant properties and act as a
sunscreen.
ACKNOWLEDGEMENTS
The authors are grateful to Edith Lpez Villafranco
and Rosario Gonzlez Valle for their technical
assistance. This work was partially financed by grant
52485 CONACYT, IN213309 PAPIIT-DGAPA-
UNAM and an internal grant from FES-Iztacala
PAPCA-UNAM.
REFERENCES
Adhami VM, Syed DN, Khan N, Afaq F. 2008.
Phytochemicals for prevention of solar ultraviolet
radiation-induced damages. Photochem Photobiol 84:
489-500.
Afaq F, Adhami VM, Ahmad N. 2003. Prevention of short-
term ultraviolet B radiation-mediated damages by
resveratrol in SKH-1 hairless mice. Toxicol Appl
Pharmacol 18: 28-37.
Afaq F, Mukhtar H. 2006. Botanical antioxidants in the
prevention of photocarcinogenesis and photoaging. Exp
Dermatol 15: 678-684.
Aguilar A, Argueta A, Cano L, Ballesteros L. 1994. Flora
Medicinal Indgena de Mxico. Vol II. Instituto
Nacional Indigenista. Mxico D. F. pp. 950
Avila JGA, Castaeda CMC, Benitez FJC, Durn DA,
Barroso VR, Martnez CG, Muoz LJL, Martnez CA,
Romo de Vivar A. 2005. Photoprotective activity of
Buddleja scordioides. Fitoterapia 76: 301-309.
Baliga MS, Katiyar SK. 2006. Chemoprevention of
photocarcinogenesis by selected dietary botanicals.
Photochem Photobiol Sci 5: 243-53.
Bissett DL, McBride JF, Hannon DP, Patrick LF. 1991.
Time-dependent decrease in sunscreen protection
against chronic photodamage in UVB-irradiated
hairless mouse skin. J Photochem Photobiol B 9: 323-
34.
El-Mahdy MA, Zhu Q, Wang QE, Wani G, Patnaik S, Zhao
Q, Arafa S, Barakat B, Mir SN, Wani AA. 2008.
Naringenin protects HaCaT human keratinocytes
against UVB-induced apoptosis and enhances the
removal of cyclobutane pyrimidine dimers from the
genome. Photochem Photobiol 84: 307-16.
Erlund I. 2004. Review of the flavonoids quercetin,
hesperetin and naringenin. Dietary sources,
bioactivities, bioavailability, and epidemiology. Nutr
Res 24: 851-874.
Kullavanijaya P, Lim HW. 2005. Photoprotection. J Am
Acad Dermatol 52: 937-958.
Matsumura Y, Ananthaswamy HN. 2004. Toxic effects of
ultraviolet radiation on the skin. Toxicol Appl
Pharmacol 195: 298-308.
Nichols JA, Katiyar SK. 2009. Skin photoprotection by
natural polyphenols: anti-inflammatory, antioxidant and
DNA repair mechanisms. Arch Dermatol Res Nov 7.
Oguma K, Katayama H, Mitani H, Morita S, Hirata T,
Ohgaki S. 2001. Determination of pyrimidine dimers in
Escherichia coli and Cryptosporidium parvum during
UV light inactivation, photoreactivation, and dark
repair. Appl Environ Microbiol 67: 46304637.
Oleszek W, Sitek M, Stochmal A, Piacente S, Pizza C,
Cheeke P. 2001. Resveratrol and other phenolics from
the bark of Yucca schidigera Roezl. J Agric Food Chem
49: 747-752.
Piacente S, Montoso P, Oleszek W, Pizza C. 2004. Yucca
schidigera bark: phenolic constituents and antioxidant
activity. J Nat Prod 67: 882-885.
Reagan-Shaw S, Mukhtar H, Ahmad N. 2008. Resveratrol
imparts photoprotection of normal cells and enhances
the efficacy of radiation therapy in cancer cells.
Photochem Photobiol 84(2): 415-421.
Rodil R, Moeder M. 2008. Development of a method for the
determination of UV filters in water samples using stir
bar sorptive extraction and thermal desorption-gas
chromatography-mass spectrometry. J Chromatogr A
1179: 81-88.
Taghipour T. 2004. Ultraviolet and ionizing radiation for
microorganism inactivation. Water Res. 38: 39403948.
Tarras-Wahlberg N, Stenhagen G, Lark O, Rosn A,
Wennberg AM, Wennerstrm O. 1999. Changes in
ultraviolet absorption of sunscreens after ultraviolet
irradiation. J Invest Dermatol 113: 547-553.
Torres P, Avila JG, Romo de Vivar A, Garca AM, Marn
JC, Aranda E, Cspedes CL. 2003. Antioxidant and
insect growth regulatory activities of stilbenes and
extracts from Yucca periculosa. Phytochemistry 64:
463-473.
Wenker E, Gottlieb HE. 1977. Carbon-13 nuclear magnetic
resonance spectroscopy of flavonoid and isoflavonoid
compounds. Phytochemistry 16: 1811-1816.
Wenzig EM, Oleszek W, Stochmal A, Kunert O, Bauer R.
2008. Influence of phenolic constituents from Yucca
schidigera bark on arachidonate metabolism in vitro. J
Agric Food Chem 56: 8885-8890.

2010 The Authors
Articulo Original | Original Article



Los remedios naturales en la prevencin y cuidado de la salud oral de
los tobas del Chaco Central (Argentina)
[Natural remedies in the prevention and oral health care of the Toba from Central Chaco (Argentina)]
Gustavo J. MARTNEZ
1,2*

1 Museo de Antropologa. Facultad de Filosofa y Humanidades. Universidad Nacional de Crdoba. Hiplito Irigoyen 174. 5000. Crdoba,
Repblica Argentina;2 CONICET.
Abstract
This contribution documents the use of natural medicines (plant and animal) in the prevention and oral health care among the Toba from Central
Chaco (Argentina). Characterized by its multiple etiologies, bucco-dental conditions are of relevant importance in the health of these communities, since they
imply 59 uses corresponding to 49 species (34 plants and 15 animals) belonging to 42 families (28 plants and 14 animals) used for this purpose. The list of
species with the highest proportion of citations are headed by native plants, highlighted by its consensus the symbolic use of the climber Clematis
montevidensis, and use of the roots of Solanum argentinum, Cucurbitella asperata and the aerial part of Schinus fasciculatus var. fasciculatus and Petiveria
alliacea var. alliacea, all for the treatment of toothache. Among the remedies of animal origin with greater consensus is indicated the use of the ashes of the
mollusk Anodontites trapesialis for the treatment of thrush and oral ulcerations.

Keywords: plant and animal pharmacopoeia; Chaco; bucco-dental diseases; ethnomedicine

Resumen
Esta contribucin documenta el uso de la farmacopea natural (vegetal y animal) en la prevencin y cuidado de la salud oral, entre los tobas del
Chaco Central (Argentina). Caracterizada por sus mltiples etiologas, las afecciones buco-dentales adquieren relevancia en la salud de estas comunidades,
documentndose 59 aplicaciones para 49 especies (34 vegetales y 15 animales) pertenecientes a 42 familias (28 vegetales y 14 animales) empleadas con este
fin. El listado de especies con mayor proporcin de citas se encuentra encabezada por plantas nativas, destacndose por su consenso el uso simblico de la
liana Clematis montevidensis, y el empleo de las races de Solanum argentinum, Cucurbitella asperata y de la parte area de Schinus fasciculatus var.
fasciculatus y de Petiveria alliacea var. alliacea, todas ellas destinadas al tratamiento de odontalgias. Entre los remedios de origen animal con mayor
consenso se seala el uso de las cenizas del molusco Anodontites trapesialis para el tratamiento de aftas y llagas bucales.

Palabras Clave: farmacopea animal y vegetal; Chaco; afecciones buco-dentales; etnomedicina.

Recibido | Received: December 14, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: February 1, 2010.
Publicado en Lnea | Published Online: March 25, 2010.
Declaracin de intereses | Declaration of interests: Authors have no competing interests.
Financiacin | Funding: Proyectos Anpcyt/ Foncyt Pict 32894 y 1612
This article must be cited as: Gustavo J. Martnez. 2010. Los remedios naturales en la prevencin y cuidado de la salud oral de los tobas del Chaco Central (Argentina). Bol
Latinoam Caribe Plant Med Aromat 9(2):109 122. {EPub March 25, 2010}.

*Contactos | Contacts:. E-mail: gustmart@yahoo.com

Martnez Los remedios naturales en la salud oral de los tobas del Chaco Central


INTRODUCCION
La documentacin y registro de usos de plantas
medicinales en el tratamiento de afecciones buco-
dentales constituye uno de los diversos tpicos que se
abordan en estudios etnobotnicos, presentndose la
mayora de las veces en forma marginal o sumaria.
Sin embargo, el empleo de la farmacopea natural en
el manejo de las dolencias vinculadas con la cavidad
oral resulta usual en las medicinas tradicionales y
alternativas, siendo considerable la cantidad de
especies implicadas en su tratamiento. Los estudios
que abordan especficamente esta temtica refieren,
por citar algunos ejemplos, el uso de 35 especies
vegetales en la India (Hebbar et al., 2004), 62
especies en Burkina Faso (Tapsoba & Deschamps,
2006), 51 especies en Mxico (Waizel-Bucay &
Martnez Rico, 2007) y ms de 100 plantas
comercializables en Chicago, Costa Rica y Colombia
(Colvard et al., 2006), as como ensayos de efectos
antimicrobianos de especies seleccionadas con este
fin (Babpour et al., 2009). Por su parte, Colvard et al.
(2006) exponen el listado de escasas publicaciones
que describen la etnografa, etnomedicina,
etnofarmacologa y/o aplicaciones basadas en
evidencias clnicas de plantas medicinales usadas
especficamente en odontologa, odontalgias y otras
afecciones orales, a la vez que sealan la ausencia de
un catlogo de referencia que describa las plantas
usadas con este fin.
En regiones en las que la atencin
odontolgica resulta inaccesible, el empleo de
remedios naturales constituye una opcin plausible,
en particular si se cuenta con un repertorio de
especies de probada eficacia farmacolgica que
posibilite su implementacin en atencin primaria. El
conocimiento de estos usos propicia la incorporacin
de la medicinas tradicionales y eventualmente la
prescripcin de etnofrmacos naturales en el sistema
local de salud, acorde con los criterios sugeridos por
la WHO (2002) para contextos de medicinas
mltiples como el que abordamos en este trabajo.
Los tobas, conocidos tambin como qom o
qomlek, son un grupo indgena integrante de la
familia lingstica Guaycur que conforman una
poblacin de bandas aliadas de unos 60.000
integrantes, cuyo hbitat se encuentra hoy en forma
mayoritaria en el Chaco Central y Austral (en las
provincias argentinas de Chaco y Formosa) y un
pequeo ncleo en el Chaco Boreal paraguayo
(ENDEPA, 1986; Arenas, 1997; Censabella, 2000)
Informaciones previas sobre la etnobotnica de
diversas parcialidades tobas puede encontrarse en los
trabajos de Franze (1925) Martnez Crovetto (1964,
1968), Vuoto P. (1981, 1999) Arenas (2000),
Martnez (2007a, 2008), Hecht et al. (2008), junto a
otros de carcter etnozoolgico (Zacaras, 1993;
Martnez Crovetto, 1995; Vuoto L., 1999; Arenas,
2003). Todos estos trabajos evidencian un gran
aprovechamiento de los recursos naturales por parte
de los nativos, y ponen de relieve la existencia de una
vasta farmacopea natural como uno de los
componentes que le da riqueza a su cultura.
El presente trabajo detalla el uso de la
farmacopea natural (vegetal y animal) en la
prevencin y cuidado de la salud oral, en particular
en el tratamiento de afecciones buco-dentales entre
los tobas del Chaco Central (Argentina), a la vez que
da cuenta del contexto etnomdico en el que ste
tiene lugar.
MATERIALES Y METODOS
rea de estudio
El rea de trabajo forma parte de la regin del
Gran Chaco, en la provincia de Chaco (Noreste de
Argentina) en las inmediaciones del ro Bermejito
(Figura 1), presentando un clima subtropical
continental con precipitaciones de entre 800 y 900
mm/ao superiores en verano -con una temperatura
promedio de 29 C- y marcada estacin seca en
invierno -con una temperatura promedio de 17 C-.
Segn sus peculiaridades fitogeogrficas corresponde
a la regin Neotropical, Dominio Chaqueo,
Provincia Chaquea, con especies propias de los
bosques del Chaco Central segn Prado (1993) o
transicin entre el Chaco Oriental o hmedo y el
Chaco Occidental o semirido, segn el criterio de
Cabrera (1994). Se caracteriza por un patrn de
vegetacin con un marcado modelado fluvial
(Morello & Admoli, 1974) y una vegetacin
climcica de bosque xerfito caducifolio, junto a
sabanas, estepas halfitas, cardonales, pajonales,
camalotales y otros tipos.
Desde el punto de vista econmico los tobas
subsisten combinando precariamente actividades
tradicionales como la caza, pesca y recoleccin, junto
a una agricultura incipiente, el manejo de ganado
caprino, la apicultura, la venta de recursos del monte
y de mano de obra asalariada comprometida en la
cosecha del algodn, as como de los ingresos que
provienen de planes de asistencia oficial.


Figura 1. rea de estudio que comprende el centro de la Provincia de Chaco, Nordeste de Argentina.



Un contexto sanitario mltiple caracteriza al
sistema local de salud, en el que coexiste el
shamanismo (desempeado por sus especialistas, los
pioxonac), la medicina domstica o casera, y la
medicina oficial en los centros de salud, a cargo de
profesionales biomdicos y agentes sanitarios tobas.
A pesar de este pluralismo, la medicina tradicional
toba no se halla incorporada an a la medicina
oficial, siendo el uso de remedios naturales y la cura
shamnica una de las primeras opciones teraputicas
a las que recurren los pobladores locales (Martnez,
2007b). Una diversidad de cuadros clnicos y
afecciones caracterizan la morbilidad en la regin, tal
como lo atestigua un Diagnstico Local de Salud
(DLS) que desarrollamos en la regin a travs de
entrevistas a los profesionales (Martnez, 2006). En
particular la atencin odontolgica resulta
prcticamente inaccesible, contando con un solo
profesional en un radio de ms de 50 km a la
redonda. El mismo DLS refiere cmo las
descalcificaciones, la presencia de caries por
debilitamiento del esmalte de los incisivos superiores,
prdida de piezas dentarias a tempranas edades,
manchas por hidroarsenicismo y odontalgias, as
como restos de races no extradas, junto con las aftas
y ulceraciones en la mucosa bucal especialmente de
los nios, constituyen algunas de las afecciones
orales ms comunes, muchas de ellas asociadas con
un desbalance nutricional, desnutricin y una dieta
escasa en protenas y lcteos, con predominio en el
consumo de farinceos. Aun cuando diversas
comunidades indgenas del Gran Chaco, entre ellas
los tobas, participaron a principios del siglo XX del
trabajo en obrajes e ingenios azucareros adoptando
saberes y prcticas de los blancos (entre ellos la
prevencin a travs del cepillado y el empleo de
dentfrico), la higiene buco-dental resulta inusual
hasta el presente.
Mtodos y tcnicas de trabajo
Como parte de un relevamiento general de la
etnobotnica mdica toba, se recolect informacin
acerca de los usos medicinales de las plantas en el
rea de estudio entre los aos 2004 al 2008. Para tal
fin se aplicaron entrevistas abiertas, extensas y
recurrentes, as como encuestas semiestructuradas a
miembros de la comunidad de distinto sexo y edad,
as como a profesionales del mbito de salud, para lo
cual se confeccion una encuesta sobre la temtica
utilizando como referencia la gua etnobotnica
propuesta por Arenas (1995). Esta informacin se
complement con la obtenida por observacin
participante. En todos los casos el material vegetal se
recolect en recorridas de campo, en compaa de los
informantes. La documentacin de la informacin se
realiz en cuaderno de campo, grabaciones digitales
y fotografas. Las muestras de referencia se
depositaron en el Museo Botnico (CORD) del
Instituto Multidisciplinario de Biologa Vegetal de la
Universidad Nacional de Crdoba. El material
vegetal fue identificado en su mayor parte por el
autor, recurrindose a la consulta de especialistas en
los taxa que presentaran dificultades, y al catlogo de
Plantas Vasculares de Argentina (Zuloaga &
Morrone, 1996, 1999) y su actualizacin electrnica
on-line para el Cono Sur (Zuloaga et al., 2008). El
material zoolgico correspondiente a los
invertebrados fue identificado por especialistas y
forman parte de la coleccin particular del autor
(Museo de Antropologa). En el caso de los
vertebrados la identificacin se realiz con
informantes mediante el empleo de fotografas e
imgenes de guas de campo, lo que fue corroborado
con bibliografa etnobiolgica especfica para la
regin del Gran Chaco (Martnez Crovetto, 1995;
Arenas, 2003).
Se realizaron seis trabajos de campo que
totalizaron ms de 100 das de estancia en
asentamientos tobas ubicados en localidades,
pertenecientes a la intendencia de Ro Bermejito
(Dpto. General Gemes, Pcia. de Chaco), en las
inmediaciones del ro homnimo siendo el Paraje El
Colchn (Figura 1) el sitio donde se realizaron la
mayor parte de las entrevistas y colectas de material
botnico. Previo a las entrevistas se inform acerca
del proyecto de investigacin y sus objetivos a
representantes y miembros de las comunidades. Las
conversaciones con especialistas y pobladores se
construyeron sobre la base de un objetivo comn:
mejorar la situacin de salud regional, incrementar el
conocimiento acerca de los remedios naturales,
recuperar saberes y prcticas tradicionales para
favorecer su circulacin y desarrollar materiales
educativos de inters regional.
Se emplearon alternativamente mtodos
cualitativos, cuantitativos y participativos en
instancias diferentes y recurrentes durante el lapso de
la investigacin, procurando enriquecer cada una de
ellas con lo generado en la otra y siguiendo el
esquema bsico de la labor etnobotnica: Trabajo de
campo y trabajo de gabinete.


- Tcnicas cualitativas: Se recurri al estudio
del contenido a los fines de interpretar en las
entrevistas abiertas y extensas la sintomatologa y
etiologas de las dolencias.
- Tcnicas cuantitativas: Se dise una
encuesta temtica semiestructurada, la que se aplic a
60 informantes, conformada tanto por especialistas
(shamanes, parteras y ancianos) como por el comn
de los miembros de la comunidad (jvenes y adultos
de ambos sexos). Se obtuvieron valoraciones
cuantitativas acerca de la cantidad de usos reportados
para cada especie (agrupadas en categoras de
frecuencia de mencin) y la proporcin de los
mismos en el total de reportes, utilizndose como
criterio de validacin la coincidencia de al menos dos
informantes para el mismo uso medicinal, esto es:
idntica aplicacin para una misma parte de una
planta, cualquiera fuera el modo de preparacin,
incluyendo datos nicos cuando stos tuvieran
soporte en otros estudios etnobotnicos desarrollados
en la regin (Scarpa, 2004). Se obtuvo el listado de
aplicaciones con mayor consenso de uso, as como el
de especies con mayor cantidad de aplicaciones
medicinales para las afecciones aqu tratadas.
Para la escritura en idioma toba se recurre al
alfabeto toba estndar (Buckwalter & Litwiller de
Buckwalter, 2001) por su amplia difusin y uso entre
los miembros de la comunidad, pudiendo consultarse
las convenciones fonticas en contribuciones
anteriores (Martnez, 2007a,b).
RESULTADOS Y DISCUSIN
Representaciones acerca de las afecciones
bucodentales
Los tobas interpretan que las afecciones
dentarias se deben a la accin de gusanos alojados en
el interior de las piezas o bien a la transgresin de
ciertos tabes. El dolor de muelas, por ejemplo,
guarda estrecha relacin con el respeto por el tiempo
de luto de un difunto. Durante esta etapa y un lapso
posterior a su muerte queda vedado entre los
familiares ms prximos el consumo de miel y carnes
de todo tipo; especial cuidado merecen los huesos de
carnes hervidas en puchero o guisos, los que deben
enterrarse o dejarse sobre el techo de las viviendas,
evitando que los perros lo coman y se les hinche la
boca propagando as esta enfermedad; de no
cumplirse esta prescripcin resulta inevitable para el
transgresor un dolor de muelas de difcil tratamiento,
cuya atencin es competencia exclusiva de los
pioxonaq. Acorde con las representaciones de los
tobas, las odontalgias se originan tambin por
consumir en la etapa de duelo algunos frutos del
monte como luaxai (Morrenia spp.), en particular si
en su interior contienen algn tipo de larva (qochil)
que ocasiona esta dolencia por contagio.
Farmacopea natural: Especies y usos medicinales
Un total de 49 especies (34 vegetales y 15
animales) pertenecientes a 42 familias (28 vegetales y
14 animales) se aplican en el tratamiento de las
afecciones bucofarngeas. Sobre 59 usos medicinales
(71 % vegetales y 29 % animales), las Tablas 1 y 2
detallan las familias, especies y aplicaciones
medicinales, junto al consenso de citas, destinadas al
tratamiento y prevencin de la salud oral.
La categora taxonmica que se encuentra
ms representada en cuanto a aplicaciones
medicinales, de acuerdo con la Figura 2, es la de las
Plantas Antfitas, (60%) lo que resulta consistente
con la gran diversidad de especies involucradas en la
misma. La categoras taxonmica de los Hongos y
lquenes, por su parte, suele estar poco representada
en estudios etnobotnicos, adquiriendo sin embargo
notoriedad en el tratamiento de las dolencias que se
abordan en este trabajo, representando un 18 % del
total de especies (3 hongos y 3 lquenes) y un 10%
del total de usos. Por su parte el Reino Animal
representa, en proporciones casi iguales de
Vertebrados e Invertebrados, un 30% del total de
usos. Entre las familias botnicas ms representadas
en cantidad de especies y usos encontramos:
Lycoperdaceae (3 especies y 3 usos), Physciaceae (2
especies y 2 usos) Asteraceae (2 especies y 2 usos),
Euphorbiaceae (2 especies y 2 usos), Solanaceae (2
especies y 2 usos) y Ranunculaceae (1 especie y 4
usos), y entre las zoolgicas, la familia
Myrmecophagidae (1 especie y 2 usos).
Figura 2. Distribucin porcentual de los usos medicinales por
grupo taxonmico.


Tabla 1.-Farmacopea vegetal empleada en afecciones bucofarngeas y dentales.
REINO
Clase
Familia
Especie
Nombre local
(Voucher)
Orig. Parte usada / Forma de
preparacin / Modo de
administracin.
Aplicacin
(Eficacia atribuida)
F.C.
FUNGII (Hongos y lquenes)
Basidiomycetes

Lycoperdaceae Lanopila bicolor (Lev.) Pat.
huaqaji l'atec
GJM 442 (CORD)

Nat. Esporas /Sin preparacin/
Tpico
Aftas y llagas bucales
(Cicatrizante, antiinflamatorio
oral)

*
Mycenastrum corium (Guers.)
Desv.
huaqaji l'atec
GJM 464 (CORD)

Tpico
oral)
*
Vascellum pampeanum (Speg.)
Homrich
huaqaji l'atec
GJM 360 (CORD)
Tpico
oral)
*
Ascomycetes

Parmeliaceae Cannomaculina pilosa
(Stizenb.) Elix & Hek
ncapeguelec 'ana 'epaq
GJM 418 (CORD)

Nat. Planta entera/Infusin o
decoccin, Incineracin
(cenizas)/ Tpico

oral)
**
Physciaceae Heterodermia albicans (Pers.)
Swinscow & Krog
GJM 417b (CORD)

(cenizas)/ Tpico

oral)
**
Physcia lopezii Moberg
GJM 417a (CORD)

(cenizas)/ Tpico
oral)
**
PLANTAE (Antfitas)
Liliopsida (Monocotyledoneae)

Dioscoreaceae Dioscorea microbotrya Griseb.
etaxat lte
GJM 469 (CORD, BAB)

Nat. Races / Sin preparacin /
Ingesta alimentaria
Fortalecer la dentadura *
Poaceae Arundo donax L.
coqta
GJM 414 (CORD)
GJM 293 (CORD)

Nat. Planta entera/Incineracin
(cenizas)/ Bebida
oral)
*
Elionurus muticus (Spreng.) O.
Kuntze
chem' auaxa
GJM 433 (CORD)
Nat. Raz/Macerado en agua /
Enjuague bucal
Odontalgias y caries dentales
(Antiodontlgico)
**

Magnoliopsida (Dicotyledoneae)

Amaranthaceae Alternanthera pungens Kunth
ta'asot
GJM 220 (CORD)
Nat. Hojas/Infusin o decoccin en
agua/ Enjuague bucal
oral)
**

Parte area/Incineracin
(cenizas)/ Tpico
oral)
**


REINO
Clase
Familia
Especie
Nombre local
(Voucher)
administracin.
Aplicacin
F.C.

Anacardiaceae Schinus fasciculatus var.
fasciculatus (Griseb.) I.M.
Johnst.
toroloquiic
GJM 21 (CORD)
Nat. Infusin o decoccin en agua/
Bebida. Tambin se acostumbra
a masticar las hojas.
Dolor de garganta
(Antibitico, calmante
bucofarngeo)
**

Parte area/Infusin o
decoccin en agua/ Enjuague
bucal. Se indica un puado de
hojas, o la corteza y gajos
obtenidos del naciente, en el
volumen de una pava, tres veces
al da.

Odontalgias
(Antiodontlgico)
***
Apiaceae Eryngium coronatum Hook. &
Arn.
ra'aloxo
GJM 344 (CORD)

Nat. Raz/ Molido o picado/
Emplasto. Se aplica un
fragmento de la raz en la
cavidad del diente afectado
Odontalgias
(Antiodontlgico)
*
Aristolochiaceae Aristolochia esperanzae Kuntze
var. esperanzae
epaq lta
GJM 542 (CORD, BAB)

Nat. Raz/Sin preparacin o en el
mate/ Mascado
Se mastica y traga un pedacito
de la raz de olor mentolado.
Dolor de garganta
bucofarngeo)
**
Asteraceae Eupatorium hecatanthum
(DC.) Baker
ronai laue
GJM 63 (CORD, BAB)
Nat. Inflorescencia, hojas/ Sin
preparacin / Tpico. Se
colocan algunas flores o bien
una hoja en las caries.

Odontalgias
(Antiodontlgico)
**
Parthenium hysterophorus L.
chemaxaraic, chimaxadaic
GJM 225 (CORD)
Nat. Raz/ Infusin o decoccin en
agua/ Emplasto. Se introduce un
trozo de raz en las caries.

Odontalgias
(Antiodontlgico)
*
Cactaceae Rhipsalis lumbricoides (Lem.)
Lem.
sallaxataxaic
GJM 428 (CORD)

Nat. Planta entera/Incineracin
(cenizas)/Tpico
oral)
*
Celastraceae Maytenus vitis-idaea Griseb.
satachec, chiqpi'
GJM 218 (CORD)
Nat. Hojas/Incineracin
(cenizas)/Tpico
oral)

*
Celtidaceae Celtis iguanaea (Jacq.) Sarg.
taxanachec
GJM 517 (CORD)

agua/ Bebida
Dolor de garganta
bucofarngeo)

**
Convolvulaceae Dichondra microcalyx (Hallier
f.) Fabris
micha ltela
GJM 286 (CORD)

Odontalgias
(Antiodontlgico)
**
Cucurbitaceae Cucurbitella asperata (Gillies
ex Hook. & Arn.) Walp.
quemoxon
GJM 280 (CORD)

Nat. Raz/Infusin o decoccin en
Odontalgias
(Antiodontlgico)
***
*
Euphorbiaceae Euphorbia serpens Kunth var.
serpens
potaxanaxaq alo'q,
qapalaxanaxaic, qoloxoloxo
Nat. Parte area/Infusin o
bucal
oral)
**


REINO
Clase
Familia
Especie
Nombre local
(Voucher)
administracin.
Aplicacin
F.C.
lauel
GJM 228 (CORD)

Sapium haematospermum Mll.
Arg.
chaxayeec
GJM 2 (CORD)

Nat. Latex/ Sin preparacin/ Tpico.
Las gotas de ltex se aplican en
forma directa en la muela
afectada.
Odontalgias
(Antiodontlgico)
*
Fabaceae Prosopis alba Griseb.
mapik
GJM 94 (CORD)
Nat. Corteza/ Incineracin (cenizas)/
Tpico
oral)
*

Corteza/ Infusin o decoccin
en agua/ Enjuague bucal
oral)
*
Lythraceae Heimia salicifolia (Kunth) Link
piaxataxai, covih lahuo
GJM 150 (CORD)

Nat. Hojas/ Infusin o decoccin en
agua / Enjuague bucal
Odontalgias
(Antiodontlgico)
*
Malvaceae Hibiscus striatus Cav.
lalaco`ja
GJM 330 (CORD)
Nat. Flores/ Incineracin (cenizas)
/Tpico
oral)

*
Meliaceae Melia azederach L.
paraso
GJM 4 (CORD)
Intr. Hojas / Infusin o decoccin
en agua/ Enjuague bucal. Se
prepara un puado de las hojas
en un jarro o se coloca un
fragmento de la hoja en las
caries.
Odontalgias
(Antiodontlgico)
**
Oxalidaceae Oxalis conorrhiza Jacq.
GJM 164 (CORD)

Nat. Parte area/Infusin o
bucal. Se emplea un puado de
plantas en una pava.
Odontalgias
(Antiodontlgico)
*
Phytolaccaceae Petiveria alliacea L. var.
alliacea
shepatoq, shipatoq
GJM 187 (CORD)
Nat. Raz, Semillas / Molido o
picado/ Emplasto. Se aplica un
emplasto de la raz en el interior
de la muela afectada. Algunos
informantes refieren con el
mismo fin el uso de la semilla.
Odontalgias
(Antiodontlgico)
**

Planta entera/ Infusin o
bucal

Odontalgias
(Antiodontlgico)
***
Ranunculaceae Clematis montevidensis Spreng.
naqolo
GJM 66 (CORD)
Nat. Hojas /Infusin o decoccin
en agua/ Enjuague bucal
oral)
**

Parte area/Sin preparacin/
Accin simblica. Se ata la
liana en la mano
correspondiente al lado de la
muela afectada, hasta que sane;
sealan el cuidado de su empleo
por un efecto custico al
contacto con la piel o la boca.
Odontalgias
(Uso simblico)
***
**

Parte area/Infusin o
bucal
Odontalgias
(Antiodontlgico)
*

Hojas/ Molido o picado/
Emplasto. Se coloca la hoja
molida con un algodn en la
cavidad del diente afectado
Odontalgias
(Antiodontlgico)
*


REINO
Clase
Familia
Especie
Nombre local
(Voucher)
administracin.
Aplicacin
F.C.
Rutaceae Citrus limon L.
limn
Intr. Frutos / Sin preparacin /
Gotas
oral)

*
Santalaceae Jodina rhombifolia (Hook. &
Arn.) Reissek
she' laue, naranja late'e
GJM 410 (CORD)

Nat. Hojas / Infusin o decoccin
en agua / Bebida
Dolor de garganta
bucofarngeo)
*
Solanaceae Jaborosa integrifolia Lam.
tapai laue
GJM 200 (CORD)
Nat. Hojas / Sin preparacin/
Emplasto. Se emplean las hojas
para "sacar la fiebre" que
ocasiona el dolor de muelas.

Odontalgias
(Antiodontlgico)
*
Solanum argentinum Bitter &
Lillo
pioq laayec
GJM 210 (CORD)
Nat. Raz y hojas/Infusin o
bucal o bebida (raz)
Odontalgias
(Antiodontlgico)
***
*

Raz / Sin preparacin/
Emplasto. Se aplica un
emplasto de la raz en el interior
de la muela o se masca un
fragmento de la misma.
Odontalgias
(Antiodontlgico)
**
Zygophyllaceae Bulnesia sarmientoi Lorentz ex
Griseb.
delliquic
GJM 608 (CORD)

Nat. Madera /Infusin o decoccin
en agua / Bebida
Dolor de garganta
bucofarngeo)

**

Tabla 2.- Farmacopea animal empleada en afecciones bucofarngeas y dentales. F.C. (Frecuencia de citas: * < 2% informantes; ** 2 - 5 %;
*** 5 - 10 %; **** 10 - 20 %; ***** > 20 %)
DIVISIN
Clase
Familia
Especie
Nombre local

administracin.
Aplicacin
F.C.
ANIMALIA (Invertebrados)
Bivalvia
Unionidae Anodontites trapesialis
coneq
Nat. Concha/ Incineracin (cenizas) /
Tpico
oral)

***
*
Gastropoda
Ampullariidae

Pomacea sp.
sapo lcooue
Nat. Huevos / Tpico Aftas y llagas bucales
oral)

**
Insecta
Apidae Trigona sp.
coilala
Nat. Miel / Ingesta alimenticia Dolor de garganta
bucofarngeo)
*
Orden
Lepidoptera
Indet. (crislida de lepidptero)
cochel

- Capullo / Incineracin
(cenizas)/ Tpico.
Odontalgias (Uso simblico) *
Mantidae Captoteryx argentina,
Stagmatoptera hyaloptera
quedenaxai'chi
Nat. Huevos /Adminculo corporal.
Se cuelga la ooteca con huevos
de un hilo hasta calmar el dolor,
puesto que se considera que ste
insecto come los "gusanos"
causantes del dolor de muelas.


DIVISIN
Clase
Familia
Especie
Nombre local

administracin.
Aplicacin
F.C.

Psychidae Oiketicus kirbyi (Guild)
cotaxat
Nat. Capullo /Adminculo corporal.
El capullo se lo emplea como
colgante del cuerpo en forma de
medalla o aro.
Capullo / Incineracin
(cenizas) / Tpico
oral)

*
Vespidae Polistes spp. (Polistes
canadensis y otras spp.)
uootel

Nat. Panal o nido /Incineracin
(cenizas) / Tpico
oral)
**
ANIMALIA (Vertebrados)
Chondrichthyes
Dasyatidae Potamotrygon sp.
lacataic
Nat. Las pas de rayas (lacataiq
lsoxanaqte) se emplean para
perforar las muelas doloridas y
eliminar el dolor.
Odontalgias

*
Reptilia
Alligatoridae Caiman latirostris chacoensis
daailoc

Nat. Accin simblica: Se muerde
un diente de yacar para
desarrollar una dentadura sana y
fuerte.

Preventivo (Uso simblico) **
Teiidae Tupinambis teguixin
(lairaxaic) (naigoxonaxa)
qolliguesaq
Nat. Grasa /Sin preparacin/ Tpico.
Se rellenan las cavidades de los
dientes afectados.

Odontalgias
(Antiodontlgico)
*
Grasa/Sin preparacin/
Fricciones y masajes.
Dolor de garganta
bucofarngeo)
*
Aves
Rheidae Rhea americana
maic
Nat. Grasa/Sin preparacin/ Tpico Aftas y llagas bucales
oral)
*
Mammalia
Canidae Canis familiaris
pioq
Se emplean los pelos para tratar
dolores de muelas originados en
la transgresin de la veda
alimentaria durante el luto.

Odontalgias
(Uso simblico)
*
Felidae Puma concolor
sauaxaic
Grasa/ Sin preparacin /
Fricciones y masajes
Dolor de garganta
bucofarngeo)

*
Myrmecopha-
gidae
Myrmecophaga tridactyla
potai

Grasa/Sin preparacin / Tpico Aftas y llagas bucales
oral)

*
Pelos / Incineracin (cenizas) /
Tpico.
Se quema el pelo de la cola
(potai laxarashet) y las cenizas
que se obtienen se aplican en la
boca de los nios,
especialmente cuando no
pueden mamar.

oral)
**


Atendiendo al nmero de especies
involucradas, los usos vegetales ms relevantes y que
ocupan la mayor atencin de las afecciones orales, de
acuerdo con la Figura 3, corresponden al tratamiento
de odontalgias (45%), aftas y llagas bucales (41 %),
dolores de garganta (12%) y preventivos de salud
dental (2%). Por su parte la farmacopea animal se
destina fundamentalmente al tratamiento de aftas y
llagas bucales (41 %), odontalgias (35%), dolor de
garganta (18%) y a la proteccin de la dentadura
(6%). Debemos sealar, asimismo que los nativos no
dan cuenta de aplicacin alguna de la farmacopea
natural para otro tipo de afecciones bucodentales
tales como la piorrea y gingivitis.
Figura 3. Cantidad de usos de la farmacopea vegetal (V) y
animal (A) de acuerdo con las diferentes aplicaciones
medicinales.

Figura 4. Distribucin porcentual de formas de aplicacin de la
farmacopea natural: (E) Usos externos; (I) Usos internos.

Las partes ms comnmente usadas para el
tratamiento de estas dolencias son porciones areas,
especialmente las hojas y races de los vegetales, y
las grasas y capullos de los animales. Estos se
preparan en su mayora en forma de infusiones o
decocciones en agua (35%), de cenizas obtenidas por
incineracin (27%) o bien se aplican en forma directa
sin que medie preparacin alguna (30%). Tanto para
la farmacopea animal y vegetal predominan las
aplicaciones externas (61%) especialmente en
forma tpica y de emplastos-, respecto de las internas
(39%), las que son usadas particularmente como
enjuagues bucales (Figura 4).
Si consideramos el origen de las especies
utilizadas, se advierte casi un uso exclusivo de
especies nativas respecto de las introducidas tanto
para farmacopea vegetal como animal, lo que da
cuenta de la relevancia del monte chaqueo como
fuente de recursos teraputicos para este grupo
humano. El listado de especies con mayor proporcin
de citas se encuentra encabezada por plantas nativas,
destacndose por su consenso el uso simblico de la
liana Clematis montevidensis, y el empleo de las
races de Solanum argentinum, Cucurbitella asperata
y de la parte area de Schinus fasciculatus var.
fasciculatus y de Petiveria alliacea var. alliacea,
todas ellas destinadas al tratamiento de odontalgias.
Con valores inferiores de consenso respecto de la
farmacopea vegetal, se destacan entre los remedios
de origen animal el uso de las cenizas del molusco
Anodontites trapsialis para el tratamiento de aftas y
llagas bucales.
Si bien muchas de las especies citadas por
sus propiedades antiodontlgicas no han sido
estudiadas desde el punto de vista fitoqumico,
existen evidencias de usos similares en otros
contextos culturales prximos y lejanos. El empleo
de esporas de hongos de la familia Lycoperdaceae
(Gasteromycetes) como cicatrizantes o
antimicrobianos, por ejemplo, ha sido sealado en
forma recurrente en trabajos etnobotnicos del Gran
Chaco (Filipov, 1994; Scarpa, 2004), y de otras
regiones del mundo (Palmese et al., 2001; Viegi et
al., 2003; Dulger, 2005), lo que sustenta su uso en la
cicatrizacin de aftas y llagas bucales. Los trabajos
de Filipov (1994) y Scarpa (2004), por su parte,
coinciden en el uso de las races de Petiveria alliacea
para el tratamiento de las odontalgias entre los pilag
y los criollos de Formosa, respectivamente.
Asimismo, y con idntica aplicacin a la prescripta
por los tobas de esta regin, los pilag emplean el
ltex de Sapium haematospermum para tratar dolores
de muelas (Filipov, 1994), y los criollos de Formosa,
el uso de Alternanthera pungens para las aftas de la
boca (Scarpa, 2004). Asimismo el empleo de Schinus
longifolius var longifolius (Anacardiaceae) como
analgsico resulta muy popular en Argentina y otras
regiones de Amrica (Filipov, 1994; Martnez &
Planchuelo, 2003; Waizel-Bucay & Martnez Rico,
2007).


Si bien, y tal como vimos anteriormente, en
el listado de aplicaciones medicinales resulta ms que
probable la existencia de especies con principios
activos de eficacia farmacolgica, tambin es posible
distinguir procedimientos o acciones en los que se
advierte una eficacia de tipo simblica, sustentada en
una lgica implcita de transferencia de propiedades
conocida como circulacin de los sntomas, en
trminos de Laplantine (1999). Tal es el caso del
empleo de la liana naqolo (Clematis montevidensis,
Ranunculaceae) atada en el brazo correspondiente al
lado de la muela afectada, hasta que sane. Con la
misma lgica teraputica se explica que la
emergencia de los dientes en los nios est
condicionada por el tratamiento que se haga de su
placenta una vez nacido, ya que cuanto ms profunda
se entierra sta, mayor ser la demora en la denticin.
De igual manera, y con un claro sentido metafrico,
existen prcticas de prevencin de la salud oral, tales
como morder la grasa del chancho moro o yolo
(Tayassu tajacu, Suidae), o los dientes de yacar o
daailoc (Caiman latirostris chacoensis,
Alligatoridae) que permiten adquirir una dentadura
fuerte y saludable por transferencia de esta cualidad
animal.
CONCLUSIONES
El significativo nmero de especies de la
farmacopea natural implicadas, que alcanza casi al
medio centenar, da cuenta de la relevancia de las
afecciones orales entre las comunidades tobas del
Chaco Central, aspecto que probablemente se vincula
a la necesidad de mitigar en forma inmediata el dolor
que caracteriza a estas dolencias. Con un predominio
en el uso de plantas, la farmacopea animal resulta
ms conspicua en cantidad de especies y usos, siendo
las odontalgias y aftas bucales las mayores
preocupaciones en orden al cuidado de la salud
bucodental. La eleccin de especies destinadas al
tratamiento de estas dolencias, a partir del amplio
listado que se presenta en este trabajo, obedece no
slo a una eventual eficacia farmacolgica, sino
tambin a aspectos simblicos implicados en la
prctica teraputica. Esto da cuenta de la vigencia de
una etnomedicina holstica, con un fuerte
componente naturalista caracterstico de grupos
tnicos que, como los tobas, an conservan en forma
ms o menos acentuada, sus prcticas de recoleccin,
caza y pesca. La dificultad a un acceso apropiado a
los profesionales de la biomedicina, y en particular a
los odontlogos, propicia la transmisin y la memoria
colectiva en torno a estos usos, algunos de ellos con
amplio consenso entre los informantes tobas y otros,
con un consenso intertnico, al compartir idnticas
aplicaciones con otros grupos del Gran Chaco, como
pilags y criollos de Formosa, provincia vecina al
rea de estudio. La dinmica etnohistrica de
contacto con estos grupos, explicara el empleo de
especies compartidas con las farmacopeas de otros
pueblos nativos de regiones vecinas, en particular con
los criollos, tal es el caso de Alternanthera pungens,
Schinus fasciculatus var. fasciculatus, Sapium
haematospermum, Melia azederach, Solanum
argentinum, Bulnesia sarmientoi, entre otras. Sin
embargo, existe tambin un ncleo de especies y usos
que al parecer, competen exclusivamente a la
etnomedicina toba, tal es el caso de los hongos y
lquenes, entre otros que ocupan un porcentaje
relevante de la farmacopea natural de este grupo
tnico. En todos los casos es evidente el empleo de
plantas nativas silvestres, sin que existan referencias
al empleo de plantas cultivadas en huertos y espacios
peridomsticos, lo que da cuenta de la relevancia del
monte nativo como fuente de remedios.
El acceso de la poblacin mundial a las ltimas
terapias clnicamente validadas, as como a
innovaciones biomdicas en el tratamiento de la salud
buco dental no resulta universal, menos an para
poblaciones rurales como las que aqu se describen.
Los preparados farmacuticos basados en ensayos
clnicos con validacin cientfica, y destinados a
tratar estas dolencias, no han reemplazado en
absoluto la persistencia en el uso de las medicinas
tradicionales y alternativas para una proporcin
importante de la poblacin mundial. De esta manera,
resulta prioritario establecer un listado apropiado de
plantas, con su identificacin, uso y eventuales dosis
estandarizadas que permitan su aplicacin en
atencin primaria de salud. Aun cuando en su
mayora las especies consideradas en este trabajo no
cuentan con estudios confirmatorios de sus
aplicaciones medicinales, el consenso de uso
asignado a las plantas con propiedades
antiodontlgicas (como Clematis montevidensis,
Solanum argentinum, Cucurbitella asperata, Schinus
fasciculatus var. fasciculatus y Petiveria alliacea var.
alliacea) permite definir un conjunto de especies para
esta regin y grupo tnico, propiciando de esta
manera la incorporacin de la medicinas tradicionales
en el sistema local de salud.


AGRADECIMIENTOS
A la comunidad toba de Ro Bermejito
(Paraje El Colchn) que me brindaron su valioso
tiempo e informacin, as como la hospitalidad y
servicialidad de las familias, pobladores e
instituciones que facilitaron mi tarea. Deseo expresar
mi gratitud a mi director el Lic. Pastor Arenas
(Conicet) por su asesoramiento permanente en mi
tarea de investigacin y a los especialistas que
identificaron (Dra. Laura Domnguez y Ctedra de
Diversidad Vegetal I: Hongos; Dra. Cecilia Strabou y
Bil. Juan M. Rodrguez: Liquenes; Bil. Liliana
Buffa y Dr. Claudio Sosa: Insecta; Dra. Alejandra
Ceballos: Invertebrados), corrigieron, confirmaron u
orientaron mis determinaciones. El presente trabajo
se realiz en el marco de los proyectos Anpcyt/
Foncyt Pict 32894 y 1612.
REFERENCIAS
Arenas P. 1995. Encuesta etnobotnica aplicada a
indgenas del Gran Chaco. Hacia una nueva carta
tnica del Gran Chaco 6: 161-178. Centro del Hombre
Antiguo Chaqueo, Las Lomitas, Formosa, Argentina.
Arenas P. 1997. Las fuentes actuales y del pasado para la
etnobotnica del Gran Chaco. Monografas del Jardn
Botnico de Crdoba 5: 17-25. Crdoba, Espaa.
Arenas P. 2000. Farmacopea y curacin de enfermedades
entre algunas etnias del Gran Chaco. En: A.G. Amat
(Coord.) Farmacobotnica y Farmacognosia en
Argentina (1980-1998). Ediciones Cientficas
Argentinas, La Plata, pp. 87- 118.
Arenas P. 2003. Etnografa y alimentacin entre los Toba
achilamole#ek y Wich-Lhuku`tas del Chaco Central
(Argentina). Edic. del autor, Buenos Aires, pp. 562.
Babpour E, Angaji A, Mahdi Angaji S. 2009.
Antimicrobial effects of four medicinal plants on
dental plaque. J Med Plant Res 3 (3): 132-137.
Buckwalter A, Litwiller de Buckwalter L. 2001.
Vocabulario toba. Edicin Revisada. Equipo
Menonita. Mennonite Board of Missions. Elkhart,
Indiana, USA, pp. 392.
Cabrera A.L. 1994. Regiones fitogeogrficas argentinas.
Enciclopedia Argentina de Agricultura y Jardinera.
Tomo II (1) Acme, Buenos Aires. pp. 85.
Censabella M. 2000. Las lenguas indgenas de la
Argentina. Una mirada actual. EUDEBA, Buenos
Aires, pp. 147.
Censo 1981. Censo y estudio de la poblacin indgena del
Paraguay, 1981. Instituto Paraguayo del Indgena
(IPA). Asuncin.
Colvard MD, Cordell GA, Villalobos R, Sancho G,
Soejarto DD, Pestle W, Echeverri TL, Perkowitz K,
Michel J. 2006. Survey of medical ethnobotanicals for
dental and oral medicine conditions and pathologies. J
Ethnopharmacol 107 (1): 134-142
Dulger B. 2005. Antimicrobial activity of ten
Lycoperdaceae. Fitoterapia 76(3-4): 352-354.
ENDEPA. 1986. Aborgenes en Argentina. Equipo
Nacional de Pastoral Aborigen. Formosa, pp. 28.
Filipov A. 1994. Medicinal plants of the Pilag of Central
Chaco. J Ethnopharmacol 44 (3): 181-193.
Franz D. 1925. Erbe medicinali del Chago e legnami
industriali argentini. Contributo delle Missioni
Francescane della Repubblica Argentina
all'Esposizione Missionaria Vaticana. Pubblicazioni
dell Istituto Cristoforo Colombo, Roma, pp. 99
Hebbar SS, Harxha VH, Shriathi V, Hegde GR. 2004.
Ehnomedicine of Dharwad district in Karnataka, India
Plants used in oral health care. J Ethnopharmacol 94
(2-3): 261-266
Hecht AC, Martnez GJ, Cneo P. 2008. Infancia toba y
mundo natural: De la atencin del malestar fsico a
las pautas de socializacin infantil. Acta Americana
16 (1): 81-106.
Laplantine F. 1999. Antropologa de la enfermedad.
Estudio etnolgico de las representaciones etiolgicas
y teraputicas en la sociedad occidental
contempornea. Ediciones del Sol, Buenos Aires, pp.
418.
Martnez GJ, Planchuelo AM. 2003. La medicina
tradicional de los criollos campesinos de Paravachasca
y Calamuchita, (Crdoba, Argentina). Scripta
Ethnologica 25: 83-116
Martnez GJ. 2006. Diagnstico Local de Salud (DLS) de
los tobas del Ro Bermejito. Cultura, Salud y Plantas
entre los tobas del Ro Bermejito. Primer informe de
avance de devolucin a las comunidades. Manuscrito.
pp. 45.
Martnez GJ. 2007a. La farmacopea natural en la salud
materno-infantil de los Tobas del Ro Bermejito.
Kurtziana 33: 39-63.
Martnez GJ. 2007b. Vienen buscando la vida (Ilotaique
nachaalataxac): Las trayectorias teraputicas de los
tobas del Ro Bermejito (Chaco). pp. 401-424. En:
Idoyaga Molina, A. (Ed.). Los caminos teraputicos y
los rostros de la diversidad. Tomo I. CAEA IUNA,
Buenos Aires, Argentina.
Martnez GJ. 2008. La farmacopea natural en la
etnomedicina de los tobas del Ro Bermejito (Chaco,
Argentina). Tesis doctoral, Facultad de Ciencias
Agropecuarias, Universidad Nacional de Crdoba,
Argentina, pp. 289.
Martnez Crovetto R. 1964. Estudios etnobotnicos. I.
Nombres de plantas y su utilidad, segn los indios
tobas del este del Chaco. Bonplandia 1: 279-333.
Martnez Crovetto R. 1968. Los indios tobas y las plantas.
Actas y Memorias del XXXVII Congreso
Internacional de Americanistas, Repblica Argentina.
Vol II, pp. 625-629.


Martnez Crovetto R. 1995. Zoonimia y etnozoologa de
los pilag, toba, mocov, mataco y vilela. Archivo de
Lenguas Indoamericanas, Coleccin Nuestra Amrica,
Facultad de Filosofa y Letras, Buenos Aires, pp. 188.
Morello J, Admoli J. 1974. Las Grandes Unidades de
Vegetacin y Ambiente del Chaco Argentino II.
Vegetacin y Ambiente de la Provincia del Chaco.
INTA, Centro de Investigaciones de Recursos
Naturales. Serie fitogeogrfica 13. Ed. Coni, pp. 130.
Palmese MT, Uncin Manganelli RE, Tomei PE. 2001. An
ethno-pharmacobotanical survey in the Sarrabus
districtsouth-east Sardinia. Fitoterapia 72: 619-643
Prado DE. 2003. What is the Gran Chaco vegetation in
South America? I. A review. Contribution to the study
of flora and vegetation of the Chaco. Candollea 48 (1):
145-172.
Scarpa GF. 2004. Medicinal plants used by the Criollos of
Northwestern Argentine Chaco. J Ethnopharmacol
91(1): 115-135.
Tapsoba H, Deschamps JP. 2006. Use of medicinal plants
for the treatment of oral diseases in Burkina Faso. J
Ethnopharmacol 104 (1-2): 68-78
Viegi L, Pieroni A, Guarrera PM, Vangelisti R. 2003. A
review of plants used in folk veterinary medicine in
Italy as basis for a databank. J Ethnopharmacol 89 (2-
3): 221-244.
Vuoto LD. 1999. Recoleccin animal entre los tobas de
Formosa. En: Aschero, C., Korstanje M.A. y Vuoto,
P. (Ed.) En los tres reinos: Prcticas de recoleccin en
el Cono Sur de Amrica. Instituto de Arqueologa y
Museo. Facultad de Ciencias Naturales e Instituto
Miguel Lillo, Universidad Nacional de Tucumn, pp.
253-260.
Vuoto PM, 1981. Plantas tiles entre los Toba-Taksk.
Entregas del Instituto Tilcara (Jujuy, Argentina) 10,
12-76. Instituto Tilcara, FFyL, Universidad de Buenos
Aires.
Vuoto PM, 1999. Recoleccin y poder. La vegetacin
entre los Toba del este del. En: Aschero, C.,
Korstanje M.A. y Vuoto, P. (Ed.) En los tres reinos:
Prcticas de recoleccin en el Cono Sur de Amrica.
Instituto de Arqueologa y Museo. Facultad de
Ciencias Naturales e Instituto Miguel Lillo.
Universidad Nacional de Tucumn, pp. 239-250.
Waizel-Bucay J., Martnez Rico M. 2007. Plantas
empleadas en odontalgias I. Revista ADM 64 (5):173-
186
WHO. 2002. Estrategia de la OMS sobre medicina
tradicional 2002-2005. OMS, Ginebra, pp. 78.
Zacaras, D., 1993. Elementos naturales y su utilizacin en
Pampa del Indio. Hacia Una Nueva Carta Etnica del
Gran Chaco 5: 215-217. Centro del Hombre antiguo
Chaqueo, Las Lomitas, Formosa, Argentina.
Zuloaga FO, Morrone O. 1996. Catlogo de las plantas
vasculares de la Repblica Argentina I. Pteridophyta,
Gymnospermae y Angiospermae
(Monocotyledoneae). Missouri Botanical Garden
Press, U.S.A., pp. 323.
Zuloaga FO, Morrone O, 1999. Catlogo de las plantas
vasculares de la Repblica Argentina II. Tomo 1:
Acanthaceae-Euphorbiaceae (Dycotyledoneae). Tomo
2: Fabaceae-Zygophyllaceae (Dycotyledoneae).
Missouri Botanical Garden Press, U.S.A., pp. 1269.
Zuloaga FO, Morrone O, Belgrano MJ. 2008. Catlogo de
las plantas vasculares del Cono Sur (Argentina, sur de
Brasil, Chile, Paraguay y Uruguay). Missouri
Botanical Garden Press. http://www2.darwin.edu.ar/
Proyectos/FloraArgentina /FA.asp. (Consultado
Diciembre 1, 2009).

2010 The Authors
2010 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 9 (2), 123-126




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-
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.
Aromatic plants from Patagonia: chemical composition and antimicrobial
activity of the essential oil of Senecio mustersii and S.subpanduratus
[Plantas aromticas de la Patagonia: Composicin qumica y actividad antimicrobiana del aceite esencial de
Senecio mustersii y S.subpanduratus]
Luz ARANCIBIA
1
, Cecilia NASPI
1
, Graciela PUCCI
2
, Mara ARCE
3

1
Ctedra de Qumica Orgnica,
2
Ctedra de Microbiologa ,
3
Ctedra de Botnica, Facultad de Ciencias Naturales, Universidad Nacional
de la Patagonia S J B, Comodoro Rivadavia, Chubut, Argentina.
Abstract
The objective of this investigation was the determination of the antimicrobial activity of two plants from Patagonia Argentina; Senecio mustersii and
Senecio subpanduratus (Asteraceae). Until the present day, no previous studies have been reported on the composition of the essential oil for these two
species of Senecio. The essential oils were obtained by hydrodistillation with a yield of 0.81% for Senecio subpanduratus and 0.71% for Senecio mustersii,
expressed as ml of essential oil per 100 g of fresh vegetable matter. The activity against bacteria and yeast was tested; Senecio mustersii showed activity
against S.aureus and Senecio subpanduratus against all tested bacteria (S.aureus, E.coli and P. aeruginosa). Senecio mustersii didnt showed antifungal
activity; meanwhile Senecio subpanduratus was active against some species of Candida.
Keywords: Essential Oils; Senecio; Antifungal activity; Antibacterial activity.
Resumen
El objetivo de la investigacin fue la determinacin de la actividad antimicrobiana de los aceites esenciales de dos especies del gnero Senecio
(Asteraceae) de la regin Patagnica: Senecio mustersii y S. subpanduratus. Hasta el momento, no se han reportado estudios sobre la composicin del aceite
esencial para estas dos especies de Senecio. Los aceites esenciales fueron obtenidos mediante hidrodestilacin logrndose un rendimiento de 0.81 % para
Senecio subpanduratus y de 0.72% para Senecio mustersii, expresado como ml de aceite esencial por cada 100 g de material vegetal fresco. Se evalu la
actividad frente a bacterias y levaduras de importancia clnica: Senecio mustersii presenta actividad antibacteriana frente a S. aureus y Senecio subpanduratus
para todas las bacterias testeadas (S.aureus, E.coli y P. aeruginosa). Senecio mustersii no present actividad antifngica, mientras que Senecio subpanduratus
actividad contra algunas especies de Candida.
Palabras Clave: Aceites Esenciales; Senecio; Actividad Antifngica; Actividad Antibacteriana.
List of Abbreviations: HRP (Herbario Regional Patagnico); CG-FID-MS (Gas chromatography-Flame ionization detector-Mass spectrum); ATCC
(American type culture collection); NIM (Nmero Instituto Malbrn);ANLIS (Administracin nacional de laboratorios e institutos de salud); MIC (minimal
inhibition concentration);

Recibido | Received: December 18, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: February 7, 2010.
Publicado en Lnea | Published Online March 25, 2010
Financiacin | Funding: Universidad Nacional de la Patagonia San Juan Bosco
This article must be cited as: Luz Arancibia, Cecilia Naspi, Graciela Pucci, Mara Arce. 2010. Aromatic plants from Patagonia: chemical composition and antimicrobial activity
of the essential oil of Senecio mustersii and S.subpanduratus. Bol Latinoam Caribe Plant Med Aromat 9(2):123 126 . EPub March 2010}. .
*Contactos | Contacts: luz@unpata.edu.ar ; cecinaspi@yahoo.com.ar
Arancibia et al. Antimicrobial activity of Senecio mustersii and S.subpanduratus


INTRODUCTION
There are about 3000 species of Senecio
around the world, mainly in hilly areas. In Argentina
there are more than 270 species, most of them in the
Andes range and in Patagonia. (Cabrera, 1971)
Senecio mustersii and S.subpanduratus
(Asteraceae) are native species that grow
spontaneously near Comodoro Rivadavia, Chubut.
Senecio subpanduratus grows in Chubut and Tierra del
Fuego meanwhile Senecio mustersii Speg. Var.
mustersii can be found in Ro Negro, southwest of
Chubut and east of Santa Cruz (Cabrera, 1971).
This genus contains essential oils on aerial
parts of the plant and also sesquiterpenes in particular
furanoeremophilanes (Bohlmann et al., 1986,
Salmeron et al., 1983; Torres et al., 1999) and
pyrrolizidine alkaloids.
The objective of this work is to study in vitro
antifungal and antibacterial activity for these species
of Senecio. Essential oils from many aromatic plants,
included Senecio have been studied because of their
chemical composition and antimicrobial activity
(Perez et al 1999, El-Shazly 2002). Thus Schinus
polygamus (Gonzalez et al.) and S. johnstonii (Malizia
et al.) from Patagonia is reported on the composition
and antimicrobial activity of the essential oil.
Plant Material
The plant material was obtained from plants at 3
Km north from Comodoro Rivadavia city, province of
Chubut (Argentina), during May of 2006. The species
were identified by botanist (M.E.Arce) and kept in the
Patagonia Regional Herbarium (Universidad Nacional
de la Patagonia San Juan Bosco) under the following
herbarium numbers: Senecio subpanduratus O.
Hoffm. HRP 6867 and Senecio mustersii Speg. Var.
Mustersii HRP 6866.
Essential oil Extraction
Fresh aerial parts of Senecio mustersii and
S.subpanduratus were cut into small pieces. The
essential oils were obtained by hydrodistillation during
4 hours in a Clevenger-type apparatus.
Gas Chromatography-Mass Spectrometry
Analyses were performed by CG-FID-MS in a
Perkin Elmer Clarus 500 provided with a unique
injector Split type (1:100 Relation) and with two
fused silica capillary columns: polyethylenglycol and
5% phenyl-95% methyl silicone, (both 60 m x 0.25
mm x 25m df). Polar column is coupled to a FID
meanwhile non polar column to a FID-mass detector
(70 eV) through an MSVent system. The carrier gas
was Helium (flow rate: 1.87 ml/min). Column
temperature was initially 90C, and then increased to
225C at 3/min rate (15 min). Samples were diluted
(10% v/v in ethanol) and 0.2 l were injected.
The constituents of the essential oils were
identified on the basis of their GC retention indices
(RI) with reference to a homologous series of n-
alkanes (C8-C20) and by comparison of their mass
spectrum with reported data (Adams, Wiley, and
Nist/EPA/NIH Mass Spectral Library).
Antibacterial and Antifungal assays
Antimicrobial and antifungal activity was
assayed against eight microorganisms including Gram
(+) and (-) bacteria and yeast: Staphylococcus aureus
(ATCC 29213), Pseudomona aeruginosa (ATCC
27853), Escherechia coli (ATCC 25299), Candida
albicans (NIM 982879), Candida tropicalis (ATCC
2000956), Candida parapsilopsis (ATCC 22019),
Candida krusei (ATCC 6258), Candida guillermondi
and Candida glabrata donated by ANLIS Malbrn
Institute.
The antimicrobial activity was performed in
solid phase by Agar Dilution Method (Wright et al,
1983; Ruhnke et al, 1996). The oil and its dilutions
(1/125, 1/250, 1/500, 1/1000 y 1/2000 v/v) were
initially mixed with sterile nutritive agar for bacteria
and 4% glucose-sabouread for yeast and then stirred
for a minute in a vortex at 3000 rpm. An inoculum of
10
6
cells per ml was mixed with the medium, bacteria
and yeast culture were incubated for 24 hours at 37C
and 48 hours at 28C respectively. The MIC endpoint
was determined visually by recording the lowest
concentration of the essential oil that prevented the
appearance of visible growth.
Essential oil Extraction
The amounts of essential oils obtained in both
Senecio species were similar. The yield was 0.81% for
Senecio subpanduratus and 0.71% for Senecio
mustersii, expressed as ml of essential oil per 100 g of
fresh vegetable matter.


Chromatography-Mass Spectrometry
A quantitative and qualitative variation between
both Senecio species was apparent. Relative
percentages of the oil constituents are showed in table
1, listed in order of elution from the used column.
Twenty four compounds were identified for
S.mustersii (95.2%) and twenty one for
S.subpanduratus (92.9%).
Table 1: Chemical composition of essential oil of S.mustersii and
S.subpanduratus (expressed as percentages).
Compounds S. mustersii S. subpanduratus
tuyene 0.1 1.7
-pinene 53.3 22.1
sabinene 1.6 23.8
myrcene 2.3 2.6
pinene 21.2 11.9
3-carene 0.2 0.4
p-cymene 1.7 8.7
limonene 1.8 1.2
perillene tr 0.2
-pinene oxide 1 0.6
-canfolenal 0.3 -
pinocarveol trans+sabinol trans 2.1 -
sabina ketone - 0.4
pinocarvone 0.4 -
Terpinen-4-ol 0.5 10.2
p-cimen-8-ol tr 0.4
-terpineol 0.4 0.7
myrtenol + myrtenal 1.3 -
myrtenal - 0.7
pinocarveol acetate trans 0.9 -
pinocarveol acetate cis 1.1 0.3
kessane 0.9 2.9
spathulenol 0.2 0.4
-oplopenone 1.2 2.5
Tcadinol 0.2 tr
Epi--murolol 0.8 0.2
-cadinol 1.7 1
TOTAL 95.2 92.9

The essential oil of S. mustersii was characterized
by -pinene (53.3%) and -pinene (21.2%) as major
components. Meanwhile in S.subpanduratus -pinene
(22.1%) and -pinene (11.9%) were also detected, in
addition to an important amount of sabinene (23.8%),
terpinen-4-ol (10.2%) and p-cymene (8.7%).
Antibacterial and Antifungal assays
The results are showed in table 2, where the
antimicrobial activity was determined by the
appearance of visible growth.
The isolated essential oil of S. subpanduratus
showed antimicrobial activity against the three
bacterial strains tested and also against C.albicans, C.
parapsilisis and C.guillermondii, a fact that may
become relevant, given the pathogenic properties of
these strains. The isolated essential oil of S. mustersii had
only antimicrobial activity against S.aureus. This
difference can be explained by the chemical
composition of the essential oils.
Table 2: Antimicrobial activity of essential oil of S.mustersii and
S.subpanduratus
Essential oil Dilutions (v/v)
Senecio mustersii Senecio subpanduratus
Bacteria
1
/
1
2
5

1
/
2
5
0

1
/
5
0
0

1
/
1
0
0
0

1
/
2
0
0
0

1
/
1
2
5

1
/
2
5
0

1
/
5
0
0

1
/
1
0
0
0

1
/
2
0
0
0

S. aureus + + - - - + + - - -
E. coli - - - - - + + - - -
P. aeruginosa - - - - - + + - - -
Yeast

C. albicans - - - - - + + + + +
C. tropicalis - - - - - - - - - -
C. parapsilosis - - - - - - - - - -
C. guillermondii - - - - - + + + + -
C. krusei - - - - - + + - - -
C. glabrata - - - - - + - - - -
(+)Antimicrobial activity. (-) No antimicrobial activity.
Antimicrobial activity of essential oils is difficult to
attribute to a specific compound, probably because of
the complexity of its composition and also for the
synergic effects that may exist between the major
components. Despite this, there are studies that explain
the antimicrobial activity of some components of the
essential oil. It has been demonstrated that and -
pinene are able not only to destroy cellular integrity,
but also inhibit respiration and ion transport processes.
They also increase the membrane permeability in yeast


cells (Magwa et al 2006, Andrews 1980, Uribe
1985).There are other studies of essential oils that
demonstrate effects also on gram negative membrane
(Helander, 1998).
CONCLUSIONS
This is the first report about chemical
composition of essential oil and biological activity of
these species of Senecio from the East Central Area of
Patagonia. The high percentage of pinenes ( and ) in
both species could play an important role in defensive
mechanism and adaptation to dessert area. The
differences in antimicrobial activity of both species of
Senecio could be related to oxygenated derivates,
which are in higher percentage in S.subpanduratus.
ACKNOWLEDGEMENTS
We would like to thank ANLIS
(Administracin Nacional de Laboratorios e Institutos
de Salud) Dr. Carlos Malbrn, for donating the
strains that were used in this investigation.
REFERENCES
Andrews RE, Parks LW, Spence KD. 1980. Some effects of
Douglas fir terpenes on certain microorganism. Appl.
Environ. Microbiol. 40: 301-304.
Bohlmann F, Zdero C, Jakupovic J, Grenz M, Castro V,
King RM, Robinson H, Vincent LPD. 1986. Further
Pyrrolizidine Alkaloids and Furanoeremophilanes from
Senecio species. Phytochemistry. 25(5): 1151-1159.
Cabrera A. 1971. Compositae, pp. 242-243. In Correa MN:
Flora Patagnica, parte 7. Coleccin Cientfica del
INTA. Buenos Aires. Argentina
De Salmeron MSA, Kavka J, Giordano OS. 1983.
Furanoeremophilanes in Senecio filaginoides and
S.pinnatus. Planta Med.. 47: 221-223.
El-Shazly A, Doral G, Wink M. 2002. Chemical
Composition and Biological Activity of the Essential
Oils of Senecio aegytius var.discoides Boiss. Z.
Naturforsch. 57 (c): 434-439.
Gonzlez S, Guerra P, Bottaro H, Demo M, Zunino M,
Zygadlo J. 2004. Aromatics plants from Patagonia,
Antimicrobial activity and chemical composition of
Schinus polygamus (Cav.) Cabrera essential oil.
Flavour Fragrance J. 19 (1): 36-39.
Helander IM, Alakomi HL, Kyosti LK, Mattiala-sndholm Y,
Pol I, Smid EJ, Gorris GM, von Wright A. 1998.
Characterization of the action of selected essential oil
components on Gram-negative bacteria. J. Agric. Food
Chem. 46: 3590-3595.
Kamatou GPP, Viljoen AM, Figueiredo AC, Tilney PM,
Van Zyl RL, Barroso JG, Pedro LG, Van Vuuren, SF.
2007. Trichomes, essential oil composition and
biological activities of Salvia albicaulis Benth. and
S.dolomitica Codd, two species from the Cape region of
South Africa. S. Afr. J. Bot. 73: 102-108.
Magwa ML, Gundidza M, Gweru N, Humphrey G. 2006.
Chemical composition and biological activities of
essential oil from the leaves of Sesuvium
portulacastrum. J Ethnopharmacol. 103: 85-89.
Malizia RA, Molli JS, Cardell DA, Retamar JA; Arancibia
LA; Arce ME. 2006. Aceite Esencial de Schinus
johnstonii Barkley. Naturalia Patagnica. 3 (1):45-48.
Perez C, Agnese AM, Cabrera JL. 1999. The essential oil of
Senecio graveolens (Compositae): Chemical
composition and antimicrobial activity test. J
Ethnopharmacol 66: 91-96.
Ruhnke M, Schmidt-Westhausen A, Engelmann E,
Trautmann M. 1996. Comparative evaluation of three
antifungal susceptibility test methods for Candida
albicans isolates and correlation with response to
fluconazole therapy. J Clin Microbiol. 34(12): 3208
3211.
Torres P, Ayala J, Grande C, Anaya J, Grande M. 1999.
Furanoeremophilane derivates from Senecio flavus.
Phytochemistry. 52: 1507-1513.
Uribe S, Ramirez T, Pena A. 1985. Effects of -pinene on
yeast membrane functions. J. Bacteriol. 161: 195-200.
Wright LR, Scott EM, Gorman SP. 1983.The sensitivity of
mycelium, arthrospores and microconidia of
Trichophyton mentagrophytes to imidazoles determined
by in-vitro tests. J. Antimicrob. Chemother. 12: 317-
327.

2010 The Authors




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-
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.
Anti-inflammatory Activity of Aristotelia chilensis Mol. (Stuntz)
(Elaeocarpaceae).
[Actividad anti-inflamatoria de Aristotelia chilensis Mol. (Stuntz) (Elaeocarpaceae).]
Carlos L CSPEDES
*1
, Julio ALARCON
1
, Jose G AVILA
2
, Antonio NIETO
3

1
Plant Biochemistry and Phytochemical Ecology Laboratory, Department of Basic Sciences, University of Bo-Bo, Chilln, Chile.
2
Phytochemical Laboratory, UBIPRO, FES-Iztacala, UNAM,
3
Laboratorio de Pruebas Biolgicas, Instituto de Qumica, UNAM.
Abstract
Context: Chilean Black-berry Aristotelia chilensis is a wild fruit that growth in southern Chile. This fruit possess a strong antioxidant activity
and is commonly used in foods and beverages in Chile. Objective: The anti-inflammatory activity of the extracts, fractions and subfractions of this fruit are
investigated here for the first time. Materials and methods: Extracts, fractions and subfractions were analyzed for their content in total phenolics and the
effects in the TPA-induced inflammation in ear of the mouse ear edema induced by single doses of TPA were investigated. In addition, the antioxidant
activity was investigated against DPPH, Crocin and TBARS. Results: The results showed that extract B, fraction F-4, and ovatifolin, quercetin, myricetin,
luteolin and diosmetin used as pattern compounds were the most active samples together with those subfractions rich in phenolic compounds. Thus, SF11-
SF15, SF16-SF20, and SF21-SF25 showed are the best subfractions inhibitors in similar form to indomethacin a known selective COX inhibitor that have an EI50
of 0.11 mg/ear. Results demonstrated that these samples strongly inhibited the induced inflammation in ear of the mouse edema in TPA inflammation model,
with EC50 values ranging from 0.3 to 11.8 g/mL. Discussion and conclusion: These findings demonstrate that the fruits and their constituents of A.
chilensis have excellent anti-inflammatory activities and thus have great potential as a source for natural health products. Additionally, these findings showed
that the flavonoids, phenolic acids and anthocyanins present in this fruit may be responsible of the antioxidant activity observed.
Keywords: Aristotelia chilensis, anti-inflammatory activity, antioxidants, DPPH, crocin, TBARS.
Resumen
Contexto: Chilean Blackberry Aristotelia chilensis es un fruto silvestre que crece en el sur de Chile. Este fruto posee una fuerte actividad antioxidante
y comnmente es usado en alimentos y bebidas en Chile. Objetivo: Se investigo la actividad anti-inflamatoria de los extractos, fracciones y subfracciones de
este fruto y son informados aqu por primera vez. Materiales y mtodos: Los extractos, fracciones y subfracciones fueron analizados por su contenido total
de fenoles y se investigo el efecto sobre la inflamacin en oreja de rata a travs de la induccin con TPA en dosis sencillas. Adems se investigo la actividad
antioxidante frente a DPPH, Crocina y TBARS. Resultados: Los resultados muestran que el extracto B, la fraccin F-4, y ovatifolina, quercetina, myricetina,
luteolina y diosmetina, que se usaron como muestras patrones, fueron las mas activas junto con aquellas subfracciones ricas en compuestos fenlicos. Asi,
SF11-SF15, SF16-SF20, y SF21-SF25 mostraron ser las mejores subfracciones inhibitorias en una forma similar a indometacina un conocido inhibidor selectivo de
COX que tiene un EI50 de 0.11 mg/oreja. Los resultados demuestran que estas muestras inhiben fuertemente la inflamacin inducida en el modelo del edema
en oreja de rata, con valores de EC50 entre 0.3 a 11.8 g/mL. Discusin y conclusin: Estos hallazgos demuestran que los frutos y sus constituyentes de A.
chilensis poseen una excelente actividad anti-inflamatoria, y as tienen un gran potencial como una fuente de productos naturales saludables. Adicionalmente,
estos hallazgos muestran que los flavonoides, cidos fenlicos y antocianinas presentes en este fruto podran ser los responsables de la actividad antioxidante
observada.
Palabras Clave: Aristotelia chilensis, anti-inflammatory activity, antioxidants, DPPH, crocin, TBARS.

Recibido | Received: November 1, 2009
Aceptado en Versin Corregida | Accepted in Corrected Version: January 4, 2010
Publicado en Lnea | Published Online March 25, 2010
Financiacin | Funding: This work was supported in part by internal grant from Department of Basic Sciences, University of Bio-Bio, Chillan, Chile.
This article must be cited as: Carlos L. Cspedes, Julio Alarcon, Jose G. Avila Acevedo, A. Nieto. 2010. Anti-inflammatory Activity of Aristotelia chilensis Mol. (Stuntz)
(Elaeocarpaceae). Bol Latinoam Caribe Plant Med Aromat 9(2):127 135. {EPub 25, March 2010}.
*Contactos | Contacts: E-mail address: ccespedes@ubiobio.cl, cespedes_leonardo@yahoo.com; Phone: +56-42-253049, Fax: +-56-42-253046
Cspedes et al. Anti-inflammatory activity of Aristotelia chilensis


INTRODUCTION
A growing body of literature points to the
importance of natural antioxidants from many
plants, which may be used to reduce cellular
oxidative damage, not only in foods, but also in
the human body (Prior et al., 2003; Haliwell and
Aruoma, 1991). This may provide protection
against chronic diseases, including cancer and
neurodegenerative diseases, inflammation and
cardiovascular disease (Prior et al., 2005).
Adverse conditions within the environment,
such as smog and U.V.-radiation, in addition to
diets rich in saturated fatty acids, increase
oxidative damage in the body. Given this
constant exposure to oxidants, antioxidants may
be necessary to counteract chronic oxidative
effects, thereby improving the quality of life
(Roberts et al., 2003; Cespedes et al., 2010).
The increasing interest in the measurement of
the antioxidant activity of different plant
samples is derived from the overwhelming
evidence of the importance of Reactive Oxygen
Species (ROS), including superoxide (O
2
),
peroxyl (ROO
), alkoxyl (RO
), hydroxyl
(OH), and nitric oxide (NO
) radicals in aging
and chronic disease (Fernandes et al., 2004).
Several methods have been developed to
measure the antioxidant activity in biological
samples, including the oxygen radical absorption
capacity (ORAC), ferric reducing antioxidant
power (FRAP), 2,2-diphenyl-1-picryl-hydrazil
(DPPH) radical scavenging and inhibition of
formation of thiobarbituric acid reactive species
(TBARS) (Taruscio et al., 2004; Schinella et al.,
2002; Prior et al., 2003; Prior et al., 2005).
The use of traditional medicine is widespread
and plants still present a large source of novel
active biological compounds with different
activities, including anti-inflammatory, anti-
cancer, anti-viral, anti-bacterial and
cardioprotective activities (Seigler 1998;
Schinella et al., 2002; Yan et al., 2002).
Berries constitute a rich dietary source of
phenolic antioxidant and bioactive properties
(Pool-Zobel et al., 1999; Smith et al., 2000).
Chilean wild black-berry Aristotelia chilensis
(Mol) Stuntz (Elaeocarpaceae), an edible black-
colored fruit, which reach its ripeness between
December to March, have a popular and very
high consume during these months in Central
and South Chile and western of Argentina.
Previously, we have reported the alkaloid
composition of the leaves of A. chilensis
(Cespedes et al., 1990; Cespedes et al., 1993;
Cespedes, 1996). The botanical characteristics
were reported previously (Cespedes et al., 1996;
2008; 2010; Silva et al., 1997).
This plant has enjoyed popularity as an
ethno-medicine for many years, used
particularly as an anti-inflammatory agent,
kidneys pains, stomach ulcers; diverse digestive
ailments (tumors and ulcers), fever and
cicatrization injuries (Bhakuni et al., 1976), and
the berries have traditionally

been consumed as
treatment for diarrhea and dysentery and the
Araucanian people prepare a liquor with an
ethanolic macerated solution that is used in
religious ritual know as machitun or
nguillatun and as daily beverages (Muoz-
Pizarro, 1966).
Up-to-date some studies reports that the juice
(an aqueous extract) from fruits of A. chilensis
has a good antioxidant activity against FRAP
analysis but not reduce endogenous oxidative
DNA damage in human colon cells (Pool-Zobel
et al., 1999), an effective capacity to inhibit the
cooper-induced LDL oxidation in vitro and the
induction of intracellular oxidative stress
induced by hydrogen peroxide in human
endothelial cells culture (Miranda-Rottmann et
al., 2002), other study report only the partial
composition of anthocyanidins constituents of
the juice (Escribano-Bailon et al., 2006), and
recently was reported the inhibitory activity
against

aldose reductase by an extract rich in
anthocyanins of this fruit (Kraft et al., 2007).
Subsequently, we have some recent reports
about the effects of MeOH extract from ripe
fruits of A. chilensis on isquemic/reperfusion
system, several antioxidant activities of that
extract and its relationship between total
phenolic levels and the cardioprotective effect
(Cespedes et al., 2008; 2010). In other recent
works, in addition to a phytochemical profile
composition by NMR, HPLC, and GC/MS
analyses, the ethanolic extract, fractions and
some phytochemicals that occurs in the fruit
were assayed against ORAC, FRAP, DPPH and


TBARS as an index of lipid peroxidation in
liposomes from brain homogenate (Cespedes et
al., 2010) and the presence of 3-hydroxyindole
in this fruit was reported together with its
antioxidant activity (Cespedes et al., 2009).
In the continuation of our general screening
program of Chilean flora with biological
activities (Cespedes et al., 2001), a re-
examination of the EtOH extract of fruits of A.
chilensis (Elaeocarpaceae) has been initiated.
Thus, in the present work, we designed to
investigate the anti-inflammatory activity in the
TPA-induced inflammation in ear of mice model
of the EtOH, acetone extracts, fractions, and
subfractions, the occurrence of phenolic
compounds, and its relationship with its
phytochemical contents (Cespedes et al., 2010;
2009) of these extracts, fractions and sub-
fractions from ripe fruits of A. chilensis.
The aim of this work was to evaluate the anti-
inflammatory activity of EtOH, acetone, ethyl
acetate and MeOH/H
2
O extracts from ripe fruits
and subfractions from SF
4
to SF
37
isolated from
F-3 and F-4 fractions (see Table 1 and scheme
1). The anti-inflammatory effect on the 12-O-
tetradecanoyl phorbol acetate (TPA)-induced
mouse ear edema test was used (Tubaro et al.,
1985; De Young et al., 1989; Paya et al., 1993).
Additionally, we are reporting the
phytochemical analysis of the bioactive fraction
F-4 and the antioxidant activity of the
subfractions.
Continuously, we are working in a more
complete metabolomic profile of the fruits and
leaves of this plant and in the evaluation of
additional biological activities of leaves.
MATERIAL AND METHODS
Plant material
Fruits of Aristotelia chilensis (Mol) Stuntz
(Elaeocarpaceae) were collected from fields at
foothills of Los Andes at the Araucanian
Region, near to Temuco City, Chile, in January,
2006. Voucher specimens are deposited at the
Herbarium (CONC) of Departamento de
Botnica, Facultad de Ciencias Naturales y
Oceanograficas, Universidad de Concepcin,
Concepcin, Chile and in the botany Collection
of University of Bio-Bio, Campus Chillan. The
collected fruits were air-dried and prepared for
extraction.
Chemicals and solvents
All reagents used were either analytical grade
or chromatographic grade, 2,2-azobis (2-
aminopropane) dihydrochloride (AAPH), 2,2-
diphenyl-1-picryl-hydrazyl (2,2-Diphenyl-1-
(2,4,6-Trinitrophenyl), DPPH), Butylated
Hydroxy Toluene (BHT), 2[3]-t-Butyl-4-
hydroxytoluene (THQ), 2[3]-tert-butyl-4-
hydroxyanisole (BHA), 2[3]-tert-
butylhydroquinone monomethyl ether (TBH),
ethylenediaminetetraacetic acid (EDTA), bovine
serum albumin, Percoll, Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid),
quercetin, Folin-Ciocalteu reagent, 2-
thiobarbituric acid (TBA), FeSO
4
, trichloroacetic
acid, gentisic acid (2,5-dihydroxybenzoic acid),
gallic acid, p-coumaric acid, o-coumaric acid,
propil-gallate, quercetin (3,3;,4,5,7-
pentahydroxyflavone), myricetin (3,3,4,5,5,7-
hexahydroxyflavone), kaempferol (3,4,5,7-
tetrahydroxyflavone), ()-catechin hydrate, (-)-
catechin gallate, (-)-gallocatechin, gallocatechin-
gallate, -carotene, saffron, crocin, sorbitol,
tricine, and trizma-hydrochloride were
purchased from Sigma-Aldrich Qumica, S.A. de
C.V., Toluca, Mexico, or Sigma, St. Louis, MO.
Glycosides of anthocyanidins (cyanidin 3,5-
diglucoside, delphinidin 3,5-diglucoside,
cyanidin, delphinidin) were purchased from
Fluka, (Fluka-Sigma-Aldrich Qumica, S. A. de
C. V., Toluca, Mexico), samples of luteolin,
diosmetin and proanthocyanidins were a gift
from Prof. Dr. David Seigler University of
Illinois at Urbana-Champaign.
Methanol, CH
2
Cl
2
, CHCl
3
, NaCl, KCl,
KH
2
PO
4
, NaHPO
4
, NaOH, KOH, HCl, sodium
acetate trihydrate, glacial acetic acid silica gel
GF
254
analytical chromatoplates, Sephadex LH-
20, silica gel grade 60, (70-230, 60A) for
column chromatography, n-hexane, and ethyl
acetate were purchased from Merck-Mexico,
S.A., Mexico. Indomethacin, quercetin,
myricetin, luteolin, diosmetin and ovatifolin
were used as pattern samples.


Apparatus
A UV Spectronic model Genesys 5
spectrophotometer was used for biological and
spectrophotometric analyses. Fluorimetric
measurements were determined with TURNER
Barnstead-Thermolyne, model Quantech S5
Fluorometer, with 420, 440, 470, 550, and 650
Turner filters. HPLC Hewlett-Packard, Series
1050, with diode array detector, and UV
detector at 254, 280, 365 and 520 nm,
column YMC C18-Pack ODS-AM-303,
AM12S05-2546 WT, 250 x 4.6 mm, ID S-
5um, 12nm; movil phase
water/methanol/acetonitrile (50:35:15),
isocratic, pressure 212 bar; it was prepared
300 L of each sample in amber vials and
injected 20 L of each sample.
Obtention of extracts, fractions, subfractions
and sample preparation
All extracts, fractions and subfractions were
obtained as described in scheme 1. The
composition of each subfraction was reported in
Cespedes et al. 2010.
Anti-inflammatory activity
The assay of TPA-induced ear edema in mice
was based on the described method (Tubaro et
al., 1985; Merlos et al., 1991; Della Loggia et
al., 1996). Groups of 5 male CD-1 mice (25-
30g) were anaesthetized with Imalgen. A
solution of 12-O-tetradecanoylphorbol-13-
acetate (TPA, 2.5 g) in acetone (10L) was
topically applied to both faces (5L each face)
of the right ear of the mice. The left are received
only acetone. Solutions of 0.05, 0.1 and 0.5 mg
in 20 L of acetone of the extracts, sub-fractions
and quercetin, ovatifolin, diosmetin, luteolin,
myricetin and indomethacine as references, these
solutions were applied to both faces of the right
ear (10L each face) 10 min after TPA
treatment. Control animals received only
acetone. Fours hours later the animals were
killed by cervical dislocation. A 9 mm diameter
plug was removed from each ear. The swelling
was assessed as the difference in weight between
the right and left ear plugs. The % inhibition of
edema was calculated by the equation: % =
(edema A edema B/edema A) x 100, where
edema A = edema induced by TPA alone and
edema B = edema induced by TPA plus sample.
Estimation of lipid peroxidation
As an index of lipid peroxidation, TBARS
levels were measured using rat brain
homogenates according to the method described
by Ng with some modifications (Ng et al.,
2000), and as is described in Dominguez et al.,
2005. Results are expressed as nanomoles of
TBARS per milligram of protein, with percent
inhibition after 30 min calculated as the
inhibition ratio (IR), where C) absorbance of the
control and E) absorbance of the test sample.
These values were plotted against the log of the
concentrations of individual extracts and
fractions, and a decrease of 50% in peroxidation
was defined as the EC
50
(Dominguez, et al.,
2005).
Reduction of the 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH)
Extracts and partitions were
chromatographed on TLC and examined for
antioxidant effects by spraying the TLC plates
with DPPH reagent. Specifically, the plates were
sprayed with 0.2% DPPH in methanol.
Quercetin and -tocopherol were used as
standards (Dominguez, et al., 2005).
Bleaching of crocin.
The solutions were placed under UV
254
light.
Following the decrease of absorbance, bleaching
of crocin and fluorescence emission at 440 and
470 nm were monitored with time each 5 min
(Cespedes et al, 2008; Dominguez et al., 2005).
Statistical analysis
Data shown in table 1 is the mean results
obtained with means of five animals and are
presented as mean standard errors of the mean
(SEM). Data were subjected to analysis of
variance (ANOVA) with significant differences
between means identified by GLM Procedures.
The results are given in the text as probability
values, with p < 0.05 adopted as the criterion of
significance, differences between treatments
means were established with a Dunnetts test.


Table 1. Amounts of phenolic content (mg/L Standard
error) from extracts, fractions and some compounds from
acetone partition B of A. chilensis and indomethacin needed
for inhibitory effect on the TPA-induced inflammation in
mice model
a
. EI=Edema Inhibition (%).
Samples
f
/ Dose EI
50
(mg/ear)
b

Indo-methacin 0.11
c

A
f
3.6
c

B
f
1.8
c

C
f
5.9
d

D
f
6.7
d

F-1 10.9
d

F-2 11.8
d

F-3 1.1
c

F-4 1.9
c

SF
4
-SF
6
6.7
d

SF
7
10.1
d

SF
8
-SF
10
11.0
d

SF
11
-SF
15
0.3
c

SF
16
-SF
20
0.9
c

SF
21
-SF
25
1.0
c

SF
26
-SF
30
3.8
c

SF
31
-SF
37
4.1
c

Quercetin 0.16
c

Ovatifolin 0.068
c

Diosmetin 0.45
c

Luteolin 0.25
c

Myricetin 0.17
c

a
Effects on ear edema of female mice CD-1. Means of five
animals in independent experiments. Data expressed as %
of the mean SD of weigh of ear. All data analyzed with t-
student test.
b
Each value correspond to concentration that
inhibits 50% of edema development during bioassay stage.
c
P < 0.05
d
P < 0.01
e
Not determined.
f
A:
Methanol/water (6:4) extract. B: Acetone extract. C: Ethyl
acetate extract. D: MeOH/H2O Residue. (Cespedes et al.,
2010)
Table 2. Amounts of Phenolic content (mg/L Standard
error) from subfractions

of A. chilensis needed to inhibit
oxidative damage by 50%
a
.
Sample
b
DPPH
c
TBARS
d Crocin
e

SF
4
-SF
6
23.8 7.9
13.4
SF
7
37.4 11.8
18.9
SF
8
-SF
10
47.6 9.9
12.0
SF
11
-SF
15
1.3 (2.2)
f
1.9
0.7
SF
16
-SF
20
2.3 (3.8)
f
2.1
0.8
SF
21
-SF
25
5.2 (7.8)
f
2.3
1.1
SF
26
-SF
30
17.1 3.9
0.9
SF
31
-SF
31
19.7 10.1
2.4
Myricetin 21.0 9.7
22.9
Quercetin 19.98 2.90
21.0
-Tocopherol 11.9 3.92
10.1
a
Values expressed as g/mL (ppm), Mean Confidence
Interval 95%, n = 3. Different letters show significant
differences at (P < 0.05), using Duncans multiple-range
test.
b
See scheme 1 for an explanation of extracts and
partitions.
c
IC
50
for inhibition of DPPH radical formation.
d

IC
50
for inhibition of peroxidation of lipids, estimated as
thiobarbituric acid reactive substances. Values are
expressed as g/mL (ppm), See Methods for details. Mean
SD, n = 3. Different letters show significant differences
at (P < 0.05), using Duncans multiple-range test.
e
IC
50
for
bleaching of crocin.
f
The values between parenthesis
correspond to the assay made with 50 M of final
concentration of DPPH. For methodology used see
Dominguez et al., 2005.
Values of extracts A, B, C, D, E,
and fractions F-1, F-2, F-3 and F-4, were reported in
Cespedes et al., 2010, here are show only SF from which
were isolated different compounds, see scheme 1.
Figure 2. HPLC-DAD of F-4, all peaks were identified comparing with data bases, patterns and authentic samples. For GC/MS
analyses of peak at 24.9 min, see Cespedes et al., 2009.



Scheme 1. Method of obtaining extracts, partitions, fractions. Fraction F-1 (Hexane 100%), fraction F-2 (hexane : ethyl acetate 1 : 1),
fraction F-3 (ethyl acetate : Methanol 1 : 1), fraction F-4 (methanol 100%). Extract E correspond water 100%. Note A: F-3 together with F-4
were collect up and chromatographed on silica-gel by Vacuum chromatography, solvent system starting with n-hexane, ethyl acetate and
increasing MeOH-H
2
O. Furthermore F
4
to F
30
were chromatographed on Sephadex LH-20 column, solvent system starting with EtOH and
going to 100% acetone. (The phytochemical composition of each subfraction in Cespedes et al., 2010).

The EC
50
values for each activity were calculated
by Probit analysis on the basis of the percentage of
inhibition obtained at each concentration of the
samples. EC
50
is the concentration producing 50%
inhibition. Completely statistical analysis was
performed by means of the MicroCal Origin 8.0
statistical and graphs PC program.
Anti-inflammatory activity.
The results of anti-inflammatory activities of
extracts A, B, C, D, fractions F-1 to F-4, and
subfractions SF
4
to SF
37
are outlined in Table 1. These
findings shows that the TPA-induced inflammation in
mouse method was well inhibited mainly by extracts
A, B, F-4, SF
11
-SF
15
, SF
16
-SF
20
, SF
21
-SF
25
and SF
26
SF
30
with EI
50
of 3.6, 1.8, 1.9, 0.3, 0.9, 1.0 and 3.8
mg/ear, respectively. Additionally, quercetin,
ovatifolin, diosmetin, luteolin, myricetin and
indomethacin showed EI
50
0.16, 0.068, 0.45, 0.25, 0.17
and 0.11 mg/ear, respectively used as pattern samples.
The bioassay was carried out between 0.01 and 15.0
mg/ear with all samples being extract B, fraction F-4,
subfractions SF
11
-SF
15
, SF
16
-SF
20
, and SF
21
-SF
25
,
ovatifolin, quercetin, myricetin, luteolin and diosmetin
the most active samples, therefore with these samples
was made a curve of dose-response, obtaining the EI
50

showed in Table 1. All samples used in this study
showed a dose-dependent anti-inflammatory activity.
These effects were compared with those produced
by the commercially available anti-inflammatory drug
indomethacin and ovatifolin (Cespedes et al., 2000),
together with quercetin, myricetin, luteolin and
diosmetin as natural compound (Cespedes et al.,
2001), (Table 1). All of compounds assayed inhibited
the TPA-induced inflammation (data not show). On
the other hand, extract B, fraction F-4, and
subfractions SF
11
-SF
15
, SF
16
-SF
20
were as well as
active as indomethacin at 0.11 mg/ear a selective
cyclo-oxygenase (COX) inhibitor (Table 1). Is
important mention that F-4 showed a very good anti-
inflammatory activity. This action could be attributed
to a synergic effect proportionated by the phenolic rich
composition observed in this fraction Fig. 2.
On the other hand, a decrease in the anti-
inflammatory activity was observed when F-1, F-2,
SF
7
, SF
8
-SF
10
, SF
26
-SF
30
, and SF
31
-SF
37
, which have
sugared components. A similar effect, but in minor
percentage was observed when was used diosmetin
instead of luteolin. Surprisingly, myricetin showed an
intermediate activity, since at 0.17 mg/ear showed a
50.0% of inhibition.


Antioxidant activities. DPPH and TBARS
evaluation
The DPPH radical scavenging assay was used first
as a screen for antioxidant components within the
primary extracts (Dominguez et al., 2005; Cespedes,
El-Hafidi, Pavon & Alarcon, 2008). The results of
antioxidant activity of extracts A, B, C, D, E, and
fractions F-1 to F-4, were published previously at
Cespedes et al., 2010. As shown in Table 2, the
subfractions SF
11
-SF
15
, SF
16
-SF
20
, and SF
21
-SF
25
had
the highest inhibitory activity against DPPH radical
formation compared to the other partitions with IC
50

values of 1.3, 2.3 and 5.2 ppm, respectively. Almost
all these samples exhibited a concentration-
dependence manner in their DPPH radical scavenging
activities, particularly SF
11
-SF
15
, and SF
16
-SF
20
which
showed the highest activity (100% inhibition) at a
concentration of 4.9 and 15.5 ppm, respectively (data
not show). This action was greater than that of -
tocopherol, which at 31.6 ppm caused only 53.8%
quenching and very similar to ferulic and p-coumaric
acids with IC
50
values of 5.1 and 7.8 ppm, respectively
(data not shown), similar performance was observed
against crocin inhibitory activity (Table 2).
In addition to pattern samples the subfractions
SF
11
-SF
15
, SF
16
-SF
20
, and SF
21
-SF
25
showed
considerable activity, quenching DPPH
radical
reduction completely (100 % of inhibition, data not
show). Nevertheless, SF
4
-SF
6
, SF
26
-SF
30
and SF
31
-
SF
37
showed a moderate activity their IC
50
values were
23.8, 17.1 and 19.7 ppm, respectively (Table 2), these
subfractions showed to have the highest concentration
of anthocyanins, and reached the 100% of inhibition at
similar concentrations than SF
16
-SF
20
, and SF
21
-SF
25
.
The lowest I
50
value for SF
11
-SF
15
, SF
16
-SF
20
, (1.3 and
2.3 ppm, respectively) than for any of the other
subfractions, might be due to a synergistic effect of the
components due to extraction procedures (mainly
gallic acid, quercetin, myricetin, delphinidin-3-
glucoside and cyaniding-3-glucoside, (scheme 1))
inside this subfraction, similar to that reported for
components of Vaccinium corymbosum and V.
angustifolium fruits (Ehlenfeldt and Prior, 2001, Smith
et al., 2000, Lo & Cheung, 2005), where the acetone
and MeOH partitions were the most active extracts.
Of the many biological macromolecules, including
carbohydrates, lipids, proteins, and DNA, that can
undergo oxidative damage in the presence of ROS,
membrane lipids are especially sensitive to oxidation
from this physiological process (Diplock et al., 1998).
For this reason, brain homogenates were used for the
investigation of lipid peroxidation as an assessment of
oxidative stress. The capacity for plant extracts to
prevent lipid peroxidation was assayed using
malondialdehyde formation as an index of oxidative
breakdown of membrane lipids, following incubation
of rat brain cortical and hearth homogenates with the
oxidant chemical species Fe
2+
. Ferrous ion both
stimulates lipid peroxidation and supports
decomposition of lipids peroxides once formed,
generating highly reactive intermediates such as
hydroxyl radicals, perferryl and ferryl species (Ko et
al., 1998). Against TBARS SF
11
-SF
15
, SF
16
-SF
20
and
SF
21
-SF
25
were most effective in similar form to
quercetin or BHT in inhibiting lipid peroxidation.
SF
11
-SF
15
had the greatest activity and reduced lipid
peroxidation in a dose-dependent manner, and proved
to be an excellent antioxidant, reflected by its low IC
50

value (1.9 ppm) when analyzed by both TBARS and
DPPH (Table 2), at the same level than quercetin and
-tocopherol whom shows IC
50
of 2.9 and 3.92 ppm,
against TBARS formation, respectively (Table 2).
When the relative contribution of each subfraction
to the total antioxidant activity was evaluated using
TBARS, all samples showed some protective effect,
all the IC
50
values of all subfractions are shown in
Table 2. SF
11
-SF
15
and SF
16
-SF
20
were the most active,
with IC
50
values of 1.9 and 2.1 ppm, respectively. It is
noteworthy that the value for SF
11
-SF
15
is very low
compared with values for flavonoids and anthocyanins
in general, as well as for myricetin or quercetin (data
not show) (Makris & Rossiter, 2001; Lo & Cheung,
2005).
CONCLUSIONS
In general these compounds that occur in these
Aristotelia species have been considered as the active
principles of many anti-inflammatory plants. Thus,
many phenolic acids, anthocyanins and flavonoids
type have shown inhibitory activities on nitric oxide
implicated in physiological and pathological process
as chronic inflammation (Matsuda et al., 2000;
Odontuya et al., 2005).
These finding shows that the anthocyanins,
flavonoids and phenolic acids may be responsible of
the anti-inflammatory activity of this fruit. We are
working in the kinetic of inhibition of these plant
extracts and compounds as anti-inflammatory and
additionally we are dissecting the sites and mechanism
of action as iNOS, COX, and TNF, among others.


ACKNOWLEDGMENTS
This work was supported in part by internal
grant from Department of Basic Sciences, University
of Bio-Bio, Chillan, Chile. We thank M. Teresa
Ramirez, and Antonio Nieto, for technical assistance;
Chemistry Institute, UNAM. The authors are indebted
to Dr. Isao Kubo, University of California at Berkeley,
US, for the great help in the correction of manuscript.
REFERENCES
Bhakuni DS, Bittner M, Marticorena C, Silva M, Weldt F,
Hoeneisen M, Hartwell JL. 1976. Screening of Chilean
plant for anticancer activity. J Nat Prod 39: 225.-243.
Cazor A, Deborde C, Moing A, Rolin D, This H. 2006.
Sucrose, glucose, and fructose extraction in aqueous
carrot root extracts prepared at different temperatures
by means of direct NMR measurements. J Agric Food
Chem 54: 4681-4686.
Cespedes CL, Jakupovic J, Silva M, Watson WH. 1990.
Indole alkaloids from Aristotelia chilensis.
Phytochemistry 29: 1354-1356.
Cespedes CL, Jakupovic J, Silva M, Tsichritzis F. 1993. A
Quinoline alkaloid from Aristotelia chilensis.
Phytochemistry 34: 881-882.
Cespedes CL. 1996. Obtencion de alcaloides indolicos tipo
aristotelia mediante cultivo de tejidos in vitro. Doctoral
thesis. University of Concepcion, Concepcion, Chile,
pp 254.
Cespedes CL, Ramirez-Apan T, Uchoa A, Calderon JS,
Hoeneisen M, Silva M. 2000. Anti-Inflammatory
activity of ovatifolin. Rev Latinoamer Qum 28: 127-
132.
Cespedes CL, Hoeneisen M, Bittner M, Becerra J, Silva M.
2001. Comparative study of ovatifolin antioxidant and
growth inhibition Activities. J Agric Food Chem 49:
4243-4251.
Cespedes CL, El-Hafidi M, Pavon N, Alarcon J. 2008.
Antioxidant and cardioprotective activities of phenolic
extracts from fruits of Chilean blackberry Aristotelia
chilensis (Elaeocarpaceae), Maqui. Food Chem 107:
820829.
Cespedes CL, Valdez-Morales M, Avila JG, El-Hafidi M,
Alarcon J, Paredes-Lopez O. 2010. Phytochemical
profile and the antioxidant activity of Chilean wild
black-berry fruits, Aristotelia chilensis (Mol) Stuntz
(Elaeocarpaceae). Food Chem 119:886-895.
Cespedes CL, Alarcon J, Valdez-Morales M, Paredes-Lopez
O. 2009. Antioxidant activity of an unusual 3-
hydroxyindole derivative isolated from fruits of
Aristotelia chilensis (Mol) Stuntz. Z Naturforsch C
64c: (9/10), 759-762.
Della Loggia R, Sosa S, Tubaro A, Morazzoni P,
Bombardelli E, Griffini A. 1996. Anti-inflammatory
activity of some Ginkgo biloba constituents and their
phospholipid-complexes. Fitoterapia 67: 257-264.
De Young LM, Kheifets

JB, Ballaron SJ, Young JM. 1989.
Edema and cell infiltration in the phorbol ester-treated
mouse ear are temporally separate and can be
differentially modulated by pharmacologic agents.
Inflamm Res 26: 335-341.
Diplock AT, Charleux JL, Crozier-Willi G, Kok FJ, Rice-
Evans C, Robefroid M, Stahl W, Via-Ribes J. 1998.
Functional food science and defence against reactive
oxidative species. Brit J Nutr 80: suppl.1, S77-S112.
Dominguez M, Nieto A, Marin JC, Keck AS, Jeffery E,
Cespedes CL. 2005. Antioxidants activities of extracts
from Barkleyanthus salicifolius (Asteraceae) and
Penstemon gentianoides (Scrophulariaceae). J Agric
Food Chem 53: 5889-5895.
Ehlenfeldt MK, Prior RL. 2001. Oxygen Radical
Absorbance Capacity (ORAC) and Phenolic and
Anthocyanin Concentrations in Fruit and Leaf Tissues
of Highbush Blueberry. J Agric Food Chem 49: 2222-
2227.
Escribano-Bailon MT, Alcalde-Eon C, Muoz O, Rives-
Gonzalo JC, Santos-Buelga C. 2006. Anthocyanins in
berries of Maqui (Aristotelia chilensis (Mol) Stuntz).
Phytochem Anal 17: 814.
Fernandes E, Costa D, Toste SA, Lima JLFC, Reis S. 2004.
In vitro scavenging activity for reactive oxygen and
nitrogen species by nonsteroidal anti-inflammatory
indole, pyrrole and oxazole derivative drugs. Free Rad
Biol Med 37: 1985-1905.
Halliwell B, Aruoma OI. 1991. DNA damage by oxygen
derived species. Its mechanism and measurement in
mammalian systems. FEBS Letts 281: 9-19.
Ko FN, Cheng ZJ, Lin CN, Teng CM. 1998. Scavenger and
antioxidant properties of prenylflavones isolated from
Artocarpus heterophyllus. Free Rad Biol Med 25: 160-
168.
Kraft T, Grace M, Yousef G, Rogers R, Raskin I, Lila MA.
2007. Phytochemical composition and aldose reductase
inhibitory activity of Aristotelia chilensis (maqui)
berries. FASEB J 21: A732-A732.
Lo KM, Cheung PCK. 2005. Antioxidant activity of extracts
from the fruiting bodies of Agrocybe aegerita var. alba.
Food Chem 89: 533-539.
Makris DP, Rossiter JT. 2001. Comparison of quercetin and
non-orthohydroxy flavonol as antioxidants by
competing in vitro oxidation reactions. J Agric Food
Chem 49: 3370-3377.
Matsuda H, Kagerura T, Toguchida I, Ueda H, Morikawa T,
Yoshikawa M. 2000. Inhibitory Effects of
sesquiterpenes from bay leaf on nitric oxide production
in lipopolysaccharide-activated macrophages: structure
requirement and role of heat shock protein induction.
Life Sci 66: 2151-2157.
Merlos M, Gomez LA, Giral M, Vericat ML, Garcia-
Rafanell J, Forn J. 1991. Effects of PAF-antagonists in


mouse ear oedema induced by several inflammatory
agents. Br J Pharmacol 104: 990-994.
Miranda-Rottmann S, Aspillaga AA, Perez DD, Vasquez L,
Martinez ALF, Leighton F. 2002. Juice and phenolic
fractions of the berry Aristotelia chilensis inhibit LDL
oxidation in vitro and project human endothelial cells
against oxidative stress. J Agric Food Chem 50: 7542
7547.
Muoz-Pizarro C (1959): Sinopsis de la Flora Chilena. Ed.
Universidad de Chile, Santiago de Chile, Chile. pp. 50-
54.
Odontuya G, Hoult JRS, Houghton PJ. 2005. Structure-
activity relationship for anti-inflammatory effect of
luteolin and its derived glycosides. Phytother Res 19:
782-786.
Paya M, Ferrandiz ML, Sanz MJ, Bustos G, Blasco R, Rios
JL, Alcaraz MJ. 1993. Study of the antioedema activity
of some seaweed and sponge extracts from the
mediterranean coast in mice. Phytother Res 7: 159
162.
Pereira GE, Gaudillere JP, Van Leeuwen C, Hilbert G,
Lavialle O, Maucourt M, Deborde C, Moing A, Rolin
D. 2005.
1
H-NMR and chemometrics to characterize
mature grape berries in four wine-growing areas in
Bordeaux, France. J Agric Food Chem 53: 63826389.
Pool-Zobel BL, Bub A, Schrder N, Rechkemmer G. 1999.
Anthocyanins are potent antioxidants in model systems
but do not reduce endogenous oxidative DNA damage
in human colon cells. Eur J Nutr 38: 227-234.
Prior RL, Hoang H, Gu L, Wu X, Bacchioca M, Howard L,
et al. 2003. Assays for hydrophilic and lipophylic
antioxidant capacity (Oxygen Radical Absorbance
Capacity (ORAC
FL
)) of plasma and other biological and
food samples. J Agric Food Chem 51: 3273-3279.
Prior RL, Wu XL, Schaich K (2005): Standardized methods
for the determination of antioxidant capacity and
phenolics in foods and dietary supplements. J Agric
Food Chem 53: 4290-4302.
Roberts WG, Gordon MH, Walker AF. 2003. Effects of
enhanced consumption of fruit and vegetables on
plasma antioxidants status and oxidative resistance of
LDL in smokers supplemented with fish oil. Eur J Clin
Nutr 57: 1303- 1310.
Schinella GR, Tournier HA, Prieto JM, Mordugovich de
Buschiazzo P, Rios JL. 2002. Antioxidant activity of
anti-inflammatory plants extracts. Life Sci 70: 1023-
1033.
Seigler DS (1998). Plant Secondary Metabolism. Kluwer
Academic Publishers, Norwell, MA.
Silva M, Bittner M, Cespedes CL, Jakupovic J. 1997. The
alkaloids of the genus Aristotelia. Aristotelia chilensis
(Mol.) Stuntz. Bol Soc Chil Quim 42: 39-47.
Smith MAL, Marley KA, Seigler DS, Singletary KW,
Meline B. 2000. Bioactive Properties of Wild Blueberry
Fruits. J Food Sci 65: 352-356.
Taruscio TG, Barney DL, Exon J. 2004. Content and profile
of flavonoids and phenolic acid compounds in
conjunction with the antioxidant capacity for a variety
of northwest Vaccinium Berries. J Agric Food Chem
52: 3169-3176.
Tubaro A, Dri P, Delbello G, Zilli C, Della Loggia R. 1985.
The Croton oil ear test revisited. Agents Actions
17:347-349.
Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A., &
Nieto, C. C. 2002. Antioxidant activities and antitumor
screening of extracts from cranberry fruit (Vaccinium
macrocarpon). J Agric Food Chem 50: 5844-5849.

2010 The Authors

I n vitro antioomycete activity of Artemisia ludoviciana extracts against
Phytophthora spp.
[Actividad antioomiceto in vitro de extractos de Artemisia ludoviciana contra Phytophthora spp]
Luz Mara DAMIAN BADILLO, Rosa Elisa MARTINEZ MUOZ, Rafael SALGADO GARCIGLIA, Mauro Manuel MARTINEZ
PACHECO
Instituto de Investigaciones Qumico Biolgicas, Universidad Michoacana de San Nicols de Hidalgo; Ed. B-3; Cd. Universitaria;
Francisco J. Mujica s/n; Col. Felicitas del Rio; Morelia, Mich. Mxico. C.P. 58060
Abstract
Artemisia ludoviciana Nutt. (Estafiate, common name) is widely used in traditional Mexican medicine to relieve pain and stomach problems, and was
studied to determine its potential antioomycete activity against Phytophthora spp. The wild oomycete isolates tested were P. cactorum, P. capsici, P.
cinnamomi, P. infestans and P. mirabilis, all of them were sensible to crude extracts of the aerial parts of the plant. From this extract was obtained a fraction
by TLC method (Rf =0.72) that contained essential oils capable of inhibiting oomycete growth with a minimum inhibitory concentration (MIC) in a range of
0.2 to 0.4 mg ml
-1
. The major compounds in the microbicidal fraction were borneol (16.28 %), camphor (7.41 %) and cis-verbenol (1.69 %). It was observed
that only a mixture of them (63:28:6.5 g ml
-1
) inhibited the growth of five Phytophthora species with a similar effect to the raw extract and the active
fraction.
Keywords: Antioomicete; Phytophthora; Borneol; Camphor; cis-Verbenol.
Resumen
Artemisia ludoviciana Nutt (Estafiate nombre comn), ampliamente usada en la medicina tradicional mexicana para aliviar el dolor y problemas
estomacales, fue estudiada para investigar la actividad antioomiceto contra Phytophthora spp. Los aislados silvestres fueron P. cactorum, P. capsici, P.
cinnamomi, P. infestans and P. mirabilis. Todos fueron sensibles al extracto crudo de la parte area de la planta. De estos extractos se obtuvo una fraccin por
TLC (Rf = 0.72) que contuvo aceites esenciales capaces de inhibir el crecimiento de los oomicetos con una concentracin mnima inhibitoria (MIC) en el
intervalo de 0.2 a 0.4 mg ml
-1
. Los compuestos mayoritarios en la fraccin microbicida fueron borneol (16.28 %), camfor (7.41 %) y cis-verbenol (1.69 %).
Se observ que nicamente una mezcla de ellos inhibi el crecimiento de las cinco especies de Phytophthora con un efecto similar al del extracto crudo y al
de la fraccin activa.
Palabras Clave: antioomiceto; Phytophthora; Borneol; Camfor; cis-Verbenol.

Recibido | Received: 19 September, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: January 15, 2010.
Publicado en Lnea | Published Online 25 March, 2010
Financiacin | Funding: This work was financed by Universidad Michoacana de San Nicolas de Hidalgo (UMSNH CIC-2.1MMP project)
This article must be cited as: Damian Badillo LM, Martnez Muoz RE, Salgado Garciglia R, Martnez Pacheco MM. 2010. In vitro antioomycete activity of Artemisia
ludoviciana extracts against Phytophthora spp. Bol. Latinoam. Caribe Plant. Med. Aromat. 9(2): 136-142. {EPub 25 March, 2010}.
*Contactos | Contacts: mpacheco@umich.mx


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-

Damin Badillo et al.
In vitro antioomycete activity of Artemisia ludoviciana extracts against Phytophthora spp.


INTRODUCTION
The Phytophthora genus is related to
heterokontas and brown gold algae. It shows different
ways of interaction with host plants and its control is
different to that applied to real fungi and they are
different phylogenetically taking into account their
classification in the Chromist (Chromalveolata)
kingdom (Van der Peer and De Wachter, 1997). It
includes more than 50 phytopathogen species for more
than 150 economically important crops and is
responsible for blight, late mildew and rooting
diseases. Depending on the species it can infect the
leaves (P. infestans and P. cactorum), roots, stem (P.
cinnamomi) and even the fruit. Interest in controlling
this pathogen was renewed when some aggressive
stocks were detected in west Mexican, in the avocado
producing region, and also because of the detection of
Mexican aggressive strain A2 (mate type) from P.
infestans that affects significantly the potato crops in
Europe and other parts of the world (Hohl and Iselin,
1984; Goodwin, 1997; Goodwin and Drenth, 1997).
All of this has motivated the search for alternative
methods to conventional chemical control, in order to
obtain efficient and eco-friendly antioomycete
substances (Damian Badillo et al., 2005). An
alternative is the use of medical plants as it is the case
of Ocimum adscendes. Their essential oil had a
protective effect against fungi in stored Capsicum
annum seed, which was more efficient than
conventional fungicides (Asthana et al., 1989). Also, it
has been reported that the raw extract from Eucalyptus
citriodora and the essential oils from other plant
species inhibited the mycelia growth in oomycetes
such as P. infestans (Schwan-Estrada, 1998; Mine
Soylu et al., 2006).
Potentially, Artemisia plants may be a source
of toxic compounds against oomycete from the
Phytophthora genus. Some species from this plant
have been widely studied from the phytochemical
point of view, mainly due to its use in traditional
medicine for stomach illnesses. Compounds such as,
camphor, germacrene D, trans-pinocarveol, -
selinene, -cariofillene, artemisia cetone, z-
epoxyocimene, crisantenyl acetate, z-epoxyocimene
and -thujone have been identified in and purified
from Artemisia annua, A. absinthium, A. santonicum
and A. spicigera. All of them show antifungal activity,
while others like arteanuine B and artemisinin, have
toxic effects against intestinal protozoon, Entamoeba
histolytica and Giardia lamblia. Other have unknown
biological functions such as the sesquiterpene lactones
from A. ludoviciana (Jakupovic et al., 1991; Juteau et
al., 2002; Ramos-Guerra, 2004; Kordali et al., 2005a).
A. ludoviciana is a widely spread species
distributed throughout Mexican territory and is
commonly known as estafiate, with medical
properties and a traditional use similar to other species
from the same genus, as described above. It has been
reported that it also has antifungal activity against
plant and vertebrates pathogens (Damin-Badillo et
al., 2008a). Therefore, in the context of using Mexican
medical plants for phytopathogen control, specifically
oomycetes, the purpose of this work was to evaluate
the in vitro antioomycete activity from A. ludoviciana
extracts against Phytophthora spp.
MATERIALS Y METHODS
Plant material
A. ludoviciana Nutt., (Asteraceae ), specimens
were collected from the estafiate crop in the Instituto
Nacional de Investigaciones Forestales Agricolas y
Pecuarias at the Uruapan Campus at Michoacan State
and were identified in the Facultad de Biologia
Herbarium, Universidad Michoacana de San Nicolas
de Hidalgo. The material plant was collected at the
early flowering stage (March-July of 2006-2007) and
was dried.
One specimen was prepared for identification
in the UMSNH herbarium (Voucher number 03309).
Material was dried at room temperature and roots,
aerial parts (stems and leaves) and flowers were
separated and pulverized, then kept protected from
direct light until the extraction process.
Plant extracts
The plant extracts were obtained according to
the Damian Badillo et al., method (2008b). Briefly, a
mixture of CH
3
Cl
3
:MeOH (1:2 v/v) was added to each
100 g of dry powder from the different plant parts and
left five days in maceration at 4 C and then filtered.
The solvent was removed and the extract was
dissolved in ethanol. To the aerial plant extract
obtained with ethyl acetate and a soxhlet equipment
was used for 2 h at 74 C, it was filtered, the solvent
removed and 1 g was dissolved in 1 mL of ethanol and
maintained at 4 C until the moment of the bioassays.
Thin layer chromatography (TLC).
A. ludoviciana chloroform-methanol extract
fractioning was carried out by thin layer


chromatography (silica gel 60, Sigma; 20 x 20 cm
plaques) with a solvent system of CH
3
Cl
3
:MeOH (2:1
v/v) in a chromatographic chamber. Plaques were
dried to room temperature and revealed with
ultraviolet light at 254 nm, marking the bands
corresponding to the fractions. The R
f
for each of the
fractions was calculated. Six different fractions were
obtained and eluted with 10 ml of a mixture of
chloroform-methanol (2:1 v/v). Solvent from each
fraction was removed using a rotary evaporator at 45
C and the residue was dissolved in 1 ml of absolute
ethanol for bioassays and methanol for gas
chromatography/mass spectrometry (GC/MS).
Essential oils: Plant extract and active fraction
analysis were performed using the Damian Badillo et
al. method (2008b), a Hewlett Packard 6890
chromatographer, with a HP 5973 mass detector, with
an Equity 5 (30m x 20 mm) capilar column. The
temperature programming condition of the oven was
from 50 to 200 C at 13 C min
-1
, 200 to 300 C and
300 C 5 min
-1
and programmed at 250 C by 2
C min
-1
. Initial injector temperature was 40 C,
increasing from 2 C to 250 C. The mass spectrum
was taken at 70 eV with a mass range from 20 to 450
amu. Compound identification was done comparing
the mass spectra and the retention time with the
spectral data basis NIST, with a reliability percentage
of 94 %.
Oomycete culture
P. cactorum, P. capsici, P. cinnamomi, P.
infestans and P. mirabilis wild strains were isolated
from sick tissues of host plants (strawberry, chili,
avocado and potato, respectively). Phytopathologist
Silvia Fernandez Pavia PhD identified them according
to the Phytophthora taxonomic keys of CABI
Biosciences Database (2003), Erwin and Ribeiro
(1996) and Cooke et al. (2000). They were grown in
dextrose potato media and once the mycelium grew,
they were maintained in potato dextrose agar (PDA)
(Difco, USA) and grown at 19 or 22 C for 7 to 15
days depending on the species.
Reagents
All substances were reactive grade and the
pure essential oils camphor, borneol and cis-verbenol,
were acquired from Sigma Co.
Growth inhibition experiment
Potato dextrose agar dishes were inoculated
with a small piece of mycelia in the center of the petri
dish and incubated from 7 to 15 days at 19 C or 22 C
depending on the oomycete tested. When mycelia
grew, 0.5 cm
2
were placed over filter paper wetted
with 10 l (0.1 mg ml
-1
) from the extracts, fractions or
diluted compounds in ethanol and were cultivated
under the conditions mentioned above. Methyl N-
(methoxyacetyl)-N-(2,6-xylyl)-D-alaninate (Ridomil
Gold EC) was used as a positive control, at a
concentration of 1 mg ml
-1
. The concentration of the
major compounds was: borneol (63 g ml
-1
), camphor
(28 g ml
-1
) and cis-verbenol (6.5 g ml
-1
). Every 12
h for the next fifteen days the inhibition diameter in
the cultures was measured, subtracting that of the
paper (10 mm).
Statistics
The results obtained are presented as the mean
SD of the inhibition zone (I % = [(C-T)C
-1
]100: I %
= relative inhibition, C = control colonial diameter, T
= colonial diameter from the treated oomycete). 100 l
of absolute ethanol or water were used as references
for comparison. The maximum growth measured was
2 cm, which was considered 100 % growth. The halo
was measured and the corresponding proportion was
calculated for each of the treatments. 100 % inhibition
corresponds to no growth at all. The minimum
inhibitory concentration from the extract and vegetable
oil required for complete control of pathogen growth
(MIC) was expressed in mg ml
-1
and classified as
biocide effect over Phytophthora spp. The minimal
oomiceticide concentration (MOC) is equal to MIC.
All the experiments were done three times
with three replicates for each treatment. The Statistic
7.0 program was used to calculate the significance of
all the data by the Tukey test (p < 0.001).
RESULTS
Screening of different extracts obtained from A.
ludoviciana was carried out to find an antioomycete
effect in this plant. It was observed that the
chloroform-methanol extract from the green parts
inhibited the growth 100 % in four of the oomycetes
and in the case of P. infestans a 60 % inhibition was
observed. Only P. capsici and P. cinnamomi were
sensitive to the extracts obtained with ethyl acetate. It
was also observed that the root extract does not
contain oomycetes growth-affecting metabolites
(Table 1). The leaf extract obtained with chloroform-
methanol was fractionated by thin layer
chromatography and six chromatographic signals were


observed, which were located by their R
f
and were
tested against five Phytophthora spp. Fraction III
inhibited the growth of all the oomycetes with a minor
MIC in a range of 0.2 to 0.4 mg ml
-1
,

relating to the
other growth inhibiting fractions, so, it was in this
fraction that we sought plant metabolites causing
Phytophthora sp growth inhibition (Table 2). The
MIC value of the positive control Ridomil Gold EC
was in a range of 0.1 to 0.25 mg ml
-1
.
To know the essential oil composition from the
fraction, it was analyzed by gas chromatography
coupled to mass spectrometer, giving as a result the
identification of compounds, mainly terpenoids. It was
observed that in contrast to fraction one, the rest of the
fractions contain: borneol (16.2 %), camphor (7.4 %)
and/or cis-verbenol (1,69 %) as major compounds
(Table 3).
To know which of the major compounds detected
on the A. ludoviciana leaf were responsible for the
inhibitory effect, the oomycete were exposed to the
purified compounds and a mixture of them. The results
showed that purified compounds in isolation did not
affect the growth of the oomycete tested. However,
with the mixture of them, the inhibitory effect on
Phytophthora sp growth was 100 % (Figure 1A), was
similar to that observed with the raw extract and
fraction III (Figure 2B).
Figure 1. Effect of essential oils from green parts from A.
ludoviciana on P. capsici mycelial growth.

A. Before A. ludoviciana effect: 1. Borneol, camphor and cis-
verbenol mixture; 2. Borneol; 3. Camphor; 4. cis-Verbenol. B.
After A. ludoviciana effect: 7. Chloroform-methanolic extract of
green parts (leaves and stems); 8. TLC-fraction III; 9. Borneol and
Camphor. The controls were 5. Ethanol and 6. Water.
Figure 2. Mass spectra of major metabolites identified from A.
ludoviciana green parts.

A, Borneol. B, Camphor. C, cis-Verbenol.
Table 1. Effect of A. ludoviciana extracts on Phytophthora spp
mycelial growth.
Tissue
Extraction
solvent
Mycelial growth
inhibition
(%)
Pcac Pcap Pcin Pinf Pmir
Flower
Ethyl
acetate
- 40 60 - -
Leaves/stems
Ethyl
acetate
- 7 - - -
Flower
Chloroform
methanol
- 90 93 - -
Leaves/stems
Chloroform
methanol
100 100 100 60 100
Susceptibility to the plant extracts were done with only one
concentration (0.1 mg ml
-1
) by the classic paper-disk agar
diffusion assay. Pcac, P. cactorum. Pcap, P. capsici. Pcin, P.
cinnamomi. Pinf, P. infestans. Pmir, P. mirabilis.


Table 2. Minimal inhibitory concentration (MIC) of the
chloroform-methanol extract fractions from the green parts of A.
ludoviciana on Phytophthora spp mycelial growth.
Fraction R
f

MIC (mgml
-1
)
Pcac Pcap Pcin Pinf Pmir
I 0.79 3.9
II 0.74 3.2 5.3
III 0.72 0.4 0.2 0.2 0.2 0.28
IV 0.70 0.5 0.7
V 0.58 0.5 1.1
VI 0.57 1.6 2.3
Susceptibility to the TLC fractions from the plant extracts were
done under similar conditions to Table 1. The concentration range
was 0.1 - 10 mg ml
-1
in the classic paper-disk agar diffusion assay.
Pcac, P. cactorum, Pcap, P. capsici, Pcin, P. cinnamomi, Pinf, P.
infestans, Pmir, P. mirabilis.
Table 3. GC/MS analysis of the microbicide TLC fractions from
the chloroform-methanol extract from A. ludoviciana green parts
TLC
fraction
Components Retention
time (min)
Relative
abundance
(%)
I Limonene 5.62 0.12
II

Camphor
Borneol
7.04
7.27
0.30
0.91
III

Eucaliptol
Terpineol
cis-verbenol
Camphor
Borneol
Mirtenal
Espatulenol
Cariofilene
derivate
Espatulenol
derivate
5.6
6.45
6.97
7.04
7.27
7.59
7.62
11.6
11.7
0.53
0.34
1.69
7.41
16.28
0.34
0.42
0.55
0.84
IV Eucaliptol
Terpineol
cis-verbenol
Camphor
Borneol
Espatulenol
Cariofilene
derivate
Espatulenol
derivate
5.67
6.47
6.97
7.04
7.27
11.60
11.68
12.13
0.26
0.33
1.29
4.27
12.51
0.44
0.61
0.21
V cis-verbenol
Camphor
Borneol
6.97
7.05
7.27
0.32
1.82
3.78
VI Camphor
Borneol
7.05
7.27
0.79
1.59
DISCUSSION
Secondary metabolites produced by plants in their
different developing steps, in their natural competition
for new ecological niches, or in their defence
mechanisms against microorganisms and predators,
are natural sources of research for alternative controls
against microorganisms causing health problems to
animals and plants, and causing biodeterioration of
different materials. In this work volatile compounds
from A. ludoviciana that inhibited Phytophthora sp.
growth were researched. The results showed that
chloroform-methanol leaf and stem extracts (green
parts) from A. ludoviciana inhibited oomycete growth.
The metabolite content in the different extracts
differed from an organ to organ, since leaves and stem
extracts showed the highest activity while those from
the roots had no apparent effect.
While the other extracts inhibited only two species
of Phytophthora, the results showed that species
variability in the same genus was significant. It
suggests that the more susceptible oomycete to this
plant species extracts were P. capsici and P.
cinnamomi even when they belong to different groups
(II and IV, respectively according to Cooke et al.,
2000) inside the phylogenetic tree of Phytophthora
genus, while the rest were not. On the other hand, P.
cactorum as well as P. infestans and P. mirabilis
belong to group I, so this difference may be due to
particular characteristics of the mentioned groups
(Cooke et al., 2000).
When the chromatographic fractions were tested
against the oomycete, only fraction III was toxic for
the five Phytophthora species. Otherwise, P. capsici
and P. infestans were sensitive to at least five
fractions. This is an interesting observation, as it
would be expected that P. infestans and P. mirabilis
would have had the same behavior because they
belong to the same group I, while P. capsici is found
in the second group (Cooke et al., 2000). A probable
explanation is the metabolite concentration and each
species sensibility to them.
This is the first report where it is showed that
borneol, camphor and cis-verbenol, the main
components of the essential oil of the aerial parts from
A. ludoviciana, have antioomycete properties.
Comparing the MIC values against the positive
control, suggest that this essential oil mixture may be
used as a versatile and potent oomiceticide agent.
Essential oils have been reported in A. dracunculus, A.
absinthium, A. santonicum and A. spicigera, which
presented a high antifungal activity against 34


phytopathogen species, among them the P. capsici
oomycete, and this activity has been attributed to
monoterpenes among which only borneol and camphor
were identified in our fraction (Meepagala et al., 2002;
Kordali et al., 2005b). Moreover, it also has been
reported that the aerial part of A. dracunculus L. var.
dracunculus, contains compounds that showed
microbicidal activity against Botrytis cinerea,
Colletotrichum gloeosporioides, C. acutatum and C.
fragariae phytopathogens, but neither correspond to
the ones identified in this work (Meepegala, et
al.,2002, 2003). Another report, related to microbicidal
activity in the Artemisia genus, refers to the aerial
extracts from A. verlotorum against the pathogenic
oomycete, Saprolegnia fera. The compounds
responsible for this activity were not mentioned
(Macchioni et al., 1999).
The mixture of borneol, camphor and cis-verbenol
is essential to obtain the antioomycetic effect, because
it is not found with the compounds alone; Shafi
(2004), reported that borneol does not have
antioomycetic activity against P. capsici. This last fact
suggests that when bioassays are being done it is
necessary to test pure compounds and the mixture of
the rest of the metabolites that are present in one
extract or the active fraction, because an effect may be
the result of the synergism of several components.
This work besides presenting the antioomycetic
effect of the chloroform-methanol extract of the green
parts of A. ludoviciana and the importance of using
mixtures of compounds generates new research aims
to identify more efficient and effective compounds, as
well as understand their mechanism of action.
CONCLUSIONS
The chloroform-methanol extract of the green parts
of A. ludoviciana, contains the secondary metabolites,
borneol, camphor and cis-verbenol. These essential
oils showed antioomycetic properties as a mixture, so
it can be affirmed that this plant is toxic to
Phytophthora spp.
ACKNOWLEDGEMENTS
This work was supported by the Universidad
Michoacana de San Nicolas de Hidalgo to the projects;
CIC-2.10-RSG y CIC-2.1-MMP. LMBD was a fellow
from UMSNH. We are grateful to C. Marquez and A.
Flores Garcia for the technical assistance on the
chemical and statistical analysis, respectively, and to
phytopathologist Sylvia Fernandez Pavia PhD from
Instituto de Investigaciones Agricolas y Forestales-
UMSNH for their donation of wild isolates from
Phytophthora sp.
REFERENCES
Asthana A, Dixit K, Tripathi NN, Dixit SN. 1989. Efficacy
of Ocimus oil against fungi attacking chilli seed during
storage. Trop Sci 29:15-20.
CABI Bioscience. 2003. Index Fungorum: Database of
fungal names. Online. CABI Bioscience,
Centraalbureau voor Schimmelcultures (CBS), and
Landcare Research.
Cooke DE, Drenth A, Duncan JM, Wagels G, Brasier CM.
2000. A molecular phylogeny of Phytophthora and
related oomycetes. Fungal Genet Biol 30:17-37.
Damian-Badillo LM, Salgado-Garciglia R, Martinez-
Pacheco MM. 2005. Efecto de extractos acuosos y no
acuosos de Heliopsis longipes, Tagetes lucida, Satureja
macrostema y Artemisia ludoviciana sobre el
crecimiento de hongos patgenos. Rev Latinoam Qum
Supl pp. 122.
Damian-Badillo LM, Espinosa Madrigal RM, Martinez
Muoz RE, Ron Echeverria OA, Salgado Garciglia R,
Flores Garcia A, Raya Gonzalez D, Martinez Pacheco
MM. 2008a. The Mexican medical plants with
antifungal properties are an economic and health
opportunity area. Pharmacologyonline 3:61-77.
Damian-Badillo LM, Salgado-Garciglia R, Martinez-Muoz
RE, Martinez-Pacheco MM. 2008b. Antifungal
propierties of som Mexican medicinal plants. Open Nat
Prod J 1:27-33.
Erwin, DC., and Ribeiro, OK. 1996. Phytophthora Diseases
Worldwide. American Phytopathological Society, St.
Paul, MN. pp 1 320.
Goodwin SB. 1997. The population genetics of
Phytophthora. Phytopathology 87:462-473.
Goodwin SB, Drenth A. 1997. Origin of the A2 mating type
of Phytophthora infestans outside Mexico.
Phytopathology 87:992-99.
Hohl HR, Iselin K. 1984. Strains of Phytophthora infestans
from Switzerland with A2 mating type behaviour. Trans
Brit Mycol Soc 83:529-530.
Jakupovic J, Tan RX, Bohlmann F, Boldt PE, Jia ZJ. 1991.
Sesquiterpene lactones from Artemisia ludoviciana.
Phytochemistry 30:1573-1577.
Juteau F, Masotti V, Bessiere JM, Dherbomez M, Viano J.
2002. Antibacterial and antioxidant activities of
Artemisia anuua essential oil. Fitoterapia 73 :532-5.
Kordali S, Kotan R, Mavi A, Cakir A, Ala A, Yildirim A.
2005a. Determination of the chemical composition and
antioxidant activity of the essential oil of Artemisia
dracunculus and of the antifungal and antibacterial
activities of Turkish Artemisia absinthium, A.
dracunculus, A. santonicum and A. spicigera essential
oils. J Agric Food Chem 53:9452-9458.


Kordali S, Cakir A, Mavi A, Kilic H, Yildirim A. 2005b.
Screening of chemical composition and antifungal and
antioxidant activities of the essential oils from three
Turkish Artemisia species. J Agric Food Chem
53:1408-1416.
Macchioni F, Perrucci S, Flamini G, Cioni PL, Morelli I.
1999. Antimycotic activity against Saprolegnia ferax of
extracts of Artemisia verlotorum and Santolina etrusca.
Phytother Res 13:242-244.
Meepagala KM, Sturtz G, Wedge DE. 2002. Antifungal
constituents of the essential oil fraction of Artemisia
dracunculus L. var. dracunculus. J Agric Food Chem
50:6989-6992.
Meepagala KM, Kuhajek JM, Sturtz GD, Wedge DE. 2003.
Vulgarone B, the antifungal constituent in the steam-
distilled fraction of Artemisia douglasiana. J Chem
Ecol 29:1771-1780.
Mine Soylu E, Soylu S, Kurt S. 2006. Antimicrobial
activities of the essential oils of various plants against
tomato late blight disease agent Phytophthora infestans.
Mycopathologia 161:119-128.
Ramos-Guerra MC, Mata-Cardenas BD, Vargas-Villareal J,
Oranday-Cardenas A, Trevio-Villareal L. 2004.
Actividad in vitro del extracto acuoso y organico de las
hojas de Artemisia ludoviciana Nutt (istafiate) sobre
Entamoeba histolytica y Giardia lamblia. Rev Salud
Publ Nutr 4:66-67.
Schwan-Estrada KRF, Stangarlin JR, Cruz MES, Bonaldo
SM, Pascholati SF. 1998. Efeito do extrato bruto de
Eucalyptus citriodora no crescimento micelial e
germinaao de esoporos de fungos fitopatogenicos.
Summa Phytopath 21:101.
Shafi PM, Geetha Nambiar MK, Clery RA, Sarma YR,
Veena SS. 2004. Composition and antifungal activity of
the oil of Artemisia nilagirica (Clark) Pamp J Essential
Oil Res 16:377-379.
Van de Peer Y, de Wachter R. 1997. Evolutionary
relationships among the eukaryotic crown taxa taking
account site-to-site rate variation in 18S rRNA. J Mol
Evol 45:619-63.

2010 The Authors


Antitumour and anti-inflammatory activities in a hydroethanolic
extract of Lindackeria paludosa, a South American shrub.
[Actividades antitumorales e anti-inflamatorias de un extracto hidroetanlico de Lindackeria paludosa,
un arbusto sudamericano.]
Ana Laura FAZIO
1
, Diana BALLN
2
, Italo M. CESARI
2
, Mara Jess ABAD
1
, Miriam ARSENAK
1
, Omar ESTRADA
3
,
Peter TAYLOR
1
.

1
Centro de Medicina Experimental,
2
Centro de Microbiologa y Biologa Celular,
3
Centro de Biofisica y Bioquimica, Instituto
Venezolano de Investigaciones Cientficas, Apartado 20632, Caracas 1020-A, Venezuela.
Abstract
Lindackeria paludosa (Benth.) Gilg is a shrub found mainly in the north of South America and is known as Sarakura in Venezuela. In the light of local
reports on the traditional use this member of the Flacourtiaceae family, we investigated the effect of a hydroethanolic extract of the bark (LP) on parameters
of the inflammatory response (tumour necrosis factor alpha [TNF-], interleukin-6 [IL-6] and nitric oxide [NO]) and its potential antitumour activity both in
vitro and in vivo. LP was notably cytotoxic for only one tumour cell line (A549, IC50 = 64 g/ml), but not for the other cell types. However, LP did inhibit
production of the inflammatory mediators, as well as the growth of primary tumours and metastases in C57BL/6 mice, but did not inhibit nuclear factor B
(NF-B) activity. Thus, LP appears to inhibit tumour growth without being directly cytotoxic to tumour cells, possibly by interfering with protumour
inflammatory processes.
Keywords: Lindackeria paludosa; cancer; metastasis; inflammation; mouse
Resumen
Lindackeria paludosa (Benth.) Gilg es un arbusto que se encuentra principalmente en el norte de Sudamrica y se conoce como Sarakura en Venezuela.
En vista de la informacin local sobre el uso en la medicina tradicional de este miembro de la familia Flacourtiaceae, investigamos el efecto de un extracto
hidroetanlico de la corteza (LP) sobre algunos parmetros de la respuesta inflamatoria (factor de necrosis tumoral alfa, interleuquina-6 y xido ntrico) y su
potencial actividad antitumoral tanto in vitro como in vivo. Se not un efecto citotxico solamente en una lnea celular tumoral (A549, IC50 = 64 g/ml), pero
no en los otros tipos de clulas. Sin embargo, LP inhibi la produccin de los mediadores inflamatorios, el crecimiento de tumores primarios y metstasis en
ratones C57BL/6, pero no inhibi la actividad del factor nuclear B. LP parece inhibir el crecimiento tumoral sin ejercer un efecto citotxico directo,
posiblemente a travs de la inhibicin de procesos inflamatorios protumorales.
Palabras Clave: Lindackeria Paludosa; cncer; metstasis; inflamacin; ratn.
List of Abbreviations: LP Lindackeria Paludosa extract; TNF- - tumour necrosis factor alpha; IL-6 - interleukin-6; NO - nitric oxide; LPS-
lipopolysaccharide; huPBMC - human peripheral blood mononuclear cells; FBS foetal bovine serum; muSplen non-adherent mouse spleen cells; muPM -
murine peritoneal macrophages; DEX dexamethasone; PAC paclitaxel; LSEC- Liver sinusoidal endothelial cells; NF-B nuclear factor B

Recibido | Received: 16 June, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: 22 March, 2010.
Publicado en Lnea | Published Online: 25 March, 2010
Declaracin de intereses | Declaration of interests: The authors have no competing interests.
Financiacin | Funding: This work was financed by IVIC
This article must be cited as: Fazio A L, Balln D, Cesari I M, Abad M J, Arsenak M, Estrada O, Taylor P. 2010 Antitumour and anti-inflammatory activities in a hydroethanolic
extract of Lindackeria paludosa, a South American shrub. . Bol Latinoam Caribe Plant Med Aromat 9 (2), 142 - 150.
*Contactos | Contacts: E-mail: ptaylor@ivic.gob.ve - Tel.: +58 212 504 1097 - Fax: +58 212 504 1086.


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-
Fazio et al.
Antitumour and anti-inflammatory activities of Lindackeria paludosa


INTRODUCTION
Lindackeria paludosa (Benth.) Gilg is a small tree or
shrub found mainly in the northern parts of South
America and is known as Sarakura in Venezuela. It
has been identified under different names such as L.
latifolia and L. maynensis (History) Although its main
use is in construction, this plant has been cited for its
medicinal use in skin diseases such as leprosy (Pupo,
1926) and to treat malaria (Fitogenticos). In
Venezuela, it has been used in the Amazon region and
the Amacuro delta as an analgesic, antidiabetic, as a
snake bite antidote and against cancer (Pedro
Maquirino personal communication). Cyanogenic
glycosides which may have anti-cancer properties
have been isolated from another plant of the same
genus, L. dentata (Jaroszewski et al., 2004).
The role of chronic inflammation in tumour
initiation and growth is well established (Coussens and
Werb, 2002) so a dual action against both
inflammation and cancer is not surprising. Studies
have shown that anti-inflammatory drugs may be
effective in cancer therapy and/or prevention (Thun et
al., 2002), and the possible mechanisms of action of
anti-inflammatory phytochemicals have been reviewed
(Surh et al., 2001).
On the basis of these leads, we investigated the
possible anti-inflammatory and antitumour effects of a
hydroethanolic extract of this plant.
Plant material and phytochemical screening
The bark of Lindackeria paludosa (Benth. ) Gilg
was collected by Mr. Pedro Maquirino near San Carlos
de Ro Negro, Amazonas State, Venezuela and
identified by Dr. Otto Huber (Universidad Central de
Venezuela) and Dr. Ernesto Medina (Instituto
Venezolano de Investigaciones Cientficas). This
shrub, from the Flacourtiaceae family, is widely
distributed throughout the Amazon Basin and is also
known in the literature as Mayna paludosa, Mayna
laxiflora, Lindackeria latifolia, Lindackeria maynensis
var laxiflora, Oncoba maynensis var laxiflora,
Carpotroche laxiflora and Carpotroche paludosa.
Aliquots were ground then macerated in a 70% ethanol
in water solution for 21 days in the dark at room
temperature. The suspension was then filtered under
sterile conditions using Whatman No. 1 filter paper
then adjusted to a stock concentration of 5 mg/ml,
which was calculated from the dry weight of a
lyophilized sample. This extract is here termed LP.
In order to identify the possible classes of
compounds present in LP, preliminary phytochemical
analysis was carried out through the treatment of this
extract with a mixture of acetone-methanol (8:2). Two
fractions were obtained, an orange solution and a
brown residue (50 mg), the acetone-methanol
insoluble fraction (AMIF). Evaporation of the orange
solution in vacuo yielded a red residue (200 mg), the
acetone-methanol soluble fraction (AMSF). The
AMSF was analyzed by thin layer chromatography on
RP18 gel plates developed with an acetonitrile-water
mixture (8:2). Spots were revealed with the following
spray-reagents: the Dragendorff reagent for alkaloids,
and a saturated 2% methanol solution of ceric sulphate
in concentrated sulphuric acid for triterpenoids and
flavonoids. The plates were hot air dried to visualize
the coloured spots (Bilia et al., 1996).
Cells and animals.
The cell lines, B16/BL6, K1735, HT29, A549,
WEHI 164 and LSEC were cultured in Dulbeccos
Modified Eagles Medium (DMEM) supplemented
with 10% heat-inactivated foetal bovine serum (FBS -
Gibco, BRL, USA), penicillin (100 Units/ml),
streptomycin (100 g/ml) and containing in addition
glucose 0.45% (HT29 cells), and L- glutamine 2 mM
(A549 cells). The origins of these cells are shown in
Table 1. Human peripheral blood mononuclear cells
(huPBMC) were obtained from healthy donors by
standard Ficoll/Hypaque gradient centrifugation and
cultured in RPMI-1640 10% FBS. Chopped spleens
from C57BL/6 mice were ground through a wire mesh
screen. After removal of detritus and lysis of red blood
cells with 0.085% sodium citrate, adherent cells were
removed by overnight incubation in plastic culture
flasks. The non-adherent cells (muSplen) were
harvested, counted and cultured in RPMI-1640 10%
FBS. Murine peritoneal macrophages (muPM) were
collected from C57BL/6 mice 4 days after a peritoneal
injection of 2 ml of 4% thioglycollate. The cells were
washed, seeded into culture flasks in RPMI-1640 10%
FBS, and non-adherent cells discarded after 3 h. The
adherent cells were then used immediately.

Fazio et al.


Female C57BL/6 mice (79 weeks old, ~20 g) were
obtained from the Animal Facility, IVIC and fed with
standard pellet diet and water ad libitum. All animal
experiments were performed according to
internationally accepted guidelines for the treatment of
animals in research.
Cytotoxicity.
Cells were plated at 2.5 - 5 x 10
4
cells / well in flat-
bottomed 96 well plates and allowed to attach for 24 h.
Different concentrations of LP up to 300 g/ml (final
concentration) were then added. Control wells were set
up containing equivalent quantities of ethanol, which
in no case exceeded 1%. No effect was observed due
to the ethanol. After a further 24 h, the number of
viable cells was assessed using the MTS/PMS
chromogenic assay (Promega Corp., USA) according
to the manufacturers instructions. The IC
50
(50%
inhibitory concentration) was calculated from the data
by linear extrapolation using an in-house programme
in Excel (Microsoft Corporation).
Inflammatory response in vitro.
Peritoneal macrophages were activated with 10
g/ml lipopolysaccharide (LPS - E. coli serotype
055:B5, Sigma, USA) for 24 h in the presence of LP,
and then the concentrations of TNF-, IL-6 and nitric
oxide (NO) were measured in the supernatants. TNF-
was quantified using the WEHI 164 cell bioassay
(Espevik and Nissen-Meyer, 1986), IL-6 with a
commercial ELISA assay (R & D Systems Inc., MN,
USA) and NO using the Griess reaction (Sandoval-
Chacn et al., 1998).
Inflammatory response in vivo.
Mice were injected intraperitoneally (i.p.) with
different doses of LPS in 100 l of PBS. After 1 h,
blood was collected by heart puncture under ether
anaesthesia. Serum was separated and assayed for the
two cytokines and NO as described above. In order to
evaluate the effect of LP on the inflammatory
response, mice were injected i.p. with 50 g LP on 3
consecutive days prior to LPS challenge.
Lung metastasis.
At day 0, mice were inoculated in the lateral tail vein
(i.v.) with 10
5
B16/BL6 cells in 100 l PBS. Two
treatment protocols with LP were performed a)
intraperitoneal (i.p.) injection of 50 g of LP in 100 l
PBS / 25% ethanol on days -2, -1 and 0, and b) i.p.
injection of the same dose of extract 5 times per week
starting from on day 0 up to day 21. Control animals
received 100 l PBS / 25% ethanol. On day 23, the
animals were sacrificed with ether; the lungs were
removed, placed for 5 min in 3% H
2
O
2
in H
2
O and
fixed in Bouin's solution. The purpose of the H
2
O
2
was
twofold: to bleach hemorrhages which could be
mistaken for metastases, and to inflate the lungs,
facilitating the evaluation of metastases under the
dissecting microscope. Animals were challenged with
LPS prior to sacrifice, in order to measure serum TNF-
and IL-6 levels as described above.
Primary tumours.
Primary tumours were induced by the subcutaneous
(s.c.) injection of 5 x 10
4
B16/BL6 cells in 100 l PBS
into the hind limb. The mice were injected i.p. with 50
g of LP 5 times per week starting from on day 0 up to
day 21. Tumour size was measured in two dimensions
with a vernier gauge. Animals were challenged with
LPS prior to sacrifice, in order to measure serum TNF-
and IL-6 levels as described above.
Statistical analysis.
Each experiment was performed at least three times
and results are expressed as the mean S.E.M.
(standard error of the mean). The unpaired Students t
test with the Welch correction was used to assess the
statistical significance of the differences.
RESULTS
Phytochemical analysis.
Thin layer chromatography of the AMSF fraction
showed violet spots on the plate with the ceric sulfate
sulphuric acid reagent indicating the presence of
triterpenes. The absence of yellow or orange spots
when the plates were sprayed with ceric sulfate and
Draggendorff reagents indicated that flavonoids and
alkaloids respectively were not present in important
amounts in this fraction. Neither could they be
detected by chromatography in the AMIF fraction.
Cytotoxicity.
LP showed no important degree of inhibition on the
cell lines, except for A549, a human lung carcinoma
line (Table 1). For most cell lines, including the
B16/BL6 melanoma line used in the in vivo
experiments, the IC
50
was above the maximum
concentration tested (300 g/ml).

Fazio et al.


Table 1. Inhibitory concentration (IC
50
) of LP on cell lines in
vitro.
Cells Origin
IC
50
(g/ml)
B16/BL6 Murine melanoma >300
HT-29 Human colon carcinoma >300
A549 Human lung carcinoma 64
K1735 Amelanotic murine melanoma >300
WEHI Murine fibrosarcoma 240
huPBMC
Human peripheral blood mononuclear
cells
261
muSplen Non-adherent mouse splenocytes >300
muPM Murine peritoneal macrophages 238
LSEC Murine liver sinusoidal endothelial cells >300
Cell viability was measured by the MTS chromogenic assay
after 24 h incubation in the presence of LP. Results are expressed
as the 50% inhibitory concentration (IC
50
)
.

Effect of LP on the inflammatory response to LPS
in vitro and in vivo.
The TNF- response of mouse peritoneal
macrophages to LPS was reduced by 50% in the
presence of 100 g/ml LP (Fig. 1), although this
reduction was not significant due to variability in the
results (P = 0.08). The IL-6 and NO responses were
significantly reduced by 65% and 68% respectively (P
< 0.001). This result was not due to a direct cytotoxic
effect as no change in the viability of these activated
cells was observed at this concentration of LP (results
not shown).

Figure 1. Inhibition by LP of the inflammatory response of
mouse peritoneal macrophages to LPS.

Cells were activated with 10 g/ml LPS for 24 h in the presence of
100 g/ml LP. TNF- , IL-6 and NO levels were then measured in
the supernatants. (mean S.E.M., n = 10). ** P < 0.001.
A similar reduction in the inflammatory response
was observed in vivo, when mice were injected i.p.
with 50 g LP on 3 consecutive days prior to challenge
with different doses of LPS (Fig. 2). The TNF, IL-6
and NO responses were reduced by 80, 30 and 67%
respectively when the animals treated with LP were
challenged with the highest dose of LPS. However this
reduction was only significant in the case of NO.

Fazio et al.


Figure 2. Inhibition by LP of the inflammatory response to LPS
in mice.

Animals were pretreated with 50 g/ml LP i.p. on 3 days, then
challenged with different doses of LPS. After 1 h, blood was
extracted and the serum assayed for TNF- , IL-6 and NO. (mean
S.E.M., n = 3). * P < 0.05.
Inhibition by LP of primary tumour growth and
metastasis in mice.
Mice were inoculated s.c. with B16/BL6 cells and
the effect of i.p. LP on primary tumour growth was
measured (Fig. 3A). At all time points after the
appearance of the tumour, there was a very significant
inhibition of tumour growth in the animals treated with
LP (P < 0.0001 at all time points). This effect was
most notable at earlier times but tumours were still
80% smaller at day 22.
Lung metastases were evaluated in the mice after i.v.
inoculation of B16/BL6 cells. Pretreatment with LP
for 3 days prior to tumour inoculation reduced the
number of lung metastases by 24% but this small
reduction was not significant (Fig. 3B P = 0.56).
However, continued treatment with LP postinoculation
very significantly reduced the number of metastases in
lung by 42% (Fig. 3C).
Figure 3. Effect of LP treatment on primary tumour growth and
metastasis in mice.

A. C57Bl/6 mice were inoculated s.c. with B16/BL6 tumour cells
to initiate a primary tumour and injected i.p. 5 times a week up to
day 21 with 50 g LP.
B. Mice were inoculated i.v. with B16/BL6 cells. Treatment with
LP consisted of 50 g i.p. on the 3 days prior to inoculation. Lung
metastases were counted on day 22.
C. Mice were inoculated i.v. with tumour cells, as in B., then
injected i.p. 5 times a week up to day 21 with 50 g LP. (mean
S.E.M., n = 10). *** P < 0.001.
Effect of LP on the inflammatory response to LPS
in tumour-bearing animals.
The results of Fig. 2 showed anti-inflammatory
activity of LP in vivo. Although it is known there may
be a general activation of the inflammatory response in
tumour-bearing animals, the basal levels of serum
TNF- and IL-6 are very low in this tumour model.
Thus in order to assess the effect of LP on the serum
TNF- and IL-6 levels in animals with either primary
tumours or metastases, we evaluated the effect of LP
on the inflammatory response to a low dose of LPS (3
g / animal) in these animals, prior to sacrifice. Figure
Fazio et al.


4 shows that both the TNF- and IL-6 responses to
low dose LPS was much greater in animals with
primary tumours than in animals without tumours
(compare with Fig. 2). In contrast, very little priming
of the inflammatory response to low dose LPS was
observed in the animals with metastasis. The TNF-
and IL-6 responses in animals with primary tumours
were greatly inhibited by LP treatment, (93 and 91%
respectively). Although TNF- levels were generally
lower in the animals with metastasis, a significant
inhibitory effect (81%) was observed after LP
treatment. The very low levels of IL-6 in the untreated
animals (6519 ng/ml) were further reduced by UT
treatment (479 ng/ml).

Figure 4. Effect of LP on the inflammatory response to LPS in
tumour-bearing mice.

C57Bl/6 mice were inoculated s.c. or i.v. with B16/BL6 tumour
cells to produce primary tumours or metastases, respectively, then
injected i.p. 5 times a week up to day 21 with 50 g LP
(corresponding to Fig 4A and 4C). One h before sacrifice, the
animals were challenged with 3 g LPS. Blood was extracted and
the serum assayed for TNF- and IL-6. (mean S.E.M., n = 10).
** P < 0.01, *** P < 0.001,
Effect of LP on the NF-B response to activation by
TNF- in HeLa cells.
The effect of LP on the NF-B response of HeLa
cells to TNF- was determined in a luciferase reporter
assay. LP showed no inhibitory effect on NF-B under
a variety of different conditions (TNF-
concentration, LP concentration, incubation time).
DISCUSSION
Cancer is a not one single pathology but rather a
group of diseases linked by the common denominator
of uncontrolled growth. As it may manifest itself in
multiple ways, from systemic leukaemia to a localized
skin lesion, these manifestations may not be
recognized to be a cancer as such (Micozzi, 2006).
This has complicated the search for new anticancer
drugs based on traditional medicine. Thus, the finding
of antitumour activity in plants has often come as a
result of their known effect on related processes such
as inflammation (Calixto et al., 2004; Middleton Jr et
al., 2000). In the case of L. paludosa, its use against
snake bites, leprosy and malaria (Willcox et al., 2004),
which all include inflammatory components led us to
consider it a suitable candidate for these experiments.
We did not find LP to exert an important direct
inhibitory on tumour cells in vitro. The MTS assay,
although commonly called a cytotoxicity assay, in fact
does not distinguish cytotoxicity from growth
inhibition. Preliminary experiments using the more
discerning Sulphorhodamine B assay, indicated that
LP is, at best, cytostatic but not cytotoxic (results not
shown). Considering these results and the dose of LP
used in the in vivo experiments, it is difficult to
conclude that the inhibitory effect seen with the
primary tumours and metastasis was due to a direct
effect on tumour cell proliferation or viability.
Although findings of cytotoxicity of plant extracts at
relatively high concentrations may perhaps lead one to
speculate on a possible direct antitumour effect in vivo,
extreme caution must be taken when extrapolating in
this way (Gertsch, 2009). Indeed there is much interest
in identifying new drugs to be used in cancer therapy
do not act directly on the tumour cell (Aggarwal et al.,
2009; Hemalswarya and Doble, 2006; Liekens et al.,
2001). We have found other plant extracts to be
effective in vivo against tumours but less so against
tumour cells in vitro (Fazio et al., 2008; Taylor et al.,
2006). However, we cannot discount the possibility
that the cytotoxic component in LP is a prodrug
which is activated by the mouses metabolism.
However, our findings on the inhibition of TNF- ,
IL-6 and NO suggest that LP may inhibit tumour
growth and metastasis through an inflammation-
related mechanism. In a previous study, we showed
that blocking TNF- with a TNF receptor construct
decreased lung metastases in tumour-inoculated mice
(Cubillos et al., 1997).
NF-B, a common factor in tumour growth and
inflammatory processes, has been proposed as a
Fazio et al.


possible target for plant-derived inhibitors (Bremner
and Heinrich, 2002) and many anti-inflammatory
agents, including terpenes which were detected in this
plant extract, appear to modulate it. However, LP did
not inhibit NF-B under the conditions of the
experiments performed here. On the other hand, there
are other inflammation-related processes which may
be investigated to explain the antitumour activity of
this plant extract (Calixto, Campos et al., 2004).
It must be kept in mind that the compounds with
anti-cancer properties in this extract are at least
partially water-soluble as all the experiments are
conducted in aqueous medium. The triterpenoids
detected in the extract might be present in glycosidic
forms, allowing solubility in both polar and nonpolar
solvents. On the other hand, it should be considered
that other classes of compounds, i.e. tannins,
carbohydrates and their derivatives polar substance
can be responsible for biological activity assayed in
this work. Further purification and evaluations in
biological systems need to be carried out in order to
determine the components and their precise
mechanism of action.
CONCLUSIONS
An anti-tumour effect of a crude extract of
Lindackeria paludosa has been described for the first
time. This extract appears to inhibit tumour growth
without being directly cytotoxic to tumour cells,
possibly by interfering with protumour inflammatory
processes.
ACKNOWLEDGEMENTS
The bark of Lindackeria paludosa and
information pertaining to its local medicinal use were
provided by Sr. Pedro Maquirino of San Carlos de Ro
Negro, Amazonas State, Venezuela. We are grateful to
Drs M Rieber and J Cardier for the B16/BL6 and
LSEC cells respectively.
REFERENCES
Aggarwal BB, Vijayalekshmi RV, Sung B. 2009. Targeting
inflammatory pathways for prevention and therapy of
cancer: Short-term friend, long-term foe. Clin Cancer
Res 15(2): 425-430.
Bilia A, Mndez J, Morelli I. 1996. Phytochemical
investigations of Licania genus. Flavonoids and
triterpenoids from Licania carii. Pharm Acta Helvet 71,
191197.
Bremner P, Heinrich M. 2002. Natural products as targeted
modulators of the nuclear factor-kappaB pathway. J
Pharm Pharmacol 54(4): 453-72.
Calixto JB, Campos MM, Otuki MF, Santos ARS. 2004.
Anti-inflammatory compounds of plant origin. Part II.
Modulation of pro-inflammatory cytokines, chemokines
and adhesion molecules. Planta Med 70(2): 93-103.
Coussens LM, Werb Z. 2002. Inflammation and cancer.
Nature 420(6917): 860-7.
Cubillos S, Scallon B, Feldmann M, Taylor P. 1997. Effect
of blocking TNF on IL-6 levels and metastasis in a
B16-BL6 melanoma/mouse model. Anticancer Res
17(3C): 2207-11.
Espevik T, Nissen-Meyer J. 1986. A highly sensitive cell
line, WEHI 164 clone 13, for measuring cytotoxic
factor/tumor necrosis factor from human monocytes. J
Immunol Meth 95(1): 99-105.
Fazio AL, Balln D, Cesari IM, Abad MJ, Arsenak M,
Taylor P. 2008. An ethanolic extract of Uncaria
tomentosa reduces inflammation and B16-BL6
melanoma growth in C57BL/6 mice. Bol Latinoam
Caribe Plant Med Aromat 7: 217-224.
Direccin Nacional de Investigacin de Recursos Genticos
(DNIRRGG) del Instituto Nacional de Investigacin y
Extensin Agraria (INIA). 1996. Per: Informe
Nacional para la Conferencia Tcnica Internacional de
la FAO sobre los Recursos Filogenticos.
http://www.inia.gob.pe/genetica/informes/Informe%20
Estado%20de%20los%20RF%20Per%C3%BA%20199
6.pdf
Gertsch J. 2009. How scientific is the science in
ethnopharmacology? Historical perspectives and
epistemological problems. J Ethnopharmacol 122(2):
177-183.
Hemalswarya S, Doble M. 2006. Potential synergism of
natural products in the treatment of cancer. Phytother
Res 20(4): 239-249.
Web Listing of Plants of the Guianas, Flacourtiaceae.
http://botany.si.edu /bdg/planthtml/ErioL.html.
Jaroszewski JW, Ekpe P, Witt M. 2004. Cyclopentanoid
cyanohydrin glucosides and amides of Lindackeria
dentata. Planta Med 70(10): 1001-1003.
Liekens S, De Clercq E, Neyts J. 2001. Angiogenesis:
Regulators and clinical applications. Biochem
Pharmacol 61(3): 253-270.
Micozzi MS. 2006. Complementary and integrative
medicine in cancer care and prevention: foundation and
evidence-based interventions. Springer Publishing
Company.
Middleton E, Kandaswami C, Theoharides TC. 2000. The
effects of plant flavonoids on mammalian cells:
Implications for inflammation, heart disease, and
cancer. Pharmacol Rev 52(4): 673-751.
Pupo JA. 1926. Tratamento especfico da lepra pelo leo de
chaulmoogra e seus derivados - estudo das
acourtiaceas do Brasil. Rio de Janeiro, Brasil-Mdico.
Fazio et al.


Sandoval-Chacn M, Thompson JH, Zhang XJ, Liu X,
Mannick EE, Sadowska-Krowicka H, Charbonnet RM,
Clark DA, Miller MJ. 1998. Antiinflammatory actions
of cat's claw: the role of NF-B. Aliment Pharmacol
Ther 12(12): 1279-1289.
Surh YJ, Chun KS, Cha HH, Seong Su H, Keum YS, Park
KK, Sang Sup L. 2001. Molecular mechanisms
underlying chemopreventive activities of anti-
inflammatory phytochemicals: Down-regulation of
COX-2 and iNOS through suppression of NF-B
activation. Mutat Res 480-481: 243-268.
Taylor P, Noriega R, Farah C, Abad MJ, Arsenak M, Apitz
R. 2006. Ajoene inhibits both primary tumor growth
and metastasis of B16/BL6 melanoma cells in C57BL/6
mice. Cancer Lett 239: 298-304.
Thun MJ, Henley SJ, Patrono C. 2002. Nonsteroidal anti-
inflammatory drugs as anticancer agents: Mechanistic,
pharmacologic, and clinical issues. J Natl Cancer Inst
94(4): 252-266.
Willcox M, Bodeker G, Rasoanaivo P. 2004. Traditional
Medicinal Plants and Malaria, CRC Press.

Publicada por | Published by: Cooperacin Latinoamericana y Caribea en Plantas Medicinales y Aromticas
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BLACPMA es una revista cientifica dedicada a las plantas medicinales, aromticas, y econmicas y a los productos naturales bioactivos.
Publica contribuciones originales en ocho reas importantes:
1. Caracterizacin de los ingredientes activos de las plantas medicinales
2. Desarrollo de mtodos para la estandarizacin para los extractos bioactivos y los productos naturales de la planta.
3. Identificacin de la bioactividad de productos naturales vegetales.
4. Identificacin de blancos y mecanismo de la actividad de productos naturales.
5. Produccin y caracterizacin genmica de la biomasa de especies medicinales.
6. Qumica y bioqumica de productos naturales bioactivos.
7. Revisiones crticas de la personalidad histrica, clnica y jurdica de plantas medicinales.
8. Aspectos agrcolas de plantas medicinales y aromticas.

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