Aceites esenciales y

extractos de Ocimum

basilicum

Jorge Hidalgo Gracia

Daniel Ramírez Ramos

Alejandro Ugena Ortiz

Objetivos

El objetivo común que tienen los estudios aquí analizados es obtener características

especiales de esta especie como puede ser:

- Identificar sus compuestos, aislarlos y determinar su capacidad como anti-

oxidante.

- Determinar la presencia de ácido chicórico en las hojas de albahaca.

- Determinar la mentona y el estragol presente en los aceites esenciales de la

albahaca cultivada en el noroeste de Irán.

- Determinar los compuestos volátiles en albahaca obtenidos tanto por micro-

extracción en fase sólida como por hidrodestilación.

- Analizar los aceites esenciales de varios cultivares de albahaca.

Introducción

La especie vegetal sobre la que se va a realizar el estudio es la denominada vul-

garmente como albahaca o alhábega, Ocimum basilicum en latín. La parte de la planta

que ha sido sometida a estudio han sido las hojas y en algún caso se ha incluido el tallo.

La albahaca es una hierba aromática de la familia de las lamiáceas, cultivada

como perenne en climas tropicales desde hace varios milenios.

Hierba anual de crecimiento bajo (entre 30-130 cm), con hojas opuestas de un

verde lustroso con un tono mucho más vivo en la parte superior, ovales u ovadas, denta-

das y de textura sedosa, que miden de 3 a 11 cm de largo por 1 a 6 cm de ancho.

Aparecerán sus espigas florales en verano, con pequeñas flores tubulares de co-

lor blanco o lavanda las cuales, a diferencia de las del resto de la familia, tienen los

cuatro estambres y el pistilo apoyados sobre el labio inferior de la corola. Su follaje es

muy aromático.

Esta planta es muy sensible a las heladas. Se cultiva únicamente por semillas a

principios o mediados de la primavera. Requiere una posición soleada, aunque en climas

de veranos muy calurosos requiere suelos fértiles, permeables y húmedos, a ser posible

con algo de sombra.

Aunque actualmente se cultivan en todo el mundo los más de cuarenta tipos de

albahaca, todo parece indicar que su origen se encuentre en India, donde se consideraba

sagrada. Desde allí se extendería por Europa gracias a las culturas griega y romana. En

antiguas civilizaciones como la egipcia, poseía un alto valor, siendo incluso uno de los

elementos utilizados en la momificación.

Al igual que otras aromáticas, como el romero o la salvia, resulta muy apropiada

para cultivarla alrededor de otros vegetales que son atacados por plagas de insectos, ya

que tiene la propiedad de ahuyentarlas.

Las hojas son la parte que se utiliza en la cocina, tanto si son frescas como secas.

Las primeras poseen mucho más aroma y sabor. Son muy apropiadas tanto para los

huevos como para pescados o guisos y son uno de los ingredientes indispensables de la

salsa pesto.

El aceite esencial de albahaca es rico en estragol, un potente carcinógeno (para

hepatomas) y genotóxico natural, en ratones y ratas. En septiembre de 2001 el Comité

Científico de la Unión Europea emitió una opinión que recomienda reducir la exposi-

ción y restringir el uso del estragol, sin poderse establecer un límite seguro para la

exposición a esta toxina de acción lenta (no hay indicios de ninguna toxicidad).

Sus aceites esenciales se utilizan también en la elaboración de licores o para

aportar aroma a sopas y guisos. También sirve como ingrediente de remedios naturales,

en infusiones, en bebidas energéticas, para prevenir la caída del cabello o para combatir

la halitosis, los dolores de cabeza, de oído, incluso para facilitar la digestión.

Metodología:

Muestras:

Las diferentes muestras de hojas usadas para el aislamiento de aglicones fueron

adquiridas en un mercado local en Split (Croacia).

Las muestras de diferentes variedades de albahaca, usadas para la búsqueda de

polifenoles, fueron adquiridas en un mercado local en Nampa (EEUU). Se conservaron

a -80ºC hasta su utilización.

Una parte de las albahacas se micorrizó con un hongo y se cultivaron en un sus-

trato de crecimiento.

Las muestras de albahaca del noroeste de Irán fueron obtenidas del distrito de

Maragheh. Se secaron a temperatura ambiente durante 4 ó 5 días y se pulverizaron de

manera homogénea.

Las muestras de tallos y hojas frescas de albahaca mexicana fueron obtenidas en

Jalisco (México). Las plantas se encontraban en floración porque es cuando la planta

tiene más aceite esencial.

Las muestras de albahaca del centro de Irán fueron obtenidas en Isfahan en épo-

ca de máxima floración.

Las muestras de las tres especies de albahaca de Papúa Nueva Guinea se obtu-

vieron de diferentes localidades de la isla.

Método de obtención:

En las muestras croatas se buscó obtener el aceite esencial por un lado y por otro

lado los aglicones volátiles libres.

Para aislar el aceite esencial se pusieron en un aparato tipo Clevenger 100 g de

planta y 500 ml de agua. El aceite esencial se obtuvo por hidrodestilación durante 3

horas y se almacenó a -20ºC.

Para aislar los aglicones volátiles libres primero se tuvo que hacer una extrac-

ción de 100 g de planta con acetato de etilo caliente durante 2 horas. El extracto obteni-

do fue secado y el sedimento eliminado por filtración. El filtrado se pasó por una croma-

tografía líquida-sólida para obtener la parte glucosídica del mismo. Por último se añadió

β-glucosidasa para romper enzimáticamente las moléculas y se extrajeron los aglicones

con n-pentano.

La extracción de las muestras utilizadas para la búsqueda de polifenoles, como el

ácido chicórico, se realizó con metanol acidificado con ácido fórmico. Los extractos

obtenidos fueron almacenados a -80ºC.

En las muestras del noroeste de Irán se buscaba obtener el aceite esencial. Para

ello se pusieron 50 g de planta en un aparato tipo Clevenger y se las sometió a hidrodes-

tilación durante 3 horas. El extracto fue secado y almacenado mediante refrigeración a

4ºC.

La extracción de las muestras mexicanas se realizó mediante microextracción en

fase sólida con fibras de PDMS/DVB o con fibras de CW/DVB. Se homogeneizaron 8 g

de material vegetal con 8 ml de agua y 1 g de NaCl y se incubaron 30 minutos a 70ºC.

Posteriormente se añadió una fibra y se mantuvo en agitación 40 minutos.

El aislamiento de los aceites esenciales de las muestras del centro de Irán se hizo

mediante hidrodestilación durante 3 horas en un aparato tipo Clevenger. El extracto fue

secado y almacenado mediante refrigeración a 4ºC.

Para obtener el aceite esencial de las muestras de Papúa Nueva Guinea se trocea-

ron las muestras y se las sometió a hidrodestilación durante 8 horas en un aparato de

destilación de cristal standard. El extracto fue secado.

Método de análisis:

Las muestras croatas se analizaron por medio de cromatografía de gases y espec-

trometría de masas. Se utilizaron dos columnas de diferente polaridad, una de metil

silicona fluida y otra de polietilenglicol. El gas portador utilizado fue el helio.

Los índices de retención fueron determinados con la co-inyección de las muestras con

una solución de las series homólogas de n-alcanos C8-C22. Los constituyentes individua-

les fueron identificados mediante la comparación de los índices de retención obtenidos

con los de la literatura científica.

La actividad antioxidante de los aglicones fue medida usando el DPPH y el

FRAP.

Los extractos usados para buscar polifenoles fueron analizados mediante croma-

tografía líquida de alta resolución (HPLC) y espectrometría de masas. Los compuestos

fenólicos fueron identificados basándose en el espectro UV, tiempo de retención, iones

moleculares y fragmentación de iones. El ácido chicórico fue identificado evaluando el

tiempo de retención, el espectro UV-vis y la información del espectrómetro de masas.

Las muestras del noroeste de Irán fueron analizadas por medio de cromatografía

de gases y espectrometría de masas. La columna capilar está rellena de fenil metil

polisiloxano. El gas portador utilizado fue el helio. La identificación de los componen-

tes se basó en la comparación de los tiempos de retención obtenidos con los de la litera-

tura científica.

Las muestras mexicanas fueron analizadas por medio de cromatografía de gases

y espectrometría de masas. Se utilizó una columna capilar polar Supelcowax-10 con

polietilenglicol de fase estacionaria. El gas portador utilizado fue el helio.

La identificación de los componentes se basó en la comparación espectral de los picos

del cromatograma obtenidos con los de la literatura científica. La cuantificación se

realizó en base al porcentaje de área de cada pico del cromatograma.

Las muestras del centro de Irán fueron analizadas por medio de cromatografía de

gases y espectrometría de masas. Se utilizó una columna capilar de sílice fundido. El

gas portador utilizado fue el helio. La identificación de los componentes se basó en la

comparación de los tiempos de retención obtenidos con los de la literatura científica.

Los índices de retención fueron determinados con la co-inyección de las muestras con

una solución de las series homólogas de n-alcanos C9-C18.

Las muestras Papúa Nueva Guinea fueron analizadas por medio de cromatogra-

fía de gases y espectrometría de masas. Se utilizó una columna capilar BPX-5. El gas

portador utilizado fue el helio. La identificación de los componentes se basó en la

comparación de los tiempos de retención obtenidos con los de la literatura científica.

Resultados

Componentes mayoritarios

Ocimum basicilum L, procedencia tailandesa

Como compuestos fenólicos predominantes, se halla el ácido rosmarínico (54%),

ácido chicórico (25%), y el ácido caftárico (8%). Este ácido está presente en todas las

muestras de hojas de albahaca, presentando un máximo de 88,5 mg en 100 g en peso

húmedo en esta albahaca tailandesa. Además, se ha detectado niveles bajos de ácido

chicórico en muestras de su tallo, a diferencia del resto de su especie. Sin embargo, no

se halló en su tallo nada de ácido caftárico. Por otro lado, no se han hallado en muestras

de hojas los derivados del ácido cafeico, ácido feruloil-tartárico, y el quercetin-

rutinósido.

Ocimum basicilum L, “italiano-genovés”

En esta especie se repite la prioridad de los compuestos, con el ácido rosmaríni-

co (54%), ácido chicórico (38%), y el ácido caftárico (1%). Se aprecia alto porcentaje

de ácido chicórico, sin embargo, no fue encontrado en sus tallos, así como el ácido

caftárico. También se han encontrado ácido feruloil-tartárico, derivados del ácido cafei-

co, ácido litospérmico, y derivados del ácido cinnámico en muestras de hojas.

Ocimum basicilum L, denominado “Petra Púrupura”

Se aprecian los mismos compuestos predominantes como ácido rosmarínico

(73%), ácido chicórico (16%), y el ácido caftárico (2%). Tampoco se ha observado

ácido chicórico en su tallo, así como ácido caftárico. En esta especie no se han encon-

trado compuestos como ácido feruloil-tartárico, derivados del ácido cafeico, ácido

litospérmico, y derivados del ácido cinnámico. Además, se han detectado grandes

cantidades de metil chavicol (52,4%) y linalool (20,1%).

Ocimum basicilum L, llamado comúnmente como albahaca dulce

De manera análoga, se detectaron ácido rosmarínico (70%), ácido chicórico

(23%), y el ácido caftárico (1%). Tampoco se aprecia ácido chicórico en su tallo,además

del ácido caftárico, ni antocianinas en muestras de hojas ni de tallos. Sin embargo, se

han encontrado derivados del ácido caffeico, ácido feruloyl-tartárico, y el quercetin-

rutinósido. El quercentin-rutinósido posee un pico menor que el resto de especies en

hojas y muestras de tallos.

Ocimum basilicum L, procedente de La Huerta, Jalisco, México

Se han detectado hasta 25 compuestos volátiles por vía cromatografía de gases-

espectrometría de masas. Se evaluaron dos fibras, Polidimetilsiloxano/Divnilbenceno

(PDMS/DVB) y Carbowax/Divinilbenceno (CW/DVB), para comparar extracción de

componentes. Serán comunes 18 compuestos entre ambas fibras. Además, se ha apre-

ciado mayor concentración en la fibra CW/DVB y se aislaron mayor número de terpe-

nos. Como compuestos mayoritarios, por orden decreciente, se destacan el cinamato de

metilo (fenilpropanoide), el linalol (monoterpeno), y el cinamato de etilo (fenilpropa-

noide). El cinamato de metilo aparece sobre el linalol con una razón de 1,5:1. En las

hojas también se hallan fenilpropanoides, monoterpenos, sesquiterpenos, y metabolitos

derivados de los ácidos grasos.

Ocimum basilicum L, procedente del noroeste de Irán

Se han detectado 47 componentes en el 97,9% del aceite total. Los compuestos

principales detectados son mentona (33,1%), estragol (21,5%), iso-neomentol (7,5%),

mentol (6,1%), mentil acetato (5,6%), pulegona (3,7%), α–cadinol (2,9%), trans-

cariofilene (2,2%), linalool (1,7%), limoneno (1,5%), germacrena D (1,4%), α-amorfena

(1,1%), metil eugenol (1%), y trans-β-farnesena (1%).

Observando los compuestos hallados en esta muestra de albahaca, se pueden

realizar las divisiones en las siguientes clases y subclases. Como monoterpenoides

(77,8%), se encuentran como subclases los monoterpenos oxigenados (75,3%) y los

hidrocarbonos monoterpenos (2,6%). Los monoterpenos oxigenados se caracterizan por

poseer grandes cantidades de metona (33,1%), estragol (21,5%), isoneomentol (7,5%),

mentol (6,1%), pulegona (3,7%) y linalool (1,7%). La mentona y el estragol compren-

den el 55% del aceite total, siendo éstos los compuestos mayoritarios en este grupo. El

limoneno (1,5%) es el único hidrocarburo monoterpeno con cantidades relativamente

altas.

Se diferencia otro grupo de sesquiterpenoides (12,8%), con subclases como los

hidrocarbonos sesquiterpenos (8,8%) y los sesquiterpenos oxigenados (4%). Esta sub-

clase de hidrocarbonos sesquiterpenos predominante consta de trans-cariofileno (2,2%),

germacreno D (1,4%), trans-β-farneseno (1,1%) y α-amorfeno (1,1%) como los com-

puestos más abundantes.

El α-Cadinol (2,9%), un sesquiterpeno oxigenado, tiene la cantidad más alta de

su subclase. Desde el punto de vista químico, los alcoholes (42%) son el grupo predo-

minante de compuestos, seguidos por las ketonas (37,8%), acetatos (5,9%), compuestos

metilados (1,1%) y óxidos (0,8%). Los principales miembros de los constituyentes

alcohólicos son estragol, isoneomentol, mentol, α-cadinol, metil eugenol y linalool.

Mentona y pulegona son los compuestos con ketona más importantes. Los compuestos

acetilados más representativos son los mentil acetato (5,6%). Finalmente, el metil

eugenol es el principal constituyente de los compuestos metilados.

Rendimiento y discusión

Se ha observado que los ácidos grasos insaturados y poliinsaturados, son los pre-

cursores de un gran número de compuestos volátiles que definen el aspecto aromático

de la planta. Los fenilpropanoides y los ésteres se encargan de dar olores afrutados; los

monoterpenos y sesquiterpenos dan aromas frescos, florales, a limón, dulces, herbáceos,

afrutados y amaderados; mientras que los aldehídos otorgan fragancias frescas, verdes,

cítricas, florales, jabonosas y oleosas. Generalmente, la albahaca puede dar olores como

limón, rosa, alcanfor, licor, amaderado y afrutado. Los fenilpropanoides se biosintetizan

a partir del ácido del ácido cinámico a través de la ruta del siquimato. Los monoterpenos

y sesquiterpenos tienen su origen biosintético en la vía del ácido mevalónico, también

conocida como ruta del mevalonato. Los ésteres y aldehídos resultan de la degradación

de los ácidos grasos insaturados y poliinsaturados.

El rendimiento de los aceites esenciales obtenidos de O. basilicum L púrpura y

O. basilicum verde fue de 0,2 y 0,5%, respectivamente. Estas diferencias pueden ser

debidas a los diferentes medios en los que crece la planta, factores genéticos, diversos

quimiotipos entres la especie, y la nutrición que lleve a cabo el espécimen en concreto.

La cantidad de citral recogido en estas muestras supera el 80,6%, compuesto

principalmente por geranial y neral, siendo éstos 2 aldehídos monoterpenos isómeros

que se producen simultáneamente, asociado con el aceite graso del limón. Sin embargo,

es en la especie Ocimum americanum donde se aprecia que estos citrales se dan en su

quimiotipo, siendo esto diferente en el caso de la albahaca, que no posee estas caracte-

rísticas y que solo comparte el linalool como componente importante. Se puede decir,

por tanto, que en la especie Ocimum basilicum está compuesta predominante por grupos

terpénicos, y por ello, son derivados de un único ácido mevalónico mediante biosíntesis.

La destilación en presencia de agua de plantas de Ocimum basilicum L., en agua

líquida, da un rendimiento del 0,7% (v/w) del peso seco de las partes aéreas, medido en

muestras de albahaca procedente de Irán. Este rendimiento no especifica si se da en el

resto de especies estudiadas.

El contenido de aglicones libres volátiles en muestra de planta seca, fue de 0,14

mg/g. Los principales aglicones encontrados son fenil-propano-eugenol (44%), chavicol

(29,5%), alcohol bencénico (5,7%), vanilina (2,9%), 2-fenil-etanol (2,7%), e isómeros

no identificados 3,7-dimetil-1,5-octadieno-3,7-diol (2,4%).

Como método de determinación de estos compuestos, cabe destacar que el ácido

chicórico fue encontrado por comparación en una muestra pura estándar, mediante

evaluación del tiempo de retención, espectro UV-visible, e información MS. El ácido

caftárico fue hallado por comparación del tiempo de retención, espectro UV-visible, ion

molecular, e iones fragmentados de uvas “Pinot noir”

Por lo general, como compuestos comunes entre esta albahaca dulce, tailandesa,

“italiano-genovés” y la “Petra Púrpura”, se ha detectado ácido litospérmico en todas

estas muestras de hoja de albahaca, pero no en tallos, aunque sí en raíces de albahaca.

Se ha encontrado además ácido 5-caffeoyl-quínico en estas albahacas pero éste y sus

isómeros (que producen ácido 1-caffeoyl-quínico, ácido 3-caffeoyl-quínico y ácido 4-

caffeoyl-quínico) no fueron hallados en las muestras.

Los tallos tienen menor variación en la composición de fenoles que las corres-

pondientes en las muestras de hojas. Se puede decir que los tallos de albahaca tienen

valores similares de fenoles totales, en comparación de sus hojas en las que surgen

diferencias. Además, es común encontrarse con compuestos de antioncianina, la cual

presenta mayor concentración en los tallos que en las hojas.

Los compuestos volátiles con glicósidos podrían ser interesantes como elemen-

tos potenciales todavía no estudiados de compuestos antioxidantes en albahaca o en

otras plantas. Está demostrado que el eugenol es el compuesto mayoritario responsable

de esta propiedad antioxidante, el cual aparece en gran cantidad en los aglicones voláti-

les (44%), lo que les dota de esta capacidad antioxidante, mucho mayor que su aceite

esencial. Esta propiedad antioxidante se da desde que los compuestos volátiles pueden

ser liberados de precursores no volátiles glicósidos, por métodos químicos o enzimáti-

cos durante el proceso de elaboración. Todo ello confirma el uso potencial de hierbas y

especias en la industria alimentaria para incrementar la vida útil de los alimentos comes-

tibles.

Albahaca

dulce

Albahaca tai-

landesa

Albahaca "italiano-

genovés"

Albahaca "Petra

Púrpura"

Hojas Tallos Hojas Tallos Hojas Tallos Hojas Tallos

Ácido caftárico

(mg/100g tejido) 16,5 - 1,93 - 3,34 - 0,44 -

Ácido chicórico

(mg/100g tejido) 51,8 - 88,5 0,3 24,8 - 11,42 -

Ácido rosmaríni-

co (mg/100g

tejido)

112 31,9 128 40,3 117 72,4 35,2 29,2

Fenoles totales

(mg/100g tejido) 208 35,9 236 50 160 76,8 50,5 31,9

Fórmulas químicas de los compuestos mayoritarios:

Ácido caftárico Ácido chicórico

Ácido rosmarínico Mentona

Estragol Chavicol

Conclusiones

La capacidad antioxidante de algunos de los compuestos de la albahaca confir-

maría un uso potencial de ésta en la industria alimentaria por su capacidad de aumentar

la vida útil de los alimentos.

La HPLC-DAD (cromatografía líquida de alto rendimiento con vector de diodos

de detección) realizada a la albahaca verifica la presencia de ácido chicorésico y ácido

caftárico en ésta. Este hallazgo proporciona beneficios potenciales para la salud de los

productos alimentarios agrícolas ya que el ácido chicorésico es mucho más fácilmente

disponible y barato en la albahaca que en las hojas de Echinacea purpurea, que se consi-

deraba tradicionalmente como la principal fuente de ácido chicorésico.

La presencia de mentona y estragol en el aceite esencial de la albahaca cultivada

en el noroeste de Irán supone diferencias sustanciales con informes anteriores, lo que

incrementaría la presencia de esta especie en las industrias alimentarias, cosméticas y

farmacéuticas.

La composición de la albahaca procedente de México está constituida por fenil-

propanoides con el cinamato de metilo como su compuesto más representativo y el

linalol es el monoterpeno más significativo. Además de comprobar que la microextrac-

ción en fase sólida seguida del análisis con cromatografía de masa-espectrometría de

masas es una técnica apropiada para obtener compuestos volátiles, se comprobó que en

las condiciones analíticas empleadas la fibra Carbowax/Divinilbenceno presentó una

mejor eficiencia de adsorción.

Cinamato de metilo Linalool Eugenol

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Faculty of Chemical Technology, Department of Biochemistry and Food Chemistry, University of Split, Teslina 10/V, 21000 Split, Croatia

Received 6 October 2005; received in revised form 23 January 2006; accepted 26 January 2006

Abstract

The present paper examines the chemical composition and antioxidant capacity of free volatile aglycones from basil compared to theiressential oil. The comparison of chemical composition of volatile aglycones with the chemical composition of essential oil reveals fourcommon compounds: eugenol, chavicol, linalool and a-terpineol. For the evaluation of the mentioned antioxidant capacities, two dif-ferent methods were performed: the 2,2 0-diphenyl-1-picrylhydrazyl radical scavenging method (DPPH) and ferric reducing/antioxidantpower assay (FRAP). DPPH method shows that free volatile aglycones possess good antioxidant properties comparable with that of theessential oil and well-known antioxidant butylated hydroxytoluene (BHT), but less than pure eugenol. The results obtained by FRAPmethod show that these compounds are some less effective antioxidants than essential oil and BHT.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Ocimum basilicum L.; Volatile aglycones; Essential oil; Chemical composition; GC–MS; Antioxidant capacity; DPPH; FRAP

1. Introduction

The antioxidants are an increasingly important ingredi-ent in food processing. Their traditional role involves, astheir name suggests, inhibiting the development of oxida-tive rancidity in fat-based foods, particularly meat anddairy products, and fried foods. The most widely used syn-thetic antioxidants in food (butylated hydroxytolueneBHT, butylated hydroxyanisole BHA) are very effectivein their role as antioxidants. However, their use in foodproducts has been failing off due to their instability, as wellas due to a suspected action as promoters of carcinogenesis(Namiki, 1990). For this reason, there is a growing interestin the studies of natural healthy (non toxic) additives aspotential antioxidants (Baratta et al., 1998; Tomainoet al., 2005).

Essential oil from aromatic and medicinal plants hasbeen known to possess biological activity, notably antibac-terial, antifungal and antioxidant properties (Baratta et al.,1998; Baratta, Dorman, & Deans, 1998).

Ocimum basilicum L. (Lamiaceae), respectively, namedbasil, is an aromatic herb that has been used traditionallyas a medicinal herb in the treatment of headaches, coughs,diarrhea, constipation, warts, worms and kidney malfunc-tions (Simon, Morales, Phippen, Vieira, & Hao, 1999). Ithas a long history as culinary herb, thanks to its foliage add-ing a distinctive flavor to many foods. It is also a source ofaroma compounds and essential oils containing biologicallyactive constituents that possess insecticidal (Deshpande& Tipnis, 1997), nematicidal (Chaterje, Sukul, Laskal, &Ghoshmajumdar, 1982), fungistatic (Reuveni, Fleisher, &Putievsky, 1984) and antimicrobial properties (Wannissorn,Jarikasem, Siriwangchai, & Thubthimthed, 2005).

Together with the essential oil, there is a growing inter-est for the study of glycosidically bound volatile com-pounds. These compounds have been extensively studied

0308-8146/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodchem.2006.01.045

* Corresponding author. Tel.: +385 21 385 633; fax: +385 21 384 770.E-mail address: olivera@ktf-split.hr (O. Politeo).

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Food Chemistry 101 (2007) 379–385

FoodChemistry

in case of grapes and wines (Gunata, Bayonove, Baumes, &Cordonnier, 1985), fruits (Schwab, Mahr, & Schreier,1989) and aromatic plants (Stahl-Biskup, Intert, Holthuij-zen, Stengle, & Schulz, 1993). To our knowledge, onlyone study was published about the chemical compositionof volatile aglycones from basil (Lang & Horster, 1977).The antioxidant capacity of basil essential oil has beenstudied several times (Baratta et al., 1998; Lee, Umano,Shibamoto, & Lee, 2005; Tomaino et al., 2005), but theantioxidant properties of glycosidically bound volatilecompounds from basil have not been studied to date. Theaim of this paper is to isolate and identify these compoundsas well as to determine their antioxidant capacity. This is apart of our investigation project, dealing with antioxidantcapacity of glycosidically bound volatile compounds fromaromatic plants (Milos, Mastelic, & Jerkovic, 2000; Rado-nic & Milos, 2003).

2. Materials and methods

2.1. Materials

Dried and chopped basil leaves (Kotanyi spice com-pany, Austria) were purchased from a local market in Split,Croatia. All of the applied chemicals were of pro analysispurity and were purchased from Fluka Chemie (Buchs,Switzerland).

2.2. Isolation of essential oil

The 100 g of plant material and 500 ml of water havebeen placed in a Clevenger type apparatus. The essentialoil was isolated by hydrodistillation for 3 h. The obtainedessential oil was separated, dried over anhydrous sodiumsulphate and stored under argon in a sealed vial, at�20 �C before usage. The voucher specimen of the basilleaves is deposited in the Laboratory of Biochemistry andFood Chemistry, Faculty of Chemical Technology, Split,Croatia.

2.3. Isolation of glycosidically bound volatile compounds

Upon the addition of internal standard, octyl-b-D-glucopyranoside, 100 g of plant material was extractedwith boiling ethyl acetate under reflux for 2 h. After perco-lation, the extract was concentrated to dryness in a rotat-ing evaporator, under reduced pressure. The residue wasdissolved in boiling water and, after cooling, the sedimentwas removed by filtration. The filtrate was subjected toliquid–solid chromatography in a glass column (150 ·20 mm) containing Amberlite XAD-2 as adsorbent(Gunata et al., 1985) at a rate of 2 ml/min. Sugars, aminoacids and proteins were removed by washing with 500 mlof distilled water. The glycosides extract was collected byeluting 100 ml of methanol. The methanolic extract con-taining the glycosides was concentrated to dryness underreduced pressure and redissolved in 2 ml of citrate-phos-

phate buffer (0.2 M, pH 5.0). The remaining volatile com-pounds were removed by liquid–liquid extraction with4 · 5 ml of n-pentane over 24 h. Prior to enzymatic hydro-lysis, TLC and GC–MS tested the absence of free volatilecompounds. Thin layer chromatography was performedon 0.2 mm precoated silica plates (Kiesegel 60, Merck)with hexane/ethyl acetate (85:15, v/v) as eluent. The vola-tile compounds were detected using 2% vanillin in concen-trated sulfuric acid.

2.4. Enzymatic hydrolysis and extraction of free volatile

aglycones

In a typical experiment, b-glucosidase from bitteralmonds (10 mg, 5–8 U/mg; Fluka) was added to the glyco-sidic extract. The enzymatic hydrolysis was realized during48 h at 37 �C. Occasionally, the mixture was shaken thor-oughly by hand. After hydrolysis, the liberated volatileaglycones were extracted from aqueous layer with4 · 5 ml of n-pentane. The combined pentane extract wasconcentrated to 0.5 ml and 2 ll was injected for GC–MSanalysis.

2.5. Gas chromatography–mass spectrometry

The analyses of the volatile compounds were run on aHewlett–Packard GC–MS system (GC 5890 series II;MSD 5971A, Hewlett–Packard, Vienna, Austria). Two col-umns of different polarity were used: a HP-101 column(Methyl silicone fluid, Hewlett–Packard; 25 m · 0.2 mmi.d., film thickness 0.2 lm) and a HP-20M column (Carbo-wax, Hewlett–Packard; 50 m · 0.2 mm i.d., film thickness0.2 lm). Oven temperature was programmed as follows:isothermal at 70 �C for 4 min, then increased to 180 �C,at a rate of 4 �C/min and subsequently held isothermalfor 15 min (for HP-20M column); isothermal at 70 �C for2 min, then increased to 200 �C, at a rate of 3 �C/minand held isothermal for 15 min (for HP-101 column). Thecarrier gas was helium (1 ml/min). The injection port tem-perature was 250 �C and the detector temperature was280 �C. Ionization of the sample components was per-formed in the EI mode (70 eV). Injected volume was 1 ll.The linear retention indices for all the compounds weredetermined by co-injection of the samples with a solutioncontaining the homologous series of C8–C22 n-alkanes(Van Den Dool & Kratz, 1963). The individual constitu-ents were identified by their identical retention indicesreferring to the compounds known from the literature data(Adams, 1995), and also by comparing their mass spectrawith spectra of either the known compounds or with thosestored in the Wiley mass spectral database (Hewlett–Pack-ard, Vienna, Austria). The aglycone concentrations werecalculated from the GC peak areas related to GC peak areaof 1-octanol (from the internal standard octyl-b-D-gluco-pyranoside). Preliminary GC–MS analysis showed theabsence of 1-octanol as potential aglycone in plantmaterial.

380 O. Politeo et al. / Food Chemistry 101 (2007) 379–385

2.6. Determination of antioxidant activity with 2,2 0-diphenyl-

1-picrylhydrazyl (DPPH) radical scavenging method

The antioxidant capacity of basil essential oil and of thevolatile aglycones was measured in terms of hydrogendonating or radical scavenging ability, using the stable rad-ical, DPPH (Brand-Williams, Cuvelier, & Berset, 1995). Anethanolic stock solution (50 ml of the antioxidant (concen-trations of stock solutions were 20, 10, 5 and 1 g/l for vol-atile aglycones (due to limited quantity of the aglyconesonly few concentration were used) and 50, 30, 20, 10, 5,1, 0.5, 0.3, 0.2, 0.1, 0.05, 0.03, 0.02, 0.01 g/l for essentialoil, BHT and eugenol) was placed in a cuvette, and 1 mlof 0.004% ethanolic solution of DPPH was added. Absor-bance measurements commenced immediately. Thedecrease in absorbance at 517 nm was determined byUV–VIS Perkin–Elmer Lambda EZ 201 spectrophotome-ter after 2 h for all samples. Ethanol was used to zero thespectrophotometer. The absorbance of the DPPH radicalwithout the antioxidant, i.e. the control, was measureddaily. Special care was taken to minimize the loss of freeradical activity of the DPPH radical stock solution (Blois,1958). All determinations were performed in triplicate. Thepercent inhibition of the DPPH radical by the samples wascalculated according to the formula of Yen & Duh (1994):

% Inhibition ¼ ððACð0Þ � AAðtÞÞ=ACð0ÞÞ � 100

where AC(0) is the absorbance of the control at t = 0 minand AA(t) is the absorbance of the antioxidant at t = 1 h.

2.7. Determination of ferric reducing antioxidant power

(FRAP assay)

Determination of ferric reducing/antioxidant powerFRAP is a simple direct test for measuring of antioxidantcapacity. This method was initially developed to assayplasma antioxidant capacity, but can be used for plantextracts too. The total antioxidant potential of the samplewas determined using a ferric reducing ability (FRAP)assay (Benzie & Strain, 1996) as a measure of ‘‘antioxidantpower’’. This assay measures the change in absorbance at593 nm owing to the formation of a blue colored Fe2+-tri-pyridyltriazine compound from colorless oxidized Fe3+

form by the action of electron donating antioxidants. Theworking FRAP reagent was prepared by mixing 10 partsof 300 mmol/l acetate buffer, pH 3.6, with 1 part of10 mmol/l TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/lhydrochloride acid and with 1 volume of 20 mmol/l ferricchloride. Freshly prepared FRAP reagent (1.5 ml) waswarmed to 37 �C and a reagent blank reading was takenat 593 nm (M1 reading). Subsequently, 50 ll of the sample(concentrations of stock solutions were 20, 10, 5 and 1 g/l)and 150 ll of deionized water were added to the FRAPreagent. Final dilution of the sample in the reaction mix-ture was 1:34. The sample was incubated at 37 �C through-out the monitoring period. The change in absorbancebetween the final reading (4-min reading) and the M1 read-

ing was selected for the calculation of FRAP values. Stan-dard curve was prepared using different concentrations(0.1–5 mmol/l) of FeCl2 · 4H2O. All solutions were usedon the day of preparation. In the FRAP assay, the antiox-idant efficiency of the antioxidant under the test was calcu-lated with reference to the reaction signal given by a Fe2+

solution of known concentration. The results were cor-rected for dilution and expressed in mmol Fe2+/l. All deter-minations were performed in triplicate.

3. Results and discussion

3.1. Chemical composition of free volatile aglycones

compared with essential oil composition

The content of free volatile aglycones in dried plantmaterial was 0.14 mg/g. As shown in Table 1, the GC–MS analysis of the aglycones revealed twenty-three com-pounds, representing 96.3% of the total aglycone fraction.Aliphatic alcohols and acids, terpene compounds, deriva-tive of phenylpropanes and derivative of norisoprenoidwere identified. The main aglycones were phenylpropa-noids eugenol (44.0%) and chavicol (29.5%). Other quanti-tatively important aglycones were benzyl alcohol (5.7%),vanillin (2.9%), 2-phenyl ethanol (2.7%) and not identifiedisomer of 3,7-dimethyl-1,5-octadiene-3,7-diol (2.4%). Theobtained results show only some qualitative similarity withthose reported by Lang & Horster (1977). They found onlyfour compounds in aglycone fraction of morphologicalundifferentiated callus and cell-suspension cultures withthymol and linalool as major ones. This is probably dueto difference in the isolation of glycosides. The aglyconessuch as aliphatic alcohols, 2-phenylethanol, benzyl alcohol,eugenol, linalool, geraniol, nerol and a-terpineol can, moreor less, be considered common in aglycone fraction ofLamiaceae family (Stahl-Biskup et al., 1993) and the euge-nol was found to be the main aglycone in most plants ofthis family (Milos et al., 2000; Radonic & Milos, 2003;Stahl-Biskup et al., 1993).

The total content of the essential oil (yield = 6.20 mg/g),determined by the gravimetric method, is 44 times higherthan that of the aglycones. Among 33 compounds identi-fied in basil essential oil, representing 97.0% of the totaloil (Table 2), monoterpene compounds, sesquiterpene com-pounds and derivative of phenylpropanoid were identified.The major compound was monoterpene alcohol linalool(28.6%). The second most important compound was phe-nylpropanoid estragole with the peak area of 21.7%. Otherimportant compounds were (E)-methyl cinnamate (14.3%),a-cadinol (7.1%), eugenol (5.9%), 1,8-cineole (4.0%),methyl eugenol (3.1%) and a-bergamotene (2.2%). Thismeans that this essential oil represents a true methyl cinna-mate chemotype (Guenter, 1965). All this supports theassumption that if alcohols and phenols are the main com-ponents of the essential oil, the corresponding aglyconescan also be detected (Stahl-Biskup & Holthuijzen, 1995).Comparing the chemical composition of the essential oil

O. Politeo et al. / Food Chemistry 101 (2007) 379–385 381

and aglycones, four compounds were established to becommon: eugenol, chavicol, linalool and a-terpineol. Ourresults show moderate qualitative correlation in the chem-ical composition of essential oil and free volatile aglyconesof this plant.

3.2. Antioxidant capacity of glycosidically bound volatile

aglycones compared with that of essential oil

The DPPH method was used to evaluate the antioxidantcapacity of basil volatile aglycones and essential oil in com-parison with known synthetic antioxidant BHT and pureeugenol. The decrease in absorbance at room temperaturewas measured every 15 min until the reaction reachedsteady state or until absorbance declined less than 10%.Typical profiles of the percent inhibition of the DPPH rad-ical by essential oil, volatile aglycones, BHT and pure euge-nol in the presence of stock solution concentration of 20 g/lare shown in Fig. 1. The percent inhibition of the DPPHradical as a function of the concentration for essentialoil, corresponding mass of eugenol in essential oil and pure

eugenol is shown in Fig. 2a, and that for volatile aglycones,corresponding mass of eugenol in volatile aglycones andpure eugenol is shown in Fig. 2b. The decrease in concen-tration of the aglycones produced reduction in their capac-ities. The same behavior was observed for the essential oilas well as BHT and pure eugenol. Antioxidant capacities inseries of concentrations of each of volatile aglycones, essen-tial oils and pure compounds were used to calculate theeffective relative concentration EC50. The amount of sam-ple, necessary to decrease the absorbance of DPPH by50% (EC50), was calculated graphically (% of inhibitionwas plotted against the logarithm of antioxidant concentra-tion in reaction system). The data given in Table 3 showthat eugenol possess the best radical scavenging capacity(EC50 = 0.096 g/l). The basil essential oil and known syn-thetic antioxidant BHT showed similar capacities

Table 1Chemical composition of free volatile aglycones isolated from basil

No. Identified compound Peakarea (%)

RIa HP-20M RIa HP-101

1 Hex-2-en-4-in-1-ol 0.2 1256 –2 3-Hexen-1-ol 1.4 1351 8383 1-Okten-3-ol 0.5 1412 9634 Linalool 0.8 1510 10845 Hotrienol 0.3 1570 –6 Lavandulol 0.7 1634 11577 a-Terpineol 0.4 1650 11728 3,7-Dimethyl-1,

5-octadiene-3,7-diolc0.4 1676 –

9 2-Butenoic acid 0.1 1709 –10 2-Hydroxymethyl

benzoate0.2 1715 –

11 Nerol 0.8 1751 125312 Geraniol 0.4 1803 125813 Benzyl alcohol 5.7 1819 109614 2-Phenyl ethanol 2.7 1844 114915 3,7-Dimethyl-1,

5-octadiene-3,7-diolc2.4 1903 1615

16 2,20-Oxybis-diacetate ethanol

1.7 1937 1303

17 Eugenol 44.0 2116 138518 Chavicol 29.5 –b –19 Phytol 0.3 –b –20 Vanillin 2.9 –b 150121 3-Hydroxy-b-damascone 0.4 – 162722 4-Hydroxy-3,

5-dimethoxybenzaldehyde0.2 – 1746

23 Hexadecanoic acid 0.3 – 1972

Total 96.3

–, not identified.a Retention indices relative to C8–C22 n-alkanes on polar HP-20M and

apolar HP-101 column.b Retention times is outside of retention times of homologous series of

C8–C22 n-alkanes (identified by MS).c Correct isomer (E or Z) is not identified. Identification is performed by

MS.

Table 2Chemical composition of basil essential oil

No. Identified compound Peak area (%) RIa HP-20M RIa HP-101

1 b-Pinene 0.1 – 9492 Limonene 0.1 1180 10053 1,8-Cineole 4.0 1185 10064 Camphor 0.5 1477 11095 Linalool 28.6 1518 10926 Bornyl acetate 0.5 1545 12527 Terpinen-4-ol 0.7 1563 11548 a-Bergamotene 2.2 1564 14079 Caryophyllene 0.3 – 1385

10 Aloaromadendrene 0.1 – 145011 Estragole 21.7 1632 117712 a-Terpineol 1.0 1653 117613 Germacrene D 0.3 1673 144414 a-Humulene 0.2 – 141715 Carvone 0.4 1685 120716 b-Cubebene 0.5 1694 105917 b-Burbonene t – 135418 b-Elemene 0.3 – 136419 c-Cadinene 0.2 1716 142620 Calamenene 0.2 – 148321 a-Amorphene 1.0 1710 147922 b-Farnesene 0.2 – 145223 D-Cadinene 0.1 1724 148624 a-Bisabolene 0.1 – 150625 (Z)-Methyl

cinnamate1.6 1900 1281

26 Methyl eugenol 3.1 1959 137827 (E)-Methyl

cinnamate14.3 2019 1364

28 Spatulenol 0.8 2066 –29 Eugenol 5.9 2105 136830 Carvacrol t 2118 181431 a-Cadinol 7.1 2120 161432 Torreyol 0.2 2173 –33 Chavicol 0.7 – –b

Total 97.0

–, not identified.t, trace (<0.1%).a Retention indices relative to C8–C22 n-alkanes on polar HP-20M and

apolar HP-101 column.b Retention times is outside of retention times of homologous series of

C8–C22 n-alkanes (identified by MS).

382 O. Politeo et al. / Food Chemistry 101 (2007) 379–385

(EC50 = 1.378 g/l and 0.908 g/l), while the capacity ofthe basil volatile aglycones was significantly lower(EC50 = 3.338 g/l). The radical scavenging capacity forthe corresponding mass of eugenol in essential oil wasshown to be comparable to the capacity for the pure euge-

nol (EC50 = 0.099 g/l), while EC50 for corresponding massof eugenol in volatile fraction was 1.950 g/l.

FRAP assay is quick and simple to perform, and the reac-tion is reproducible and linearly related to the molar concen-tration of the antioxidant present. It is based on comparison

0

25

50

75

100

0 30 60 90 120

time (min)

AI

(%)

essential oil

volatileaglycones

BHT

eugenol

Fig. 1. Typical profile of the percent inhibition of the DPPH radical in the presence of 20 g/l of basil volatile aglycones, basil essential oil, BHT and pureeugenol.

0

25

50

75

100

0.001 0.01 0.1 1 10 100

log conc. (g/l)

AI

(%)

eugenol

volatile aglycones

equiv. mass of eugenol involatile aglycones

0

25

50

75

100

0.001 0.01 0.1 1 10 100

log conc. (g/l)

AI

(%)

eugenol

essential oil

equiv. mass of eugenol inessent. oil

(a)

(b)

Fig. 2. Antioxidant capacity for total essential oil, corresponding mass of eugenol in essential oil and pure eugenol (a) as well as volatile aglycones,corresponding mass of eugenol in volatile aglycones and pure eugenol (b), measured by DPPH method.

O. Politeo et al. / Food Chemistry 101 (2007) 379–385 383

of the total amount of antioxidant to the reducing capacity ofthe sample. Absorbance changes are linear over a wide con-centration range of antioxidant mixture. The reducing power(FRAP) of the liberated volatile aglycones and the essentialoil, as well as BHT and pure eugenol, as determined byFRAP assay, is shown in Fig. 3. As in the previous method,decrease in concentration caused a reduction in the reducingpower for all of the applied samples. The highest reducingpower (FRAP) shows samples of pure eugenol. The reducingpower of the volatile aglycones is comparable, but less thanthe reducing power of essential oil and BHT. A hierarchy inthe reducing capacity of samples could be observed as well:eugenol > BHT > basil essential oil > basil volatileaglycones.

Among the compounds identified in the essential oil andvolatile aglycones from basil, eugenol was considered themain contributor of the antioxidant capacity. The antioxi-dant capacity of eugenol (Dorman, Surai, & Deans, 2000;Nagababu & Lakshmaiah, 1992; Satoh, Ida, Sakagami,Tanaka, & Fusisawa, 1998) has been reported earlier. Sur-prisingly, in spite of much higher percentage of eugenol involatile aglycones (44.0%) than in essential oil (5.9%), theessential oil shows higher antioxidant capacity. The com-parison of the plots in Fig. 2a shows that the antioxidantcapacity of pure eugenol (EC50 = 0.096 g/l) is more potent

than that of the total essential oil (EC50 = 1.378 g/l). Butthe comparison of the plot for pure eugenol with the plotcorresponding to equivalent mass of eugenol in total essen-tial oil (EC50 = 0.099 g/l) shows very similar antioxidantcapacities for both in all concentration range. This findingclearly suggests that the antioxidant capacity of total essen-tial oil is due only or mainly to the presence of eugenol(5.9%) in its chemical composition and that other constitu-ents do not have significant effect on eugenol capacity. Theplots presented in Fig. 2b show that the antioxidant capac-ity of total aglycones (EC50 = 3.338 g/l) is also mainly dueto the presence of eugenol (44%) in its composition(EC50 = 1.950 g/l), but it is obvious that other aglyconesantagonize antioxidant capacity compared to pure eugenolcapacity (EC50 = 0.096 g/l). It could be explained withsmaller antioxidant capacity of volatile aglycones com-pared to total essential oil. However, the DPPH resultsshow that glycosidically bound basil aglycones possess agood free radical scavenging capacity comparable withbasil essential oil and well-known antioxidant butylatedhydroxytoluene (BHT), but less than pure eugenol. Theresults obtained by FRAP method show that these com-pounds were some less effective antioxidants than essentialoil and BHT. The observed differences can be explained bydifferent solvent polarity in the two assays. It is known thatsubstrate polarity does not affect to DPPH scavengingcapacity (Koleva, van Beek, Linssen, de Groot, & Evstati-eva, 2002).

Glycosidically bound volatile compounds could be inter-esting as hidden potential of antioxidant compounds in basilor in other plants. Since volatile compounds can be releasedfrom nonvolatile glycoside precursors by enzymatic orchemical pathways during manufacturing process, thesecompounds can be considered as potential precursors ofantioxidant substances in plant material and may contributeto the total antioxidant capacity of plants. This confirms the

Table 3Radical scavenging of basil volatile aglycones, basil essential oil, BHT andpure eugenol determined with DPPH method

Antioxidant EC50a

Volatile aglycones 3.338Essential oil 1.378BHT 0.908Eugenol 0.096Equivalent mass of eugenol in essential oil 0.099Equivalent mass of eugenol in volatile aglycones 1.950

a Concentration (g/l) for a 50% inhibition.

Fig. 3. The reducing power of the basil volatile aglycones, essential oil plus BHT and pure eugenol by using FRAP method.

384 O. Politeo et al. / Food Chemistry 101 (2007) 379–385

potential use of herbs and spices in the food industry toincrease the shelf life of foodstuffs. The antioxidant proper-ties of glycosidically bound volatile compounds from otherplants merit being the objective of future researches.

Acknowledgement

This work was supported by the Ministry of Science,Education and Sports of the Republic of Croatia, Project0011-003.

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O. Politeo et al. / Food Chemistry 101 (2007) 379–385 385

Chicoric acid found in basil (Ocimum basilicum L.) leaves

Jungmin Lee a,*, Carolyn F. Scagel b

a United States Department of Agriculture, Agricultural Research Service, PWA, Horticultural Crops Research Unit Worksite, 29603 U of I Lane, Parma, ID 83660, USAb United States Department of Agriculture, Agricultural Research Service, Horticultural Crops Research Unit, Corvallis, OR 97330, USA

a r t i c l e i n f o

Article history:Received 28 August 2008Received in revised form 18 December 2008Accepted 19 December 2008

Keywords:PhenolicsCichoric acidCaffeic acid derivativesDicaffeoyltartaric acidLamiaceaeGlomus intraradices

a b s t r a c t

This is the first report to identify the presence of chicoric acid (cichoric acid; also known as dicaffeoyltar-taric acid, which is a caffeic acid derivatized with tartaric acid) in basil leaves. Rosmarinic acid, chicoric acidand caftaric acid (in the order of most abundant to least; all derivatives of caffeic acid) were identified infresh basil leaves. Rosmarinic acid was the main phenolic compound found in both leaves and stems. Chic-oric acid was not detected in sweet basil stems, although a small amount was present in Thai basil stems.Other cinnamic acid monomers, dimers and trimers were also found in minor quantities in both stems andleaves. Basil polyphenolic contents were determined by blanched methanol extraction, followed by HPLC/DAD analysis. The characterization of the polyphenolics found in the basil extracts were performed byHPLC/DAD/ESI–MS/MS and co-chromatographed with purchased standard. The influence of inoculationwith an arbuscular mycorrhizal fungus (AMF), Glomus intraradices, on plant phenolic composition wasstudied on two basil cultivars,‘Genovese Italian’ and ‘Purple Petra’. Inoculation with AMF increased totalanthocyanin concentration of ‘Purple Petra’ but did not alter polyphenolic content or profile of leavesand stems, of either cultivar, compared to non-inoculated plants. In the US diet, basil presents a more acces-sible source of chicoric acid than does Echinacea purpurea, in which it is the major phenolic compound.

Published by Elsevier Ltd.

1. Introduction

Culinary herbs have been reported to possess antioxidant activ-ities (Yanishlieva, Marinova, & Pokorny, 2006) suggesting that theymight have potential human health benefits. Basil (family Lamia-ceae) is a popular herb in the US and Mediterranean diets. Basil’simportance as a culinary herb, its historic usage, essential oil com-position and phenolics have been well reviewed by Makri andKintzios (2008). Basil has shown antioxidant and antimicrobialactivities due to its phenolic and aromatic compounds (Gutierrez,Barry-Ryan, & Bourke, 2008; Hussain, Anwar, Sherazi, & Przybylski,2008; Javanmardi, Khalighi, Kashi, Bais, & Vivanco, 2002;Yanishlieva et al., 2006).

The main phenolics reported in basil are phenolic acids and fla-vonol-glycosides (Javanmardi et al., 2002; Jayasinghe, Gotoh, Aoki,& Wada, 2003; Kivilompolo & Hyotylainen, 2007; Kosar, Dorman, &Hiltunen, 2005; Nguyen & Niemeyer, 2008; Tada, Murakami,Omoto, Shimomura, & Ishimaru, 1996). The presence of caffeic acidderivatives (phenolic acid class) has been reported in basil(Jayasinghe et al., 2003; Kivilompolo & Hyotylainen, 2007; Nguyen& Niemeyer, 2008; Tada et al., 1996; Toussaint, Smith, & Smith,

2007), but complete phenolic profiles of basil have not been re-ported. Of the caffeic acid derivatives in basil, the present studyis the first to identify the presence of chicoric and caftaric acidsin basil, respectively the second and third major phenolic acidspresent in basil leaves.

Chicoric acid is the dominant phenolic reported in Echinacea pur-purea (Molgaard, Johnsen, Christensen, & Cornett, 2003; Perry,Burgess, & Glennie, 2001) and has been found in all parts (flowerheads, leaves, stems and root) of the E. purpurea plant ( Molgaardet al., 2003). Echinacea (family Asteraceae) has been reported to havepotential antioxidant, anti-inflammatory, antiviral and immuno-stimulating properties, arising from the naturally occurringalkamides, caffeic acid derivatives, polysaccharides and glycopro-teins (Barnes, Anderson, Gibbons, & Philipson, 2005; Charvat, Lee,Robinson, & Chamberlin, 2006; Dalby-Brown, Barsett, Landbo,Meyer, & Molgaard, 2005; Molgaard et al., 2003; Perry et al., 2001).However, in a well-summarized and detailed review of Echinaceaspecies’ possible health benefits, its effectiveness was no better thanthat of a placebo (Barnes et al., 2005 and references therein).

Chicoric acid itself has been reported to inhibit HIV integrase(Charvat et al., 2006) and to exhibit antioxidant activities(Dalby-Brown et al., 2005). As dietary supplements, Echinacea her-bal extracts have been very popular (US annual sales estimated at$100–200 million during the years 2000–2006; Tilburt, Emanuel, &Miller, 2008) but, for US consumers, basil represents a more readilyavailable and inexpensive source of these compounds.

0308-8146/$ - see front matter Published by Elsevier Ltd.doi:10.1016/j.foodchem.2008.12.075

* Corresponding author. Tel.: +1 208 722 6701x282; fax: +1 208 722 8166.E-mail addresses: jungmin.lee@ars.usda.gov (J. Lee), carolyn.scagel@ars.usda.gov

(C.F. Scagel).

Food Chemistry 115 (2009) 650–656

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Phenolic production in a plant can be affected by biotic and abi-otic factors (Hussain et al., 2008; Lee & Martin, 2009; Toussaintet al., 2007; Yao, Zhu, & Zeng, 2007). Colonization by arbuscularmycorrhizal fungi (AMF) is known to improve plant nutrient up-take and use (Clark & Zeto, 2000), and stress tolerance (Smith &Read, 1997). AMF colonization can also alter or enhance phenolicproduction within the host plant (Toussaint et al., 2007; Ganz,Kailis, & Abbott, 2002). The relationship between AMF colonizationand the phenolic compounds produced in plants is still not wellunderstood (Toussaint, 2007; Yao et al., 2007).

The objectives of this study were to better identify the phenoliccompounds found in the aerial portions of the fresh basil plant,and to determine the impact of AMF on plant phenolics within twocultivars. The polyphenolic contents of basil samples were deter-mined using a blanched methanol extraction procedure, followedby HPLC/DAD analysis. The characterization of the polyphenolicsfound in the basil extracts was performed by HPLC/DAD/ESI–MS/MS.

2. Materials and methods

2.1. Plant materials

Common sweet basil and Thai basil (Ocimum basilicum, specificcultivar names unknown) were purchased from a local market inNampa, ID, USA. Edible portions (mainly leaves) were separatedfrom stems, and both fractions were then stored frozen at �80 �Cprior to extraction. Purchased sweet basil will be referred to assweet basil, for clarification hereafter, to distinguish it from theother (sweet) basil samples of the AMF portion of this study.Two basil cultivars (Genovese Italian and Purple Petra) were usedto examine the effect of inoculation with AMF on plant phenolicproduction.

Echinacea whole plant herbal supplement extract was pur-chased from a local shop (Nampa, ID, USA) and analyzed by HPLC.The Echinacea herbal extract contained E. purpurea, according tothe manufacturer’s label. All chemicals for polyphenolic extractionand HPLC analysis were obtained from Sigma Chemical Co. (St.Louis, MO, USA) unless indicated otherwise. Solvents and chemi-cals for this investigation were of analytical and high performanceliquid chromatography (HPLC) grade. Purified chicoric acid stan-dard was purchased from Indofine Chemical Company, Inc.(Hillsborough, NJ, USA).

2.2. AMF inoculum

The AMF, Glomus intraradices, originally obtained from NativePlants Incorporated (Salt Lake City, UT, USA), was propagated inpot cultures on roots of bunching onion (Allium cepa L. ‘WhiteLisbon’) grown in a 1:1 mixture of Willamette Valley alluvial siltloam and river sand (8 mg kg�1 available phosphorus, pH 6.3) forfive months. Inoculum consisted of a mixture of the soil substrate,extraradical hyphae and spores, and colonized root segments(<2 mm in length). Propagule numbers (10 propagules g�1 of soilsubstrate) in the inoculum used in this study were estimated usingthe MPN (most probable number) method (Woomer, 1994).

2.3. Plant culture and AMF inoculation

Surface-sterilized (30% sodium hypochlorite for 10 min, fol-lowed by a sterile water rinse) basil seeds were sown in 4 in. pots(Gage Dura Pot #GDP400) containing a steam-pasteurized (60 �Cfor 30 min) 3:1 mixture of Willamette Valley alluvial silt loamand vermiculite (Horticultural Vermiculite, SunGro Horticulture,Bellevue, WA, USA). Available P in the growing substrate was3 mg kg�1, and pH was 6.2. One-half of the plants were inoculated

with AMF by hand-mixing 52 g of the AMF inoculum into thegrowing substrate in each pot before sowing seeds (AMF treat-ment). The same quantity of sterile (121 �C, 15 min) inoculumwas mixed into the growing substrate for the remainder of theplants before sowing seeds (control). Containers were placed in aglass house and watered as needed. Supplemental light(�720 lmol PAR m�2 s�1) was provided for 16 h d�1 by high-pres-sure multi-vapour lamps. Day/night temperatures were controlledat �24/15 �C during the experiment. After cotyledons had fully ex-panded, plants were fertilized weekly with 50 ml of a liquid fertil-izer (Peters Professional, Scotts Company, Maysville, OH, USA)containing 380 mg N l�1, 150 mg P l�1, 380 mg P l�1, 64 mg S l�1,100 mg Ca l�1, 36 mg Mg l�1, 4.6 mg Fe l�1, 18 mg Cl l�1, 0.55 mgMn l�1, 0.37 mg B l�1, 0.024 mg Zn l�1, 0.06 mg Cu l�1, and0.006 mg Mo l�1. Plants were pruned back, to three nodes, sixweeks after germination and flowers that began to develop nineweeks after germination were removed from plants.

2.4. Plant harvest and colonization assessment

All plants were destructively harvested 16 weeks after germina-tion and separated into leaves, stems, and roots. Substrate was re-moved from roots by washing and samples of roots were taken forassessing AMF colonization. Samples of leaves and stems were ta-ken for phenolic analyses (described below). The samples of freshroots were cleared and stained, using a modified procedure of Phil-lips and Hayman (1970), in which lacto-phenol was replaced withlacto-glycerin, and assessed for AMF colonization. AMF coloniza-tion was measured on �1 cm sections of root samples, using theBiermann and Linderman (1980) method.

2.5. Sample preparation and extraction for polyphenolic analysis

Frozen samples were liquid nitrogen-powdered and extractedwith acidified methanol (0.1% formic acid, v/v) as described in Leeand Finn (2007) with the following modification: An IKA M20 Uni-versal mill (IKA works Inc., Wilmington, NC, USA) was used to grindfrozen samples. Frozen powder (5 g) with the first methanol addi-tion was blanched in a boiling water bath for 5 min, then immedi-ately chilled in an ice bath for 10 min. This chilled mixture wascentrifuged and the pellet was re-extracted, two additional times,with acidified methanol, as described in Lee and Finn (2007). Meth-anol was evaporated with a RapidVap Vacuum Evaporation System(Labconco Corp., Kansas City, MO, USA) at 40 �C, and re-dissolved inwater to a final volume of 25 ml. Aqueous extracts were stored at�80 �C prior to further analyses.

To examine the importance of inactivating native enzymes inphenolic retention, sets of sweet basil leaves were powdered andthen extracted, with and without the initial blanching step (intriplicates).

2.6. Total anthocyanins (TACY) and total phenolics (TP) determination

TACY were analyzed as described in Lee, Durst, and Wrolstad(2005) and TP were determined as described by Waterhouse(2002). TACY absorbance was measured at 520 and 700 nm, andexpressed as mg cyanidin-3-glucoside 100 g�1 tissue. TP absor-bance was taken at 765 nm and was expressed as mg gallicacid 100 g�1 tissue. A SpectraMax M2 microplate reader (Molecu-lar Devices Corp., Sunnyvale, CA, USA) was used. Both measure-ments were conducted in duplicate.

2.7. HPLC/DAD and HPLC/DAD/ESI–MS/MS analyses

Basil and Echinacea herbal extracts were analyzed as describedin Lee and Finn (2007). Briefly, solid phase C18 (Sep-Pak Plus;

J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650–656 651

Waters Corp. Milford, MA, USA) clean-up, prior to HPLC analysis,was conducted (Lee & Finn, 2007). HPLC/DAD (HP1100; AgilentTechnologies, Inc., Palo Alto, CA, USA) and HPLC/DAD/ESI–MS/MS(MS was an XCT ion trap mass spectrometer, Agilent Technologies,Inc.) system and conditions were performed, using a previouslypublished method (Lee & Finn, 2007). ESI was operated in negativeionization mode. Phenolic acids were quantified as caffeic acid at320 nm. Flavonol-glycoside was quantified as quercetin-rutinosideat 370 nm. Both classes of phenolics were quantified, based onexternal standards. Phenolic compounds were identified, basedon UV–visible (UV–vis) spectra, retention time, molecular ionsand fragmentation ions. When available, authentic standards wereused to aid the identification of phenolics (namely, caffeic acid,chicoric acid, quercetin-rutinoside, and rosmarinic acid). A sumof the individual phenolics (determined by HPLC) will be referredto as total polyphenolics, to distinguish these values from TP in thispaper. Phenolics other than anthocyanins will be referred to aspolyphenolics in the following sections for conciseness.

2.8. Statistical analysis

Statistica for Windows version 7.1 (StatSoft Inc., Tulsa, OK, USA)was used for t-test calculations and one-way analysis of variance(ANOVA) for all measurements (a = 0.05). The Pearson product mo-ment correlation coefficient (r) was used to assess the relationshipbetween the TP obtained via spectrophotometer and total poly-phenolics by HPLC (a = 0.05).

3. Results and discussion

3.1. Identification and quantification of phenolics from basil

Three major phenolic compounds were found in purchasedfresh sweet and Thai basil leaf samples (Tables 1–3), and in sam-ples of ‘Genovese Italian’ and ‘Purple Petra’ (Table 4). Most of thebasil phenolics were derivatives of caffeic acid. The dominant phe-nolic in all samples, regardless of plant tissue or cultivar (Tables 2–4), was rosmarinic acid (peak 11; Table 1 and Fig. 1A). Rosmarinicacid (caffeic acid dimer) has been found as the dominant phenolicin basil by numerous other researchers ( Javanmardi et al., 2002;Jayasinghe et al., 2003; Nguyen & Niemeyer, 2008). Rosmarinicacid is also the main phenolic acid in many other herbs of the fam-ily Lamiaceae, namely, oregano, sage, thyme, rosemary, spearmint,and marjoram (Fecka & Turek, 2008; Kivilompolo & Hyotylainen,2007).

The second most dominant phenolic, and previously unidenti-fied in basil leaves, was chicoric acid (peak 7; Table 1 and

Fig. 1A). Identity of chicoric acid in basil leaves (Fig. 1A) was con-firmed by comparison with a purchased pure standard (Fig. 1C)and E. purpurea herbal extracts (Fig. 1B), by evaluating retentiontime, UV–vis spectra (Fig. 2), and MS information (Table 1). TheUV–vis spectra of chicoric acid from the three samples (sweet basilleaves, E. purpurea herbal extract, and pure standard) are overlaidin Fig. 2. Chicoric acid was present in all basil leaves examined (Ta-bles 2 and 4), with Thai basil leaf samples having the highest levelof chicoric acid (88.5 mg 100 g�1 fresh weight; Table 2). Making di-rect comparisons between chicoric acid concentrations in basilleaves to concentrations reported in E. purpurea is complicatedby other findings being expressed as mg g�1 of dry weight or mlof extract (Bergeron, Gafner, Batcha, & Angerhofer, 2002; Molgaardet al., 2003; Perry et al., 2001). A small level of chicoric acid wasfound in Thai basil stems (Table 2). Chicoric acid was not detectedin stems of sweet basil, ‘Genovese Italian’, or ‘Purple Petra’.

The third major phenolic identified was caftaric acid (caffeoyl-tartaric acid). This is the first report identifying caftaric acid in basilleaves, though tartaric acid has been reported (Leal et al., 2008).Identification of caftaric acid (peak 2 in Table 1 and Fig. 1A) wasconfirmed by comparing retention time, UV–vis spectra, molecularion, and fragmented ions to caftaric acid from ‘Pinot noir’ grapes(Lee & Martin, 2009). The basil caftaric acid peak was also com-pared to the caftaric acid peak of commercially-available E. purpu-rea herbal extracts (Fig. 1B), since they have been reported tocontain caftaric acid (Bergeron et al., 2002; Perry et al., 2001). Caf-taric acid was not detected in any stem samples from basil (Tables2 and 4).

We suspect that the potency of fraction VI from basil leaves, de-scribed by Jayasinghe et al. (2003), as having the most antioxidantactivity, was due to chicoric acid. Though unidentified, Jayasingheet al. (2003) suspected the dominant peak in fraction VI to be a caf-feoyl derivative. Results presented by Nguyen and Niemeyer(2008) showed two other major 330 nm absorbers (present in theirbasil samples with much higher peak areas than caffeic acid),which the authors did not identify. We suspect that these uniden-tified peaks were caftaric acid and chicoric acid.

Jayasinghe et al. (2003) reported the presence of numerous phe-nolic compounds in basil leaves (�20 compounds). We did not findsimilar variation in phenolic composition in our basil leaf samples,possibly due to differences in cultivars, plant-growing conditions,sample preparation methods and analytical conditions. Our datashow some variation in phenolic composition between the basilcultivars. Peaks 1, 4, 6, and 9 were present in extracts from sweetbasil leaves but not Thai basil leaf samples (Table 2). Similarly,peaks 2, 4, 6, 8, and 10 were present in extracts from leavesof ‘Genovese Italian’ but not ‘Purple Petra’. Additionally, the

Table 1Phenolics found in market-purchased basil leaves (Ocimum basilicum) from an unnamed cultivar of sweet basil. Of the basil cultivars examined in this study, this cultivarcontained the most diverse assortment of compounds. ESI was operated in negative mode.

Peaksa Compounds Molecular ions [M-H]� Fragmented ions (m/z) kmax (nm)b

1 Caffeic acid derivative 359 197, 179 [caffeic acid-H]� 3352 Caftaric acid 311 179 [caffeic acid-H]-, 149 [tartaric acid-H]� 330, 300s3 Cinnamyl malic acid 295 163 [M-H-malic acid]� = [coumaric acid-H]�, 131 [malic acid-H]�, 113, 119 315, 295s4 Feruloyl tartaric acid 324 282, 193 [ferulic acid-H]�, 149 [tartaric acid-H]- 330, 300s5 Caffeic acid 179 135 320, 290s6 Caffeic acid derivative 179 149, 135 330, 300s7 Chicoric acid 472 309 [M-H-tartaric acid]�, 291 [M-H-caffeic acid]�, 179 [caffeic acid-H]� 330, 300s8 Lithospermic acid 536 491 330, 300s9 Quercetin-rutinoside 609 301 [M-H-rutinoside]� = [quercetin-H]� 36010 Cinnamic acid derivative 359 161 335, 285s11 Rosmarinic acid 359 161 330, 290s

a Peaks listed in the order of elution. Peak 2 was identified with the aid of ‘Pinot noir’ grape extracts (Lee & Martin, 2009). Peaks 5, 7, 9, and 11 were co-chromatographedwith purchased standards. All other peaks (1, 3, 4, 6, 8, and 10) were tentatively identified.

b s: shoulder. Peaks 1–8 and 10–11 were quantified as caffeic acid. Peak 9 was quantified as quercetin-rutinoside.

652 J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650–656

Jayasinghe et al. (2003) results were based on extractions fromdried basil leaves, compared to fresh basil samples analyzed inour study. As fresh leaves contain more water, concentrations ofphenolics would be lower when expressed on a fresh weight basis,and a decrease in concentration may have contributed towardsfewer compounds being identified in our study.

E. purpurea has been reported to be a major human dietarysource of chicoric acid (Nusslein, Kurzmann, Bauer, & Kreis,2000), but our data from basil and the reports of chicoric acid iniceberg lettuce (Lactuca sativa L.; Baur, Klaiber, Koblo, & Carle,2004) and chicory (Cichorium intybus L.- endives, radicchio,Innocenti et al., 2005) indicate that this is clearly not the case.Iceberg lettuce and chicory are within the Asteraceae, and basil isamong the Lamiaceae. Olah, Radu, Mogosan, Hanganu, and Gocan(2003) found chicoric acid in a herb, known as Cat’s whiskers(Orthosiphon aristatus) in the Lamiaceae. Chicoric acid might bepresent in other food sources, but has so far been unidentifieddue to its rapid degradation by native enzymes, and the difficultyin obtaining a purified standard for comparison (Nusslein et al.,2000; Perry et al., 2001). Identification of chicoric acid in other foodsources may be important because it has been reported tohave stronger antioxidant activity than has rosmarinic acid(Dalby-Brown et al., 2005; Jayasinghe et al., 2003).

Several minor peaks were identified from leaf and stem samples(Table 1 and Fig. 1A). All minor peaks were at concentrations of lessthan 6.6 mg 100 g�1. Quercetin-rutinoside was a minor peak of thesweet basil leaf and stem samples, which had been reported in O.basilicum (Grayer et al., 2002) and other Ocimum species by Grayer,Kite, Abou-Zaid, and Archer (2000), Grayer et al. (2002). Lithosper-mic acid (a caffeic acid trimer) was detected in all basil leaf samples,but not in stems, though it has been found in basil roots (Tada et al.,

Table 2Phenolic composition of the market-purchased basil (Ocimum basilicum) leaves and stems from two unnamed cultivars of sweet and Thai basil.

Componentsa Sweet basil Thai basil

Leaves Stems Leaves Stems

TACY ndb nd 2 (0) 13 (0)TP 523 (15)c 244 (4) 605 (6) 231 (1)Polyphenolics

Peak 2: caftaric acid 16.5 (1.18) nd 1.93 (0.08) ndPeak 7: chicoric acid 51.8 (0.65) nd 88.5 (2.03) 0.30 (0.04)Peak 11: rosmarinic acid 112 (1.86) 31.9 (4.74) 128 (1.50) 40.3 (1.05)Other minor compounds 28.3 (0.23) 3.98 (0.77) 17.6 (1.44) 9.41 (0.06)

Total polyphenolics 208 (3.50) 35.9 (5.51) 236 (2.41) 50.0 (1.10)

a Samples were split into leaf and stem fractions before extraction and chemical analyses. All values expressed on a fresh weight basis. TACY: total anthocyanins (mg ofcyanidin-glucoside � 100 g�1 tissue). TP: total phenolics (mg gallic acid � 100 g�1 tissue). Polyphenolics: polyphenolic compounds as determined by HPLC (mg � 100 g�1 oftissue). Other minor compounds: sum of peaks 1, 3, 4, 5, 6, 8, 9, and 10 (sweet basil leaves); 5, 9, and 10 (sweet basil stems); 3, 5, 8, and 10 (Thai basil leaves); 3, 5, 9, and 10(Thai basil stems). Total polyphenolics: phenolics other than anthocyanins.

b nd: not detected.c Means (n = 3) followed by standard errors in parentheses.

Table 3Comparison of phenolic composition of market-purchased basil (Ocimum basilicum)leaves from an unnamed sweet basil cultivar after extraction with (blanched) andwithout (non-blanched) an initial blanching step before phenolic analyses byspectrophotometer and HPLC.

Componenta Blanched extracts Non-blanched extracts

TP 523a (15)b 478b (1)Polyphenolics

Peak 2: caftaric acid 16.5a (1.18) 14.6a (2.45)Peak 7: chicoric acid 51.8a (0.65) 46.9b (1.34)Peak 11: rosmarinic acid 112a (1.86) 89.6b (1.71)Other minor compounds 28.3a (0.23) 24.6b (0.68)

Total polyphenolics 208a (3.50) 176b (5.78)

a All values expressed on a fresh weight basis. TP: total phenolics (mg gallic acid �100 g�1 tissue). Polyphenolics: phenolic compounds, as determined by HPLC (mg �100 g�1 of tissue). Other minor compounds: sum of peaks 1, 3, 4, 5, 6, 8, 9, and 10.Total polyphenolics: phenolics other than anthocyanins.

b Means (n = 3) followed by standard errors in parentheses. Means followed bydifferent lower-case letters within a row are significantly different (p 6 0.05, t-test).

Table 4Summary of phenolic concentration and composition in leaves and stems of two basil cultivars (Ocimum basilicum ‘Genovese Italian’ and ‘Purple Petra’) inoculated (AMF) or not(control) with the arbuscular mycorrhizal fungus, Glomus intraradices.

Componenta ‘Genovese Italian’ ‘Purple Petra’

Control AMF Control AMF

Leaves Stems Leaves Stems Leaves Stems Leaves Stems

TACY ndb nd nd nd 65a (2) 41a (5) 88b (7) 48a (5)TP 644ac (79) 376a (48) 489a (63) 335a (31) 325a (21) 172a (12) 390a (27) 190a (25)Polyphenolics

Peak 2: caftaric acid 3.34a (1.30) nd 1.94a (0.45) nd 0.44a (0.08) nd 0.36a (0.11) ndPeak 7: chicoric acid 24.8a (3.63) nd 19.4a (1.84) nd 11.42a (1.49) nd 10.9a (1.99) ndPeak 11: rosmarinic acid 117a (19.4) 72.4a (9.25) 80.5a (8.78) 60.3a (2.56) 35.2a (2.93) 29.2a (3.86) 39.4a (3.36) 36.6a (5.32)Other minor compounds 14.7a (2.77) 4.47a (0.19) 10.7a (0.88) 4.33a (0.64) 3.46a (0.42) 2.66a (0.43) 3.23a (0.19) 2.96a (0.14)

Total polyphenolics 160a (25.4) 76.8a (9.24) 112a (11.7) 64.6a (2.42) 50.5a (4.38) 31.9a (3.90) 53.9a (5.12) 39.6a (5.31)Fresh Weight (g per plant) 20.2a (2.7) 16.6a (1.8) 15.7a (1.1) 15.4a (0.4) 22.9a (2.7) 9.8a (0.6) 21.8a (1.1) 9.5a (1.4)

a All phenolic concentrations are expressed on a fresh weight basis. TACY: total anthocyanins (mg of cyanidin-glucoside � 100 g�1 tissue). TP: total phenolics (mg gallicacid � 100 g�1 tissue). Polyphenolics: polyphenolic compounds, as determined by HPLC/DAD (mg � 100 g�1 of tissue). Other minor compounds: sum of peaks 3, 4, 5, 6, 8, and 10(‘Genovese Italian’ leaves); 5 and 10 (‘Genovese Italian’ stems); 5 and 9 (‘Purple Petra’ leaves); 5 and 10 (‘Purple Petra’ stems). Total polyphenolics: phenolics other thananthocyanins determined by HPLC.

b nd: not detected.c Means (n = 4) followed by standard errors in parentheses. Means within a cultivar and tissue followed by different lower-case letters within a row are significantly

different (p 6 0.05, one-way ANOVA).

J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650–656 653

1996). 5-Caffeoylquinic acid has been reported in basil (Jayasingheet al., 2003), but 5-caffeoylquinic acid and its isomers (isomerizedas described by Nagels, Van Dongen, DeBrucker, & DePooter, 1980that produced 1-caffeoylquinic acid, 3-caffeolylquinic acid, and 4-caffeoylquinic acid) were not found in these samples. Chromato-grams extracted from a total ion current (TIC) MS scan chromato-gram, in the negative ion mode at m/z 353, revealed weakchromatographic peaks, but they were not at the correspondingretention times of the caffeoylqunic acid isomers (Lee & Finn,2007; Nagels et al., 1980). Other cinnamic acid derivatives werealso present in minor quantities (Table 1 and Fig. 1A). Peaks otherthan caftaric, chicoric, and rosmarinic acids were combined andlisted as other minor compounds (Tables 2–4). All basil stem frac-tions had lower phenolic concentrations and less variation in phe-nolic composition than did the corresponding cultivar’s leaf sample.

No anthocyanins were detected in sweet basil leaves or stems(Table 2). Thai basil stems had higher concentrations of TACY than

leaves, as expected from visual observations (Thai basil leaves hadno visual purple colour and the petiole had a slight purple hue)(Table 2). Thai basil leaves had higher levels of TP than sweet basilleaves. The purchased basil stems had similar levels of TP.

Some studies (Nguyen & Niemeyer, 2008; Toussaint et al., 2007)have only reported the concentration of caffeic acid and rosmarinicacid in basil leaves, leading to a potential underestimation of basilphenolic content by HPLC. The results from our studies indicatethat, if only caffeic and rosmarinic acids were measured, approxi-mately 25–50% of the other individual phenolics in leaves and 4–18% of those in stems, would not be accounted for, based onmg 100 g�1 of fresh weight.

3.2. Effect of blanching prior to chemical extraction of sweet basilleaves

During preliminary research (data not shown), we observedbrowning of basil tissue during sample preparation for phenolicanalyses, similar to that described by others (Dogan, Turan, Dogan,Arslan, & Alkan, 2005); therefore we decided to evaluate the effectof an initial blanching step prior to chemical extraction of basil tis-sue. The TP value for blanched extracts was 9.0% higher than that ofnon-blanched extracts (Table 3). Blanched extracts had higher lev-els of chicoric acid (9.6%), rosmarinic acid (19.9%), other minorcompounds (13.1%) and total polyphenolics by HPLC (15.7%) thandid non-blanched extracts. Although not statistically significant,caftaric acid was higher in blanched extracts, as well. These ob-served differences between blanched and non-blanched extractsmight be greater in studies utilizing procedures with extendedextraction times that do not include liquid nitrogen powdering ororganic solvent. Extended extraction time, as well as higher tem-peratures, favours native enzymatic degradation of phenolics andcould contribute toward large differences in phenolic concentra-tions. Perry et al. (2001) showed a greater than 50% loss in pheno-lics when Echinacea was initially extracted with water only, whichis more favourable for native enzyme activity than is organic sol-vent extraction.

Time (min)

A

B

C

1

7

7

7

2

3 4 5 6 9 10

11

Abs

orba

nce

(mA

U)

8

2

10 20 30 40 50 60

Fig. 1. Chromatogram of market-purchased basil (Ocimum basilicum) leaf extract from an unnamed sweet basil cultivar (A), commercially-available Echinacea purpurea herbalextract (B), and chicoric acid standard (C) monitored at 320 nm. Peak assignments are listed in Table 1. All other peaks were monitored and quantified at 320 nm and 370 nm.

240 260 280 300 320 340 360 380

Abs

orba

nce

(mA

U)

Wavelength (nm)

basil Echinacea purpurea chicoric acid

Fig. 2. Superimposed UV–vis spectra chicoric acid peaks from market-purchasedbasil (Ocimum basilicum) leaves from an unnamed sweet basil cultivar, commer-cially-available Echinacea purpurea extract, and a pure chicoric acid standard.

654 J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650–656

An initial blanching step, prior to extraction or processing, hasoften been included with samples known to turn brown rapidly,e.g. potatoes (Rodriguez-Saona, Giusti, & Wrolstad, 1998). Blanch-ing has also been used to denature (deactivate) native enzymes (i.e.polyphenoloxidase) in basil (Dogan et al., 2005) and blueberries(Lee, Durst, & Wrolstad, 2002). In our study, it is possible thatblanching facilitated phenolic extraction compared to non-blanched samples. Nusslein et al. (2000) and Dogan et al. (2005)demonstrated how chicoric acid (E. purpurea) and rosmarinic acid(basil) rapidly degrade in the presence of active native enzymesin analytical preparations. Susceptibility of basil phenolics to oxi-dation can be minimized, using an initial blanching step to maxi-mize phenolic extraction and retention prior to analysis.

3.3. Influence of analysis method on TP values

The TP and total polyphenolic values we obtained from basil,using different methods (spectrophotometer vs. HPLC), were sig-nificantly correlated (market-purchased basil: r = 0.991, p 6 0.05;AMF-study basil: r = 0.934, p 6 0.05). TP values from spectrophoto-metric analyses tended to be higher than the total polyphenolicvalues determined by HPLC/DAD, as previously observed (Lee &Finn, 2007; Lee & Martin, 2009). Higher TP values from spectropho-tometric analysis are partially due to the interference of com-pounds found in plant samples (Waterhouse, 2002).

3.4. Effects of AMF on phenolic composition of basil

Plants inoculated with AMF had high (>67%) root colonizationand non-inoculated plants showed no sign of AMF colonization.Inoculation of basil with AMF had no effect on fresh weight ofleaves or stems (Table 4), or dry weight of leaves, stems, or roots(data not shown). Leaves of ‘Purple Petra’ plants inoculated withAMF had significantly higher TACY than had non-inoculated plants,although the same trend was observed in ‘Purple Petra’ stems, thisdifference was not statistically significant (Table 4). Inoculation ofbasil with AMF had no influence on TP, individual polyphenoliccompounds, or total polyphenolics (Table 4).

Copetta, Lingua, and Berta (2006) tested three AMFs (G. mosseaeBEG 12, Gigaspora margarita BEG 34, and Gi. rosea BEG 9) and dem-onstrated AMF altered essential oil composition of ‘Genovese’ basil,depending on the AMF isolated used for inoculation. The authorssuggested that the increase in essential oil production in Gi. roseainoculated plants may have been linked to higher plant biomass(Copetta et al., 2006). In our study, the isolate of AMF and phospho-rus (P) concentration in the fertilizer used in this study were se-lected to grow plants with similar fresh weights, thus eliminatingthe effects of differences in composition due to biomass. Thereforeincreased TACY between inoculated and non-inoculated ‘PurplePetra’ plants were a consequence of inoculation.

Mycorrhizal fungi are well known for improving plant P uptake(Smith & Read, 1997). One symptom of plants under P-stress is theaccumulation of anthocyanins in the leaves; therefore, there is ahigher probability that non-mycorrhizal plants would have higheranthocyanin concentrations than would mycorrhizal plants. Toler,Morton, and Cumming (2005), however, reported that mycorrhizalsorghum had higher concentrations of anthocyanins than had non-mycorrhizal plants, but only when plants were under stress fromelevated concentrations of copper (Cu) in the growing substrate.Several authors have speculated that many of the physiologicaladvantages of mycorrhiza are only measurable when plants aregrowing under physiologically stressful conditions (Smith & Read,1997). In our study, inoculated and non-inoculated plants con-tained similar amounts of nutrients (data not shown) and wereprovided with similar amounts of water. Therefore, increased TACY

in inoculated ‘Purple Petra’ plants were probably not a result of in-creased water or nutrient stress.

Using the same AMF species, Toussaint et al. (2007) reportedthat inoculation of ‘Genova’ basil resulted in no significant differ-ences in concentrations of rosmarinic acid or caffeic acid in shoots(combination of stems and leaves), compared to non-inoculatedplants when oven-dried samples from 7-week-old plants wereanalyzed using HPLC. Interestingly, they also reported variationin responses of basil to different isolates of AMF. Plants inoculatedwith G. caledonium or G. mosseae had higher concentrations of caf-feic acid in shoots than had non-inoculated plants, and the re-sponse was independent of the effects of AMF on plant Pnutrition. In our study, lack of response in basil phenolics to AMFinoculation may be a result of the isolate of AMF used in the studyand its compatibility with the cultivars of basil.

Ganz et al. (2002) found that AMF (mixture of G. invermaium,Acaulospora laevis, and Scutellospora calospora; strains not re-ported) inoculated olive plants had lower phenolic content thanhad non-AMF-inoculated plants, in both leaves and roots. Basil rootphenolic composition was not the focus of this study. In the future,it would be interesting to determine whether root phenolics werealtered due to AMF colonization, as reported by Toussaint et al.(2007) for rosmarinic acid and caffeic acid when basil plants wereinoculated with specific AMF isolates.

The effects of AMF inoculation on variation in basil phenolicsdue to cultivar, AMF isolate and colonization, and plant age willbe explored in the future, as interest in this research has increasedand there have been few studies in this field (Toussaint et al.,2007). Plant secondary metabolites actively participate in plant-microbe interactions, such as those in AMF-plant symbioses(Zhi-Lin, Chuan-Chao, & Lian-Quing, 2007). Enhancement of sec-ondary metabolite accumulation in plants is of great importancein production of culinary herbs with antioxidant activity. The rela-tionship between AMF and phenolic content alteration, withinvarious plants, has been reviewed by Yao et al. (2007) andToussaint (2007) and both agree that numerous factors needclarification in order to better define the relationship betweenAMF and its host plant’s secondary metabolite production.

4. Conclusions

To the best of our knowledge, this is the first paper to identifythe presence of chicoric acid and caftaric acid in basil. The presenceof chicoric acid has now been reported in plants of the family Aster-aceae (Echinacea, iceberg lettuce, and chicory) and Lamiaceae (basiland Cat’s whiskers). This is the first report of chicoric acid in basil,and only the second to find chicoric acid in plants of the Lamiaceaefamily.

The response of basil phenolics to inoculation with AMF appearsto depend on cultivar, polyphenolic class, plant tissue, and mayalso depend on AMF isolate.

Regarding the potential health benefits of agricultural food-stuffs, this study provides relevant information related to the die-tary availability of chicoric acid. Chicoric acid is much more readilyavailable from common and inexpensive basil leaves than from E.purpurea extracts, which was traditionally considered to be themain source of chicoric acid.

Acknowledgements

We thank Chris Rennaker, Jesse Mitchell, Anne Davis, and RoseJepson of the USDA-ARS for technical assistance. This work wasfunded by USDA-ARS CRIS numbers 5358-21000-041-00D and5358-12210-003-00D.

J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650–656 655

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chemija. 2010. vol. 21. No. 1. P. 59–62© lietuvos mokslų akademija, 2010© lietuvos mokslų akademijos leidykla, 2010

Menthone- and estragole-rich essential oil of cultivated Ocimum basilicum L. from Northwest Iran

Mohammad Bagher Hassanpouraghdam1*,

Abbas Hassani2,

Mohammad Safi Shalamzari3

1 Department of Horticultural Sciences, Faculty of Agriculture, University of Maragheh, Maragheh 55181-83111, Iran

2 Department of Horticultural Sciences, Faculty of Agriculture, Urmian University, Urmia 5715944931, Iran

3 Research Service Laboratory, Faculty of Chemistry, University of Tabriz, Tabriz, Tabriz 51666, Iran

* corresponding author. e-mail: hassanpouraghdam@gmail.com

The hydrodistilled essential oil from aerial parts of cultivated Ocimum basilicum L. plants from Northwest Iran was analyzed by gas chromatog-raphy / mass spectrometry. Forty seven components were identified, com-prising 97.9% of total oil. Monoterpenoids (77.8%) prevailed among the essential oil components, followed by the lesser share of sesquiterpenoids (12.8%). Oxygenated monoterpenes (75.3%) were the predominant compo-nents of oil with menthone (33.1%), estragol (21.5%), isoneomenthol (7.5%), menthol (6.1%) and pulegone (3.7%) as the main compounds. Limonene (1.5%) was the only highlighted monoterpene hydrocarbon. Sesquiterpene hydrocarbons (8.8%) were the second subclass of essential oil constituents with trans-caryophyllene (2.2%), germacrene D (1.4%), trans-β-farnesene (1.1%) and α-amorphene (1.1%) as their main ones. α-Cadinol (2.9%) – an oxygenated sesquiterpene – comprised notable amounts in the essential oil. An acetylated compound, namely menthyl acetate (5.6%), showed traceable amounts in the volatile oil profile. Methyl eugenol, a compound with highly appreciated amounts from most previous reports, comprised only one per-cent of oil. In total, the chemical and percentage composition of oil from cultivated O. basilicum L. from Northwest Iran was characterized as a new menthone / estragole type capable of providing these oxygenated monoter-penes for related food and pharmaceutical industries.

Key words: Ocimum basilicum L., Lamiaceae, essential oil constituents, GC/MS, menthone, estragole, isoneomenthol

INTRODUCTION

Common basil or sweet basil (Ocimum basilicum L. Fam: Lamiaceae or Labiateae) is an annual herbaceous plant with common morphological characteristics of the mint family, reaching 15–45 cm in height [1, 2]. This plant has obovate serrated opposite leaves and fragrant white or violet com-pound terminate flowers [2, 3]. Sweet basil is a cosmopoli-tan herb and aromatic plant grown nearly in all parts of the world. This plant is native of Iran and commonly grows in Azerbaijan provinces [2].

Sweet basil is a multipurpose plant with divergent applica-tions in perfume, food, cosmetic and pharmaceutical indus-tries [4]. Medicinally, this plant and its essential oil have long

been used as immunostimulant [5], sedative, hypnotic, local anesthetic [6], anticonvulsant, antitussive, diuretic [2], car-minative, galactogogue, stomachic, spasmodic [7], vermifuge [8] and platelet anti-aggregant [9]. Furthermore, different bio-logical activities such as nematicidal, fungistatic, antifungal [5, 8], insecticidal, pesticidal [4], antiviral [7], insect repellent and antioxidant [8], have been reported for this plant. Sweet basil plant and its preparations have been used in food and oral / dental products [7], fragrances, and to treat nausea, dys-entery, mental fatigue, colds, rhinitis [8], decrease of plasma lipid content, clear heartburn, to soothe the nerves, remove heat and eliminate toxins [9], as a first aid treatment for wasp stings and snake bites [8] and in traditional rituals [4].

The chemical composition of basil essential oil has been investigated since the 1930s [10], and by now more than 200 chemical components have been identified. Lawrence [11]

Mohammad Bagher Hassanpouraghdam, Abbas Hassani, Mohammad Safi Shalamzari60

has found that the main constituents of the volatile oil of basil are synthesized via two distinct biochemical pathways: phenylpropanoids like chavicol, eugenol and methyl eugenol by the shikimic acid pathway, and terpenes such as linalool by the cytosolic mevalonic acid pathway. Basil essential oil is commercially termed as essence de basilic [2].

The compositional analysis of the essential oil of sweet basil has revealed a comprehensive diversity in the oil com-ponents, and the different chemovarieties have been reported from various regions of the world. Koba et al. [4] reported four chemotypes of estragol, linalool / estragol, methyl eugenol and methyl eugenol / (E)-anethol from Togo. Seven chemo-types with major components greater than 50%, namely me-thyl chavicol, linalool, geraniol, linalool / methyl cinnamate, linalool / geraniol, methyl cinnamate / linalool and eug-enol / linalool, were characterized from Sudan [12]. Linalool, linalool / eugenol, methyl chavicol, methyl chavicol / linalool, methyl eugenol / linalool, methyl cinnamate / linalool and bergamotene have been reported as the major chemotypes of O. basilicum from Mississippi, USA [13]. Italian cultivars of sweet basil were categorized in three chemotypes of linalool, linalool / methyl chavicol and linalool / eugenol [14]. Methyl eugenol and α-cubebene were reported as the main compo-nents of sweet basil oil from Turkey [8]. In a previous study from Iran, methyl chavicol and linalool were the principle components of basil oil [7]. Zamfirache et al. [5] introduced germacrene D and β-elemene as the main constituents of basil oil from Hungary. Meanwhile, linalool, (Z) cinnamic acid me-thyl ester, estragol, eugenol, 1,8-cineol, bergamotene, methyl cinnamate, α-cadinol and limonene have been listed as major and predominant constituents of sweet basil oil from China, Croatia, Israel, Republic of Guinea, Nigeria, Egypt, Pakistan and Malaysia [6, 9, 12, 15–21].

However, only limited studies have been conducted so far on the compositional analysis of O. basilicum L. from North-west Iran. The aim of the present study was to characterize for the first time the volatile oil composition of an endemic-cultivated O. basilicum herb from Northwest Iran.

EXPERIMENTAL

Plant material sampling and preparation. The flowering above-ground parts of endemic purple green-leaved culti-vated O. basilicum L. plants from Maragheh district in North-west Iran were harvested in early summer 2008. The plant specimen was identified by a plant taxonomist, and a voucher specimen was deposited in the Herbarium of the Faculty of Agriculture, University of Maragheh, Iran. The collected plant material from about 20 individual plants as spontaneous rep-resentatives of the local native population were dried at room temperature (about 30 °C) for 4–5 days until constant weight. The air-dried plant material was mixed and pulverized to ob-tain a homogeneous fine-grade material.

Recovery of essential oil. A sample (50 g) of air-dried powdered plant material was extracted by the hydrodistilla-

tion technique within 3 hours in an all-glass Clevenger type apparatus. The extracted crude essential oil was dried over anhydrous sodium sulphate and stored in a hermetically sealed glass flask with a rubber lid, covered with aluminum foil to protect the contents from photo-conversion and kept under refrigeration at 4 °C until analysis. Extraction was car-ried out in triplicate.

Gas chromatography / mass spectrometry. The analy-sis of the oil was carried out using a GC (Agilent Technolo-gies 6890N) connected to a mass-selective detector (MSD, Agilent 5973B) equipped with an non-polar Agilent HP-5-MS (5%-phenyl methyl polysiloxane) capillary column (30 m × 0.25 mm i. d. and 0.25 µm film thickness). The carrier gas was helium with a linear velocity of 1 ml/min. The oven temperature was set at 50 °C for 2 min, then programmed un-til 110 °C at the rate of 10 °C/min with a hold time of 3 min, again heated to 200 °C at the rate 10 °C/min with a 2-min hold time, and finally increased at the rate 20 °C/min to 280 °C and kept isothermal at this temperature for 2 min. The injector and detector temperatures were 300 °C and 200 °C, respectively. Injection mode, split; split ratio, 1 : 100, volume injected, 4 µl of the oil. The MS operating parameters were as follows: ionization potential, 70 eV; interface temperature, 200 °C; and acquisition mass range, 50–800.

Identification and quantification of constituents. The relative percentage amounts of the essential oil components were evaluated from the total peak area (TIC) by the appara-tus software. The identification of components in the volatile oil was based on a comparison of their mass spectra and re-tention time with literature data and by computer matching with NIST and WILEY library as well as by comparison of the fragmentation pattern of the mass spectral data with those reported in the literature [22].

RESULTS AND DISCUSSION

Water-distillation of the Ocimum basilicum L. plants provid-ed a greenish yellow lighter than water liquid with a yield of 0.7% (v/w) of the dry weight of aerial parts. The identified essential oil components, their percentage composition, re-tention indices and molecular formulae as well as the main classes, subclasses and chemical groups are listed in Tables 1 and 2, respectively. Forty seven components were identi-fied in the O. basilicum oil, accounting for 97.9% of total oil. Monoterpenoids (77.8%) were the chief class of components, followed by a minor share of sesquiterpenoids (12.8%) and some other components (Table 2). Oxygenated monoterpe-nes (75.3%) were found to be the major components of the essential oil; they are characterized by the presence of high amounts of menthone (33.1%), estragol (21.5%), isoneom-enthol (7.5%), menthol (6.1%), pulegone (3.7%) and lina-lool (1.7%). Menthone and estragol (sum 54.6%) comprised about 55% of the total oil. Limonene (1.5%) was the only representative of monoterpene hydrocarbons with relatively high amounts (Table 1). Sesquiterpene hydrocarbons (8.8%)

61Menthone- and estragole- rich essential oil of cultivated Ocimum basilicum L. from Northwest Iran

Ta b l e 1 . Essential oil composition of Ocimum basilicum L. from Iran

Compound RI Molecular formula %α-Pinene 0 939 C10H16 0.1Sabinene 0 975 C10H16 0.1β-Pinene 0 979 C10H16 0.3

β-Myrcene 0 991 C10H16 0.13-Octanol 0 991 C8H18O 0.1

α-Phellandrene 1 003 C10H16 0.1p-Cymene 1 025 C10H14 0.1

Limonene 1 029 C10H16 1.51,8-Cineole 1 031 C10H18O 0.2

(Z)-β-Ocimene 1 037 C10H16 0.1γ-Terpinene 1 060 C10H16 0.2Fenchone 1 078 C10H16O 0.3

Linalool 1 097 C10H18O 1.7cis-Rose oxide 1 108 C10H18O 0.2

Camphor 1 146 C10H16O 0.3Menthone 1 153 C10H18O 33.1

Menthol 1 172 C10H20O 6.1Iso-neomenthol 1 187 C10H20O 7.5

Estragol 1 196 C10H12O2 21.5Pulegone 1 237 C10H12O2 3.7Chavicol 1 250 C9H10O 0.1

Piperitone 1 253 C10H16O 0.3Isopulegol acetate 1 278 C12H20O2 0.3

Menthyl acetate 1 295 C12H22O2 5.6Carvacrol 1 299 C10H14O 0.2

α-Cubebene 1 351 C15H24 0.1Eugenol 1 359 C10H12O2 0.1

α-Copaene 1 377 C15H24 0.5β-Bourbonene 1 388 C15H24 0.1

β-Cubebene 1 388 C15H24 0.3β-Elemene 1 391 C15H24 0.5

Methyl eugenol 1 404 C11H14O2 1trans-Caryophyllene 1 419 C15H24 2.2

trans-α-Bergamotene 1 435 C15H24 0.7α-Guaiene 1 440 C15H24 0.1

trans-β-Farnesene 1 457 C15H24 1.1Germacrene D 1 485 C15H24 1.4α-Amorphene 1 485 C15H24 1.1(E)-β-Ionone 1 489 C13H20O 0.1

Bicyclogermacrene 1 500 C15H24 0.5cis-Calamene 1 540 C15H24 0.2Spathulenol 1 578 C15H24O 0.4

Caryophyllene oxide 1 583 C15H24O 0.4Muurolol 1 646 C15H26O 0.1

β-Eudesmol 1 651 C15H26O 0.2α-Cadinol 1 654 C15H26O 2.9Phytol 1 943 C20H40O 0.1

Total 97.9

Compounds are reported according to their elution order on non-polar column.

Ta b l e 2 . Main classes, subclasses and chemical groups of Ocimum basili-cum L. essential oil constituents from Iran

Class, subclass and chemical group of compound %Monoterpenoids 77.8

Monoterpene hydrocarbons 2.6Oxygenated monoterpenes 75.3

Sesquiterpenoids 12.8Sesquiterpene hydrocarbons 8.8Oxygenated sesquiterpenes 4

Others 7.3Total identified 97.9

Chemical groupsAlcohols 42Ketones 37.8Acetates 5.9

Methylated compounds 1.1Oxides 0.8

subclass (Table 1). From the chemical point of view, alcohols (42%) were the predominant group of compounds, followed by ketones (37.8%), acetates (5.9%), methylated compounds (1.1%) and oxides (0.8%) (Table 2). Estragol, isoneomenthol, menthol, α-cadinol, methyl eugenol and linalool were the principle members of the alcoholic constituents. Menthone and pulegone were the most important ketone compounds (Tables 1 and 2). The highlighted representative of acetylated compounds was menthyl acetate (5.6%). Methyl eugenol was the principal constituent of methylated compounds.

The essential oil of O. bsilicum has been the subject of former studies [4–9, 12–21]. Taking into account the chemi-cal profile, especially the monoterpenoidal profile of the present study and reports of other scientists from different countries, it seems that there are meaningful qualitative and quantitative differences among volatile oil components. These differences are more pronounced in regard to mentho-ne, menthol, isoneomenthol and pulegone since these oxy-genated monoterpenes are characteristic of mint plants and their high amounts in basil oil are unfamiliar. To our know-ledge, the presence of such high amounts of menthone and other menthane skeleton compounds in sweet basil volatile oil have not yet been reported. Meanwhile, a high amount of limonene chemotaxonomically relates O. basilicum to Ruta-ceae plants. On the other hand, low amounts of methyl eug-enol and linalool weaken the chemo-similarity of the study plant with most of the above-cited literature [4–9, 12–21]. In total, it is noteworthy that, although O. basilicum from dif-ferent localities has been thoroughly investigated with re-gard to volatile oil composition, the results of our findings are supportive of the concept that continuing the chemical inventory of this plant is still a major scientific interest to encourage the comprehensive exploitation of this valuable medicinal and aromatic plant. These great chemical varia-tions from diverse localities seem to be due to the divergent climatological and geographical conditions (light quality and quantity, soil characteristics, water and nutrient availability,

were the main subclass of 15 carbon sesquiterpenoidal com-pounds with trans-caryophyllene (2.2%), germacrene D (1.4%), trans-β-farnesene (1.1%) and α-amorphene (1.1%) as the most abundant components. α-Cadinol (2.9%), an oxygenated sesquiterpene, had the highest amount of its

Mohammad Bagher Hassanpouraghdam, Abbas Hassani, Mohammad Safi Shalamzari62

temperature fluctuation) as well as different genetical factors such as subspecies, natural hybridization and chemovariety. Furthermore, the effects of varied growing conditions like fertilization, irrigation regime, weed control and other minor factors on plant and its biochemical potential are inevitable. These different conditions and options regularly modify the photosynthesis capability and hence interactive relationship between the primary and secondary metabolism of plants and lead to the biosynthesis of distinct end-products and chemical components from the same initial substrates of aromatic principles, i. e. phenylalanine for phenylpropanoids and geranyl pyrophosphate for terpenoids. This trend, beside different harvesting time, plant parts used for extraction and the extraction protocol, strongly affect the chemical profile and eventually the biological activity of aromatic herbs and their suitability in related industries. It is possible that O. ba-silicum L. plants studied in the present experiment might be a unique chemotype of this plant owing to its distinct vola-tile oil composition. However, this plea requires comparative studies based on detailed phytochemical assays.

CONCLUSIONS

In brief, the chemical composition of the essential oil of cul-tivated O. basilicum L. plant from Northwest Iran was charac-terized by the presence of appreciable amounts of menthone and estragole. The results showed substantial chemical profile differences between the present study and previous reports. However, it can be noted that O. basilicum plants studied in the current study could be a good source of these oxygenated monoterpenes in supplying the increasing demands of food, cosmetic and pharmaceutical industries.

Received 2 November 2009 accepted 23 November 2009

References

1. a. Zarghari, Medicinal Plants (in Persian), Tehran University Publications, iran (1997).

2. v. mozaffarian, A Dictionary of Iranian Plant Names (in Persian), Farhang moaaser Publishing company, iran (2004).

3. a. Ghahreman, Plant Systematics: Cormophytes of Iran (in Persian), iran University Press, iran (1993).

4. a. Koba, P. W. Poutouli, c. Raynaud, j. P. chaumont, K. Sanda, Bangladesh J. Pharmacol., 4, 1 (2009).

5. m. m. Zamfirache, i. Burzo, Z. Olteanu, S. Dunca, S. Surdu, e. Truta, m. Stefan, c. m. Rosu, An. St. Univ. Al. L. Cuza. Iasi, 4, 35 (2008).

6. m. ismail, Pharm. Biol., 44(8), 619 (2006). 7. S. e. Sajjadi, Daru, 14(3), 128 (2006). 8. m. Ozcan, j. c. chalchat, Czech J. Food Sci., 20(6), 223

(2002). 9. j. W. Zhang, S. K. li, W. j. Wu, Molecules, 14, 273 (2009). 10. X. chang, P. G. alderson, c. j. Wright, Environ. Exp. Bot.,

63, 216 (2008). 11. B. m. lawrance, in: B. m. lawrance, B. D. mokheyee,

B. j. Willis (eds.), Developments in Food Sciences, Flavour and Fragrances; A World Perspective, elsevier, the Nether-lands (1988).

12. a. h. N. abduelrahman, e. a. alhussein, N. a. i. Osman, a. h. Nour, Int. J. Chem. Technol., 1(1), 1 (2009).

13. v. D. Zheljazkov, a. N. callahan, c. l. cantrell, J. Agric. Food Chem., 56(1), 241 (2007).

14. m. marotti, R. Piccaglia, e. Giovanelli, J. Agric. Food Chem., 44(12), 3926 (1996).

15. e. Werker, e. Putievsky, U. Ravid, N. Dudai, i. Katzir, Ann. Bot., 71, 43 (1993).

16. O. Politeo, m. jukic, m. milos, Croat. Chem. Acta, 79(4), 545 (2006).

17. S. m. Keita, c. vincent, j. P. Schmit, a. Belanger, Flavour Frag. J., 15(5), 339 (2000).

18. a. a. Kasali, a. O. eshilokun, S. adeola, P. Winterhalter, h. Knapp, B. Bonnlander, Flavour Frag. J., 20(1), 45 (2004).

19. a. j. hussein, F. anwar, S. T. h. Sherazi, R. Prybylski, Food Chem., 108(3), 986 (2008).

20. j. c. chalchat, m. m. Ozcan, Food Chem., 110(2), 501 (2008).

21. S. R. vani, S. F. cheng, c. h. chuah, Am. J. Appl. Sci., 6(3), 523 (2009).

22. R. P. adams, Identification of Essential Oil Components by Gas Chromatography / Quadrupole Mass Spectroscopy, allured Publishing corp., carol Stream, il, USa (2004).

Mohammad Bagher Hassanpouraghdam, Abbas Hassani, Mohammad Safi Shalamzari

MENTONO IR ESTRAGOLIO TURINTIS ETERINIS ALIEJUS IŠ ŠIAURĖS VAKARŲ IRANE KULTIVUOJAMO OCIMUM BASILICUM L.

S a n t r a u k aEterinis aliejus, hidrodistiliavimo būdu išskirtas iš Šiaurės vaka-rų Irane kultivuojamų Ocimum basilicum L. augalų, buvo tiria-mas dujų chromatografijos-masių spektrometrijos metodu. Buvo identifikuoti 47 komponentai, kartu sudarantys 97,9 % eterinio aliejaus. Tarp komponentų rasta 77,8 % monoterpenoidų ir 12,8 % seskviterpenoidų. Apskritai tirtasis eterinis aliejus yra apibūdinamas kaip naujo mentono–estragolio tipo aliejus, kuris gali būti panau-dotas maisto ir farmacijos pramonėje.

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RedalycSistema de Información Científica

Red de Revistas Científicas de América Latina, el Caribe, España y Portugal

González-Zúñiga, Juan Antonio; González-Sánchez, Héctor Manuel; González-

Palomares, Salvador; Rosales-Reyes, Tábata; Andrade-González, Isaac

Microextracción en fase sólida de compuestos volátiles en albahaca (Ocimum basilicum

L.)

Acta Universitaria, vol. 21, núm. 1, enero-abril, 2011, pp. 17-22

Universidad de Guanajuato

Guanajuato, México

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Acta Universitaria

ISSN (Versión impresa): 0188-6266

vargase@quijote.ugto.mx

Universidad de Guanajuato

México

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U n i v e r s i d a d d e G u a n a j u a t o

Microextracción en fase sólida de compuestos volátiles en albahaca (Ocimum basilicum L.)Juan Antonio González-Zúñiga*, Héctor Manuel González-Sánchez**, Salvador González-Palomares**, Tábata Rosales-Reyes**, e Isaac Andrade-González***

Palabras clave:SPME; volátiles; albahaca; cinamato de metilo.

Keywords: SPME; volatile; basil; methyl cinnamate.

RESUMEN

Los compuestos volátiles de la albahaca (Ocimum basilicum L.) fueron extraídos mediante la microextracción en fase sólida (SPME) y analizados con cromatografía de gases-espec-trometría de masas (GC-MS). Se evaluaron dos fibras, Polidimetilsiloxano/Divinilbenceno (PDMS/DVB, 65 µm) y Carbowax/Divinilbenceno (CW/DVB, 65 µm), para comparar la extracción de componentes. Entre los 25 compuestos volátiles recuperados en la albaha-ca, se identificaron fenilpropanoides, monoterpenos, sesquiterpenos, ésteres, y aldehídos. Hubieron diferencias significativas (P < 0,05) entre las fibras analizadas. La comparación de las dos fibras mostró que la extracción con la fibra CW/DVB es aparentemente mejor tanto en el número de componentes aislados como en la concentración total de los com-puestos. Cuantitativamente, el componente más importante fue el cinamato de metilo, seguido por el linalol.

ABSTRACT

Volatile compounds of basil (Ocimum basilicum L.) were extracted with solid phase microex-traction (SPME) and analyzed with gas chromatography-mass spectrometry (GC-MS). Two SPME fiber coatings, Polydimethylsiloxane/Divinylbenzene (PDMS/DVB, 65 µm) and Carbo-wax/Divinylbenzene (CW/DVB, 65 µm) were evaluated in order to compare the extraction of components. Among the 25 volatile compounds detected were phenylpropanoids, monoter-penes, sesquiterpenes, esters, and aldehydes. There were significant (P < 0,05) differences between the two analyzed fibers: with CW/DVB fiber is apparently superior with respect to the number of components isolated as well as the total concentration of compounds. Quan-titatively, the most important component was methyl cinnamate, followed by linalool.

*Unidad de Ciencias de los Alimentos. Instituto Tecnológico Superior de La Huerta (ITSH). Av. Rafael Palomera No. 161, col. El Maguey, C.P. 48850. La Huerta, Jalisco, México. Correo elec-trónico: chava1142@hotmail.com**Laboratorio de Biotecnología. Universidad de Guadalajara (UdG). Centro Universitario de la Costa Sur (CUCSur). Av. Independencia Nacional No. 151. C.P. 48900. Autlán de Navarro, Jalisco, México. Correo electrónico: chava1142@yahoo.com.mx***Planta Piloto de Procesos Agroindustriales. Instituto Tecnológico de Tlajomulco, Jalisco (ITTJ). Km. 10 carr. San Miguel Cuyutlán. C.P. 45640. Tlajomulco de Zúñiga, Jalisco, México.

Recibido: 13 de septiembre de 2010Aceptado: 12 de noviembre de 2010

INTRODUCCIÓN

La albahaca (Ocimum basilicum L., Lamiaceae) es una hierba anual, tiene gran aprecio por los consumidores debido a sus propiedades medicinales, así como también por su aroma y sabor característico (Koba et al., 2009). En México, el cultivo de esta planta se distribuye en casi todo el país y es ampliamente usada en desór-denes digestivos, problemas inflamatorios y enfermedades respiratorias (Argueta y Cano, 1994; Salazar, 2003). Actualmente se le reconoce al aceite esencial de la planta entera una cierta acción sobre el sistema digestivo y el sistema neurovegetativo (Arvy y Gallouin, 2007). La albahaca también tiene una diversidad de usos en alimentos, cosméticos, perfumes, productos orales y dentales, licores, pesticidas y medicinas (Lachowicz et al., 1997; Murillo et al., 2004; Arvy y Gallouin, 2007; Koba et al., 2009).

El género Ocimum, colectivamente llamado albahaca, crece en varias re-giones del mundo (Simon et al., 1999) e incluye un gran número de especies, subespecies y variedades que son producto de una abundante polinización cruzada (Lawrence, 1988). Por estas razones, se ha investigado extensa-mente la composición química del aceite esencial de la albahaca en diferen-tes países (Fleisher, 1981; Lachowicz et al., 1996; Viña y Murillo, 2003; Lee

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Vol. 21 No. 1 Enero-Abril 2011 18

et al., 2005; Koba et al., 2009), encontrándose que ésta varía mucho según el origen geográfico de la planta (Arvy y Gallouin, 2007), el estado de desarrollo vegeta-tivo (Fleisher, 1981), las condiciones agronómicas en su producción (Miele et al., 2001), y las variedades cul-tivadas (Lachowicz et al., 1997; Viña y Murillo, 2003). Sin embargo, hay pocos datos disponibles acerca de la caracterización química de las especies del género Ocimum cultivadas en México.

Los aceites esenciales contienen compuestos aro-máticos muy volátiles, los cuales son responsables de los olores y sabores característicos de las plan-tas (Padrini y Lucheroni, 1997; González-Palomares y Vázquez-García, 2008). En la albahaca, el carácter aromático de cada variedad se origina principalmente por una mezcla compleja de monoterpenos, sesqui-terpenos, fenilpropanoides (Fleisher, 1981; Lachowicz et al., 1997; Miele et al., 2001; Wossa et al., 2008; Koba et al., 2009), ácidos orgánicos (Argueta y Cano, 1994), alcoholes, aldehídos, cetonas y ésteres (Lee et al., 2005). Varios compuestos aromáticos, tales como metil-chavicol (estragol), cinamato de metilo, metil-eugenol, eugenol, linalol y geraniol, han sido reporta-dos como los mayores componentes de los aceites de O. basilicum (Sajjadi, 2006; Wossa et al., 2008). Estas diferencias en la composición química, permitieron la clasificación de la albahaca en quimiotipos [conocidos por los nombres según el origen geográfico (Simon et al., 1999)] con base en los componentes predominan-tes (Lawrence, 1988) o a los compuestos detectados en una cantidad superior al 20 % (Miele et al., 2001; Wossa et al., 2008). Actualmente se han añadido nue-vos quimiotipos y subtipos a la clasificación estableci-da por Lawrence en 1988 (Wossa et al., 2008).

El análisis por cromatografía de gases acoplada a espectrometría de masas (GC-MS) ha permitido cono-cer la composición química y la abundancia relativa de los principales componentes de los aceites de O. basilicum (Acosta et al., 2003). Para lo cual, previa-mente se realiza la extracción de los compuestos volá-tiles a través de métodos como la microextracción en fase sólida (SPME), que es muy popular en la actua-lidad (Fan y Sokorai, 2002; Marín y Céspedes, 2007; Reineccius, 2007; González-Palomares et al., 2009, González-Palomares et al., 2010a).

Las virtudes de la SPME son ampliamente acla-madas e incluyen que es relativamente económica, rápida, no utiliza disolventes en la preparación de la muestra y es razonablemente sensible a la recupera-ción de compuestos volátiles (Pawliszyn, 1997; Beau-lieu y Lea, 2006; Marín y Céspedes, 2007; Reineccius, 2007). En esta técnica, una fibra revestida con uno o

más polímeros de extracción remueve por adsorción los analitos de la muestra (e.g. los compuestos aro-máticos), y luego es insertada directamente dentro del sistema GC-MS para la desorción térmica y el análisis (Marín y Céspedes, 2007; Reineccius, 2007; González-Palomares et al., 2009). La combinación de SPME y GC-MS ha sido exitosamente aplicada en la extracción de compuestos orgánicos volátiles y semi-volátiles de diversas muestras (Vas y Vékey, 2004; González-Palo-mares et al., 2010a).

La escasez de información científica acerca de la composición química de la albahaca cultivada en Ja-lisco, México, generó como objetivo del trabajo: deter-minar los componentes aromáticos más abundantes de la albahaca (Ocimum basilicum L.) procedente de La Huerta, Jalisco, mediante la evaluación de dos fibras de extracción de compuestos volátiles por microex-tracción en fase sólida y cromatografía de gases- es-pectrometría de masas. La información generada ser-virá como antecedente básico para trabajos futuros dirigidos a la búsqueda de los compuestos volátiles de plantas popularmente empleadas en Jalisco, México, como es el caso de la albahaca.

MATERIALES Y MÉTODOS

Muestras de albahaca: Las hojas y tallos frescos de albahaca (Ocimum basilicum L.), con 80 % de hume-dad, se colectaron de un campo de producción de La Huerta, Jalisco, México. El material vegetal cosechado provino de plantas en época de floración. En esta eta-pa fenológica es cuando la albahaca tiene un mayor contenido de aceite esencial (Fleisher, 1981).

Microextracción en fase sólida (SPME): Se em-plearon dos fibras de extracción, polidimetilsiloxa-no/divinilbenceno (PDMS/DVB, 65 µm) y carbowax/divinilbenceno (CW/DVB, 65 µm). Las fibras se acon-dicionaron considerando las instrucciones provistas por el fabricante: 30 min a 260 ºC para PDMS/DVB y 30 min a 250 ºC para CW/DVB. Después del acondi-cionamiento, se corrieron las fibras blanco para con-firmar la limpieza del sistema GC-MS. La jeringa, fi-bras y viales para SPME se obtuvieron de la Compañía Supelco (Bellefonte, PA, USA).

Las muestras de albahaca se homogeneizaron a temperatura ambiente durante 20 segundos. Los compuestos volátiles de las muestras homogeniza-das fueron extraídos por el método de SPME (Cue-vas-Glory et al., 2008), con modificaciones (Gonzá-lez-Palomares et al., 2009; González-Palomares et al., 2010a). Se transfirieron 8 g del homogenizado de albahaca, 1 g de NaCl y 8 mL de agua desioinizada

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CompuestoConcentración (% de área)

PDMS/DVB: CW/DVB:Fenilpropanoides

Cinamato de etilo 3,11 4,11 Cinamato de metilo 8,50 15,00

Monoterpenos

Linalol 6,00 10,50Neral -- 0,90α-Pineno -- 0,63β-Pineno -- 0,44β-Mirceno -- 0,32p-Cimeno -- 0,45α-Terpineno 1,60 2,00

Sesquiterpenos

β-Cariofileno 0,93 1,70α-Cadineno -- 0,51

Ésteres

Acetato de isoamilo 0,23 1,10Acetato de hexilo 0,92 2,15Acetato de etilo 1,10 1,41Acetato de octilo 1,17 2,05Acetato de bencilo 1,19 1,852-metilbutirato de etilo 1,25 2,21Miristato de etilo 1,21 1,35Benzoato de etilo 0,97 0,99Hexanoato de etilo -- 1,95

Aldehídos

Nonanal 0,94 0,81Hexanal 0,91 0,90Decanal 0,45 1,54(E)-2-Octenal 0,47 0,70(E)-2-Nonenal 0,59 1,89Total de compuestos: 18 25

Tabla 1. Compuestos volátiles de albahaca identificados mediante dos fibras de extracción a través de SPME/GC-MS.

(Barnsted E-pure) a un vial de 40 mL, el cual se selló herméticamente por medio de una septa PTFE-silicón. El vial se incubó a 70 ºC en un termobaño con agita-ción durante 30 min. Transcurrido este tiempo, la fibra para SPME se insertó en el espacio de cabeza del vial, manteniéndose la temperatura y la agitación durante 40 min. La incubación con temperatura y agitación sirvió para promover el equilibrio entre los analitos en el espacio de cabeza del vial, la muestra y el polímero de la fibra, y así obtener una mayor concentración de compuestos volátiles en la fibra. Al terminar el tiempo de extracción, se retiró del vial la fibra con los com-puestos volátiles adsorbidos y se insertó en el puer-to de inyección de un cromatógrafo de gases con un tiempo de desorción de 5 min. Este proceso se realizó con cinco repeticiones en las mismas condiciones.

Cromatografía de gases-espectrometría de ma-sas (GC-MS): Los compuestos volátiles aislados de la albahaca por SPME, se analizaron en un GC-MS Hewlett-Packard 6890/5973 (Agilent Technologies, Palo Alto, CA, USA), equipado con una columna ca-pilar polar Supelcowax-10 (Supelco, Bellefonte, PA, USA) de 30 m de largo x 0,25 µm de diámetro interno y con fase estacionaria de polietilenglicol. Las condi-ciones empleadas durante el análisis de las muestras fueron: temperatura del inyector y del detector de 190 °C y 240 ºC, respectivamente. Se estableció una temperatura inicial del horno de 40 ºC, mantenida por 5 min hasta llegar a una temperatura final de 250 ºC con incrementos de 5 ºC por minuto. El gas acarrea-dor fue helio grado cromatográfico (INFRA S.A.), con un flujo de 0,8 mL/min (González-Palomares et al., 2009; González-Palomares et al., 2010b).

Identificación y cuantificación de compuestos volátiles: Los compuestos volátiles de albahaca se identificaron por comparación espectral de los picos del cromatograma de iones totales de las muestras con los compuestos de referencia de la biblioteca Wiley 275L instalada en el GC-MS. La cuantificación se rea-lizó con base en el porcentaje de área de cada pico del cromatograma correspondiente a cada compuesto vo-látil de la albahaca (González-Palomares et al., 2009; González-Palomares et al., 2010b).

Análisis estadístico: Se evaluaron las fibras de ex-tracción utilizadas durante la SPME, con base en el número y concentración de compuestos volátiles ais-lados. Las concentraciones totales de compuestos vo-látiles de la albahaca se obtuvieron del promedio de las cinco repeticiones (n = 5) realizadas en la micro-extracción con las fibras PDMS/DVB y CW/DVB. Los datos generados fueron sujetos a análisis estadístico

a través de la prueba de “t de Student” y considera-dos significativos con P < 0,05 (González-Palomares et al., 2010b).

RESULTADOS Y DISCUSIÓN

En albahaca (Ocimum basilicum L.) procedente de La Huerta, Jalisco, México, se identificaron y cuantifi-caron 25 compuestos volátiles vía GC-MS. En el ais-lamiento de los componentes aromáticos mediante SPME, se emplearon dos fibras: PDMS/DVB y CW/DVB. Del total de los compuestos extraídos, 18 fueron comunes para ambas fibras (tabla 1).

Los valores representan el promedio de cinco repeticiones (n=5).

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Se encontraron diferencias significativas (P < 0,05) al comparar entre si las fibras de extracción, tanto en el número de componentes identificados (18 con la fi-bra PDMS/DVB y 25 con la fibra CW/DVB) como en la concentración total: 31,54 % con la fibra PDMS/DVB y 57,46 % con la fibra CW/DVB. Además, con la fibra PDMS/DVB se aislaron menos terpenos que con la fibra CW/DVB. Estas discrepancias pueden de-berse a la composición de cada fibra y a la afinidad de los compuestos de la muestra a la matriz adsorbente. En el tequila, la fibra PDMS/DVB permitió muy baja cuantificación de terpenos, la cual mejoró al adicionar 100 % de NaCl, con temperatura y tiempo de extrac-ción de 25 ºC y 30 min, respectivamente (Peña-Álvarez et al., 2006). En el aislamiento de los componentes de la miel, la fibra PDMS/DVB tuvo mayor eficiencia que la CW/DVB (Cuevas-Glory et al., 2008). Para Costa et al., (2001) la fibra CW/DVB permitió la clasificación de los compuestos volátiles del café con base en el ori-gen geográfico. Klimánková et al., (2008) obtuvieron perfiles de compuestos volátiles de albahaca bastante similares entre las fibras PDMS/DVB y CW/DVB.

En el actual estudio, los componentes extraídos de la albahaca se agruparon con base en sus característi-cas estructurales en fenilpropanoides, monoterpenos, sesquiterpenos, ésteres y aldehídos. Los compuestos volátiles pertenecientes a los tres primeros grupos co-incidieron con los reportados en otros trabajos de in-vestigación (Lachowicz et al., 1996; Özcan y Chalchat, 2002; Viña y Murillo, 2003; Lee et al., 2005; Chang et al., 2009). Estos autores han tabulado en la albahaca más compuestos, sin embargo, debido a que fueron aislados por hidrodestilación o por destilación con va-por, posibles artefactos o productos de degradación están en algunas de estas listas. De otros estudios, que utilizaron SPME, correspondieron los compuestos más abundantes (Reyes et al., 2007) y algunos mono y sesquiterpenos (Klimánková et al., 2008).

Con las dos fibras de extracción utilizadas, el cina-mato de metilo (fenilpropanoide), el linalol (monoter-peno), y el cinamato de etilo (fenilpropanoide) fueron los compuestos de mayor concentración. El cinamato de metilo y el linalol, han sido reportados por otros autores como los mayores componentes (Lachowicz et al., 1997; Acosta et al., 2003; Viña y Murillo, 2003; Reyes et al., 2007), o dentro de los mayores constitu-yentes del aroma (Lee et al., 2005; Muñoz et al., 2007; Politeo et al., 2007; Klimánková et al., 2008) en dife-rentes variedades de O. basilicum. Lee et al., (2005) también detectaron al cinamato de etilo aunque en baja concentración. De acuerdo con Lawrence (1988),

el componente predominante es el que determina el quimiotipo de la albahaca. En la presente investiga-ción, el cinamato de metilo fue el principal compo-nente, seguido por el linalol (razón 1,5:1). En algunas variedades de O. basilicum se han encontrado pro-porciones iguales o similares entre estos compuestos (Lachowicz et al., 1997; Viña y Murillo, 2003) y se es-tableció que eran del quimiotipo “cinamato de metilo”, subtipo “cinamato de metilo> linalol” (Viña y Murillo, 2003). Posiblemente dicho quimiotipo y subtipo sean también los que corresponden a la albahaca utilizada en este trabajo. No obstante, se requieren investiga-ciones posteriores para confirmar tal información.

En las hojas de la albahaca se encuentran gran-des cantidades de fenilpropanoides, monoterpenos y sesquiterpenos, así como metabolitos derivados de los ácidos grasos. Estos grupos de compuestos, indivi-dualmente y en combinación, imparten un distintivo aroma y sabor (Gang et al., 2001). Los ácidos grasos insaturados y poliinsaturados son precursores de un gran número de compuestos volátiles que son impor-tantes para definir el carácter aromático, entre ellos, los ésteres y los aldehídos (Christensen et al., 2007). Los ésteres y los aldehídos han sido escasamente re-portados en albahaca, sin embargo, en este estudio el mayor número de los compuestos extraídos corres-ponden a estos dos grupos. En la albahaca se han registrado un amplio rango de aromas: a limón, rosa, alcanfor, licor, amaderado y afrutado (Simon et al., 1999). Los compuestos encontrados en esta investi-gación proporcionan diversas notas: los fenilpropano-ides y los ésteres, aromas afrutados (Christensen et al., 2007); los monoterpenos y sesquiterpenos proveen olores frescos, florales, a limón, dulces, herbáceos, afrutados y amaderados (Tamura et al., 2001), y los aldehídos, notas frescas, verdes, cítricas, florales, ja-bonosas y oleosas (Rouseff y Perez-Cacho, 2007). Los compuestos volátiles se forman de los constituyentes mayores de las plantas a través de varias rutas bioquí-micas (Christensen et al., 2007). Los fenilpropanoides se biosintetizan a partir del ácido cinámico a través de la ruta del siquimato. Los monoterpenos y sesquiter-penos tienen su origen biosintético en la vía del ácido mevalónico, también conocida como ruta del mevalo-nato (Viña y Murillo, 2003; Wossa et al., 2008). Los ésteres y aldehídos resultan de la degradación de los ácidos grasos insaturados y poliinsaturados (Chris-tensen et al., 2007).

La composición química de la albahaca puede ver-se influenciada por la variedad, las condiciones agro-climatológicas (Lachowicz et al., 1997; Viña y Murillo,

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2003), el estatus nutricional de las plantas (Özcan y Chalchat, 2002; Sajjadi, 2006), el estado de desarrollo vegetativo (Fleisher, 1981), y la parte de la planta ana-lizada (Muñoz et al., 2007; Klimánková et al., 2008). Las variedades de Ocimum basilicum L., exhiben dis-tinto carácter aromático, características morfológicas, composición química en los aceites esenciales, y qui-miotipos (Simon et al., 1999). Varios métodos analíti-cos han sido desarrollados para determinar los cons-tituyentes volátiles de los aceites esenciales, aunque algunos de ellos pueden ocasionar cambios químicos en los compuestos o pérdida de la mayoría de los volá-tiles (Klimánková et al., 2008). Aún cuando la extrac-ción de los compuestos volátiles se realice mediante el mismo método, hay factores que pueden variar los resultados. Particularmente, en la SPME, el tipo y es-pesor del recubrimiento de la fibra usada, la adición de un electrolito (e.g. una sal), las condiciones de ex-tracción (tiempo y temperatura), y la temperatura del puerto de inyección para la desorción de los analitos de la fibra (Vas y Vékey, 2004). Las variaciones encon-tradas en el aislamiento de compuestos volátiles de la albahaca, entre este proyecto y otras investigaciones pueden deberse a la variedad de las plantas, al méto-do y las condiciones de extracción utilizadas.

Es importante recordar que ningún método para aislar el aroma de una planta da una identificación completa de los compuestos aromáticos presentes en ella (Reineccius, 2007; González-Palomares y Váz-quez-García, 2008). La composición del aroma es una mezcla compleja de sustancias que aunque lleva en sí misma la huella del vegetal del que procede, cada planta tiene su esencia particular, la cual es única e irreproducible (Padrini y Lucheroni, 1997; González-Palomares et al., 2009).

La sensibilidad de la SPME usada en este trabajo fue comparable con métodos convencionales como la hidrodestilación o la destilación con vapor en cuan-to a la extracción de los fenilpropanoides, monoter-penos y sesquiterpenos. Según la literatura citada en los resultados, dentro de estos grupos de compuestos se encuentran los mayores constituyentes del aroma. En la SPME, las variaciones en la composición quí-mica de la albahaca con respecto a reportes previos, principalmente estuvieron dadas por la variedad, el recubrimiento de la fibra, la adición de sal, el tiempo y la temperatura de extracción. Con el fin de ampliar y complementar la información obtenida de la albahaca, se justifican investigaciones posteriores de los com-puestos volátiles y así dirigir la información obtenida hacia posibles aplicaciones.

CONCLUSIONES

El perfil químico de Ocimum basilicum L., procedente de La Huerta, Jalisco, México está constituido princi-palmente por fenilpropanoides y monoterpenos, cuyos principales compuestos representantes son el cina-mato de metilo y el linalol, respectivamente.

La técnica de SPME/GC-MS representa un método apropiado para la extracción de compuestos volátiles de la albahaca. En las condiciones analíticas emplea-das, la fibra CW/DVB presentó una mejor eficiencia de adsorción de compuestos volátiles.

AGRADECIMIENTOS

Al Consejo Estatal de Ciencia y Tecnología de Jalisco (COECYTJAL), por el apoyo económico para realizar esta investigación. A la doctora Lya Esther Sañudo Guerra, doctora Martha Vergara Fregoso, maestra Ruth Catalina Perales Ponce y a la maestra Martha Daniela Concepción García Moreno, autoridades de la Dirección General de Investigación de la Secretaría de Educación Jalisco (SEJ), por las sugerencias en el de-sarrollo del trabajo.

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128

ANALYSIS OF THE ESSENTIAL OILS OF TWO CULTIVATED

BASIL (OCIMUM BASILICUM L.) FROM IRAN

SEYED EBRAHIM SAJJADI

Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

ABSTRACT

The chemical compositions of the essential oils of Ocimum basilicum L. cv. purple and Ocimum basilicum L. cv. green cultivated in Iran were investigated by GC-MS. Twenty constituents (98.5% of the total oil) were identified in the volatile oil of O. basilicum L. cv. Purple. The main constituents found in the oil were methyl chavicol (52.4%), linalool (20.1%), epi-α-cadinol (5.9%) and trans-α-bergamotene (5.2%). In the volatile oil of O. basilicum L. cv. green, twelve components were characterized representing 99.4% of the total oil. Methyl chavicol (40.5%), geranial (27.6%), neral (18.5%) and caryophyllene oxide (5.4%) were the major components. Methyl chavicol is the dominant constituent in each of the two oils. Although the oil of green basil was characterized by a highccontent (46.1%) of citral (neral and geranial), citral was not detected in the oil of purple basil oil. Keywords: Ocimum basilicum, Lamiaceae, Essential oil, Methyl chavicol, Citral

INTRODUCTION The genus Ocimum comprises more than 150 species and is considered as one of the largest genera of the Lamiaceae family (1). Ocimum basilicum L. (sweet basil) is an annual herb which grows in several regions all over the world. The plant is widely used in food and oral care products. The essential oil of the plant is also used as perfumery (2). The leaves and flowering tops of sweet basil are used as carminative, galactogogue, stomachic and antispasmodic medicinal plant in folk medicine (3, 4). Antiviral and antimicrobial activities of this plant have also been reported (5, 6). There are many cultivars of basil which vary in their leaf color (green or purple), flower color (white, red,ppurple) and aroma (7). Ocimum spp. contain a wide range of essential oils rich in phenolic compounds and a wide array of other natural products including polyphenols such as flavonoids and anthocyanins (8). The chemical composition of basil oil has been the subject of considerable studies. There is extensive diversity in the constituents of the basil oils and several chemotypes have been established from various phytochemical investigations. However, methyl chavicol, linalool, methyl cinnamate, methyleugenol, eugenol and geraniol are reported as major components of the oils of different chemotypes of O. basilicum (9-11). The present study describes the composition of the essential oils of two sweet basil cultivated in Iran.

MATERIALS AND METHODS

Plants Material Aerial parts of cultivated O. basilicum L. cv. purple and O. basilicum L. cv. green at full flowering stage were collected from Isfahan in Sep of 2004 at an altitude of 1570m. The plants were identified at the Botany Department of the Faculty of Sciences, Isfahan University, Isfahan, Iran and voucher specimens have been deposited in the Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran (N0. 1114 and 1115).

Isolation of the Oils Plants material was hydrodistilled in a clevenger-type apparatus for 3h according to the method recommended in the British Pharmacopoeia (12). The volatile oils were dried over anhydrous sodium sulphate and stored in sealed vials at 4° C until analysis. The yield of the oils was calculated based on dried weight of plant materials.

GC-MS Analysis GC-MS analysis was carried out on a Hewlett-Packard 6890 gas chromatograph fitted with a fused silica HP-5MS capillary column (30 m × 0.25 mm; film thickness 0.25 µm). The oven temperature was programmed from 60°-280°C at 4°C/min. Helium was used as carrier gas at a flow rate of 2 mL/min. The gas chromatograph was coupled to a Hewlett-Packard 6890 mass selective detector. The MS operating parameters were ionization voltage, 70 eV; and ion source temperature, 200°C.

Correspondence: S. Ebrahim Sajjadi, Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran, E-mail: sajjadi@pharm.mui.ac.ir

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Essential oils of two cultivated basil from Iran 129

Table 1. Percentage composition of the essential oils of Ocimum basilicum L. cv. purple and Ocimum basilicum L. cv. green cultivated in Iran

No Compound Composition (%) RI Purple Basil Green Basil 1 1-octen-3-ol 979 0.4 0.3 2 6-methyl-5-hepten-2-one 987 0.4 3 1,8-cineole 1035 2.4 4 fenchone 1089 0.5 0.3 5 linalool 1100 20.1 6 camphor 1146 0.6 7 terpinen-4-ol 1180 0.8 8 methyl chavicol 1203 52.4 40.5 9 neral 1244 18.5

10 geranial 1274 27.6 11 trans-caryophyllene 1419 1.2 1.6 12 trans-α-bergamotene 1437 5.2 0.8 13 α-humulene 1455 0.5 1.1 14 germacrene-D 1482 1.8 15 bicyclogermacrene 1496 0.9 16 germacrene-A 1504 0.7 17 γ-cadinene 1514 1.8 18 trans-α-bisabolene 1544 1.1 19 spathulenol 1579 0.9 20 caryophyllene oxide 1584 1.4 5.4 21 humulene epoxide II 1610 0.3 1.8 22 1,10-di-epi-cubenol 1616 0.5 23 epi-α-cadinol 1643 5.9 24 β-eudesmol 1652 0.2

RI= retention indices on HP-5 capillary column. %: Calculated from TIC data. Identification of components of the volatile oils were based on retention indices and computer matching with the Wiley275.L library, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature (13, 14). Retention indices (RI) values were measured on HP-5MS column. For RI calculation, a mixture of homologues n-alkanes (C9-C18) wasuused,uunder the same chromatographic conditions which was used for the analysis of the essential oils.

RESULTS AND DISCUSSION The yield of the essential oils obtained from aerial parts of O. basilicum L. cv. purple and O. basilicum L. cv. green were 0.2% and 0.5% (v/w) respectively. Results of the GC-MS analysis of the oils are shown in Table 1, where the components are listed in order of their elution from the HP-5MS column. Twenty compounds of the oil of O. basilicum L. cv. purple and twelve components of O. basilicum L. cv. green oil were identified (98.5% and 99.4% of the total oils respectively). The main constituents found in the oil of O. basilicum L. cv. purple were methyl chavicol (52.4%), linalool (20.1%), epi-α-cadinol (5.9%), trans-α-bergamotene (5.2%) and 1,8-cineole (2.4%). In the oil of O. basilicum L. cv. green, methyl chavicol (40.5%), geranial (27.6%), neral (18.5%), caryophyllene oxide (5.4%) and humulene epoxide II (1.8%) were the major

components. In O. basilicum from Bangladesh, linalool and geraniol are reported as the main components (15). In the oils, obtained from aerial parts of O. basilicum grown in Colombia and Bulgaria, linalool and methyl cinnamate are reported as major components of volatile oils respectinely (16, 17). Linalool and methyl eugenol are the main components of the essential oils of O. basilicum cultivated in Mali (11) and Guinea (18). The observed differences may be probably due to different environmental and genetic factors, different chemotypes and the nutritional status of the plants as well as other factors that can influence the oil composition. Mixture of methyl chavicol and linalool comprise 72.5% of the oil of O. basilicum L. cv. purple. The results of this study indicate that the composition of volatile oil of purple balm cultivated in Iran is similar to those which are reported from Nigeria (19), Benin (20) and Togo (21). On the other hands, geranial and neral were not detected in the oil of purple balm and the green basil was characterized by high content (46.1%) of citral (geranial and neral). For determination of probable chemotypes further investigations would be required.

ACKNOWLEDGMENTS The author would like to acknowledge Mr. I. Mehregan for identification of plants material and Mrs. A. Jamshidi for her technical help.

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Seyed Ebrahim Sajjadi 130

REFERENCES 1. Evans WC. Trease and Evans’ pharmacognosy. London: W.B. Saunders Company; 1996. p. 48. 2. Bauer K, Garbe D, Surburg H. Common fragrance and flavor materials. 3rd edition, Weinheim: Wiley-

VCH; 1997. p. 171. 3. Chiej R. The Macdonald encyclopedia of medicinal plants. London: Macdonald and Co (Publishers)

Ltd.; 1988. p. 207. 4. Duke JA. CRC handbook of medicinal herbs. Boca Raton: CRC Press; 1989. p. 333. 5. Chiang LC, Cheng PW, Chiang W, Lin CC. Antiviral activity of extracts and selected pure

constituents of Ocimum basilicum. Cli Exp Pharmacol Physiol 2005; 32: 811-816. 6. Baratta MT, Dorman HJD, Deans SG, Figueiredo AC, Barroso JG, Ruberto G. Antimicrobial and

antioxidant properties of some commercial essential oil. Flav Fragr J 1998; 13: 235-234. 7. Morales MR, Simon JE. New basil selections with compact inflorescences for the ornamental market,

In: Janick J (ed.), Progress in new crops. Arlington: ASHS Press; 1996. p. 543-546. 8. Phippen WB, Simon JE. Anthocyanins in basil (Ocimum basilicum L.). J Agr Food Chem 1998; 46:

1734–1738. 9. Grayer RJ, Kite GC, Goldstone FJ, Bryan SE, Paton A, Putievsky E. Infraspecific taxonomy and

essential oil chemotypes in sweet basil, Ocimum basilicum. Phytochemistry 1996; 43: 1033-1039. 10. Marotti M, Piccaglia R, Giovanelli E. Differences in essential oil composition of basil (Ocimum

basilicum L.) Italian cultivars related to morphological characteristics. J Agr Food Chem 1996; 44: 3926-3929.

11. Chalchat JC, Garry RP, Sidibe L, Marama M. Aromatic plants of Mali (I): Chemical composition of essential oils of Ocimum basilicum L. J Essent Oil Res 1999; 11: 375-380.

12. British pharmacopoeia. Vol. 2, London: HMSO; 1988. p. A137-A138. 13. Adams RP. Identification of essential oil components by gas chromatography /mass spectroscopy.

Illinois: Allured Publ. Corp.; 1995. p. 69-351. 14. Swigar AA, Silverstein RM. Monoterpenes. Infrared, mass, 1H-NMR, 13C-NMR spectra and kovats

indices. Wisconsin: Aldrich Chemical Company Inc.; 1981. p. 1-121. 15. Mondello L, Zappia G, Cotroneo A, Bonaccorsi I, Chowdhury JU, Usuf M, Dugo G. Studies on the

chemical oil-bearing plants of Bangladesh. Part VIII. Composition of some Ocimum oils, O. basilicum L. var. purpurascens; O. sanctum L. green; O. sanctum L. purple; O. americanum L., citral type; O. americanum L., camphor type. Flav Fragr J 2002; 17: 335-340.

16. Vina A, Murillo E. Essential oil composition from twelve varieties of basil (Ocimum spp) grown in Colombia. J Brazil Chem Soci 2003; 14: 744-749.

17. Jirovetz L, Buchbauer G. Analysis, chemotype and quality control of the essential oil of new cultivated basil (Ocimum basilicum L.) plant from Bulgaria. Scientia Pharmaceutica 2001; 69: 85-89.

18. Keita SM, Vincent C, Schmit JP, Belanger A. Essential oil composition of Ocimum basilicum L., O. gratissium L. and O. suave L. in the Republic of Guinea. Flav Fragr J 2000; 15: 339-341.

19. Kasali AA, Eshilokun AO, Adeola S, Winterhalter P, Knapp H, Bonnlander, Koenig WA. Volatile oil composition of new chemotype of Ocimum basilicum L. from Nigeria. Flav Fragr J 2004; 20: 45-47.

20. Moudachirou M, Yayi E, Chalchat JC, Lartigue C. Chemical features of some essential oils of Ocimum basilicum L. from Benin. J Essent Oil Res 1999; 11: 779-782.

21. Sanda K, Koba K, Nambo P, Gaset A. Chamical investigation of Ocimum species growing in Togo. Flav Fragr J 1998; 13: 226-232.

25

Volatile Chemical Constituents of three Ocimum species (Lamiaceae) from

Papua New Guinea

Stewart W Wossa*1, Topul Rali

2 and David N Leach

3

1University of Goroka, P. O. Box 1078, Goroka, Papua New Guinea.

2Chemistry Department, University of Papua New Guinea, P. O. Box 320, University, Papua New Guinea 3Centre for Phytochemistry and Pharmacology, Southern Cross University, NSW, Australia

*Corresponding author, e-mail: wossas@uog.ac.pg

ABSTRACT Fresh aerial parts of three species of basil, Ocimum basilicum, O. tacilium and O. canum were subjected to exhaustive

hydrodistillation to afford pale yellow coloured oils in 1.0, 0.7 and 0.01 percent yields respectively. Detailed chemical

evaluation by GC and GC/MS revealed O. basilicum to be composed of a total of eleven components representing 100

percent of the total oil composition. Neral (36.1 %) and geranial (44.5 %) were found to be the major components.

Ocimum tacilium was found to be composed of five components representing 99.8 % of the total oil composition with

estragole (96.6 %) being the major component. Five components were observed in O. canum, representing 72.3 percent of

the total oil composition with eugenol (35.3 %) and linalool (27.2 %) as the major components. The high citral (neral +

geranial) content in O. basilicum suggests that it belong to the citral chemotype while O. tacilium belong to the estragole

chemotype and O. canum belong to the eugenol chemotype.

1 INTRODUCTION The genus Ocimum belongs to the family Lamiaceae

and is comprised of more than 50 species of herbs and

shrubs distributed in tropical and subtropical regions of

Asia, Africa and the Americas. Most members of this

family such as Hyptis, Thymus, Origanum, Salvia and

Mentha species are considered economically useful

because of their basic natural characteristics as essential oil

producers. These essential oils are composed primarily of

monoterpenes and sesquiterpenes (Lawrence, 1993) and

have been the subject of extensive studies due to their

economic importance.

The individual species within the genus Ocimum have

been observed to show significant variation in the aromatic

character as well as morphological features. Such

observations have been attributed to the abundant cross-

pollination that occurs within this genus resulting in

considerable degrees of variation in the genotypes, hence

diversity in growth characteristics, leaf size, flower colour,

physical appearance and aroma (Lawrence 1988).

Consequently, high diversity of species, subspecies,

varieties and chemotypes are evident in this genus, each

having distinct aromatic characters, morphological features

and chemical composition in the essential oil distillates.

Such difference in the essential oil compositions in O.

basilicum from different geographical localities led to the

classification of basil into chemotypes on the basis of the

prevalent chemical components (Lawrence, 1992) or

components having composition greater than 20 percent

(Grayer et al. 1996). Four main chemotypes and numerous

other sub-chemotypes were established on the basis of the

structural features of the main constituents as belonging to

either the phenylpropanoid group (methyl chavicol,

eugenol, methyleugenol and methyl cinnamate) or the

terpenic group (linalool and geraniol), which are derived

from the shikimic acid and the mevalonic acid biosynthetic

pathways respectively. Other latter studies on the basils

from other geographical regions have added new

chemotypes to that list based on the established

classification scheme (Lawrence, 1992; Grayer, 1996).

Some of such chemotypic entries include terpenen-4-ol

type from O. canum and thymol type from O. gratissimum

(Sanda et al. 1998; Yusuf et al. 1998; Keita et al. 2000);

geranyl acetate type from O. minimum (Ozcan and

Chalchat, 2002); citral and camphor types from O.

americanum (Mondello et al. 2002); and p-cymene type

from O. suave (Keita et al. 2000). A report on the chemical

constituents in O. canum from Rwanda indicated the oil to

be composed of 60-90 percent linalool (Ntezurubanza et

al. 1985).

There is a substantial wealth of literature on the

chemical composition and biological activities of basil.

The chemical compositions in the basils studied are

composed mainly of monoterpenes or sesquiterpenes with

predominant features representing the terpenic chemotype

group such as linalool and geraniol or the phenylpropenic

chemotype groups, while the observed biological activities

are attributable to either the individual components within

the matrix of the oil or due to a synergistic effect of the

components (Lachowicz et al. 1998; Sinha and Gulani,

1990; Holm, 1999; Vasudaran et al. 1999; Carleton et al.

1992; Svoboda et al. 2003). The prospect of further

developing and using essential oils exhibiting broad-

spectrum biological activities holds promise in medicine

and agriculture, owing to its low mammalian toxicity,

biodegradability, non-persistence in the environment and

affordability.

In spite of such wide-ranging studies on the essential

oil composition in the Ocimum species, no data are

available on the Papua New Guinean (PNG) cultivars of

basil. As part of an ongoing research program to identify

and document the chemical constituents in the essential

oils from the diversity of aromatic flora of PNG, we report

herein a complete analysis of the essential oils obtained

from the aerial parts of O. basilicum, O. tacilium and O.

canum collected respectively from Waigani in NCD, Isan

(Kabwum District) in Morobe and Tabubil in the Western

Provinces of PNG.

10.1071/SP08003

The South Pacific Journal of Natural Science, Volume 26, 2008

26

2 MATERIALS AND METHODS The fresh leaves of Ocimum basilicum, O. tacilium and

O. canum were collected from different localities in PNG

in 2004 and the voucher specimens deposited at the

University of Papua New Guinea (UPNG) Herbarium in

Port Moresby. The fresh leaves were cut into small pieces

and subjected to exhaustive hydrodistillation over an 8-

hour period in an all-glass standard distillation apparatus.

The pure oil obtained was dried over anhydrous

magnesium sulphate and analyzed by gas chromatography

coupled to a mass spectrometer.

The oil sample was injected in hexane using the

GC/MS on an Agilent 6890 gas chromatograph, equipped

with a split/splitless injector and a 7963 Mass Selective

Detector (MSD). Chromatography was performed on a

BPX-5 capillary column (50m x 0.22mm and 1.0 µm film

thickness – SGE, Melbourne) terminated at the MSD

operating at: transfer temperature: 310oC; ionization 70

eV, source temperature: 230oC; quadrupole temperature

150oC and scanning a mass range 35-550 m/z. The injector

temperature was 250oC and the carrier gas was helium at

23.10 psi and an average velocity of 28 cm/sec to the

MSD. The column oven was programmed as follows:

initial temperature: 50oC; initial time 1.0 min; program rate

4oC/min; final temperature 300

oC; final time 10 min.

The individual compounds in the oil were identified on

the basis of their retention indices relative to known

compounds, and further by comparison of their mass

spectra with the authentic compounds or published spectral

data (Adams, 1995).

3 RESULTS AND DISCUSSIONS Hydrodistilled aerial parts of O. basilicum, O. tacilium

and O. canum afforded pale yellow colored oils in 1.0, 0.4

and 0.01 percent yields respectively. GC/MS analysis of

the oil indicated O. basilicum to be composed of 11

components; O. tacilium with 6 components and O. canum

with 5 components as presented in Table 1. The major

components of O. basilicum were geranial (44.5 %) and

neral (36.1 %). The other important components identified

were linalool (6.0 %), cis-α-bisabolene (3.8 %) and nerol

(3.3 %) whilst other monoterpenes made up the remainder.

Estragole (96.6 %) was found to be the major component

of O. tacilium whilst the major components in O. canum

were eugenol (35.3 %), linalool (27.2 %) and 1,8-cineole

(5.6 %).

Table 1 Retention Index (RI) and percentage composition of the components of the Ocimum basilicum, O. tacilium

and O. canum

Percentage Composition (% Area)

Chemical Constituents

Retention

Index (RI)

O. basilicum

O. tacilium

O. canum

1,8-cineole 1058 - - 5.6

linalool 1110 6.0 0.4 27.2

estragole (methyl chavicol) 1227 - 96.6 -

octyl acetate 1234 0.7 - -

nerol 1242 3.3 - -

neral 1261 36.1 - -

cis-isocitral 1265 0.7 - -

geranial 1288 44.5 0.4 -

trans-isocitral 1292 1.3 - -

neryl acetate 1367 0.7 - -

eugenol 1393 - - 35.3

β-caryophyllene 1464 1.4 - -

α-farnescene 1466 1.4 - -

cis-α-bisabolene 1565 3.8 0.8 -

bicyclosesquiphellandrene 1555 - - 2.6

cis-α-bisabolene 1565 3.8 0.8 -

γ-cadinene 1558 - - 1.6

3-methoxy cinnamaldehyde 1629 - 1.6 -

Geranial and neral, the two co-occuring isomeric

monoterpene aldehydes, collectively referred to as citral

are commonly associated with the lemon grass oil

(Cymbopogon citratus.). In this study, the total citral

content in O. basilicum was found to be 80.6 %.

Interestingly, this composition is comparable to that as

reported from the lemon grasses Cymbopogon citratus

(Poaceae) oil from PNG by Sino and coworkers (1992) and

Wossa and co-workers (2004) containing 68 and 91

percent citral composition respectively. The citral

Volatile Chemical Constituents of three Ocimum species: Wossa et al.

27

chemotype in basil have been reported to occur in high

proportion in a cultivar of O. americanum species,

however none has been reported from O. basilicum.

Furthermore, the major components reported in other

cultivars of O. basilicum were not found in this cultivar

except linalool, suggesting that this cultivar is of the citral

chemotype in accordance with the proposed classification

schemes (Lawrence, 1992; Grayer, 1996).

O. tacilium, on the other hand is an estragole rich

cultivar with comparably higher estragole content, while

O. canum is a eugenol-linalool rich cultivar. It was also

noted that linalool was present in all the three species of

basil while geranial and cis-α-bisabolene occurred in O.

basilicum and O. tacilium. Eugenol and 1,8-cineole

occurred only in O. canum. The other monoterpenes

occurred in traces and in various proportions of

composition.

On the basis of the chemical biogenesis as proposed

earlier (Lawrence, 1992; Grayer et al. 1996), O. basilicum

is composed predominantly of the terpenic group and is

therefore derived from a single mevalonic acid

biosynthetic pathway. Likewise, O. tacilium is composed

predominantly of estragole, belonging to the

phenylpropanoid group and is therefore derived through

the shikimic acid pathway. O. canum, on the other hand, is

composed of eugenol and linalool, which have been

categorized as belonging to the phenylpropanoid and

terpenic groups respectively. Eugenol and linalool in O.

canum were found to be in quantities greater than 20

percent, which suggests that the biogenetic mechanisms

that operate in the production of the components in O.

canum are dual in nature.

4 ACKNOWLEDGEMENT The authors are grateful to Mr. Pius Piskaut of the

University of PNG Herbarium for plant description and

identification, the UPNG Research Council for the

research grant and scholarship (to SWW) and Mr. Jones

Hiaso for commenting on the draft manuscript.

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