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Estudio de la eficacia antibitica de un extracto

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BARNI, Mara V.;FONTANALS, Adriana;MORENO, Silvia

Estudio de la eficacia antibitica de un extracto etanlico de Rosmarinus officinalis L.
contra Staphylococcus aureus en dos modelos de infeccin en piel de ratn
Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, Vol. 8,
Nm. 3, mayo, 2009, pp. 219-233
Sociedad Latinoamericana de Fitoqumica
Chile

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2009 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 8 (3), 219 - 223
BLACPMA ISSN 0717 7917

Artculo Original | Original Article

Estudio de la eficacia antibitica de un extracto etanlico de Rosmarinus

officinalis L. contra Staphylococcus aureus en dos modelos de infeccin
en piel de ratn
[Study of the antibiotic efficacy of an ethanolic extract from Rosmarinus officinalis against Staphylococcus aureus
in two skin infection models in mice]
Mara V. BARNI, Adriana FONTANALS y Silvia MORENO*

Fundacin Instituto Leloir I.I.B.B.A-CONICET, Patricias Argentinas 435, C1405FFX, Ciudad de Buenos Aires, Argentina.

Abstract

In this paper we examined the antibacterial efficacy of an ethanolic extract of Rosmarinus officinalis L. containing a high amount of antioxidant
polyphenols against the pathogenic bacteria Staphylococcus aureus in two skin infection models in mice, superficial and subcutaneous. Results obtained
showed that the rosemary extract containing 2.3% of polyphenols had bacteriostatic activity against S. aureus on the skin infection. Using a double
concentration of bioactive polyphenols in the treatment (4.6% of polyphenols), a total bacterial growth inhibition on the skin was suggested since we did not
recover any bacteria viable in the skin mouse. A similar effect was observed in two skin models. Our results also showed that the antibacterial efficacy of the
bioactives of rosemary is comparable with the antibiotic action of the well recognized antibiotic fusidic acid. All findings indicated that bioactive polyphenols
of rosemary are able to carry out bactericide as well as bacteriostatic action against S. aureus in the skin mouse.

Keywords: Rosmarinus officinalis L;, Antimicrobial activity; Superficial skin infection model in mouse; Subcutaneous skin infection model in mouse.

Resumen

En este trabajo evaluamos la eficiencia antibacteriana de un extracto etanlico de Rosmarinus officinalis L. que contiene altas concentraciones de
polifenoles antioxidantes, en dos modelos de infeccin en piel de ratn: superficial y subcutneo contra la bacteria patgena Staphylococcus aureus. Los
resultados obtenidos muestran que el extracto de romero que contiene 2,3% de polifenoles bioactivos present una accin bacteriosttica contra S. aureus
sobre la piel del ratn, mientras que ensayando una doble concentracin de polifenoles bioactivos (4,6%) se observ una inhibicin total del crecimiento
bacteriano. En los dos modelos de infeccin experimentados se observaron efectos similares. Los datos obtenidos tambin demuestran que la eficacia
antibacteriana del extracto de romero es comparable con la accin del antibitico comercial cido fusdico. Los resultados indican que los polifenoles
bioactivos del romero, dependiendo de su concentracin, pueden ejercer in vivo acciones bacteriostticas y bactericidas contra S. aureus.

Palabras Clave: Rosmarinus officinalis L.; Actividad antimicrobiana, Modelo de infeccin superficial en piel de ratn, Modelo de infeccin subcutneo en
piel de ratn.

Recibido | Received: December 30, 2008.

Aceptado en Versin Corregida | Accepted in Corrected Version: May 18, 2009.
Publicado en Lnea | Published Online: May 29, 2009
Declaracin de Intereses | Declaration of interests: authors have no competing interests.
Financiacin | Funding: This work was supported by Consejo Nacional de Investigaciones Cientficas y Tcnicas (CONICET) and ANPCyT, proyecto PICT2005-35401
This article must be cited as: Mara V. Barni, Adriana Fontanals y Silvia Moreno. 2009. Estudio de la eficacia antibitica de un extracto etanlico de Rosmarinus officinalis L.
contra Staphylococcus aureus en dos modelos de infeccin en piel de ratn. Bol Latinoam Caribe Plant Med Aromat 8(3):219 223. {EPub May 29, 2009}.

*Contactos | Contacts: E-mail: smoreno@leloir.org.ar

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

This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. (http://creativecommons.org/licenses/by-nc-nd/3.0/ )
which permits to copy, distribute and transmit the work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work.
Any of these conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights.

Este es un articulo de Acceso Libre bajo los terminos de una licencia Creative Commons Atribucion-No Comercial-No trabajos derivados 3.0 Internacional (http://creativecommons.org/licenses/by-nc-
nd/3.0/deed.es) Usted es libre de copiar, distribuir y comunicar pblicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los crditos de la obra de la manera especificada por el autor
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alterar, transformar o generar una obra derivada a partir de esta obra. Al reutilizar o distribuir la obra, tiene que dejar bien claro los trminos de la licencia de esta obra. Alguna de estas condiciones puede no
aplicarse si se obtiene el permiso del titular de los derechos de autor Nada en esta licencia menoscaba o restringe los derechos morales del autor.
Barni et al. Eficacia antibitica de Rosmarinus officinalis L. contra Staphylococcus aureus en ratones
INTRODUCCIN
Las enfermedades infecciosas representan en la
actualidad una importante causa de mortalidad en
humanos, especialmente en los pases en desarrollo.
Actualmente la emergencia de resistencia a
antibiticos de uso comn en la clnica, se ha
incrementado en algunas bacterias patgenas
(Cabrera Cao et al., 2005) y es un problema particular
en pacientes infectados con Staphylococcus aureus
debido al aumento de variantes resistentes a -
lacatamasas, aminoglicsidos (Styers et al., 2006).
Esta bacteria puede ingresar al cuerpo a travs de
heridas, quemaduras, lceras o puede ser trasportada
a travs de sondas o cnulas en los pacientes
hospitalarios, pudiendo llegar al torrente sanguneo y
as diseminarse en el organismo, comprometiendo el
corazn, la sangre o los huesos (Naimi et al., 2003).
Adems, recientemente se encontraron bacterias
resistentes en mbitos hospitalarios que fueron
transmitidas en la comunidad (Kollef et al., 2006).
En consecuencia, dichos sucesos impulsan la
bsqueda de nuevas drogas para controlar las
bacterias resistentes. La obtencin de nuevos agentes
antimicrobianos de origen vegetal es una estrategia
competente en la farmacologa actual (Cowan et al.,
1999). Se sabe que las plantas bajo condiciones de
estrs bitico y abitico sintetizan metabolitos
secundarios que incluyen flavonas, taninos,
antocianinas y terpenos (mono-di y sesqui terpenos)
para adaptarse al medio que las rodea (Demming-
Adams et al., 2002, Kliebenstein, 2004). Algunos de
estos compuestos presentan actividad antimicrobiana
(Lewis et al., 2006).
Las plantas pertenecientes a la familia Lamiaceae
crecen en zonas ridas y de alta irradiacin UV, por
lo tanto acumulan una gran cantidad de metabolitos
secundarios, en particular el gnero Rosmarinus
officinalis L. es una fuente importante de compuestos
antioxidantes de naturaleza polifenlica que presenta
propiedades biolgicas como antibacteriano, anti-
inflamatorio y antitumoral. Previamente reportamos
que la eficacia antimicrobiana in vitro de diferentes
extractos de romero estandarizados en el contenido
de cido rosmarnico, cido carnsico y carnosol
dependa de la composicin de sus polifenoles
(Moreno et al., 2006). El objetivo de este trabajo es
estudiar la eficacia antibacteriana in vivo de un
extracto etanlico de R. officinalis contra la bacteria
patgena Staphylococcus aureus, en dos modelos de
infeccin en piel de ratn comparando su potencia
www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (3) 2009 | 220
antimicrobiana con el antibitico comercial cido
fusdico.
MATERIALES Y MTODOS
Material vegetal
El material vegetal fue cosechado en marzo del
2008 en el Valle de Lerma (Salta, Argentina). El
extracto de R. officinalis L. fue obtenido a partir de
hojas secas por extraccin etanlica y se determin
por HPLC la composicin de polifenoles (cido
carnsico, carnosol y cido rosmarnico) como se
describi previamente (Moreno et al., 2006). El
extracto de romero seco se almacen a -20 C.
Preparacin de inculo bacteriano
Se prepar un cultivo bacteriano de
Staphylococcus aureus ATCC 25923 en caldo
Mueller Hinton (DIFCO) que se creci durante 20
horas a 37 C con una agitacin de 250 rpm, luego se
midi la concentracin bacteriana por densidad ptica
a 625 nm y se diluy a una concentracin final de 0,5
Mc Fardland correspondiente a 1 x 108 UFC
(unidades formadoras de colonias)/mL.
Animales y modelos de infeccin en piel de ratn
Se utilizaron ratones hembras BALB/cAnNCrl de
2 meses de edad y peso variable (19-21 g), criados en
la Fundacin Instituto Leloir. El protocolo de trabajo
fue aprobado por el bioterio del Instituto Leloir, y
cumple con el reglamento interno vigente y con la
legislacin nacional (Disposicin 6344/96 de la
Administracin Nacional de Medicamentos,
Alimentos y Tecnologa Mdica y Resolucin
617/2002 del Servicio Nacional de Sanidad y Calidad
Agroalimentaria). Responsable a cargo del bioterio
Adriana Fontanals.
Los ratones fueron anestesiados va intra-
peritoneal con una dosis de 100 mg/kg de ketamina
(Ketonal, Richmond) y 5 mg/kg de clorhidrato de
xilacina (Xilacina 20, Richmond). Luego se procedi
a rasurar con gillette la parte dorsal izquierda del
animal para eliminar el pelaje.
Se establecieron dos modelos de infeccin en piel
de ratn:
Modelo de infeccin superficial. Se utiliz el
modelo de infeccin superficial previamente
descripto por Kugelberg et al. (2005), con algunas
modificaciones. Se removieron las primeras capas
drmicas del rea de la piel de ratn rasurada, con
tres pasajes de hoja de afeitar de acero inoxidable
Barni et al. Eficacia antibitica de Rosmarinus officinalis L. contra Staphylococcus aureus en ratones
para facilitar la infeccin bacteriana. Bajo campana
de flujo laminar se colocaron 5 L del inculo
bacteriano (5 x 105 bacterias) sobre una superficie de
piel de 2 cm2. Pasados 5 min, para dar lugar a la
absorcin de las bacterias en la piel del ratn, se
coloc un apsito estril sobre el rea inoculada.
Modelo de infeccin subcutnea: Como describi
Gisby et al. (2000), se estableci el nmero de
bacterias adsorbidas en 1 cm de hilo de ciruga en
ensayos in vitro (7,5 x 104 bacterias). Para llevar a
cabo la infeccin subcutnea se colocaron hilos de
ciruga de 10 cm en tubos con 5 mL del inculo
bacteriano (1 x 108 UFC/mL) y se dejaron incubar a
temperatura ambiente durante 30 min. Luego cada
hilo inoculado se enhebr en una aguja de ciruga.
Esta se introdujo en la parte rasurada del ratn de
modo de atravesar la dermis sin llegar al msculo
carnoso y dejando 1 cm de hilo bajo la piel. Luego se
anudaron y se cortaron los extremos del hilo. Para
permitir la penetracin de las cremas de tratamiento
se realiz un incisin con bistur por debajo de la
sutura en la misma direccin que el hilo (Gisby et al.,
2000). Posteriormente se coloc un apsito estril
sobre la herida.
Tratamientos
El extracto seco de R. officinalis L., empleado
para la preparacin de los tratamientos, contiene un
porcentaje total de compuestos fenlicos de 23%, que
comprende un 20% de diterpenos fenlicos (cido
carnsico 10% y su derivado carnosol 10%) y un 3%
de cido rosmarnico, determinados por HPLC.
Se realizaron dos preparaciones del extracto de
romero que contienen diferentes concentraciones de
compuestos polifenlicos bioactivos: 2,3% (p/p) y
4,6% (p/p), respectivamente. Para ello, se emulsion
de manera homognea el extracto de R. officinalis L.
con crema base hidrosoluble comercial.
El placebo consisti en la aplicacin de la crema
base hidrosoluble sin ningn agregado, y se consider
como control negativo. Como control positivo se
utiliz el antibitico tpico comercial, cido fusdico
(FUSIMED, Laboratorio Cassar) al 0,5; 1 y 2%.
Las dosis aplicadas fueron de 20 mg/2 cm2 de piel.
Ensayos y diseo experimental
Para el modelo de infeccin subcutneo se
realizaron 2 ensayos que difieren en el diseo
temporal y cantidad de dosis aplicadas. Uno consisti
en la aplicacin de 2 dosis a las 4 y 6 h post-
inoculacin y la recuperacin de bacterias de la piel a
www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (3) 2009 | 221
las 8 h post-inoculacin (Fig. 1A). En el otro ensayo
se aplic una nica dosis de tratamiento a las 4 h
post-inoculacin y la recuperacin de bacterias se
realiz a las 6 h post-inoculacin (Fig. 1B).
Para el modelo de infeccin superficial se realiz
el ensayo que consisti en la aplicacin de 2 dosis a
las 4 y 6 h post-inoculacin y la recuperacin de
bacterias se realiz a las 8 h post-inoculacin (Fig.
1A).
La primera dosis de los tratamientos se aplic a
las 4 h post-inoculacin como se describi previa-
mente (Gisby et al., 2000) dado que la colonizacin
bacteriana se establece dentro de dicho perodo.
Figura 1. Diseo experimental. A - Modelo de infeccin
subcutneo y superficial (2 dosis). B - Modelo de infeccin
subcutneo (1 dosis).
Por lo tanto, para evaluar la accin antibitica del
extracto de romero se realizaron 3 tipos de ensayos
diferentes en total, considerando los dos modelos de
infeccin, variable tiempo y dosis aplicadas. En cada
ensayo se incluyeron los controles: placebo, sin
tratamiento y el control positivo (cido fusdico a tres
concentraciones).
Recuperacin de bacterias de la piel de ratn
La determinacin de la concentracin bacteriana
post-tratamiento en ambos modelos se realiz segn
Gisby et al. (2000). Se retir con bistur el rea de 2
cm2 de la piel tratada, se coloc en un tubo con 1 mL
de caldo Mueller Hinton donde se homogeneiz con
un grinder. Luego se realizaron diluciones del
homogenato en solucin fisiolgica y se sembraron
alcuotas de 100 L en placas de Petri con agar
Mueller Hinton. Las placas se incubaron a 37 C
durante 24 h y posteriormente se determinaron las
UFC/mL.
Se realiz un control de inculo recuperando las
bacterias de la piel del ratn sin tratamientos previos
para confirmar su viabilidad en la piel.
Barni et al. Eficacia antibitica de Rosmarinus officinalis L. contra Staphylococcus aureus en ratones

Anlisis estadstico
Se utiliz el programa Info Stat para el anlisis
estadstico. El test t de Student ( = 0,001) se aplic
a muestras independientes para cada grupo de
tratamiento (n = 6).
El promedio del nmero de bacterias (UFC)
recuperadas de la piel de ratn (n = 6) en cada
tratamiento con su respectiva barra de error se
transform a log10 UFC/herida.
RESULTADOS Y DISCUSIN
Se utilizaron dos modelos de infeccin en piel de
ratn para determinar la actividad antibitica del
extracto de R. officinalis. El modelo de infeccin
subcutneo fue ensayado para estudiar el efecto del
extracto de romero sobre infecciones que se producen
a niveles intradrmicos, como por ejemplo las
infecciones producidas en heridas quirrgicas. Por el
contrario, el modelo superficial fue diseado para
estudiar infecciones provocadas en zonas drmicas
expuestas al ambiente.
En el modelo de infeccin superficial los
promedios de concentracin bacteriana recuperados
de la piel de ratn para cada tratamiento expresados
en UFC/herida fueron: placebo (1,55 x 104 5,47 x
102), no tratado (1,25 x 104 8,99 x 102), extracto de
R. officinalis 2,3% (2,02 x 101 4,12), extracto de R.
officinalis 4,6% (0), cido fusdico 0,5% (1,27 x 101
1,63), cido fusdico 1,0% (0), cido fusdico 2,0%
(0). Mientras que para el modelo de infeccin
subcutneo con el diseo experimental (Fig. 1A), los
promedios para cada tratamiento fueron: placebo
(1,95 x 104 5,47 x 102), no tratado (1,72 x 104
5,61 102), extracto de R. officinalis 2,3% (2,57 x 102
22,03), extracto de R. officinalis 4,6% (0), cido
fusdico 0,5% (1,78x 101 3.33), cido fusdico 1,0%
(0), cido fusdico 2,0% (0).
En el tercer ensayo, modelo de infeccin
subcutneo con el diseo experimental (Fig. 1B) los
resultados fueron: placebo (1,02 x 104 5,63 x 102),
no tratado (1,10 x 104 1,74 103), extracto de R.
officinalis 2,3% (1,21 x 102 30,09), extracto de R.
officinalis 4,6% (0), cido fusdico 0,5% (1,27 x 102
27), cido fusdico 1,0% (4,40 x 101 3,52), cido
fusdico 2,0% (0).
Para los 3 ensayos se determin que los resultados
individuales de los tratamientos: placebo y no tratado,
no fueron significativamente diferentes ( = 0,001).
En cada ensayo se compararon los resultados
individuales de los tratamientos: extracto de R.
officinalis 2,3% y cido fusdico 0,5% y se obtuvo
que no fueron significativamente diferentes ( =
0,001) para el modelo de infeccin superficial y para
el subcutneo con una dosis, mientras que para el
modelo subcutneo con dos dosis si fueron
significativamente diferentes (P<0,001).

Tabla 1. Actividad antibitica in vivo del extracto de R. officinalis.

log10 UFC/herida

Tratamiento Modelo de infeccin Modelo de infeccin Modelo de infeccin

superficial subcutneo subcutneo
(2 dosis)* (2 dosis)* (1 dosis)**

Extracto de R. officinalis (%)

2,3 1,30 0,24 2,41 0,27 2,07 0,08
4,6 - - -

cido fusdico (%)

0,5 1,11 0,04 1,24 0,16 2,10 0,07
1 - - 1,64 0,03
2 - - -

Placebo 4,19 0,38 4,29 0,30 4,01 0,02

No Tratado 4,10 0,35 4,23 0,24 4,00 0,02
* Se realiz segn diseo experimental A. ** Se realiz segn diseo experimental B.
( - ) Ausencia de crecimiento bacteriano.
Los tratamientos fueron realizados con al menos n = 6 animales. cido fusdico, control positivo.

www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (3) 2009 | 222
Barni et al. Eficacia antibitica de Rosmarinus officinalis L. contra Staphylococcus aureus en ratones
Se determin que los resultados de los tratamientos
con extracto de R. officinalis 2,3% y 4,6%, lograron
ser significativamente diferentes (P<0,001) a los
obtenidos con el tratamiento placebo para los tres
ensayos realizados.
El extracto de R. officinalis 2,3% logra reducir
entre 2-3 log10 UFC/herida segn el modelo de
infeccin y diseo experimental utilizado, logrando
una accin bacteriosttica sobre S. aureus (Tabla 1).
Mientras que el tratamiento con extracto 4,6% logra
inhibir el crecimiento bacteriano en su totalidad
ejerciendo un efecto bactericida en los tres ensayos
evaluados. Es importante destacar que el mismo
efecto se logra por la aplicacin de una nica dosis en
el modelo subcutneo.
Estos resultados demuestran que el extracto vegetal
presenta una accin antibitica eficaz in vivo contra
la infeccin de la bacteria patgena S. aureus.
CONCLUSIN
Los resultados evidenciaron la accin
bacteriosttica del extracto de R. officinalis que
contiene un 2,3% de polifenoles activos y la accin
bactericida del extracto que contiene un 4,6% de
polifenoles activos contra la bacteria patgena S.
aureus en los dos modelos de infeccin en piel de
ratn. Adems se estableci que el extracto de
romero tuvo una potencia antibitica in vivo
comparable a la del cido fusdico. Los sistemas
ensayados, de carcter preclnico, aportan
informacin cientfica y son tiles para la evaluacin
de los compuestos vegetales como potenciales drogas
teraputicas que podran ser competentes con los
antibiticos de uso comn que actualmente ya no son
efectivos contra las bacterias resistentes. Por dicha
razn es pertinente la evaluacin que hoy en da se
lleva a cabo en nuestro laboratorio, sobre los
mecanismos de accin de los compuestos activos que
componen el extracto de R. officinalis L. y el estudio
de la accin antibitica de los bioactivos combinados
con otras drogas, para aumentar su eficiencia
antimicrobiana sobre un amplio espectro bacteriano.
www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (3) 2009 | 223
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 2009 The Authors
2009 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 8 (3), 224 - 233
BLACPMA ISSN 0717 7917

Artculo Original | Original Article

The effect of Minthostachys verticillata essential oil on the immune

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[Efecto del aceite esencial de Minthostachys verticillata sobre la respuesta inmune de pacientes alrgicos a caros
del polvo]
Laura CARIDDI1,*, Marina MOSER1, Melisa ANDRADA1, Mirta DEMO1, Julio ZYGADLO2, Liliana SABINI1, Ana
MALDONADO1

1
Department of Microbiology and Immunology, Universidad Nacional de Ro Cuarto, Ro Cuarto, Crdoba, Argentina
2
Faculty of Exact, Physical and Natural Sciences, Universidad Nacional de Crdoba, Crdoba, Argentina.

Abstract

The essential oil from leaves of Minthostachys verticillata was obtained by steam distillation and analyzed by gas chromatography. Peripheral blood
mononuclear cells from allergic patients were stimulated with essential oil. By application of MTT or 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium
bromide colorimetric test the proliferation was assayed. In lymphocyte cultures CD4+, CD8+ and B cells were quantified, and in supernatants, IFN- and IL-
13. The liberation of -hexosaminidase enzyme was determined for basophils with essential oil added and challenged with allergen and its effects were
compared with those of anti-allergic drugs. The main constituents identified were pulegone, menthone and limonene. Essential oil increased absolute values
of CD4+, CD8+ and B cells (p<0.002), stimulated IFN- synthesis and reduced IL-13 levels. Essential oil diminished -hexosaminidase liberation by basophils
(p<0.0001), with effects majors to those of the drugs tested. Essential oil stimulated the Th1 deviation and reduced -hexosaminidase enzyme liberation by
basophils from allergic patients.

Keywords: Minthostachys verticillata; Essential oil; Immunomodulator; Basophil degranulation; -hexosaminidase.

Resumen

El aceite esencial de hojas de M. verticillata fue obtenido por destilacin en corriente de vapor y analizado por cromatografa gaseosa. Clulas
mononucleares de sangre perifrica de pacientes alrgicos, fueron estimuladas con aceite esencial. La proliferacin fue ensayada mediante mtodo
colorimtrico del MTT o 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide. Se cuantificaron clulas CD4+, CD8+ y B en cultivos de linfocitos y
en sobrenadantes, IFN- e IL-13. Se determin la liberacin de la enzima -hexosaminidasa de basfilos adicionados de aceite esencial, y desafiados con el
alergeno. Los efectos del aceite esencial fueron comparados con los de drogas antialrgicas. Los principales constituyentes identificados fueron pulegona,
mentona y limoneno. Aceite esencial increment los valores de clulas CD4+, CD8+ y B (p<0.002), estimul la sntesis de IFN- y redujo los niveles de IL-13.
El aceite esencial disminuy la liberacin de -hexosaminidasa de basfilos (p<0.0001) con mayores efectos que los de las drogas ensayadas. Aceite esencial
estimul la desviacin Th1 y redujo la liberacin de la enzima -hexosaminidasa de basfilos de pacientes alrgicos.

Palabras Clave: Minthostachys verticillata; Aceite esencial; Inmunomodulador; Desgranulacin de basfilos; -hexosaminidasa.

Recibido | Received: March 19, 2009.

Aceptado en Versin Corregida | Accepted in Corrected Version: May 18, 2009.
Publicado en Lnea | Published Online: May 25, 2009
Declaracin de Intereses | Declaration of interests: authors have no competing interests.
Financiacin | Funding: This work was supported by grants from PICTOR 8/20325 BID 1728/OC-AR, Agencia Crdoba Ciencia-Nacin, Resolution #222, years 2007/09, and
SeCyT, Resolution #425, years 2007/09, from Universidad Nacional de Ro Cuarto and CONICET.
This article must be cited as: Laura Cariddi, Marina Moser, Melisa Andrada, Mirta Demo, Julio Zygadlo, Liliana Sabini, Ana Maldonado. 2009. The effect of Minthostachys
verticillata essential oil on the immune response of patients allergic to dust mites. Bol Latinoam Caribe Plant Med Aromat 8(3):224 233. {EPub May 25, 2009}.

*Contactos | Contacts: E-mail: lcariddi@exa.unrc.edu.ar; Tel.: 54-0358-4676433; Fax: 54-0358-4676231.

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

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which permits to copy, distribute and transmit the work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work.
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Cariddi et al.
INTRODUCTION
Allergic pathologies occur at an incidence of up to
30% in the population. Allergic patients are generally
treated with corticoids and/or antihistamines, which
constitute the anti-inflammatory therapies of choice
because they block many inflammatory routes that are
abnormally activated in this type of pathology (Barnes,
2001). Nevertheless, these methods are not always
effective and can produce several adverse effects.
Corticoids suppress both the innate inflammatory
response and cellular immunity, inhibiting synthesis of
IL-1, on behalf of macrophages, and of IL-2, on behalf
of T cells. A correlation has also been discovered
between their anti-lymphoproliferative effects and
capacity to produce apoptosis in lymphocyte
populations (Winiski et al., 2007).
Antihistamines can produce diverse adverse effects
such as anemia or reduced platelets, reduced white
cells and bone marrow failure (Leonardi et al., 2007).
In addition, some allergic patients display no
response to treatment with these drugs, which, in
conjunction with the fact that this resistance has also
been observed in other inflammatory diseases (Barnes,
2001), justifies the search for new immunomodulators.
A quarter of the drugs used in industrialized countries
are obtained from the Plant Kingdom. Current
investigations aim to find plant/herbal medicines
whose therapeutic indices: permit phytomedicinal
application, are low-priced and do not bring about
adverse reactions or undesirable indirect effects, in
order to be administered as an alternative or
complementary therapy for conventional treatments.
Several in vitro or in vivo investigations have
demonstrated that different extracts or pure
compounds derived from medicinal plants can
modulate the Th1/Th2 response, towards the Th1
profile, producing a beneficial effect for allergic
patients (Na et al., 2002; Ko et al., 2004; Lee et al.,
2006; Park et al., 2009).
Minthostachys verticillata (Griseb.) Epling,
(Labiatae) commonly referred to as peperina', has a
wide geographical distribution and is known for its
ethno-medicinal properties. It is used as a digestive, a
sedative, an anti-spasmodic, a stimulant and a
bronchial-dilating agent (Ratera & Ratera, 1980).
Previous studies have revealed that the essential oil
derived from this herb presents antimicrobial activity
against some staphylococcal strains and antiviral
properties against HSV-1, strain RC/79 of PrV and
Herpes Suis Virus (Primo et al., 2001). Furthermore, in
www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (3) 2009 | 225
Immunomodulatory effects of Minthostachys verticillata essential oil
an in vitro study carried out on patients allergic to
environmental fungi, essential oil, in concentrations of
mg/mL, revealed mitogenic properties similar to those
of PWM on human lymphocytes (Cariddi et al., 2005),
were able to stimulate CD8+ T cells, increased levels
of IFN- and demonstrated anti-allergic properties
majors to those of dexamethasone and theophylline
(Cariddi et al., 2006).
The purpose of this investigation was to determine
if the essential oil from M. verticillata, at concentra-
tions smaller than previously evaluated, can modulate
the Th1/Th2 response in allergic patients with history
of allergies and asthma, and to verify if it maintains its
inhibiting properties on basophil degranulation in
these patients, comparing its effects with those of
conventional anti-allergic drugs.
MATERIALS AND METHODS
Plant samples
Green leaves and thin stems of Minthostachys
verticillata (Griseb.) Epling (Labiatae) were collected,
during morning hours, from the city Santa Rosa in the
Crdoba province, Argentina, in April 2004. The plant
was identified by Dr. Margarita Grosso, professor in
the Area of Botany of the Universidad Nacional de Ro
Cuarto, and a voucher specimen was stored in the
RCV (Ro Cuarto Vasculares) herbarium as file #1955.
The morphological characterization of the plant was
executed macro-and microscopically to confirm the
identity of these specimens. The aerial parts of the
plant were made up of the leaves and parts of the stem.
The oil was isolated from the aerial parts.
Oil isolation
To prepare the essential oil (EO), 60 g of ground
plant material were hydro-distilled for 3 h using a
Clevenger-type apparatus, yielding 4.8% of the oil.
The oil was separated from the aqueous phase, dried
over anhydrous Na2SO4 and stored in the dark at -20
C until use (Senatore, 1998). In order to perform the
immunological in vitro assays, various concentrations
were obtained, and the oil was emulsified in
dimethylsulfoxide (DMSO) and diluted in Roswell
Park Memorial Institute (RPMI-1640) medium.
Identification and quantification of EO compounds
by Gas chromatography (GC)
Quantification of components present in the oil
sample was made by measuring the area under each
peak of the chromatogram (Zygadlo et al., 1996;
Cariddi et al.
Cariddi et al., 2007). Briefly, analytical GC was
performed on a Shimadzu GC-R1A gas chromato-
graph fitted with a DB5 capillary column (30 x 0.25
m). Carrier gas N2, flow rate 1.5 mL/min, split mode.
Oven temperature programmed from 40260 C at 3
C/min. Injector temp 280 C. Detector used FID,
temperature 300 C. The identification of the
compounds was made comparing their retention times
against standard pure drugs injected in the same
conditions.
Human blood sample
The sample included peripheral blood mononuclear
cells (PBMCs) and basophils from 57 allergic patients,
27 male and 30 female (ages 1 to 30, average value:
17) with positive prick tests for allergy to dust mites
(Dermatophagoides pteronyssinus, Dermatophagoides
farinae). This study was approved by the Universidad
Nacional de Ro Cuarto Institutional Review Board. In
accordance with ethical standards, parents of underage
children were properly informed of the study and
signed an agreement authorizing the test. The
characteristics of these patients are shown in Table 1.
Skin prick testing was performed using a standard
technique (single-headed lancet technique) and
standard commercial solutions (International
Pharmaceutical Immunology SA). All tests were
performed by a single operator, an allergist doctor.
Each patient underwent skin prick testing for
allergens. Saline solutions and histamine were used as
negative and positive controls, respectively. The size
of the wheal was measured after 15 min and a positive
result was noted if the wheal size was at least 3 mm
greater than the negative saline control (Barbaud
2001). The allergen extracts were provided by
International Pharmaceutical Immunology, S.A.
(Alicante, Spain) Dropper vials were used, containing
3 mL of the antigen extract in 50% glycerin solution,
plus 0.42% phenol each (concentration = 10.000
PNU/mL). The in vitro assays were performed with
the same extracts. Ten milliliters of venous peripheral
blood were obtained from each patient and stored in
sterile tubes containing heparin.
PBMCs cultures
The effects of different concentrations (6 x 103,
600, 60, 6, 0.6, 0.06 and 0.006 g/mL) of EO on the
proliferation of lymphocyte from healthy individuals
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Immunomodulatory effects of Minthostachys verticillata essential oil
were evaluated. A dose-response curve was construc-
ted in a previous in vitro assay. EO visibly
demonstrated lymphoproliferative capacity, with dose-
dependency. The concentration of 6 g/mL obtained
constituted the highest Proliferation Index (PI)
PBMCs were isolated from blood samples using
Ficoll-Hypaque (Hystopaque-1077; Sigma, St. Louis,
US) centrifugation. The cellular proliferation assays
were carried out with the Vybrant MTT Cell
Proliferation Assay Kit (Molecular Probes Invitrogen
Detection Technologies, Eugene, Oregon, USA). Cells
(2 x 105/mL), with a final volume of 200 l, were
cultured in 96-well sterile microplates containing
RPMI-1640, to which was added 25 mM Hepes, 2 mM
L-glutamine, 5% FCS, 50 mM 2-ME, 100 g/mL
streptomycin and 100 g/mL penicillin (Merck).
Cultures were stimulated with 10 g/mL PHA, 5
g/mL ConA and 6 g/mL oil. Control lymphocyte
cultures were performed using only media.
Cells were incubated during 72 h at 37 C with 5%
CO2 in a humid atmosphere. An aliquot of 100 L of
fresh RPMI-1640 and 10 L of MTT (3,(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl tetrazolium bromide)
solution (1 mg/mL of MTT in PBS 0.01 M pH 7.2)
was added to each well and the plate was incubated at
37 C with 5% CO2 for 4 h. After incubation, the plate
was centrifuged and supernatants, removed. DMSO
(50 L) was added to each well in order to dissolve the
formazan crystals that resulted from the conversion of
the salt (MTT). The plate was read in a UV
spectrophotometer (Labsystems Multiskan MS) at
570/690 nm wave lengths.
The cellular expansion reached by the classic
mitogens was compared with that produced by EO by
calculating the PI according to the following formula:
PI = stimulated cells/ non-stimulated cells > 1.20
(Tuchscherer et al., 2002).
CD4+ and CD8+ T cells and B cells quantification
by immunofluorescence (IF)
The CD4+ and CD8+ T cells present in the EO-
stimulated cultures were counted with the indirect IF
technique using anti-human CD4 and anti-human
(Sigma, St. Louis, US) CD8 monoclonal antibodies
(Sigma, St. Louis, US), and B cells were counted with
direct IF using anti-human -globulin-FITC (Sigma,
St. Louis, US) (Edidin, 1989).
Cariddi et al. Immunomodulatory effects of Minthostachys verticillata essential oil

Table 1. Characterization of select allergic patients, including: age in years, symptoms, total IgE values and skin prick testing results.
Skin prick testing with dust mite

N clinical Age Symptoms IgE D. pteronyssinus D. farinae

history (years) (UI/mL)*
5 2 As Rh 618 ++ +++
9 5 Rh Ecz 190 +++ +
10 5 As Rh 790 +++ ++
12 6 As Rh Sin Pr 1133 ++ +++
13 6 As Rh 1664 ++ +
14 7 Rh, As 1236 ++ +
15 8 Rh Si 365 + +
16 8 As Rh 760 ++++ ++
18 9 As Rh 1000 + +
20 11 Rh As 1321 ++ ++
21 11 As Rh 786 ++ ++
23 12 Rh Si 420 ++ ++
27 15 Rh 460 +++ +++
28 17 Rh Ecc 350 + +
33 19 As Rh 512 ++++ ++
35 21 As Rh Ecz 122 + +
36 21 As Rh Ecz 122 + +
38 23 Rh Ecz 280 + +
39 23 As Rh 139 + +
41 26 As Rh 141 + +
44 27 As 512 + +
45 27 As RhEcz 123 + +
47 28 Rh Ecz 790 + +
50 29 As Ecz Rh 1560 + +
51 29 As Rh Sin Pr 1133 ++ +++
52 29 As Rh 355 + +
53 29 Rh, As 236 ++ +
54 30 As Ecz Rh 568 + +
55 30 As Rh Sin Pr 1233 ++ +++
56 30 As Rh 965 + +
57 30 Rh, As 1236 ++ +

As - Asthma; Rh - Rhinitis; Ecz - Eczema; Pr - Prurigo; Si - Sinusitis. D. Pteronyssinus - Dermatophagoides pteronyssinus; D. Farinae -
Dermatophagoides farinae. Prick test: scale from + to ++++ according to Bousquet (1988).
* IgE was measurement by ELISA Kit (Iema Well de Radim, Rome, Italy)

6 g/mL of EO, plus allergen. As a control, these

IFN- and IL-13 measurement
IFN- and IL-13 synthesis was quantified in EO-
stimulated supernatants of PBMC cultures from
allergic patients, collected at 60 h incubation to
optimize the detection of cytokine levels. IFN-
detection was performed using a commercial Human
Interferon Gamma (Hu- IFN-) ELISA kit (PBL
Biomedical Laboratories, USA) and the lower limit of
detection for the cytokine assay was less than 2 pg/mL
(Kelder et al., 1986). IL-13 detection was performed
using a commercial EHIL13 Human Interleukin-13
ELISA Kit (Pierce Endogen, France) and the lower
limit of detection for the cytokine assay was less than
7 pg/mL (Ly et al., 2005). This assay was carried out
with PBMC cultures stimulated with: a) allergen alone
(a suspension of dust mite at 10.000 PNU/mL) and b)
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cytokines were quantified in PBMC cultures with
media alone. Each instance was triplicated.
-hexosaminidase enzyme release assay
The effects of different concentrations (160, 80, 40,
20, 10 and 1 g/mL) of EO on the release of - hexo-
saminidase enzyme were evaluated constructing a
dose-response curve in a previous assay. It was
observed that EO reduced the levels of enzyme
released in a dose-dependent way. The concentration
of 10 g/mL was that which produced the highest
diminution of the release of enzyme.
Basophils of peripheral blood were obtained by
density gradient using 6% of dextran 70 with NaCl
0.9%. The interphase that contained the cells was
centrifuged and re-suspended in RPMI-1640. An
Cariddi et al.
aliquot from the cellular suspension was taken and the
cells were dyed with Toluidine Blue that it dyes only
basophils. The cellular count was realized in camera
Fuch Rosenthal in optical microscope. The concen-
tration of basophils was increased 100 to 200 times
after the treatment with dextran (from 1-2 to 128-256
cells in 64 fields). It had contamination by other PMN,
such as neutrophils and eosinophils.
Fifty microliters of the basophil suspension at a
concentration of 1 x 105 cells/mL were placed in each
well of the 96-well microplate and pre-incubated for
15 min at 37 C with the specific allergen alone (dust
mite suspension), and the allergen plus: a) 0.04 mg/mL
dexamethasone (Sidus); b) 0.2 mg/mL theophylline
(Phoenix); c) 0.2 mg/mL disodium cromoglicate
(Phoenix); d) 0.05 mg/mL ipratropium bromide
(Altana Pharma), or e) 10 g/mL essential oil. The
negative control values were obtained using RPMI-
1640 and the cells alone, without any allergen
stimulation, in order to view unspecific spontaneous
degranulation. In addition, montelukast (0.04 mg/mL)
(Merck) was used as a negative control of -
hexosaminidase enzyme inhibition because it acts in
the late phase of the allergic reaction, inhibiting
leukotriene receptors. Blank values were obtained
using just the media or DMSO. Following incubation,
50 L of the chromogenic substrate for the enzyme -
hexosaminidase (4-nitrophenyl-N-acetyl--D-glucosa-
minide) (Sigma-Aldrich, Inc, St. Louis, USA) 1
mmol/L, was added to each well and dissolved in 0.1
mol/L of citrate buffer, pH 5. The system was
incubated at 37 C for 1 h. The reaction was stopped
with 200 L of carbonate buffer 0.1 M, pH 10.5 per
well. The product of the cleavage (4-nitrophenol) was
interpreted by reader ELISA (Labsystems Multiskan
MS) at 405 nm (Shibata et al., 1996). The percentage
of - hexosaminidase release inhibited was calculated
according to the formula: Inhibition % = (A B) x
100/A, where A is the amount of -hexosaminidase
released by basophils in the presence of the specific
allergen alone and B, in the presence of the allergen
aggregates from the anti-allergic drugs or oil (Na et
al., 2002).
Statistical analysis
All of the values obtained in the assays carried out
during this investigation were expressed in averages
and standard deviation. The parameters were evaluated
using the program GraphPad Prism version 4.0 (Inc.
San Diego, USA, 2004) and compared with the
parametric test t-Student for twin samples. Statistical
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Immunomodulatory effects of Minthostachys verticillata essential oil
differences were considered significant when the value
was p <0.05.
RESULTS
Identification and quantification of essential oil
compounds
GC analysis revealed a composition not
significantly different than those reported previously
by the authors (Zygadlo et al., 1996; Cariddi et al.,
2007). The main components were pulegone 63.0%,
menthone 16.4% and limonene 1.9%, accompanied by
other minor terpenoid components such as -pinene
(0.2%), -pinene (0.3%) and 1,8-cineole (0.1%) were
found in lower quantities in accordance with previous
reports.
Lymphocyte response to stimulation with essential
oil
EO (6 g/mL) stimulated the proliferation of
lymphocyte from allergic patients, revealing
significant statistical differences in comparison with
the cultures lacking stimulus (p<0.0001) and no
differences when compared to PHA (p=0.1396) or
ConA (p=0.1749). The PI calculated for EO, PHA or
ConA, were > 1.20, indicating cellular proliferation
(Fig. 1).
Absolute values of CD4+ and CD8+ T cells and B
cells in cellular cultures stimulated with EO revealed
an increase (p<0.002 to each one of cell types) in
regard to the cultures without stimulus (Table 2).
In supernatants of EO-stimulated cultures, values of
IFN- revealed an increase in regard to cultures
without stimulus (p<0.001) and cultures stimulated
with allergen alone (p<0.0001) (Fig. 2). IL-13 levels
were greater in the cultures stimulated with allergen
than in cultures without stimulus (p<0.0001). In
allergen-stimulated cultures with EO, the levels of IL-
13 were visibly reduced in regard to cultures
stimulated with the allergen alone (p<0.0001) (Fig. 2).
Effects of essential oil on basophil degranulation
Basophils of all the allergen-challenged patients
released high amounts of -hexosaminidase enzyme in
comparison with the values of enzyme released
spontaneously (p<0.01) (Fig. 3). The anti-allergic
drugs, excluding montelukast, reduced the in vitro
levels of released enzyme in comparison with
basophils challenged with the allergen alone (p<0.02)
(Fig. 3). This result demonstrated that the plant
fraction did not reveal allergenic effects in vitro. The
Cariddi et al. Immunomodulatory effects of Minthostachys verticillata essential oil

addition of EO to allergen-challenged basophils disodium cromoglicate and 0.05mg/mL of ipratropium

reduced -hexosaminidase liberation to the same bromide (p<0.05 for each one) (Fig. 3). The per-
levels as those of the spontaneous release of the centage of inhibition of -hexosaminidase released
enzyme. The inhibiting activity of the oil at 10 g/mL under different treatments is shown in Table 3 and the
on the enzyme was higher than the one produced by essential oil is significantly active.
standard drugs at concentrations: 0.04 mg/mL of dexa-
methasone, 0.2 mg/mL of theophylline, 0.2 mg/mL of

Tabla 2. Average absolute values + SD of total leukocytes from allergic patients and T CD4+, T CD8+ and B cells in cultures without
stimulus (W/S) or stimulated with essential oil (EO).
n Total W/S cultures Cultures stimulated with EO
leukocytes/mm3 CD4+ CD8+ B cells CD4+ CD8+ B cells

22 6432.6 + 1321,6 1677 + 65 714 + 60 705 + 57 2174 + 82 988 + 60 916 + 61

Table 3. Percentage + SD of inhibition of -hexosaminidase enzyme release from basophils of allergic patients by anti-allergic drugs or
essential oil.

Anti-allergice drugs or essential oil

n=57
dexa theo crom Ip EO

Inhibition (%) 44.8 + 8 47.6 + 10 44.7 + 7 46.9 + 5 54.2 + 7

Dexa - dexamethasone; theo - theopylline; crom - disodium cromoglicate; Ip - ipratropium bromide; EO - essential oil.

Figure 1. Proliferation of lymphocytes from allergic patients Figure 2. Quantification of IFN- and IL-13 in supernatants of
(n=57), stimulated with 6 g/mL essential oil (EO) from M. lymphocyte cultures from allergic patients (n=38).
verticillata, according to MTT colorimetric assay.
IL-13
200
allergic patients e
IFN-
2.5
c
concentration (pg/ml)

d 150 c f
b
Lymphocyte proliferation

2.0
(OD 570/690 nm)

100
1.5 a b
a
50 d
1.0

0.5 0
/S

/S
L)
en

en

en
m
W

W
rg

rg

rg
g/
le

le

le

0.0
m
al

al

al
(6
W/S

+
PHA (10 ug)

ConA (5 ug)

EO (6 ug)

EO

L)
m
g/
m
(6
EO

IFN- was quantified in lymphocyte cultures without stimulus or

PHA (10 g/mL) or ConA (5 g/mL) were used as positive stimulated with 6 g/mL essential oil (EO) from M. verticillata.
controls of proliferation, and lymphocyte cultures without IL-13 was quantified in lymphocyte cultures without stimulus,
stimulus, as negative controls. W/S, without stimuli; a vs d p < stimulated with allergen alone or stimulated with allergen and
0.0001; b vs d p = 0.1396; c vs d p = 0.1749; PI (PHA) = 1.81 + with 6 g/mL EO from M. verticillata added. W/S, without
0.12; PI (ConA) = 1.90 + 0.11; PI (EO) = 1.87 + 0.10 stimuli; a vs b p= 0.1332; a vs c p<0.001; b vs c p<0.0001, d vs e
p<0.0001; d vs f p<0.0001, e vs f p<0.0001.

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Cariddi et al. Immunomodulatory effects of Minthostachys verticillata essential oil

Figure 3. The amount of -hexosaminidase enzyme released by types of enzymatic routes which involve Ras-Map
allergen-challenged basophils from allergic patients (n=57) was kinases, calcium-dependent PLCl and PLClDAG,
reduced by essential oil (EO) and several anti-allergic drugs,
excluding montelukast. key participants in cellular proliferation, differen-
tiation and apoptosis (Crespo & Gutkind, 2004).
c i
According to some authors in vitro studies of
80
allergic patients have shown increased values of
CD4+ T cells (1500-1750 cells/mm3), differentiating
-hexosaminidase released (%)

70
*
60
d f towards the Th2 profile; a decrease in CD8+ T cells
e
50 g (370-520 cells/mm3) (Bratke et al., 2006) and high
40 values of B cells (480-650 cells/mm3) (Mohamed et
h al., 2003) in comparison with non-allergic
30 a
20
b individuals. In this study EO (6 g/mL) increased in
vitro absolute values for both T CD4+ and CD8+ cells
10
subpopulations in patients allergic to dust mites,
0
indicating that this plant fraction stimulated cellular
k

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EO

immunity. These data are compatible with those from

on
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S

ba + E

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ph

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M

g+
r

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+d
bl

+c
s+ + a

s+ rg+

ba rg+
D

so

s+ ler

+
ba erg

a previous in vitro study in which high EO

ba erg

rg
ba

le

le
s

al

le
l

al

al
al

s+
al

al

concentrations (mg/mL) increased values of CD8+ T

s+

s+
ba

ba

ba

cells in both patients allergic to environmental fungi

Basophils were incubated with EO alone, without allergen, and
no significant liberation of the enzyme was observed. Cells
without the addition of allergen and cells with addition of
dimethylsulfoxide (DMSO) were also evaluated as controls.
a vs c p< 0.0001; a vs b p = 0.3373; b vs c p <0.002; c vs d p <
0.01; c vs e p < 0.002; c vs f p< 0.01; c vs g p < 0.002; c vs h p
< 0.0001; c vs i p = 0.8245; h vs * p = ns; h vs i p<0.0001; d
vs i p < 0.01; e vs i p< 0.002; f vs i p< 0.02; g vs i p<0.003.
blank, media alone; DMSO, dimethylsulfoxide; bas, basophils;
allerg, allergen ; dexa, dexamethasone ; theo, theopylline ; crom,
disodium cromoglicate; iprat, ipratropium bromide; EO, essential
oil; mont, montelukast
DISCUSSION
In the present study, the essential oil from M.
verticillata was composed mainly by mono-and
sesquiterpenes. Zygadlo et al. (1996) analyzed oils of
M. verticillata of different geographic areas from
Argentina. They found that the main components are
terpenes and seem to be divided in three chemotypes:
carvone, thymol-carvacrol and predominantly
pulegone-menthone. Our results agree those of De
Feo et al. (1998), Gonzlez Pereyra et al. (2005),
Cariddi et al. (2007), Maldonado et al. (2007), who
studied M. verticillata oil and reported the
composition.
At optimal concentrations, EO from M.
verticillata was able to stimulate in vitro the
lymphocyte proliferation in patients allergic to dust
mites with effects similar to those of PHA or ConA.
These lectins bond specifically to carbohydrate
components, including proteins of the TcR-CD3
complex. This produces the activation of several
and healthy individuals alike (Cariddi et al., 2006).
The EO also increasing the absolute values of B
cells. Previously we demonstrated that EO had effects
similar to those of PWM, mitogen of B and T cells,
on lymphocyte proliferation in both allergic and
healthy individuals (Cariddi et al., 2005). These
results correspond with those of this investigation,
where we demonstrated that EO was able to stimulate
both T and B cells.
Treatment with the EO increased the levels of
IFN-, which is why it is suggested that the EO-ac-
tivated CD4+ T cells in this study could be Th1 cells,
which produce IFN-. EO was able to stimulate in
vitro CD8+ T cells as well, also producers of IFN-.
In addition, we observed that EO reduced IL-13
values in lymphocyte cultures co-stimulated with the
allergen, as compared to cultures stimulated with
allergen alone. These data demonstrated that EO mo-
dulated the in vitro Th1/Th2 response, favoring Th1
deviation. Anti-allergic therapies aim to redirect the
profile of Th2 cells towards Th1, increasing IFN-
synthesis and decreasing IL-4 and IL-13 values, thus
reducing IgE levels (Bang & Plosker, 2004). It was
confirmed that the vegetal derivative acts on the
immune response by means of the stimulation of the
Th1 profile. In previous studies this was deduced of
the cuantification of IFN- and LT CD8+ (Cariddi et
al., 2006, 2007; Maldonado et al., 2007).
-hexosaminidase, an acid hydrolase, is released
along with histamine when basophils are activated.
Therefore, -hexosaminidase is accepted as a
degranulation marker (Shibata & Yagi, 1996).

www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (3) 2009 | 230
Cariddi et al.
During the -hexosaminidase enzyme release
assay, we confirmed that the anti-allergic drugs
tested, excluding montelukast, were able to reduce
the levels of enzyme released, as compared to
basophils challenged with allergen alone.
Dexamethasone, in addition to bonding to the
cytoplasmic specific receptor that enters the nucleus
and attaches itself to the DNA, works by stimulating
cAMP production, inhibiting the degranulation
process in basophils and mast cells (Fukui, 2008).
Theophylline acts as inhibitor of phosphodiesterase.
This enzyme deactivates cAMP forming icAMP,
which favors cell degranulation (Lipworth, 2005).
Cromoglycate is a basophil and mast cell membrane
stabilizer, as it prevents their degranulation (Gotua et
al., 2008). Ipratropium inhibits the liberation of
chemical mediators, increased by acetylcholine, by
blocking cholinergic receptors on the surface of
basophils and mast cells (Spooner et al., 2003). In
contrast, montelukast, a leukotriene receptor
antagonist (Kawai et al., 2008), which was used in
this study as a negative control for inhibition of the
immediate reaction, did not decrease the levels of -
hexosaminidase released by basophils challenged
with allergen, as expected in this model.
EO, at a concentration of 10 g/mL, reduced the
levels of -hexosaminidase released by basophils,
with effects majors to the anti-allergic drugs tested,
excluding montelukast, which is why it has been
suggested that this plant fraction could act upon cell
membranes with a mechanism similar to that of one
of these drugs. These results demonstrated that EO
maintained its inhibiting properties on basophil
degranulation at high (Cariddi et al., 2006) and small
concentrations alike.
Some of main components of EO identified by GC
could be responsible for its biological activity.
Numerous investigations demonstrate the protective
effects of terpenes on cell membranes. These
substances also carry out a regulating activity on T
cells, the synthesis of cytokines and inhibition of the
apoptosis processes (Yeh et al., 2006; Ashibe et al.,
2007).
Essential oils are complex mixtures of numerous
molecules and their biological effects are the result of
a synergy between all of them or the reflection of
main molecules present at high levels according to
analysis by GC (Bakkali et al., 2008).
According to the aforementioned, EO could have
displayed protective effects on cell membranes when
added to the basophils. This indicates that the
www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (3) 2009 | 231
Immunomodulatory effects of Minthostachys verticillata essential oil
increase in basophil membrane permeability may be
an essential trigger for the release of -hexo-
saminidase. The terpenes from EO may have acted
upon basophil membranes, avoiding the disturbance
induced by the specific allergen. There would not be
modifications related at least to types of allergens
both tried (fungi and dust mite) (Gonzlez Pereyra et
al., 2005; Cariddi et al., 2006, 2007; Maldonado et
al., 2007). Moreover the active concentrations seem
to be much lower than previously thought, as this
work found activity in the range 6 mg/ml at 0.006
g/mL, in contrast with our previous tests at only 150
mg/mL (Cariddi et al., 2005).
CONCLUSION
The EO from leaves of Minthostachys verticillata
at small concentrations acted as a mitogen, increasing
proliferation of T and B cells from allergic patients.
As an immunomodulator, it favored Th1 deviation by
increasing IFN- synthesis and decreasing IL-13 pro-
duction. EO maintained its inhibiting effects on baso-
phil degranulation, reducing the levels of -hexo-
saminidase released.
Additional studies are being realized along the
same line of investigation to reveal if the terpenes
identified can have therapeutic applications in
allergies. In the future, these products could be
applied in alternative or complementary therapies
with patients who do not respond to conventional
treatment.
ACKNOWLEDGEMENTS
We would like to thank Mnica Wagner for the
English version.
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