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Boletín Latinoamericano y del Caribe

de Plantas Medicinales y Aromáticas ISSN 0717 7917

Achyrocline satureioides

Volumen 9, Número 2, Marzo de 2010 Editoriales | Editorials

BARANDA (Chile) Haití, tierra de esperanza.

CESPEDES y ALARCON (Chile) La ciencia chilena se pone de pie.

Artículos | Articles

DELAZAR, et al. (Reino Unido) Ornithogalum cuspidatum Bertol. bulbs, a source of free radical scavengers and phytosterols.

RETTA et al. (Argentina) Diferenciación de las especies Achyrocline satureioides, A. flaccida y Gnaphalium gaudichaudianum por sus perfiles cromatográficos.

GARCÍA-BORES et al. (México) Photoprotective activity and general toxicity of Yucca periculosa stilbenes.

MARTÍNEZ et al. (Argentina) Los remedios naturales en la prevención 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. (México) 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 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (2), i

BLACPMA ISSN 0717 7917

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en esta licencia menoscaba o restringe los derechos morales del autor.

EDITOR JEFE | EDITOR IN CHIEF

José L. Martínez (Santiago, Chile)

EDITORES CIENTIFICOS | SCIENTIFIC EDITORS

José María 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 (Rosário, Argentina)

Jeannette Gavillán (San Juan, Pto Rico)

Leonora Mendoza (Santiago, Chile)

Horacio Olivo (Iowa, USA)

Edgar Pastene (Concepción, Chile)

Verónica Rivas (Monterrey, México)

Gabriela Ricciardi (Chaco, Argentina)

Luis A. Simeoni (Brasília, Brasil)

Beatriz Varela (Buenos Aires, Argentina)

EDITOR HONORARIO | HONORARY EDITOR

Jorge Rodríguez Chanfreau (La Habana, Cuba)

CONSEJO EDITORIAL | EDITORIAL ADVISORY BOARD

Julio Alarcón (Chillán, Chile)

Talal Aburjai (Amman, Jordan)

Arnaldo Bandoni (Buenos Aires, Argentina)

Elizabeth Barrera (Santiago, Chile)

Armando Cáceres (Guatemala, Guatemala)

Salvador Cañigueral (Barcelona, España)

Bruce Cassels (Santiago, Chile)

Geoffrey Cordell (Illinois, USA)

Rosa Degen (Asunción, Paraguay)

Marco Dehesa (Quito, Ecuador)

Olga Lock (Lima, Perú)

Rodolfo Juliani (New Jersey, USA)

Patricia Landázuri (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 Morón (La Habana, Cuba)

Patrick Moyna (Montevideo, Uruguay)

Pulok Mukkerjee (Jadavpur, India)

Luca Rastrelli (Salerno, Italia)

Vicente Martínez (Guatemala, Guatemala)

John A. O. Ojewole (Natal, Sudafrica)

Edison Osorio (Medellín, Colombia)

Mahendra Rai (Maharashtra, India)

Elsa Rengifo (Iquitos, Perú)

José Luis Ríos (Valencia, España)

Lionel Robineau (Pointe à Pitre, Guadalupe)

Gloria Saavedra (Cochabamba, Bolivia)

Marcelo Wagner (Buenos Aires, Argentina)

Talal Zari (Arabia Saudita)

© 2010 The Authors

© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (2), 84


Editorial

www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. 9 (2) 2010 | 84

Haití, tierra de esperanza [Haiti, land of hope]

Benito BARANDA.1,* 1. América 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 caído la catedral con su

arzobispo muerto), y aun en puerto príncipe se respira polvo y se siente ya ese olor a cuerpos

descompuestos. Todo lo avanzado con dedicación y

sacrificio en los últimos años, 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 príncipe, 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 está

inutilizable), y la demuestran en sus deseos de trabajar y levantarse nuevamente, de reconstruir con

sus manos la ciudad y de alzarlas también para alabar

y ayudar a otros.

En ese territorio los jóvenes profesionales voluntarios y voluntarias de América Solidaria están

dejando nuevamente parte de sus vidas, en años

pasados 70 de ellos y ellas donaron su tiempo, cariño y profesión a personas con nombre, a familias y

comunidades, hoy nuevamente retoman el servicio y

ya se alistan 10 para sumarse a otros 3 que están allá, se espera que en el 2010 se llegue a tener

simultáneamente 40 voluntarios colaborando con la

reconstrucción y desarrollo de Haití, fortaleciendo

sus instituciones y colaborando con la consolidación

de la red solidaria que los mismos haitianos han creado.

Creemos y seguiremos convencidos que es posible

superar las injusticias y la pobreza en América, en especial en los países más pobres y con mayores

grados de exclusión social, y específicamente en

nuestro subcontinente latinoamericano, juntos lograremos en las próximas décadas reescribir la

historia y que mejor que recomenzarla en el primer

país libre de nuestro continente (y seguramente en el

primero de personas procedentes de áfrica 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 soñar 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 más bien con la promoción de la iniciativa personal y colectiva tendiente al bien

común. Para ello es necesario poner nuestra

inteligencia y voluntad al servicio de los demás, no de nosotros mismos ni de nuestros propios deseos de

poder o de acumulación de bienes, sino más 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 niños y niña nacida aquí

goce de la libertad y de las oportunidades para desarrollarse en plenitud, con felicidad, amor e

igualdad.

© 2010 The Authors

© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (2), 85-86


Editorial


La ciencia chilena se pone de pie [Chilean science gets up]

Carlos L CESPEDES1, Julio ALARCON2 1Laboratorio de Bioquímica Vegetal y Fitoquímica-Ecológica. ; 2Laboratorio de Síntesis y Biotransformación de Productos Naturales.

Departamento de Ciencias Básicas, Universidad del Bío-Bío, Av. Andrés Bello s/n, Casilla 447, CP 3780000, Chillán, Chile Enviado: 2010-03-17

Para la comunidad científica de Chile, el

comienzo de este año 2010 ha sido muy duro, durante la madrugada del 27 de Febrero 2010, ocurrió un

fuerte terremoto grado 8.8 que sacudió las regiones

quinta, sexta, séptima, octava, novena, y región 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), Valparaíso (Católica de Valparaíso), Talca

(Universidad de Talca), Chillan (Universidad del

Bío-Bío), Concepción (Universidad de Concepción) y Temuco (Universidad Católica de Temuco,

Universidad de la Frontera) resultaron con graves

pérdidas, destrozos de infraestructura, material de

trabajo (vidrio, reactivos y solventes), y de valiosos equipos. Así, la comunidad científica chilena lamenta

la terrible perdida del edificio de la Facultad de

Ciencias Químicas de la Universidad de Concepción, el que resulto completamente destruido tanto por el

terremoto como por el casi inmediato incendio de

prácticamente todo el edificio. Lo más lamentable es la perdida de mucha investigación, algo que muy

probablemente tomara algunos años en recuperarse.

La comunidad científica en Chile, espera que la

agencia estatal para la ciencia y la tecnología (Comisión Nacional de Investigación Científica y

Tecnológica/CONICYT) de las garantías y el apoyo

para recuperar equipos y materiales, algo imperioso y fundamental para poder comenzar y/o reiniciar las

investigaciones, y así poder volver a obtener las

evidencias científicas que permitan recomenzar con el chequeo de hipótesis, y obtención de datos que

permitan responder en parte a las numerosas

preguntas científicas que se habían generado en las

investigaciones que estaban en curso.

La región centro-sur incluye a las Universidades:

Católica de Valparaíso (UCV), de Valparaíso (UV), de Santiago de Chile (USACH), de Talca

(UTALCA), del Bío-Bío (UBB: en sus dos Campus

Chillan y Concepción), de Concepción (UDEC, en sus tres Campus: Concepción, Chillan y Los

Ángeles), Católica de la Santísima Concepción

(UCSC), Católica de Temuco (UCT), de la Frontera

de Temuco (UFRO) y la Austral de Valdivia (UAV). Todas estas universidades con algún tipo de daños,

pero las que resultaron con más daños fueron las

UDEC, UBB y UTALCA.

Figura 1: Imágenes de la devastación causada por el terremoto en

las instalaciones universitarias de la Región del Bío-Bío.

.

Céspedes y Alarcón La ciencia chilena se pone de pie


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

Todas estas universidades poseen en conjunto una productividad científica significativa, sumando entre

toda el 33% nacional, datos que no hacen sino

evidenciar el potencial de la investigación científica

de las regiones, que con fuerza contribuyen al desarrollo científico y tecnológico de Chile

Finalmente, este esfuerzo de trabajo

mancomunado y solidario requiere del apoyo de las autoridades (Intendentes, Gobernadores, Alcaldes,

CORFO, CONICYT, Ministerio de Educación-Div.

Educ. Superior), para que unidos no solo nos pongamos de pie (de lo cual estamos más 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 tenía antes de este terrible terremoto y tsunami. Esta experiencia es

el mejor ejemplo de que se debe apoyar

incondicionalmente a la ciencia básica, pilar

fundamental en los avances del conocimiento de la naturaleza. Los conocimientos adquiridos a través de

la investigación científica, no siempre tienen o

poseen una aplicación mediata y/o inmediata, sino

que muchas veces se aplican a largo plazo, como por ejemplo las investigaciones en sismología, y es en

este minuto cuando resulta relevante el conocimiento

adquirido por nuestros colegas de la Universidad de Chile, expertos en sismología, quienes prácticamente

hacen investigaciones científicas en este campo del

conocimiento con un mínimo de apoyo.

“La universidad está dañada pero no está en el

suelo ni devastada”

Rector Sergio Lavanchy, Universidad de Concepción, Diario El Sur, 9 de Marzo 2010.

© 2010 The Authors

© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (2), 87 - 92


Articulo Original | Original Article







apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra. Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada


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 DELAZAR1 Ehsan NAZIFI 1,2 Ali MOVAFEGHI 2 Hossein NAZEMIYEH1 Salar HEMMATI 1 Lutfun NAHAR3 Satyajit D. SARKER4,*

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 análisis GC-MS del extracto metanólico (MeOH) de los bulbos de Ornithogalum cuspidatum llevo a la identificación 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 extracción en fase sólida se evaluaron mediante el ensayo del DPPH y la fracción 40% MeOH-H2O exhibió 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 fracción 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 Versión Corregida | Accepted in Corrected Version: December 2, 2009.

Publicado en Línea | Published Online: March 19, 2010. Declaración de intereses | Declaration of interests: Authors have no competing interests. Financiación | 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: [email protected] ; Tel: +44 (0)1902 322578 ; Fax: +44 (0)1902 322496.

mailto:[email protected]

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 C18 Sep-Pak

cartridge (10 g) using a step gradient of H2O:MeOH and DCM to obtain seven fractions 1-5, H2O: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

µL) 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 3°C/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 µL 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 C18H12N5O6, 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 C18H12N5O6, 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 RC50 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. RC50 values of the MeOH extract and its fractions in the DPPH assay, and their total phenolics content (TPC)

Extract and fractions RC50 (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





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 C27H46O 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 C28H48O 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 C27H46O2 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 C29H48O 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 C28H48O 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 C29H50O 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 C29H48O 55 (62.7), 69 (41), 83 (100), 129 (41), 255 (21.9), 394 (16.1), 484 (13.3)

-Sitosterol 116.5 10.73 414 C29H50O 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 C29H48O 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 C29H46 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 C28H48O 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 C29H50O 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 C29H48O 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 C27H42O2 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 C29H50O 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 C29H46O 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 RC50 (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.

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Diferenciación de las especies Achyrocline satureioides, A. flaccida y

Gnaphalium gaudichaudianum por sus perfiles cromatográficos [Differentiation of the species Achyrocline satureioides, A. flaccida and Gnaphalium gaudichaudianum by their

chromatographic profiles]

Daiana RETTA1; Rocío FERNANDEZ PENUTO1; María CORREA1; Martha GATTUSO2;

Susana GATTUSO2; Arnaldo BANDONI1 1 Cátedra de Farmacognosia-IQUIMEFA. Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires-CONICET. Junín

956, 2º piso. C1113AAD Buenos Aires, Argentina. 2 Cátedra de Farmacobotánica, Facultad de Ciencias Bioquímicas y Farmacéuticas,

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 realizó el estudio de los perfiles cromatográficos por capa fina y cromatografia gaseosa de muestras de inflorescencias de Achyrocline satureioides,

A. flaccida y Gnaphalium gaudichaudianum de Argentina. Los perfiles por cromatografía 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, éstas no se encuentran o son de menor intensidad. Los perfiles obtenidos por cromatografía de gases de las fracciones volátiles 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 técnica por TLC facilita la correcta identificación de estas especies, en forma sencilla y rápida.

Palabras Clave: Achyrocline satureioides; Achyrocline flaccida; Marcela; Gnaphalium gaudichaudianum; CCF; CG.

Recibido | Received: October, 15, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: January 4, 2010.

Publicado en Línea | Published Online 25 March 2010 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | Funding: This work was financed by UBACYT B014 and PICTR -0284 This article must be cited as: Daiana Retta; Rocío Fernandez Penuto; Cecilia Correa; Martha Gattuso; Susana Gattuso; Arnaldo Bandoni. 2010. Diferenciación de las especies

Achyrocline satureioides, A. flaccida y Gnaphalium gaudichaudianum por sus perfiles cromatográficos. Bol Latinoam Caribe Plant Med Aromat 9(2):93 – 99. {EPub 25 March

2010 }.

*Contactos | Contacts: e-mail: [email protected]


Retta et al. Diferenciación cromatografica de A. satureioides, A. flaccida y G. gaudichaudianum

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

INTRODUCCION

El género Achyrocline (Asteraceae)

comprende unas 15-20 especies y ocurre

principalmente en América del Sur y Central, con

extensión a África tropical y zonas montañosas de la

Isla de Madagascar (Giangualani, 1976; Nesom,

1990). En Argentina habitan ocho especies,

distribuidas principalmente en el norte y centro del

país (Zuloaga y Morrone, 1999). Es bien conocida la

dificultad que presenta la correcta identificación de

éstas especies, dado que poseen caracteres

morfológicos muy semejantes entre sí (Giangualani,

1976).

La especie más utilizada, debido a la amplia

distribución en nuestro país, y de mayor interés

comercial es Achyrocline satureioides (Lam.) DC. Es

una planta herbácea, perenne, tomentosa, que se

conoce con los nombres vulgares de “marcela”,

“marcelita”, “marcela blanca”. Sus partes aéreas e

inflorescencias son tradicionalmente usadas como

digestivas, antiinflamatorias, antiespasmódicas,

antidiabéticas y antiasmáticas (Ratera y Ratera, 1980;

Toursarkissian, 1980). En Argentina no solamente es

usada en medicina tradicional y en la formulación de

fitoterápicos, sino también en la elaboración de

productos alimenticios, dado que posee un intenso

aroma que recuerda al levístico y su sabor es amargo.

En Uruguay se comercializan además, productos

cosméticos, debido a sus propiedades antioxidantes y

antiinflamatorias.

La demanda actual, en la región, está cubierta

por la colecta de material silvestre, lo que lleva

muchas veces a que ésta se encuentre mezclada con

otras especies similares, como Achyrocline flaccida

(Weinm.) DC. con la cual comparte parcialmente su

área de distribución y es también conocida como

“marcela” o “marcela amarilla”. Sus inflorescencias

son principalmente empleadas como digestivas,

antiespasmódicas, febrífugas, tónico, antihelmínticas

(Hieronymus, 1882; Parodi, 1886). Otra especie

contaminante o adulterante es Gnaphalium

gaudichaudianum DC. (Asteraceae), conocida como

“vira vira” o “marcelita”. Es también empleada en

trastornos digestivos y su morfología es similar a las

dos especies de Achyrocline antes mencionadas

(Martínez Crovetto, 1981).

Existen estudios botánicos comparativos de

identificación de dichas especies (Amat 1988; Gattuso

et al., 1998, 2008; Petenatti et al., 2004) donde se

describen los caracteres diacríticos de diferenciación,

pero dado que éstos resultan sutiles, suele presentar

cierta dificultad la identificación de las mismas.

Esto planteó la necesidad de encontrar una

técnica analítica sencilla, rápida, accesible que permita

obtener mayores evidencias para la identificación de

estos materiales, en particular cuando se presentan en

polvo o triturados.

El análisis por cromatografía en capa fina

(TLC) de la especie A. satureioides se encuentra

descripta en la Farmacopea Brasilera (2003), faltando

el análisis cromatográfico de A. flaccida y G.

gaudichaudianum.

Por lo expuesto, se propuso la búsqueda de

una técnica por TLC, que permita determinar los

perfiles cromatográficos 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

recolección e identificación botánica 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

cátedra de Farmacobotánica, de la Facultad de

Ciencias Bioquímicas y Farmaceúticas de la

Universidad Nacional del Rosario.

Extracción Extracción: 2 g de material vegetal se

extrajeron por maceración en hexano, por 24 h. Se

filtró y se llevó a sequedad en evaporador rotatorio a

presión reducida a temperatura no mayor a 40 ºC y se

reconstituyó con 2 ml de hexano.

Cromatografía en capa fina Se emplearon cromatoplacas de sílica gel

Merck ® como fase estacionaria. Como fase móvil se

empleó una mezcla de Tolueno: Acetato de etilo:

Ácido acético (9:1: III). La siembra se realizó en

banda de 1 cm. La distancia de desarrollo fue de 10

cm. El revelado se realizó por aspersión con el

reactivo de anisaldehído sulfúrico y posterior

aplicación de calor, en estufa a 105 ºC. La observación

se realizó a la luz natural.



Cromatografía de Gases (GC-FID-MS) Los aceites esenciales fueron extraídos de 100

gramos de las inflorescencias desecadas a temperatura

ambiente de cada especie, por hidrodestilación durante

3 horas usando una trampa tipo Clevenger. El producto

obtenido se secó con sulfato de sodio anhidro y

almacenó a 2 °C hasta su análisis.

Para el análisis de los aceites esenciales se

utilizó un GC-FID-MS Perkin Elmer Clarus 500,

equipado con un inyector automático (relación 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 μm de espesor de fase. La columna polar se

conectó a un detector FID, mientras que la no polar se

conectó a un FID y a un detector de masa cuadrupolar

(70 eV), mediante un sistema de venteo (MSVent™).

La fase móvil fue Helio, flujo constante de 1.87

mL/min. La temperatura fue programada de acuerdo al

siguiente gradiente: 90º-225ºC a 3ºC/min, luego

isotérmico por 15 min. El inyector y ambos FID se

usaron a 255ºC y 275ºC, respectivamente. El volumen

de inyección fue 0.2 μL de una solución etanólica al

10%. La temperatura de la línea de transferencia y la

de la fuente iónica fueron 180ºC y 150ºC

respectivamente; el rango de masas buscado fue 40-

300 Da (10 scan/segundo).

La identificación de los constituyentes se

realizó por comparación de sus índices de retención

(relativos al de los n-alcanos C8-C20) obtenidos en

ambas columnas, con los de compuestos de referencia.

Además, cada espectro de masa obtenido fue

comparado con los de las bases de datos electrónicas

tradicionales (Adams, 2007, Wiley/NBS, 2008) y con

los desarrollados en nuestro laboratorio a partir de

patrones o aceites esenciales de composición química

conocida. El análisis cuantitativo se realizó usando el

método de porcentaje de áreas, sin considerar alguna

corrección por factores de respuesta. Se consideró para

cada constituyente la respuesta más baja entre las

obtenidas en las dos columnas usadas.

Tabla 1: Datos de recolección e identificación del material

vegetal.

Muestra Especie Lugar de recolección

S1/1554 A. satureioides Dpto. Punilla- Córdoba

S2/1631 A. satureioides Río Espinillo- Córdoba

S3/1892 A. satureioides Villa Ventana- Buenos Aires

S4/1627 A. satureioides Icho Cruz- Córdoba

S5/1626 A. satureioides Los Reartes- Córdoba

S6/1629 A. satureioides Santa Rosa de Calamuchita- Córdoba

S7/1894 A. satureioides Dique La Florida- San Luis

S8/1895 A. satureioides Yacanto de Calamuchita-

Córdoba

S9/1890 A. satureioides Coronel Rosales- Buenos Aires

F1/1546 A. flaccida Dpto. Colón- Entre Ríos

F2/1660 A. flaccida Santo Tomé- Corrientes

F3/1662 A. flaccida Jardín América- Misiones

F4/1664 A. flaccida Ruta 127, límite Entre Ríos y

Corrientes

F5/1587 A. flaccida Pronunciamiento- Entre Ríos

F6/1663 A. flaccida Cuña Pirú- Misiones

F7/1559 A. flaccida Dpto. San Javier- Misiones

F8/1661 A. flaccida San José- Misiones

G1/1632 G. aff. gaudichaudianum Los Reartes- Córdoba

G2/1628 G. gaudichaudianum Santa Rosa de Calamuchita- Córdoba

G3/1633 G. aff. gaudichaudianum Los Reartes- Córdoba

G4/1625 G. aff. gaudichaudianum Villa General Belgrano-

Córdoba

G5/1936 G. gaudichaudianum Santa Rosa de Calamuchita-

Córdoba

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 Río Espinillo- Córdoba



Figura1: Cromatografía en capa fina de muestras de A. satureioides (el círculo indica la banda de interés).

S1 S2 S3 S4 S5 S6 S7 S8 S9

Figura2: Cromatografía en capa fina de muestras de A. flaccida (el círculo indica la banda de interés).

F1 F2 F3 F4 F5 F6 F7 F8

Figura 3: Cromatografía en placa fina de muestras de G. gaudichaudianum (el círculo indica la banda de interés).

G1 G2 G3 G4 G5 G6 G7 G8 G9



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

(A) A. satureioides

(B) A. flaccida

(C) G. gaudichaudianum



RESULTADOS

Cromatografía en capa fina En las figuras 1, 2 y 3 se muestran los perfiles

cromatográficos obtenidos de cada especie.

De los perfiles de las muestras de A.

satureioides y A. flaccida se observó que los mismos

son semejantes. Pero existe una diferencia que podría

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

prácticamente no es detectada.

Ambas especies presentan algunas bandas en

común: 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, éstas 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 también una banda rosada en Rf 0.6.

Cromatografia de Gases (GC-FID-MS) Los perfiles obtenidos por cromatografía de

gases de las fracciones volátiles 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 estadísticamente significativa por el

análisis de varias muestras de cada especie.

DISCUSIÓN Y CONCLUSIONES

Si bien la identificación primaria del material

vegetal se realiza por medio de estudios botánicos, en

el caso de la muestras de “marcelas” esto resulta

bastante difícil, dado que las características que las

diferencias son sutiles y requieren de un estudio

bastante minucioso y preciso del material, dando lugar

a posibles errores en la identificación, en particular si

esta es realizada por personal no entrenado. Por otro

lado, el empleo de técnicas cromatográficas, como

TLC, resulta más sencillo, está ampliamente

difundido, es de fácil acceso, económico y

reproducible.

Si bien, ambos estudios son complementarios,

hasta el momento solo se encontraba descripto el

análisis 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

permitiría discriminarlas entre sí. Además, en dicha

norma se emplea Celulosa como fase estacionaria, con

la que se suele obtener menor resolución de las bandas

presentes en las muestras que con Sílica gel.

Como parte de la búsqueda y optimización de

un sistema cromatográfico que caracterizara estas

especies, se probaron distintos sistemas y distintos

tipos de extractos. Partiendo de extractos realizados

con etanol al 80% y sistemas cromatográficos

específicos para compuestos de mayor polaridad, no se

evidenciaron diferencias significativas en los perfiles

de las especies de Achyrocline, por lo tanto se decidió

partir de un extracto de menor polaridad. La técnica

que aquí se propone permite diferenciar las tres

especies y junto con la identificación botánica permite

obtener resultados más sólidos, aportando mayores

evidencias, que contribuyen a minimizar la ocurrencia

de errores en la identificación del material.

Por otro lado, dado que el extracto que se

emplea para la siembra es hexánico, se cree que

muchas, aunque no todas las bandas detectadas,

pueden corresponder a los componentes volátiles

presentes en el aceite esencial de dichas especies. El

estudio de aceites esenciales por cromatografía

gaseosa de muestras de A. satureioides y A. flaccida

provenientes de Argentina ya ha sido reportado, no

encontrándose 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 cromatografía de gases de las fracciones

volátiles 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 útil que sigue siendo el empleo de la

cromatografía en capa fina como criterio de

identificación 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 químicas observadas no se encuentran

correlacionadas con diferencias en la identificación.

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 químicas en

muestras de esta especie. A fin de profundizar en

dichas diferencias, el análisis de los componentes

volátiles de esta especie se encuentra bajo estudio por

nuestro grupo de trabajo.

Para concluir podemos decir que esta técnica

resulta de utilidad para discriminar estas especies tan

similares entre sí, 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.

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florales de A. satureioides, A. flaccida y G.

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© 2010 The Authors








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Photoprotective activity of Yucca periculosa polyphenols [Actividad fotoprotectora de los polifenoles de Yucca periculosa]

Ana María GARCÍA-BORES1,4, Christian BELLO1, Yukiko CAMPOS1, José del Carmen BENITEZ2, Saul FLORES3, Margarita CANALES1, Tzasná HERNÁNDEZ1, José Guillermo AVILA ACEVEDO1

1Laboratorio de Fitoquímica, UBIPRO, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autónoma de México,

Tlalnepantla 54090, Edo. de México, México; 2 Laboratorio 1, UMF, Facultad de Estudios Superiores-Iztacala, Universidad Nacional

Autónoma de México, Tlalnepantla 54090, Edo. de México, México; 3 Laboratorio de Recursos Naturales, UBIPRO, Facultad de Estudios

Superiores-Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla 54090, Edo. de México, México 4Posgrado en Ciencias

Biológicas, Universidad Nacional Autónoma de México

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 metanólico 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 radiación UV, siendo el MS y el resveratrol los que poseen mayores propiedades fotoprotectoras. El factor de protección solar (FPS) de las sustancias se evaluó en cobayos y mediante un estudio histopatológico de la piel irradiada con UV.

Se determinó que todas las sustancias previenen del daño histológico en la piel inducido por la UV, además 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 Versión Corregida | Accepted in Corrected Version: December 21, 2009.

Publicado en Línea | Published Online 25 March 2010 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | 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 María García-Bores, Christian Bello, Yukiko Campos, José del Carmen Benitez, Saul Flores, Margarita Canales, Tzasná Hernández, José

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: [email protected]; Tel.: + 52-5-623-11-36; Fax + 52-5-623-12-25;


García-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 México. 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 earth’s 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 Zapotitlán de las

Salinas, Puebla, México 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 CHCl3:MeOH mixtures.

Fractions of CHCl3: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 1H 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 107

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/cm2 (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/cm2 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 cm2 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/cm2. 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/cm2 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/cm2)

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, R2=0.93). B With protection, OMC () (K= 0.13, R2=0.99); MeOH extract () (K= 0.12, R2=0.99); 1: Naringenin (o) (K= 0.25, R2=0.99); 2: Resveratrol () (K= 0.09, R2=0.93); 3: MS, trans-3, 3’, 5, 5’-tetrahydroxy-4’-methoxystilbene (■) (K=

0.07, R2=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)

Lo

g #

su

rviv

ors

0

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50 60 70 80 90 100

Time (minutes)

Lo

g #

su

rviv

ors

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 (107)

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 (6–4) 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 (λmax 288 nm), resveratrol (λmax 305 nm)

and MS (λmax 316 nm). In addition, resveratrol and MS

have molar extinction coefficients higher than the

OMC ( 37 800 M-1

cm-1

and 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: λmax 310 nm, 24 000M-

1cm

-1; cis: λmax 301 nm, 12600M

-1cm

-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 López Villafranco

and Rosario González 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.

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© 2010 The Authors



Articulo Original | Original Article







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Los remedios naturales en la prevención 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. MARTÍNEZ1,2*

1 Museo de Antropología. Facultad de Filosofía y Humanidades. Universidad Nacional de Córdoba. Hipólito Irigoyen 174. 5000. Córdoba, República 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 contribución documenta el uso de la farmacopea natural (vegetal y animal) en la prevención y cuidado de la salud oral, entre los tobas del

Chaco Central (Argentina). Caracterizada por sus múltiples etiologías, las afecciones buco-dentales adquieren relevancia en la salud de estas comunidades,

documentándose 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 proporción de citas se encuentra encabezada por plantas nativas, destacándose por su consenso el uso simbólico de la

liana Clematis montevidensis, y el empleo de las raíces de Solanum argentinum, Cucurbitella asperata y de la parte aérea 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 señala 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 Versión Corregida | Accepted in Corrected Version: February 1, 2010.

Publicado en Línea | Published Online: March 25, 2010. Declaración de intereses | Declaration of interests: Authors have no competing interests. Financiación | Funding: Proyectos Anpcyt/ Foncyt Pict 32894 y 1612

This article must be cited as: Gustavo J. Martínez. 2010. Los remedios naturales en la prevención 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: [email protected]


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


INTRODUCCION

La documentación y registro de usos de plantas medicinales en el tratamiento de afecciones buco-

dentales constituye uno de los diversos tópicos que se

abordan en estudios etnobotánicos, presentándose la

mayoría 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 específicamente esta temática 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 México (Waizel-Bucay & Martínez Rico, 2007) y más de 100 plantas

comercializables en Chicago, Costa Rica y Colombia

(Colvard et al., 2006), así 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 etnografía, etnomedicina,

etnofarmacología y/o aplicaciones basadas en

evidencias clínicas de plantas medicinales usadas

específicamente en odontología, odontalgias y otras afecciones orales, a la vez que señalan la ausencia de

un catálogo de referencia que describa las plantas

usadas con este fin. En regiones en las que la atención

odontológica resulta inaccesible, el empleo de

remedios naturales constituye una opción plausible,

en particular si se cuenta con un repertorio de especies de probada eficacia farmacológica que

posibilite su implementación en atención primaria. El

conocimiento de estos usos propicia la incorporación de la medicinas tradicionales y eventualmente la

prescripción de etnofármacos naturales en el sistema

local de salud, acorde con los criterios sugeridos por la WHO (2002) para contextos de medicinas

múltiples como el que abordamos en este trabajo.

Los tobas, conocidos también como qom o

qoml’ek, son un grupo indígena integrante de la familia lingüística Guaycurú que conforman una

población de bandas aliadas de unos 60.000

integrantes, cuyo hábitat se encuentra hoy en forma mayoritaria en el Chaco Central y Austral (en las

provincias argentinas de Chaco y Formosa) y un

pequeño núcleo en el Chaco Boreal paraguayo (ENDEPA, 1986; Arenas, 1997; Censabella, 2000)

Informaciones previas sobre la etnobotánica de

diversas parcialidades tobas puede encontrarse en los trabajos de Franze (1925) Martínez Crovetto (1964,

1968), Vuoto P. (1981, 1999) Arenas (2000),

Martínez (2007a, 2008), Hecht et al. (2008), junto a

otros de carácter etnozoológico (Zacarías, 1993; Martínez 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

prevención 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 etnomédico en el que éste

tiene lugar.

MATERIALES Y METODOS

Área de estudio El área de trabajo forma parte de la región del

Gran Chaco, en la provincia de Chaco (Noreste de

Argentina) en las inmediaciones del río Bermejito (Figura 1), presentando un clima subtropical

continental con precipitaciones de entre 800 y 900

mm/año superiores en verano -con una temperatura

promedio de 29 ºC- y marcada estación seca en invierno -con una temperatura promedio de 17 ºC-.

Según sus peculiaridades fitogeográficas corresponde

a la región Neotropical, Dominio Chaqueño, Provincia Chaqueña, con especies propias de los

bosques del Chaco Central según Prado (1993) o

transición entre el Chaco Oriental o húmedo y el Chaco Occidental o semiárido, según el criterio de

Cabrera (1994). Se caracteriza por un patrón de

vegetación con un marcado modelado fluvial

(Morello & Adámoli, 1974) y una vegetación climácica de bosque xerófito caducifolio, junto a

sabanas, estepas halófitas, cardonales, pajonales,

camalotales y otros tipos. Desde el punto de vista económico los tobas

subsisten combinando precariamente actividades

tradicionales como la caza, pesca y recolección, 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 algodón, así como de los ingresos que provienen de planes de asistencia oficial.



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



Un contexto sanitario múltiple caracteriza al

sistema local de salud, en el que coexiste el shamanismo (desempeñado por sus especialistas, los

pi’oxonac), la medicina doméstica o casera, y la

medicina oficial en los centros de salud, a cargo de

profesionales biomédicos y agentes sanitarios tobas. A pesar de este pluralismo, la medicina tradicional

toba no se halla incorporada aún a la medicina

oficial, siendo el uso de remedios naturales y la cura shamánica una de las primeras opciones terapéuticas

a las que recurren los pobladores locales (Martínez,

2007b). Una diversidad de cuadros clínicos y afecciones caracterizan la morbilidad en la región, tal

como lo atestigua un Diagnóstico Local de Salud

(DLS) que desarrollamos en la región a través de

entrevistas a los profesionales (Martínez, 2006). En particular la atención odontológica resulta

prácticamente inaccesible, contando con un solo

profesional en un radio de más de 50 km a la redonda. El mismo DLS refiere cómo las

descalcificaciones, la presencia de caries por

debilitamiento del esmalte de los incisivos superiores, pérdida de piezas dentarias a tempranas edades,

manchas por hidroarsenicismo y odontalgias, así

como restos de raíces no extraídas, junto con las aftas

y ulceraciones en la mucosa bucal especialmente de los niños, constituyen algunas de las afecciones

orales más comunes, muchas de ellas asociadas con

un desbalance nutricional, desnutrición y una dieta escasa en proteínas y lácteos, con predominio en el

consumo de farináceos. Aun cuando diversas

comunidades indígenas del Gran Chaco, entre ellas

los tobas, participaron a principios del siglo XX del trabajo en obrajes e ingenios azucareros adoptando

saberes y prácticas de los blancos (entre ellos la

prevención a través del cepillado y el empleo de dentífrico), la higiene buco-dental resulta inusual

hasta el presente.

Métodos y técnicas de trabajo Como parte de un relevamiento general de la

etnobotánica médica toba, se recolectó información acerca de los usos medicinales de las plantas en el

área de estudio entre los años 2004 al 2008. Para tal

fin se aplicaron entrevistas abiertas, extensas y recurrentes, así como encuestas semiestructuradas a

miembros de la comunidad de distinto sexo y edad,

así como a profesionales del ámbito de salud, para lo

cual se confeccionó una encuesta sobre la temática utilizando como referencia la guía etnobotánica

propuesta por Arenas (1995). Esta información se

complementó con la obtenida por observación

participante. En todos los casos el material vegetal se recolectó en recorridas de campo, en compañía de los

informantes. La documentación de la información se

realizó en cuaderno de campo, grabaciones digitales

y fotografías. Las muestras de referencia se depositaron en el Museo Botánico (CORD) del

Instituto Multidisciplinario de Biología Vegetal de la

Universidad Nacional de Córdoba. El material vegetal fue identificado en su mayor parte por el

autor, recurriéndose a la consulta de especialistas en

los taxa que presentaran dificultades, y al catálogo de Plantas Vasculares de Argentina (Zuloaga &

Morrone, 1996, 1999) y su actualización electrónica

on-line para el Cono Sur (Zuloaga et al., 2008). El

material zoológico correspondiente a los invertebrados fue identificado por especialistas y

forman parte de la colección particular del autor

(Museo de Antropología). En el caso de los vertebrados la identificación se realizó con

informantes mediante el empleo de fotografías e

imágenes de guías de campo, lo que fue corroborado con bibliografía etnobiológica específica para la

región del Gran Chaco (Martínez Crovetto, 1995;

Arenas, 2003).

Se realizaron seis trabajos de campo que totalizaron más de 100 días de estancia en

asentamientos tobas ubicados en localidades,

pertenecientes a la intendencia de Río Bermejito (Dpto. General Güemes, Pcia. de Chaco), en las

inmediaciones del río homónimo siendo el Paraje El

Colchón (Figura 1) el sitio donde se realizaron la

mayor parte de las entrevistas y colectas de material botánico. Previo a las entrevistas se informó acerca

del proyecto de investigación y sus objetivos a

representantes y miembros de las comunidades. Las conversaciones con especialistas y pobladores se

construyeron sobre la base de un objetivo común:

mejorar la situación de salud regional, incrementar el conocimiento acerca de los remedios naturales,

recuperar saberes y prácticas tradicionales para

favorecer su circulación y desarrollar materiales

educativos de interés regional. Se emplearon alternativamente métodos

cualitativos, cuantitativos y participativos en

instancias diferentes y recurrentes durante el lapso de la investigación, procurando enriquecer cada una de

ellas con lo generado en la otra y siguiendo el

esquema básico de la labor etnobotánica: Trabajo de campo y trabajo de gabinete.



- Técnicas cualitativas: Se recurrió al estudio

del contenido a los fines de interpretar en las entrevistas abiertas y extensas la sintomatología y

etiologías de las dolencias.

- Técnicas cuantitativas: Se diseñó una

encuesta temática semiestructurada, la que se aplicó a 60 informantes, conformada tanto por especialistas

(shamanes, parteras y ancianos) como por el común

de los miembros de la comunidad (jóvenes y adultos de ambos sexos). Se obtuvieron valoraciones

cuantitativas acerca de la cantidad de usos reportados

para cada especie (agrupadas en categorías de frecuencia de mención) y la proporción de los

mismos en el total de reportes, utilizándose como

criterio de validación la coincidencia de al menos dos

informantes para el mismo uso medicinal, esto es: idéntica aplicación para una misma parte de una

planta, cualquiera fuera el modo de preparación,

incluyendo datos únicos cuando éstos tuvieran soporte en otros estudios etnobotánicos desarrollados

en la región (Scarpa, 2004). Se obtuvo el listado de

aplicaciones con mayor consenso de uso, así como el de especies con mayor cantidad de aplicaciones

medicinales para las afecciones aquí tratadas.

Para la escritura en idioma toba se recurre al

alfabeto toba estándar (Buckwalter & Litwiller de Buckwalter, 2001) por su amplia difusión y uso entre

los miembros de la comunidad, pudiendo consultarse

las convenciones fonéticas en contribuciones anteriores (Martínez, 2007a,b).

RESULTADOS Y DISCUSIÓN

Representaciones acerca de las afecciones bucodentales

Los tobas interpretan que las afecciones dentarias se deben a la acción de gusanos alojados en

el interior de las piezas o bien a la transgresión de

ciertos tabúes. El dolor de muelas, por ejemplo,

guarda estrecha relación 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 más próximos 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 así esta enfermedad; de no

cumplirse esta prescripción resulta inevitable para el

transgresor un dolor de muelas de difícil tratamiento, cuya atención es competencia exclusiva de los

pi’oxonaq. Acorde con las representaciones de los

tobas, las odontalgias se originan también por consumir en la etapa de duelo algunos frutos del

monte como luaxai (Morrenia spp.), en particular si

en su interior contienen algún tipo de larva (qochi’l)

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 bucofaríngeas. 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 prevención de la salud oral.

La categoría taxonómica que se encuentra

más representada en cuanto a aplicaciones medicinales, de acuerdo con la Figura 2, es la de las

Plantas Antófitas, (60%) lo que resulta consistente

con la gran diversidad de especies involucradas en la

misma. La categorías taxonómica de los Hongos y líquenes, por su parte, suele estar poco representada

en estudios etnobotánicos, 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 líquenes) 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 botánicas más 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 zoológicas, la familia

Myrmecophagidae (1 especie y 2 usos).

Figura 2. Distribución porcentual de los usos medicinales por grupo taxonómico.



Tabla 1.-Farmacopea vegetal empleada en afecciones bucofaríngeas y dentales.

REINO Clase Familia

Especie

Nombre local (Voucher)

Orig. Parte usada / Forma de preparación / Modo de administración.

Aplicación (Eficacia atribuida)

F.C.

FUNGII (Hongos y líquenes) Basidiomycetes Lycoperdaceae Lanopila bicolor (Lev.) Pat.

huaqajñi l'atec GJM 442 (CORD)

Nat. Esporas /Sin preparación/ Tópico

Aftas y llagas bucales (Cicatrizante, antiinflamatorio oral)

*

Mycenastrum corium (Guers.) Desv.




*

Vascellum pampeanum (Speg.) Homrich




*

Ascomycetes Parmeliaceae Cannomaculina pilosa

(Stizenb.) Elix & Hek

ncapeguelec 'ana 'epaq GJM 418 (CORD)

Nat. Planta entera/Infusión o decocción, Incineración (cenizas)/ Tópico


**

Physciaceae Heterodermia albicans (Pers.)

Swinscow & Krog

ncapeguelec 'ana 'epaq GJM 417b (CORD)

Nat. Planta entera/Infusión o

decocción, Incineración (cenizas)/ Tópico

Aftas y llagas bucales

(Cicatrizante, antiinflamatorio oral)

**

Physcia lopezii Moberg

ncapeguelec 'ana 'epaq GJM 417a (CORD)

Nat. Planta entera/Infusión o decocción, Incineración (cenizas)/ Tópico


**

PLANTAE (Antófitas) Liliopsida (Monocotyledoneae) Dioscoreaceae Dioscorea microbotrya Griseb.

etaxat lte GJM 469 (CORD, BAB)

Nat. Raíces / Sin preparación / Ingesta alimentaria

Fortalecer la dentadura *

Poaceae Arundo donax L.

coqta GJM 414 (CORD) GJM 293 (CORD)

Nat. Planta entera/Incineración

(cenizas)/ Bebida

Aftas y llagas bucales


*

Elionurus muticus (Spreng.) O. Kuntze

chem' auaxa GJM 433 (CORD)

Nat. Raíz/Macerado en agua / Enjuague bucal

Odontalgias y caries dentales (Antiodontálgico)

**

Magnoliopsida (Dicotyledoneae) Amaranthaceae Alternanthera pungens Kunth

ta'asot GJM 220 (CORD)

Nat. Hojas/Infusión o decocción en agua/ Enjuague bucal


**

Parte aérea/Incineración (cenizas)/ Tópico


**



REINO Clase Familia

Especie




F.C.

Anacardiaceae Schinus fasciculatus var.

fasciculatus (Griseb.) I.M. Johnst.

toroloquiic GJM 21 (CORD)

Nat. Infusión o decocción en agua/ Bebida. También se acostumbra a masticar las hojas.

Dolor de garganta (Antibiótico, calmante bucofaríngeo)

**

Parte aérea/Infusión o decocción en agua/ Enjuague

bucal. Se indica un puñado de hojas, o la corteza y gajos obtenidos del naciente, en el volumen de una pava, tres veces al día.

Odontalgias (Antiodontálgico)

***

Apiaceae Eryngium coronatum Hook. & Arn.

ra'aloxo GJM 344 (CORD)

Nat. Raíz/ Molido o picado/ Emplasto. Se aplica un fragmento de la raíz en la

cavidad del diente afectado


*

Aristolochiaceae Aristolochia esperanzae Kuntze var. esperanzae

epaq ltaá GJM 542 (CORD, BAB)

Nat. Raíz/Sin preparación o en el mate/ Mascado Se mastica y traga un pedacito de la raíz de olor mentolado.


**

Asteraceae Eupatorium hecatanthum

(DC.) Baker

ronai’ laue GJM 63 (CORD, BAB)

Nat. Inflorescencia, hojas/ Sin

preparación / Tópico. Se colocan algunas flores o bien una hoja en las caries.

Odontalgias

(Antiodontálgico) **

Parthenium hysterophorus L.

chemaxaraic, chimaxadaic GJM 225 (CORD)

Nat. Raíz/ Infusión o decocción en agua/ Emplasto. Se introduce un trozo de raíz en las caries.


*

Cactaceae Rhipsalis lumbricoides (Lem.) Lem.

sallaxataxaic GJM 428 (CORD)

Nat. Planta entera/Incineración (cenizas)/Tópico


*

Celastraceae Maytenus vitis-idaea Griseb.

satachec, chiqpi' GJM 218 (CORD)

Nat. Hojas/Incineración (cenizas)/Tópico


*

Celtidaceae Celtis iguanaea (Jacq.) Sarg.

taxanachec GJM 517 (CORD)

Nat. Hojas/Infusión o decocción en agua/ Bebida


**

Convolvulaceae Dichondra microcalyx (Hallier f.) Fabris

micha ltela GJM 286 (CORD)

Nat. Hojas/Infusión o decocción en agua/ Enjuague bucal


**

Cucurbitaceae Cucurbitella asperata (Gillies ex Hook. & Arn.) Walp.

quemoxon GJM 280 (CORD)

Nat. Raíz/Infusión o decocción en agua/ Enjuague bucal


****

Euphorbiaceae Euphorbia serpens Kunth var. serpens

potaxanaxaq alo'q, qapalaxanaxaic, qoloxoloxo

Nat. Parte aérea/Infusión o decocción en agua/ Enjuague

bucal

Aftas y llagas bucales (Cicatrizante, antiinflamatorio

oral)

**



REINO Clase Familia

Especie




F.C.

lauel GJM 228 (CORD)

Sapium haematospermum Müll.

Arg.

chaxayeec GJM 2 (CORD)

Nat. Latex/ Sin preparación/ Tópico. Las gotas de látex se aplican en forma directa en la muela afectada.


*

Fabaceae Prosopis alba Griseb.

mapik GJM 94 (CORD)

Nat. Corteza/ Incineración (cenizas)/ Tópico


*

Corteza/ Infusión o decocción en agua/ Enjuague bucal


*

Lythraceae Heimia salicifolia (Kunth) Link

piỹaxataxai, covih lahuo GJM 150 (CORD)

Nat. Hojas/ Infusión o decocción en agua / Enjuague bucal


*

Malvaceae Hibiscus striatus Cav.

lalaco`jña GJM 330 (CORD)

Nat. Flores/ Incineración (cenizas) /Tópico


*

Meliaceae Melia azederach L. paraíso GJM 4 (CORD)

Intr. Hojas / Infusión o decocción en agua/ Enjuague bucal. Se prepara un puñado de las hojas

en un jarro o se coloca un fragmento de la hoja en las caries.


**

Oxalidaceae Oxalis conorrhiza Jacq. GJM 164 (CORD)

Nat. Parte aérea/Infusión o decocción en agua/ Enjuague bucal. Se emplea un puñado de plantas en una pava.


*

Phytolaccaceae Petiveria alliacea L. var.

alliacea

shepatoq, shipatoq GJM 187 (CORD)

Nat. Raíz, Semillas / Molido o

picado/ Emplasto. Se aplica un emplasto de la raíz en el interior de la muela afectada. Algunos informantes refieren con el mismo fin el uso de la semilla.

Odontalgias

(Antiodontálgico) **

Planta entera/ Infusión o decocción en agua/ Enjuague bucal


***

Ranunculaceae Clematis montevidensis Spreng.

naqolo GJM 66 (CORD)

Nat. Hojas /Infusión o decocción en agua/ Enjuague bucal


**

Parte aérea/Sin preparación/ Acción simbólica. Se ata la liana en la mano correspondiente al lado de la muela afectada, hasta que sane;

señalan el cuidado de su empleo por un efecto cáustico al contacto con la piel o la boca.

Odontalgias (Uso simbólico)

*****

Parte aérea/Infusión o decocción en agua/ Enjuague bucal


*

Hojas/ Molido o picado/ Emplasto. Se coloca la hoja

molida con un algodón en la cavidad del diente afectado


*



REINO Clase Familia

Especie




F.C.

Rutaceae Citrus limon L. limón

Intr. Frutos / Sin preparación / Gotas


*

Santalaceae Jodina rhombifolia (Hook. & Arn.) Reissek

she' laue, naranja late'e GJM 410 (CORD)

Nat. Hojas / Infusión o decocción en agua / Bebida


*

Solanaceae Jaborosa integrifolia Lam.

tapañi laue GJM 200 (CORD)

Nat. Hojas / Sin preparación/ Emplasto. Se emplean las hojas para "sacar la fiebre" que ocasiona el dolor de muelas.


*

Solanum argentinum Bitter & Lillo

pioq laayec GJM 210 (CORD)

Nat. Raíz y hojas/Infusión o decocción en agua/ Enjuague bucal o bebida (raíz)


****

Raíz / Sin preparación/ Emplasto. Se aplica un emplasto de la raíz en el interior de la muela o se masca un fragmento de la misma.


**

Zygophyllaceae Bulnesia sarmientoi Lorentz ex Griseb.

delliquic GJM 608 (CORD)

Nat. Madera /Infusión o decocción en agua / Bebida

Dolor de garganta (Antibiótico, calmante

bucofaríngeo)

**

Tabla 2.- Farmacopea animal empleada en afecciones bucofaríngeas y dentales. F.C. (Frecuencia de citas: * < 2% informantes; ** 2 - 5 %; *** 5 - 10 %; **** 10 - 20 %; ***** > 20 %)

DIVISIÓN Clase Familia

Especie

Nombre local

Orig. Parte usada / Forma de

preparación / Modo de administración.

Aplicación

(Eficacia atribuida)

F.C.

ANIMALIA (Invertebrados) Bivalvia Unionidae Anodontites trapesialis

coneq Nat. Concha/ Incineración (cenizas) /

Tópico Aftas y llagas bucales (Cicatrizante, antiinflamatorio oral)

****

Gastropoda Ampullariidae

Pomacea sp.

sapo lco’oue’ Nat. Huevos / Tópico Aftas y llagas bucales


**

Insecta Apidae Trigona sp.

coilala Nat. Miel / Ingesta alimenticia Dolor de garganta

(Antibiótico, calmante

bucofaríngeo)

*

Orden Lepidoptera

Indet. (crisálida de lepidóptero)

cochel

- Capullo / Incineración (cenizas)/ Tópico.

Odontalgias (Uso simbólico) *

Mantidae Captoteryx argentina, Stagmatoptera hyaloptera

quedenaxai'chi

Nat. Huevos /Adminículo corporal. Se cuelga la ooteca con huevos de un hilo hasta calmar el dolor, puesto que se considera que éste

insecto come los "gusanos" causantes del dolor de muelas.




DIVISIÓN Clase Familia

Especie

Nombre local



F.C.

Psychidae Oiketicus kirbyi (Guild)

cotaxat Nat. Capullo /Adminículo corporal.

El capullo se lo emplea como colgante del cuerpo en forma de medalla o aro.


Capullo / Incineración (cenizas) / Tópico


*

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

uootel

Nat. Panal o nido /Incineración (cenizas) / Tópico


**

ANIMALIA (Vertebrados) Chondrichthyes Dasyatidae Potamotrygon sp.

lacataic Nat. Las púas de rayas (lacataiq

lsoxanaqte) se emplean para

perforar las muelas doloridas y eliminar el dolor.

Odontalgias

*

Reptilia Alligatoridae Caiman latirostris chacoensis

da’ail’oc

Nat. Acción simbólica: Se muerde un diente de yacaré para desarrollar una dentadura sana y fuerte.

Preventivo (Uso simbólico) **

Teiidae Tupinambis teguixin

(lairaxaic) (naigoxonaxa) qolliguesaq

Nat. Grasa /Sin preparación/ Tópico. Se rellenan las cavidades de los dientes afectados.


*

Grasa/Sin preparación/ Fricciones y masajes.


*

Aves Rheidae Rhea americana

mañic Nat. Grasa/Sin preparación/ Tópico Aftas y llagas bucales


*

Mammalia Canidae Canis familiaris

pioq Se emplean los pelos para tratar

dolores de muelas originados en la transgresión de la veda alimentaria durante el luto.

Odontalgias (Uso simbólico)

*

Felidae Puma concolor

sauaxaic Grasa/ Sin preparación /

Fricciones y masajes Dolor de garganta (Antibiótico, calmante bucofaríngeo)

*

Myrmecopha-gidae

Myrmecophaga tridactyla

potai

Grasa/Sin preparación / Tópico Aftas y llagas bucales (Cicatrizante, antiinflamatorio oral)

*

Pelos / Incineración (cenizas) / Tópico. Se quema el pelo de la cola (potai’ laxarashet) y las cenizas que se obtienen se aplican en la boca de los niños, especialmente cuando no

pueden mamar.


**



Atendiendo al número de especies

involucradas, los usos vegetales más relevantes y que ocupan la mayor atención 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 protección de la dentadura

(6%). Debemos señalar, asimismo que los nativos no

dan cuenta de aplicación 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. Distribución porcentual de formas de aplicación de la farmacopea natural: (E) Usos externos; (I) Usos internos.

Las partes más comúnmente usadas para el

tratamiento de estas dolencias son porciones aéreas,

especialmente las hojas y raíces de los vegetales, y

las grasas y capullos de los animales. Estos se

preparan en su mayoría en forma de infusiones o decocciones en agua (35%), de cenizas obtenidas por

incineración (27%) o bien se aplican en forma directa

sin que medie preparación alguna (30%). Tanto para la farmacopea animal y vegetal predominan las

aplicaciones externas (61%) – especialmente en

forma tópica 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 chaqueño como fuente de recursos terapéuticos para este grupo

humano. El listado de especies con mayor proporción

de citas se encuentra encabezada por plantas nativas, destacándose por su consenso el uso simbólico de la

liana Clematis montevidensis, y el empleo de las

raíces de Solanum argentinum, Cucurbitella asperata

y de la parte aérea 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 antiodontálgicas no han sido

estudiadas desde el punto de vista fitoquímico, existen evidencias de usos similares en otros

contextos culturales próximos y lejanos. El empleo

de esporas de hongos de la familia Lycoperdaceae (Gasteromycetes) como cicatrizantes o

antimicrobianos, por ejemplo, ha sido señalado en

forma recurrente en trabajos etnobotánicos 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

cicatrización de aftas y llagas bucales. Los trabajos de Filipov (1994) y Scarpa (2004), por su parte,

coinciden en el uso de las raíces de Petiveria alliacea

para el tratamiento de las odontalgias entre los pilagá y los criollos de Formosa, respectivamente.

Asimismo, y con idéntica aplicación a la prescripta

por los tobas de esta región, los pilagá emplean el

látex 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

analgésico resulta muy popular en Argentina y otras

regiones de América (Filipov, 1994; Martínez & Planchuelo, 2003; Waizel-Bucay & Martínez Rico,

2007).



Si bien, y tal como vimos anteriormente, en

el listado de aplicaciones medicinales resulta más que probable la existencia de especies con principios

activos de eficacia farmacológica, también es posible

distinguir procedimientos o acciones en los que se

advierte una eficacia de tipo simbólica, sustentada en una lógica implícita de transferencia de propiedades

conocida como circulación de los síntomas, en

términos 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 lógica terapéutica se explica que la

emergencia de los dientes en los niños esté

condicionada por el tratamiento que se haga de su

placenta una vez nacido, ya que cuanto más profunda se entierra ésta, mayor será la demora en la dentición.

De igual manera, y con un claro sentido metafórico,

existen prácticas de prevención de la salud oral, tales como morder la grasa del chancho moro o yolo

(Tayassu tajacu, Suidae), o los dientes de yacaré o

da’ail’oc (Caiman latirostris chacoensis, Alligatoridae) que permiten adquirir una dentadura

fuerte y saludable por transferencia de esta cualidad

animal.

CONCLUSIONES El significativo número 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

más 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 elección de especies destinadas al

tratamiento de estas dolencias, a partir del amplio listado que se presenta en este trabajo, obedece no

sólo a una eventual eficacia farmacológica, sino

también a aspectos simbólicos implicados en la

práctica terapéutica. Esto da cuenta de la vigencia de una etnomedicina holística, con un fuerte

componente naturalista característico de grupos

étnicos que, como los tobas, aún conservan en forma más o menos acentuada, sus prácticas de recolección,

caza y pesca. La dificultad a un acceso apropiado a

los profesionales de la biomedicina, y en particular a los odontólogos, propicia la transmisión y la memoria

colectiva en torno a estos usos, algunos de ellos con

amplio consenso entre los informantes tobas y otros, con un consenso interétnico, al compartir idénticas

aplicaciones con otros grupos del Gran Chaco, como

pilagás y criollos de Formosa, provincia vecina al

área de estudio. La dinámica etnohistórica de contacto con estos grupos, explicaría 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 también un núcleo de especies y usos

que al parecer, competen exclusivamente a la

etnomedicina toba, tal es el caso de los hongos y líquenes, entre otros que ocupan un porcentaje

relevante de la farmacopea natural de este grupo

étnico. 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

peridomésticos, lo que da cuenta de la relevancia del monte nativo como fuente de remedios.

El acceso de la población mundial a las últimas

terapias clínicamente validadas, así como a

innovaciones biomédicas en el tratamiento de la salud buco dental no resulta universal, menos aún para

poblaciones rurales como las que aquí se describen.

Los preparados farmacéuticos basados en ensayos clínicos con validación científica, y destinados a

tratar estas dolencias, no han reemplazado en

absoluto la persistencia en el uso de las medicinas

tradicionales y alternativas para una proporción importante de la población mundial. De esta manera,

resulta prioritario establecer un listado apropiado de

plantas, con su identificación, uso y eventuales dosis estandarizadas que permitan su aplicación en

atención primaria de salud. Aun cuando en su

mayoría 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

antiodontálgicas (como Clematis montevidensis, Solanum argentinum, Cucurbitella asperata, Schinus

fasciculatus var. fasciculatus y Petiveria alliacea var.

alliacea) permite definir un conjunto de especies para esta región y grupo étnico, propiciando de esta

manera la incorporación de la medicinas tradicionales

en el sistema local de salud.



AGRADECIMIENTOS

A la comunidad toba de Río Bermejito (Paraje El Colchón) que me brindaron su valioso

tiempo e información, así 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 investigación y a los especialistas que identificaron (Dra. Laura Domínguez y Cátedra de

Diversidad Vegetal I: Hongos; Dra. Cecilia Strabou y

Biól. Juan M. Rodríguez: Liquenes; Biól. Liliana Buffa y Dr. Claudio Sosa: Insecta; Dra. Alejandra

Ceballos: Invertebrados), corrigieron, confirmaron u

orientaron mis determinaciones. El presente trabajo

se realizó en el marco de los proyectos Anpcyt/ Foncyt Pict 32894 y 1612.

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Aromatic plants from Patagonia: chemical composition and antimicrobial activity of the essential oil of Senecio mustersii and S.subpanduratus

[Plantas aromáticas de la Patagonia: Composición química y actividad antimicrobiana del aceite esencial de Senecio mustersii y S.subpanduratus]

Luz ARANCIBIA 1, Cecilia NASPI 1, Graciela PUCCI 2, María ARCE 3 1Cátedra de Química Orgánica, 2Cátedra de Microbiología ,3Cátedra de Botánica, 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 didn´t 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 investigación fue la determinación de la actividad antimicrobiana de los aceites esenciales de dos especies del género Senecio (Asteraceae) de la región Patagónica: Senecio mustersii y S. subpanduratus. Hasta el momento, no se han reportado estudios sobre la composición del aceite esencial para estas dos especies de Senecio. Los aceites esenciales fueron obtenidos mediante hidrodestilación lográndose 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 evaluó la actividad frente a bacterias y levaduras de importancia clínica: 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 presentó actividad antifúngica, mientras que Senecio subpanduratus actividad contra algunas especies de Candida.

Palabras Clave: Aceites Esenciales; Senecio; Actividad Antifúngica; Actividad Antibacteriana.

List of Abbreviations: HRP (Herbario Regional Patagónico); CG-FID-MS (Gas chromatography-Flame ionization detector-Mass spectrum); ATCC (American type culture collection); NIM (Número Instituto Malbrán);ANLIS (Administración nacional de laboratorios e institutos de salud); MIC (minimal inhibition concentration);

Recibido | Received: December 18, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: February 7, 2010. Publicado en Línea | Published Online March 25, 2010 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | Funding: Universidad Nacional de la Patagonia San Juan Bosco This article must be cited as: Luz Arancibia, Cecilia Naspi, Graciela Pucci, María 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: [email protected] ; [email protected]



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 Río 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 25μm 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 90ºC, and then increased to 225ºC at 3º/min rate (15 min). Samples were diluted (10% v/v in ethanol) and 0.2 μl 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 Malbrán 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 106 cells per ml was mixed with the medium, bacteria and yeast culture were incubated for 24 hours at 37ºC and 48 hours at 28ºC 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

1/125

1/250

1/500

1/1000

1/2000

1/125

1/250

1/500

1/1000

1/2000

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 (Administración Nacional de Laboratorios e Institutos de Salud) “Dr. Carlos Malbrán”, 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 Patagónica, parte 7. Colección Científica 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.

González 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 Patagónica. 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








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Anti-inflammatory Activity of Aristotelia chilensis Mol. (Stuntz) (Elaeocarpaceae).

[Actividad anti-inflamatoria de Aristotelia chilensis Mol. (Stuntz) (Elaeocarpaceae).]

Carlos L CÉSPEDES*1, Julio ALARCON 1, Jose G AVILA2 , Antonio NIETO3 1Plant Biochemistry and Phytochemical Ecology Laboratory, Department of Basic Sciences, University of Bío-Bío, Chillán, Chile. 2Phytochemical Laboratory, UBIPRO, FES-Iztacala, UNAM, 3Laboratorio de Pruebas Biológicas, Instituto de Química, 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 μg/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 comúnmente 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 aquí por primera vez. Materiales y métodos: Los extractos, fracciones y subfracciones fueron analizados por su contenido total

de fenoles y se investigo el efecto sobre la inflamación en oreja de rata a través de la inducción con TPA en dosis sencillas. Además se investigo la actividad

antioxidante frente a DPPH, Crocina y TBARS. Resultados: Los resultados muestran que el extracto B, la fracción 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 fenólicos. 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 inflamación inducida en el modelo del edema

en oreja de rata, con valores de EC50 entre 0.3 a 11.8 μg/mL. Discusión y conclusión: Estos hallazgos demuestran que los frutos y sus constituyentes de A.

chilensis poseen una excelente actividad anti-inflamatoria, y así tienen un gran potencial como una fuente de productos naturales saludables. Adicionalmente,

estos hallazgos muestran que los flavonoides, ácidos fenólicos y antocianinas presentes en este fruto podrían 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 Versión Corregida | Accepted in Corrected Version: January 4, 2010

Publicado en Línea | Published Online March 25, 2010 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | 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. Céspedes, 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: [email protected], [email protected]; Phone: +56-42-253049, Fax: +-56-42-253046



Céspedes 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 (O2˙ˉ),

peroxyl (ROO), alkoxyl (RO), hydroxyl (OH˙ˉ), 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 (Muñoz-

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/H2O extracts from ripe fruits and subfractions from SF4 to SF37 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 Botánica, Facultad de Ciencias Naturales y

Oceanograficas, Universidad de Concepción,

Concepción, 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), FeSO4, 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 Química, 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 Química, 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, CH2Cl2, CHCl3, NaCl, KCl,

KH2PO4, NaHPO4, NaOH, KOH, HCl, sodium acetate trihydrate, glacial acetic acid silica gel

GF254 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 µL of each sample in amber vials and

injected 20 µL 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 EC50 (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 α-tocopherol were used as

standards (Dominguez, et al., 2005).

Bleaching of crocin. The solutions were placed under UV254 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 Dunnett’s 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 modela. EI=Edema Inhibition (%).

Samplesf / Dose EI50 (mg/ear)b

Indo-methacin 0.11 c Af 3.6 c Bf 1.8 c Cf 5.9 d Df 6.7 d

F-1 10.9 d

F-2 11.8 d

F-3 1.1 c

F-4 1.9 c

SF4-SF6 6.7 d

SF7 10.1 d

SF8-SF10 11.0 d

SF11-SF15 0.3 c

SF16-SF20 0.9 c

SF21-SF25 1.0 c

SF26-SF30 3.8 c

SF31-SF37 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.

Sampleb DPPHc TBARSd Crocine

SF4-SF6 23.8 7.9 13.4

SF7 37.4 11.8 18.9

SF8-SF10 47.6 9.9 12.0

SF11-SF15 1.3 (2.2)f 1.9 0.7

SF16-SF20 2.3 (3.8)f 2.1 0.8

SF21-SF25 5.2 (7.8)f 2.3 1.1

SF26-SF30 17.1 3.9 0.9

SF31-SF31 19.7 10.1 2.4

Myricetin 21.0 9.7 22.9

Quercetin 19.98 2.90 21.0

α-Tocopherol 11.9 3.92 10.1

aValues expressed as g/mL (ppm), Mean Confidence

Interval 95%, n = 3. Different letters show significant differences at (P < 0.05), using Duncan’s multiple-range test. b See scheme 1 for an explanation of extracts and partitions. c IC50 for inhibition of DPPH radical formation. d IC50 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 Duncan’s multiple-range test. e IC50 for bleaching of crocin. fThe values between parenthesis correspond to the assay made with 50 μM 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-H2O. Furthermore F4 to F30 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 EC50 values for each activity were calculated

by Probit analysis on the basis of the percentage of

inhibition obtained at each concentration of the

samples. EC50 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 SF4 to SF37 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, SF11-SF15, SF16-SF20, SF21-SF25 and SF26–

SF30 with EI50 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 EI50 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 SF11-SF15, SF16-SF20, and SF21-SF25, ovatifolin, quercetin, myricetin, luteolin and diosmetin

the most active samples, therefore with these samples

was made a curve of dose-response, obtaining the EI50

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 SF11-SF15, SF16-SF20 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,

SF7, SF8-SF10, SF26-SF30, and SF31-SF37, 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 SF11-SF15, SF16-SF20, and SF21-SF25 had

the highest inhibitory activity against DPPH radical formation compared to the other partitions with IC50

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 SF11-SF15, and SF16-SF20 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 IC50 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

SF11-SF15, SF16-SF20, and SF21-SF25 showed considerable activity, quenching DPPH radical

reduction completely (100 % of inhibition, data not

show). Nevertheless, SF4-SF6, SF26-SF30 and SF31-SF37 showed a moderate activity their IC50 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 SF16-SF20, and SF21-SF25.

The lowest I50 value for SF11-SF15, SF16-SF20, (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 SF11-SF15, SF16-SF20 and SF21-SF25 were most effective in similar form to

quercetin or BHT in inhibiting lipid peroxidation.

SF11-SF15 had the greatest activity and reduced lipid

peroxidation in a dose-dependent manner, and proved to be an excellent antioxidant, reflected by its low IC50

value (1.9 ppm) when analyzed by both TBARS and

DPPH (Table 2), at the same level than quercetin and α-tocopherol whom shows IC50 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 IC50 values of all subfractions are shown in

Table 2. SF11-SF15 and SF16-SF20 were the most active, with IC50 values of 1.9 and 2.1 ppm, respectively. It is

noteworthy that the value for SF11-SF15 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.

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© 2010 The Authors




In vitro antioomycete activity of Artemisia ludoviciana extracts against Phytophthora spp.

[Actividad antioomiceto in vitro de extractos de Artemisia ludoviciana contra Phytophthora spp]

Luz María DAMIAN BADILLO, Rosa Elisa MARTINEZ MUÑOZ, Rafael SALGADO GARCIGLIA, Mauro Manuel MARTINEZ PACHECO

Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo; Ed. B-3; Cd. Universitaria; Francisco J. Mujica s/n; Col. Felicitas del Rio; Morelia, Mich. México. 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 µg·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 común), 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 aérea de la planta. De estos extractos se obtuvo una fracción por

TLC (Rf = 0.72) que contuvo aceites esenciales capaces de inhibir el crecimiento de los oomicetos con una concentración mínima inhibitoria (MIC) en el

intervalo de 0.2 a 0.4 mg·ml-1

. Los compuestos mayoritarios en la fracción microbicida fueron borneol (16.28 %), camfor (7.41 %) y cis-verbenol (1.69 %).

Se observó que únicamente una mezcla de ellos inhibió el crecimiento de las cinco especies de Phytophthora con un efecto similar al del extracto crudo y al

de la fracción activa.

Palabras Clave: antioomiceto; Phytophthora; Borneol; Camfor; cis-Verbenol.

Recibido | Received: 19 September, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: January 15, 2010.

Publicado en Línea | Published Online 25 March, 2010 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | 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, Martínez Muñoz RE, Salgado Garciglia R, Martínez 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: [email protected]





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Damián 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, β-

selinene, β-cariofillene, artemisia cetone, z-epoxyocimene, crisantenyl acetate, z-epoxyocimene

and β-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 (Damián-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 CH3Cl3: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 ºC, it was filtered, the solvent

removed and 1 g was dissolved in 1 mL of ethanol and maintained at 4 ºC 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 CH3Cl3: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 Rf 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 µl (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 μg·ml

-1), camphor

(28 μg·ml-1) and cis-verbenol (6.5 μg·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 µl

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 Rf 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 Rf MIC (mg·ml-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.

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© 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 hidroetanólico de Lindackeria paludosa,

un arbusto sudamericano.]

Ana Laura FAZIO1, Diana BALLÉN2, Italo M. CESARI2, María Jesús ABAD1, Miriam ARSENAK1, Omar ESTRADA3, Peter TAYLOR1.

1 Centro de Medicina Experimental, 2 Centro de Microbiología y Biología Celular, 3 Centro de Biofisica y Bioquimica, Instituto

Venezolano de Investigaciones Científicas, 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 μg/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 κB

(NF-κB) 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 Sudamérica y se conoce como Sarakura en Venezuela.

En vista de la información local sobre el uso en la medicina tradicional de este miembro de la familia Flacourtiaceae, investigamos el efecto de un extracto

hidroetanólico de la corteza (LP) sobre algunos parámetros de la respuesta inflamatoria (factor de necrosis tumoral alfa, interleuquina-6 y óxido nítrico) y su

potencial actividad antitumoral tanto in vitro como in vivo. Se notó un efecto citotóxico solamente en una línea celular tumoral (A549, IC50 = 64 μg/ml), pero

no en los otros tipos de células. Sin embargo, LP inhibió la producción de los mediadores inflamatorios, el crecimiento de tumores primarios y metástasis en

ratones C57BL/6, pero no inhibió la actividad del factor nuclear κB. LP parece inhibir el crecimiento tumoral sin ejercer un efecto citotóxico directo,

posiblemente a través de la inhibición de procesos inflamatorios protumorales.

Palabras Clave: Lindackeria Paludosa; cáncer; metástasis; inflamación; ratón.

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-κB – nuclear factor κB

Recibido | Received: 16 June, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: 22 March, 2010.

Publicado en Línea | Published Online: 25 March, 2010 Declaración de intereses | Declaration of interests: The authors have no competing interests. Financiación | Funding: This work was financed by IVIC

This article must be cited as: Fazio A L, Ballén 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: [email protected] - Tel.: +58 212 504 1097 - Fax: +58 212 504 1086.





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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 (Fitogenéticos). 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 Río Negro, Amazonas State, Venezuela and

identified by Dr. Otto Huber (Universidad Central de Venezuela) and Dr. Ernesto Medina (Instituto

Venezolano de Investigaciones Científicas). 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 Dulbecco’s

Modified Eagle’s Medium (DMEM) supplemented

with 10% heat-inactivated foetal bovine serum (FBS - Gibco, BRL, USA), penicillin (100 Units/ml),

streptomycin (100 μg/ml) and containing in addition

glucose 0.45% (HT29 cells), and L- glutamine 2 mM

(A549 cells). 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.



Female C57BL/6 mice (7–9 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 µg/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 manufacturer’s instructions. The IC50 (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

µg/ml lipopolysaccharide (LPS - E. coli serotype

055:B5, Sigma, USA) for 24 h in the presence of LP,

and then the concentrations of TNF-α, IL-6 and nitric

oxide (NO) were measured in the supernatants. TNF-α was quantified using the WEHI 164 cell bioassay

(Espevik and Nissen-Meyer, 1986), IL-6 with a

commercial ELISA assay (R & D Systems Inc., MN, USA) and NO using the Griess reaction (Sandoval-

Chacón et al., 1998).

Inflammatory response in vivo.

Mice were injected intraperitoneally (i.p.) with

different doses of LPS in 100 μl of PBS. After 1 h, blood was collected by heart puncture under ether

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 μg 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 105

B16/BL6 cells in 100 µl PBS. Two treatment protocols with LP were performed a)

intraperitoneal (i.p.) injection of 50 μg of LP in 100 µl

PBS / 25% ethanol on days -2, -1 and 0, and b) i.p. injection of the same dose of extract 5 times per week

starting from on day 0 up to day 21. Control animals

received 100 μl PBS / 25% ethanol. On day 23, the animals were sacrificed with ether; the lungs were

removed, placed for 5 min in 3% H2O2 in H2O and

fixed in Bouin's solution. The purpose of the H2O2 was

twofold: to bleach hemorrhages which could be mistaken for metastases, and to inflate the lungs,

facilitating the evaluation of metastases under the

dissecting microscope. Animals were challenged with LPS prior to sacrifice, in order to measure serum TNF-

α and IL-6 levels as described above.

Primary tumours. Primary tumours were induced by the subcutaneous

(s.c.) injection of 5 x 104 B16/BL6 cells in 100 µl PBS

into the hind limb. The mice were injected i.p. with 50

μg of 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-

α 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 Student’s 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 IC50 was above the maximum concentration tested (300 μg/ml).



Table 1. Inhibitory concentration (IC50) of LP on cell lines in vitro.

Cells Origin IC50 (μg/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 (IC50).

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 μg/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 µg/ml LPS for 24 h in the presence of 100 μg/ml LP. TNF- α, 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 μg 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.



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

Animals were pretreated with 50 μg/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- α, 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 μg LP. B. Mice were inoculated i.v. with B16/BL6 cells. Treatment with LP consisted of 50 μg 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 μg 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

μg / animal) in these animals, prior to sacrifice. Figure



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 μg LP (corresponding to Fig 4A and 4C). One h before sacrifice, the animals were challenged with 3 μg LPS. Blood was extracted and

the serum assayed for TNF- α and IL-6. (mean S.E.M., n = 10).

** P < 0.01, *** P < 0.001,

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

The effect of LP on the NF-κB response of HeLa

cells to TNF-α was determined in a luciferase reporter assay. LP showed no inhibitory effect on NF-κB under

a variety of different conditions (TNF- α

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 mouse’s metabolism.

However, our findings on the inhibition of TNF- α, 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-α with a TNF receptor construct

decreased lung metastases in tumour-inoculated mice

(Cubillos et al., 1997). NF-κB, a common factor in tumour growth and

inflammatory processes, has been proposed as a



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-κB 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 Río

Negro, Amazonas State, Venezuela. We are grateful to

Drs M Rieber and J Cardier for the B16/BL6 and LSEC cells respectively.

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inflammatory pathways for prevention and therapy of

cancer: Short-term friend, long-term foe. Clin Cancer Res 15(2): 425-430.

Bilia A, Méndez J, Morelli I. 1996. Phytochemical

investigations of Licania genus. Flavonoids and

triterpenoids from Licania carii. Pharm Acta Helvet 71,

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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.

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Coussens LM, Werb Z. 2002. Inflammation and cancer.

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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.

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tomentosa reduces inflammation and B16-BL6

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la FAO sobre los Recursos Filogenéticos.

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Publicada por | Published by: Cooperación Latinoamericana y Caribeña en Plantas Medicinales y Aromáticas

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Descripción

BLACPMA es una revista cientifica dedicada a las plantas medicinales, aromáticas, y económicas y a los productos naturales bioactivos.

Publica contribuciones originales en ocho áreas importantes:

1. Caracterización de los ingredientes activos de las plantas medicinales

2. Desarrollo de métodos para la estandarización para los extractos bioactivos y los productos naturales de la planta.

3. Identificación de la bioactividad de productos naturales vegetales.

4. Identificación de blancos y mecanismo de la actividad de productos naturales.

5. Producción y caracterización genómica de la biomasa de especies medicinales.

6. Química y bioquímica de productos naturales bioactivos.

7. Revisiones críticas de la personalidad histórica, clínica y jurídica de plantas medicinales.

8. Aspectos agrícolas de plantas medicinales y aromáticas.

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Farmacólogos, Etnofarmacologos, farmacéuticos, fitoquímicos, químicos orgánicos, toxicólogos, botánicos y antropólogos, entre otros.

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