Aceites esenciales y extractos de Ocimum basilicum

Jorge Hidalgo Gracia Daniel Ramrez Ramos Alejandro Ugena Ortiz

Objetivos
El objetivo comn que tienen los estudios aqu analizados es obtener caractersticas especiales de esta especie como puede ser: Identificar sus compuestos, aislarlos y determinar su capacidad como antioxidante. Determinar la presencia de cido chicrico en las hojas de albahaca. Determinar la mentona y el estragol presente en los aceites esenciales de la albahaca cultivada en el noroeste de Irn. Determinar los compuestos voltiles en albahaca obtenidos tanto por microextraccin en fase slida como por hidrodestilacin. Analizar los aceites esenciales de varios cultivares de albahaca.

Introduccin
La especie vegetal sobre la que se va a realizar el estudio es la denominada vulgarmente como albahaca o alhbega, Ocimum basilicum en latn. La parte de la planta que ha sido sometida a estudio han sido las hojas y en algn caso se ha incluido el tallo. La albahaca es una hierba aromtica de la familia de las lamiceas, 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 ms vivo en la parte superior, ovales u ovadas, dentadas y de textura sedosa, que miden de 3 a 11 cm de largo por 1 a 6 cm de ancho. Aparecern sus espigas florales en verano, con pequeas flores tubulares de color 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 aromtico. Esta planta es muy sensible a las heladas. Se cultiva nicamente por semillas a principios o mediados de la primavera. Requiere una posicin soleada, aunque en climas de veranos muy calurosos requiere suelos frtiles, permeables y hmedos, a ser posible con algo de sombra.

Aunque actualmente se cultivan en todo el mundo los ms de cuarenta tipos de albahaca, todo parece indicar que su origen se encuentre en India, donde se consideraba sagrada. Desde all se extendera por Europa gracias a las culturas griega y romana. En antiguas civilizaciones como la egipcia, posea un alto valor, siendo incluso uno de los elementos utilizados en la momificacin. Al igual que otras aromticas, 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 ms 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 carcingeno (para hepatomas) y genotxico natural, en ratones y ratas. En septiembre de 2001 el Comit Cientfico de la Unin Europea emiti una opinin que recomienda reducir la exposicin y restringir el uso del estragol, sin poderse establecer un lmite seguro para la exposicin a esta toxina de accin lenta (no hay indicios de ninguna toxicidad). Sus aceites esenciales se utilizan tambin en la elaboracin de licores o para aportar aroma a sopas y guisos. Tambin sirve como ingrediente de remedios naturales, en infusiones, en bebidas energticas, para prevenir la cada del cabello o para combatir la halitosis, los dolores de cabeza, de odo, incluso para facilitar la digestin.

Metodologa:
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 bsqueda de polifenoles, fueron adquiridas en un mercado local en Nampa (EEUU). Se conservaron a -80C hasta su utilizacin.

Una parte de las albahacas se micorriz con un hongo y se cultivaron en un sustrato de crecimiento. Las muestras de albahaca del noroeste de Irn fueron obtenidas del distrito de Maragheh. Se secaron a temperatura ambiente durante 4 5 das y se pulverizaron de manera homognea. Las muestras de tallos y hojas frescas de albahaca mexicana fueron obtenidas en Jalisco (Mxico). Las plantas se encontraban en floracin porque es cuando la planta tiene ms aceite esencial. Las muestras de albahaca del centro de Irn fueron obtenidas en Isfahan en poca de mxima floracin. Las muestras de las tres especies de albahaca de Papa Nueva Guinea se obtuvieron de diferentes localidades de la isla.

Mtodo de obtencin:
En las muestras croatas se busc obtener el aceite esencial por un lado y por otro lado los aglicones voltiles 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 hidrodestilacin durante 3 horas y se almacen a -20C. Para aislar los aglicones voltiles libres primero se tuvo que hacer una extraccin de 100 g de planta con acetato de etilo caliente durante 2 horas. El extracto obtenido fue secado y el sedimento eliminado por filtracin. El filtrado se pas por una cromatografa lquida-slida para obtener la parte glucosdica del mismo. Por ltimo se aadi -glucosidasa para romper enzimticamente las molculas y se extrajeron los aglicones con n-pentano. La extraccin de las muestras utilizadas para la bsqueda de polifenoles, como el cido chicrico, se realiz con metanol acidificado con cido frmico. Los extractos obtenidos fueron almacenados a -80C.

En las muestras del noroeste de Irn se buscaba obtener el aceite esencial. Para ello se pusieron 50 g de planta en un aparato tipo Clevenger y se las someti a hidrodestilacin durante 3 horas. El extracto fue secado y almacenado mediante refrigeracin a 4C. La extraccin de las muestras mexicanas se realiz mediante microextraccin en fase slida 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 70C. Posteriormente se aadi una fibra y se mantuvo en agitacin 40 minutos. El aislamiento de los aceites esenciales de las muestras del centro de Irn se hizo mediante hidrodestilacin durante 3 horas en un aparato tipo Clevenger. El extracto fue secado y almacenado mediante refrigeracin a 4C. Para obtener el aceite esencial de las muestras de Papa Nueva Guinea se trocearon las muestras y se las someti a hidrodestilacin durante 8 horas en un aparato de destilacin de cristal standard. El extracto fue secado.

Mtodo de anlisis:
Las muestras croatas se analizaron por medio de cromatografa de gases y espectrometra 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 ndices de retencin fueron determinados con la co-inyeccin de las muestras con una solucin de las series homlogas de n-alcanos C8-C22. Los constituyentes individuales fueron identificados mediante la comparacin de los ndices de retencin obtenidos con los de la literatura cientfica. La actividad antioxidante de los aglicones fue medida usando el DPPH y el FRAP. Los extractos usados para buscar polifenoles fueron analizados mediante cromatografa lquida de alta resolucin (HPLC) y espectrometra de masas. Los compuestos fenlicos fueron identificados basndose en el espectro UV, tiempo de retencin, iones moleculares y fragmentacin de iones. El cido chicrico fue identificado evaluando el tiempo de retencin, el espectro UV-vis y la informacin del espectrmetro de masas.

Las muestras del noroeste de Irn fueron analizadas por medio de cromatografa de gases y espectrometra de masas. La columna capilar est rellena de fenil metil polisiloxano. El gas portador utilizado fue el helio. La identificacin de los componentes se bas en la comparacin de los tiempos de retencin obtenidos con los de la literatura cientfica. Las muestras mexicanas fueron analizadas por medio de cromatografa de gases y espectrometra de masas. Se utiliz una columna capilar polar Supelcowax-10 con polietilenglicol de fase estacionaria. El gas portador utilizado fue el helio. La identificacin de los componentes se bas en la comparacin espectral de los picos del cromatograma obtenidos con los de la literatura cientfica. La cuantificacin se realiz en base al porcentaje de rea de cada pico del cromatograma. Las muestras del centro de Irn fueron analizadas por medio de cromatografa de gases y espectrometra de masas. Se utiliz una columna capilar de slice fundido. El gas portador utilizado fue el helio. La identificacin de los componentes se bas en la comparacin de los tiempos de retencin obtenidos con los de la literatura cientfica. Los ndices de retencin fueron determinados con la co-inyeccin de las muestras con una solucin de las series homlogas de n-alcanos C9-C18. Las muestras Papa Nueva Guinea fueron analizadas por medio de cromatografa de gases y espectrometra de masas. Se utiliz una columna capilar BPX-5. El gas portador utilizado fue el helio. La identificacin de los componentes se bas en la comparacin de los tiempos de retencin obtenidos con los de la literatura cientfica.

Resultados
Componentes mayoritarios
Ocimum basicilum L, procedencia tailandesa Como compuestos fenlicos predominantes, se halla el cido rosmarnico (54%), cido chicrico (25%), y el cido caftrico (8%). Este cido est presente en todas las muestras de hojas de albahaca, presentando un mximo de 88,5 mg en 100 g en peso hmedo en esta albahaca tailandesa. Adems, se ha detectado niveles bajos de cido

chicrico en muestras de su tallo, a diferencia del resto de su especie. Sin embargo, no se hall en su tallo nada de cido caftrico. Por otro lado, no se han hallado en muestras de hojas los derivados del cido cafeico, cido feruloil-tartrico, y el quercetinrutinsido. Ocimum basicilum L, italiano-genovs En esta especie se repite la prioridad de los compuestos, con el cido rosmarnico (54%), cido chicrico (38%), y el cido caftrico (1%). Se aprecia alto porcentaje de cido chicrico, sin embargo, no fue encontrado en sus tallos, as como el cido caftrico. Tambin se han encontrado cido feruloil-tartrico, derivados del cido cafeico, cido litosprmico, y derivados del cido cinnmico en muestras de hojas. Ocimum basicilum L, denominado Petra Prupura Se aprecian los mismos compuestos predominantes como cido rosmarnico (73%), cido chicrico (16%), y el cido caftrico (2%). Tampoco se ha observado cido chicrico en su tallo, as como cido caftrico. En esta especie no se han encontrado compuestos como cido feruloil-tartrico, derivados del cido cafeico, cido litosprmico, y derivados del cido cinnmico. Adems, se han detectado grandes cantidades de metil chavicol (52,4%) y linalool (20,1%). Ocimum basicilum L, llamado comnmente como albahaca dulce De manera anloga, se detectaron cido rosmarnico (70%), cido chicrico (23%), y el cido caftrico (1%). Tampoco se aprecia cido chicrico en su tallo,adems del cido caftrico, ni antocianinas en muestras de hojas ni de tallos. Sin embargo, se han encontrado derivados del cido caffeico, cido feruloyl-tartrico, y el quercetinrutinsido. El quercentin-rutinsido posee un pico menor que el resto de especies en hojas y muestras de tallos. Ocimum basilicum L, procedente de La Huerta, Jalisco, Mxico Se han detectado hasta 25 compuestos voltiles por va cromatografa de gasesespectrometra de masas. Se evaluaron dos fibras, Polidimetilsiloxano/Divnilbenceno (PDMS/DVB) y Carbowax/Divinilbenceno (CW/DVB), para comparar extraccin de componentes. Sern comunes 18 compuestos entre ambas fibras. Adems, se ha apreciado mayor concentracin en la fibra CW/DVB y se aislaron mayor nmero de terpe-

nos. Como compuestos mayoritarios, por orden decreciente, se destacan el cinamato de metilo (fenilpropanoide), el linalol (monoterpeno), y el cinamato de etilo (fenilpropanoide). El cinamato de metilo aparece sobre el linalol con una razn de 1,5:1. En las hojas tambin se hallan fenilpropanoides, monoterpenos, sesquiterpenos, y metabolitos derivados de los cidos grasos. Ocimum basilicum L, procedente del noroeste de Irn 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%), cadinol (2,9%), transcariofilene (2,2%), linalool (1,7%), limoneno (1,5%), germacrena D (1,4%), -amorfena (1,1%), metil eugenol (1%), y trans--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 comprenden el 55% del aceite total, siendo stos los compuestos mayoritarios en este grupo. El limoneno (1,5%) es el nico 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 subclase de hidrocarbonos sesquiterpenos predominante consta de trans-cariofileno (2,2%), germacreno D (1,4%), trans--farneseno (1,1%) y -amorfeno (1,1%) como los compuestos ms abundantes. El -Cadinol (2,9%), un sesquiterpeno oxigenado, tiene la cantidad ms alta de su subclase. Desde el punto de vista qumico, los alcoholes (42%) son el grupo predominante de compuestos, seguidos por las ketonas (37,8%), acetatos (5,9%), compuestos metilados (1,1%) y xidos (0,8%). Los principales miembros de los constituyentes alcohlicos son estragol, isoneomentol, mentol, -cadinol, metil eugenol y linalool. Mentona y pulegona son los compuestos con ketona ms importantes. Los compuestos

acetilados ms representativos son los mentil acetato (5,6%). Finalmente, el metil eugenol es el principal constituyente de los compuestos metilados.

Rendimiento y discusin
Se ha observado que los cidos grasos insaturados y poliinsaturados, son los precursores de un gran nmero de compuestos voltiles que definen el aspecto aromtico de la planta. Los fenilpropanoides y los steres se encargan de dar olores afrutados; los monoterpenos y sesquiterpenos dan aromas frescos, florales, a limn, dulces, herbceos, afrutados y amaderados; mientras que los aldehdos otorgan fragancias frescas, verdes, ctricas, florales, jabonosas y oleosas. Generalmente, la albahaca puede dar olores como limn, rosa, alcanfor, licor, amaderado y afrutado. Los fenilpropanoides se biosintetizan a partir del cido del cido cinmico a travs de la ruta del siquimato. Los monoterpenos y sesquiterpenos tienen su origen biosinttico en la va del cido mevalnico, tambin conocida como ruta del mevalonato. Los steres y aldehdos resultan de la degradacin de los cidos grasos insaturados y poliinsaturados. El rendimiento de los aceites esenciales obtenidos de O. basilicum L prpura 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 genticos, diversos quimiotipos entres la especie, y la nutricin que lleve a cabo el espcimen en concreto. La cantidad de citral recogido en estas muestras supera el 80,6%, compuesto principalmente por geranial y neral, siendo stos 2 aldehdos monoterpenos ismeros que se producen simultneamente, asociado con el aceite graso del limn. 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 caractersticas y que solo comparte el linalool como componente importante. Se puede decir, por tanto, que en la especie Ocimum basilicum est compuesta predominante por grupos terpnicos, y por ello, son derivados de un nico cido mevalnico mediante biosntesis. La destilacin en presencia de agua de plantas de Ocimum basilicum L., en agua lquida, da un rendimiento del 0,7% (v/w) del peso seco de las partes areas, medido en muestras de albahaca procedente de Irn. Este rendimiento no especifica si se da en el resto de especies estudiadas.

El contenido de aglicones libres voltiles en muestra de planta seca, fue de 0,14 mg/g. Los principales aglicones encontrados son fenil-propano-eugenol (44%), chavicol (29,5%), alcohol bencnico (5,7%), vanilina (2,9%), 2-fenil-etanol (2,7%), e ismeros no identificados 3,7-dimetil-1,5-octadieno-3,7-diol (2,4%). Como mtodo de determinacin de estos compuestos, cabe destacar que el cido chicrico fue encontrado por comparacin en una muestra pura estndar, mediante evaluacin del tiempo de retencin, espectro UV-visible, e informacin MS. El cido caftrico fue hallado por comparacin del tiempo de retencin, espectro UV-visible, ion molecular, e iones fragmentados de uvas Pinot noir Por lo general, como compuestos comunes entre esta albahaca dulce, tailandesa, italiano-genovs y la Petra Prpura, se ha detectado cido litosprmico en todas estas muestras de hoja de albahaca, pero no en tallos, aunque s en races de albahaca. Se ha encontrado adems cido 5-caffeoyl-qunico en estas albahacas pero ste y sus ismeros (que producen cido 1-caffeoyl-qunico, cido 3-caffeoyl-qunico y cido 4caffeoyl-qunico) no fueron hallados en las muestras. Los tallos tienen menor variacin en la composicin de fenoles que las correspondientes en las muestras de hojas. Se puede decir que los tallos de albahaca tienen valores similares de fenoles totales, en comparacin de sus hojas en las que surgen diferencias. Adems, es comn encontrarse con compuestos de antioncianina, la cual presenta mayor concentracin en los tallos que en las hojas. Los compuestos voltiles con glicsidos podran ser interesantes como elementos potenciales todava no estudiados de compuestos antioxidantes en albahaca o en otras plantas. Est demostrado que el eugenol es el compuesto mayoritario responsable de esta propiedad antioxidante, el cual aparece en gran cantidad en los aglicones voltiles (44%), lo que les dota de esta capacidad antioxidante, mucho mayor que su aceite esencial. Esta propiedad antioxidante se da desde que los compuestos voltiles pueden ser liberados de precursores no voltiles glicsidos, por mtodos qumicos o enzimticos durante el proceso de elaboracin. Todo ello confirma el uso potencial de hierbas y especias en la industria alimentaria para incrementar la vida til de los alimentos comestibles.

Albahaca dulce

Albahaca tailandesa Tallos

-

Albahaca "italianogenovs" Hojas

3,34

Albahaca "Petra Prpura" Hojas

0,44

Hojas Tallos Hojas cido caftrico (mg/100g tejido) cido chicrico (mg/100g tejido) cido rosmarnico (mg/100g tejido) Fenoles totales (mg/100g tejido)
208 35,9 236 112 31,9 128 51,8 88,5 16,5 1,93

Tallos
-

Tallos
-

0,3

24,8

11,42

40,3

117

72,4

35,2

29,2

50

160

76,8

50,5

31,9

Frmulas qumicas de los compuestos mayoritarios:

cido caftrico

cido chicrico

cido rosmarnico

Mentona

Estragol

Chavicol

Cinamato de metilo

Linalool

Eugenol

Conclusiones
La capacidad antioxidante de algunos de los compuestos de la albahaca confirmara un uso potencial de sta en la industria alimentaria por su capacidad de aumentar la vida til de los alimentos. La HPLC-DAD (cromatografa lquida de alto rendimiento con vector de diodos de deteccin) realizada a la albahaca verifica la presencia de cido chicorsico y cido caftrico en sta. Este hallazgo proporciona beneficios potenciales para la salud de los productos alimentarios agrcolas ya que el cido chicorsico es mucho ms fcilmente disponible y barato en la albahaca que en las hojas de Echinacea purpurea, que se consideraba tradicionalmente como la principal fuente de cido chicorsico. La presencia de mentona y estragol en el aceite esencial de la albahaca cultivada en el noroeste de Irn supone diferencias sustanciales con informes anteriores, lo que incrementara la presencia de esta especie en las industrias alimentarias, cosmticas y farmacuticas. La composicin de la albahaca procedente de Mxico est constituida por fenilpropanoides con el cinamato de metilo como su compuesto ms representativo y el linalol es el monoterpeno ms significativo. Adems de comprobar que la microextraccin en fase slida seguida del anlisis con cromatografa de masa-espectrometra de masas es una tcnica apropiada para obtener compuestos voltiles, se comprob que en las condiciones analticas empleadas la fibra Carbowax/Divinilbenceno present una mejor eficiencia de adsorcin.

Bibliografa
Ttulo: Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Autores: O. Politeo, M. Jukic, M. Milos. Publicacin: Food Chemistry 101 (2.007) 379385.

Ttulo: Chicoric acid found in basil (Ocimum basilicum L.) leaves. Autores: Jungmin Lee, Carolyn F. Scagel. Publicacin: Food Chemistry 115 (2.009) 650656.

Ttulo: Menthone- and estragole-rich essential oil of cultivated Ocimum basilicum L. from Northwest Iran. Autores: Mohammad Bagher Hassanpouraghdam, Abbas Hassani, Mohammad Safi Shalamzari. Publicacin: Chemija (2.010) vol. 21. No. 1. P. 5962.

Ttulo: Microextraccin en fase slida de compuestos voltiles en albahaca (Ocimum basilicum L.) Autores: Gonzlez-Ziga, Juan Antonio; Gonzlez-Snchez, Hctor Manuel; Gonzlez-Palomares, Salvador; Rosales-Reyes, Tbata; AndradeGonzlez, Isaac Publicacin: Acta Universitaria, vol. 21, nm. 1, enero-abril, 2011, pp. 17-2

Ttulo: Analysis of the essential oils of two cultivated basil (Ocimum basilicum L.) from Iran Autores: Seyed Ebrahim Sajjadi Publicacin: Daru Volume 14, No. 3, 2006

Ttulo: Volatile Chemical Constituents of three Ocimum species (Lamiaceae) from Papua New Guinea Autores: Stewart W Wossa, Topul Rali and David N Leach Publicacin: The South Pacific Journal of Natural Science, Volume 26,2008

Food Chemistry
Food Chemistry 101 (2007) 379385 www.elsevier.com/locate/foodchem

Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil
O. Politeo *, M. Jukic, M. Milos
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 their essential oil. The comparison of chemical composition of volatile aglycones with the chemical composition of essential oil reveals four common compounds: eugenol, chavicol, linalool and a-terpineol. For the evaluation of the mentioned antioxidant capacities, two different methods were performed: the 2,2 0 -diphenyl-1-picrylhydrazyl radical scavenging method (DPPH) and ferric reducing/antioxidant power assay (FRAP). DPPH method shows that free volatile aglycones possess good antioxidant properties comparable with that of the essential oil and well-known antioxidant butylated hydroxytoluene (BHT), but less than pure eugenol. The results obtained by FRAP method show that these compounds are some less eective antioxidants than essential oil and BHT. 2006 Elsevier Ltd. All rights reserved.
Keywords: Ocimum basilicum L.; Volatile aglycones; Essential oil; Chemical composition; GCMS; Antioxidant capacity; DPPH; FRAP

1. Introduction The antioxidants are an increasingly important ingredient in food processing. Their traditional role involves, as their name suggests, inhibiting the development of oxidative rancidity in fat-based foods, particularly meat and dairy products, and fried foods. The most widely used synthetic antioxidants in food (butylated hydroxytoluene BHT, butylated hydroxyanisole BHA) are very eective in their role as antioxidants. However, their use in food products has been failing o due to their instability, as well as due to a suspected action as promoters of carcinogenesis (Namiki, 1990). For this reason, there is a growing interest in the studies of natural healthy (non toxic) additives as potential antioxidants (Baratta et al., 1998; Tomaino et al., 2005).

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

Essential oil from aromatic and medicinal plants has been known to possess biological activity, notably antibacterial, antifungal and antioxidant properties (Baratta et al., 1998; Baratta, Dorman, & Deans, 1998). Ocimum basilicum L. (Lamiaceae), respectively, named basil, is an aromatic herb that has been used traditionally as a medicinal herb in the treatment of headaches, coughs, diarrhea, constipation, warts, worms and kidney malfunctions (Simon, Morales, Phippen, Vieira, & Hao, 1999). It has a long history as culinary herb, thanks to its foliage adding a distinctive avor to many foods. It is also a source of aroma compounds and essential oils containing biologically active 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 interest for the study of glycosidically bound volatile compounds. 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

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O. Politeo et al. / Food Chemistry 101 (2007) 379385

in case of grapes and wines (Gunata, Bayonove, Baumes, & Cordonnier, 1985), fruits (Schwab, Mahr, & Schreier, 1989) and aromatic plants (Stahl-Biskup, Intert, Holthuijzen, Stengle, & Schulz, 1993). To our knowledge, only one study was published about the chemical composition of volatile aglycones from basil (Lang & Ho rster, 1977). The antioxidant capacity of basil essential oil has been studied several times (Baratta et al., 1998; Lee, Umano, Shibamoto, & Lee, 2005; Tomaino et al., 2005), but the antioxidant properties of glycosidically bound volatile compounds from basil have not been studied to date. The aim of this paper is to isolate and identify these compounds as well as to determine their antioxidant capacity. This is a part of our investigation project, dealing with antioxidant capacity of glycosidically bound volatile compounds from aromatic plants (Milos, Mastelic, & Jerkovic, 2000; Radonic & Milos, 2003). 2. Materials and methods 2.1. Materials Dried and chopped basil leaves (Kotanyi spice company, Austria) were purchased from a local market in Split, Croatia. All of the applied chemicals were of pro analysis purity 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 have been placed in a Clevenger type apparatus. The essential oil was isolated by hydrodistillation for 3 h. The obtained essential oil was separated, dried over anhydrous sodium sulphate and stored under argon in a sealed vial, at 20 C before usage. The voucher specimen of the basil leaves is deposited in the Laboratory of Biochemistry and Food Chemistry, Faculty of Chemical Technology, Split, Croatia. 2.3. Isolation of glycosidically bound volatile compounds Upon the addition of internal standard, octyl-b-Dglucopyranoside, 100 g of plant material was extracted with boiling ethyl acetate under reux for 2 h. After percolation, the extract was concentrated to dryness in a rotating evaporator, under reduced pressure. The residue was dissolved in boiling water and, after cooling, the sediment was removed by ltration. The ltrate was subjected to liquidsolid 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, amino acids and proteins were removed by washing with 500 ml of distilled water. The glycosides extract was collected by eluting 100 ml of methanol. The methanolic extract containing the glycosides was concentrated to dryness under reduced pressure and redissolved in 2 ml of citrate-phos-

phate buer (0.2 M, pH 5.0). The remaining volatile compounds were removed by liquidliquid extraction with 4 5 ml of n-pentane over 24 h. Prior to enzymatic hydrolysis, TLC and GCMS tested the absence of free volatile compounds. Thin layer chromatography was performed on 0.2 mm precoated silica plates (Kiesegel 60, Merck) with hexane/ethyl acetate (85:15, v/v) as eluent. The volatile compounds were detected using 2% vanillin in concentrated sulfuric acid. 2.4. Enzymatic hydrolysis and extraction of free volatile aglycones In a typical experiment, b-glucosidase from bitter almonds (10 mg, 58 U/mg; Fluka) was added to the glycosidic extract. The enzymatic hydrolysis was realized during 48 h at 37 C. Occasionally, the mixture was shaken thoroughly by hand. After hydrolysis, the liberated volatile aglycones were extracted from aqueous layer with 4 5 ml of n-pentane. The combined pentane extract was concentrated to 0.5 ml and 2 ll was injected for GCMS analysis. 2.5. Gas chromatographymass spectrometry The analyses of the volatile compounds were run on a HewlettPackard GCMS system (GC 5890 series II; MSD 5971A, HewlettPackard, Vienna, Austria). Two columns of dierent polarity were used: a HP-101 column (Methyl silicone uid, HewlettPackard; 25 m 0.2 mm i.d., lm thickness 0.2 lm) and a HP-20M column (Carbowax, HewlettPackard; 50 m 0.2 mm i.d., lm thickness 0.2 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 isothermal for 15 min (for HP-20M column); isothermal at 70 C for 2 min, then increased to 200 C, at a rate of 3 C/min and held isothermal for 15 min (for HP-101 column). The carrier gas was helium (1 ml/min). The injection port temperature was 250 C and the detector temperature was 280 C. Ionization of the sample components was performed in the EI mode (70 eV). Injected volume was 1 ll. The linear retention indices for all the compounds were determined by co-injection of the samples with a solution containing the homologous series of C8C22 n-alkanes (Van Den Dool & Kratz, 1963). The individual constituents were identied by their identical retention indices referring to the compounds known from the literature data (Adams, 1995), and also by comparing their mass spectra with spectra of either the known compounds or with those stored in the Wiley mass spectral database (HewlettPackard, Vienna, Austria). The aglycone concentrations were calculated from the GC peak areas related to GC peak area of 1-octanol (from the internal standard octyl-b-D-glucopyranoside). Preliminary GCMS analysis showed the absence of 1-octanol as potential aglycone in plant material.

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2.6. Determination of antioxidant activity with 2,2 0 -diphenyl1-picrylhydrazyl (DPPH) radical scavenging method The antioxidant capacity of basil essential oil and of the volatile aglycones was measured in terms of hydrogen donating or radical scavenging ability, using the stable radical, DPPH (Brand-Williams, Cuvelier, & Berset, 1995). An ethanolic stock solution (50 ml of the antioxidant (concentrations of stock solutions were 20, 10, 5 and 1 g/l for volatile aglycones (due to limited quantity of the aglycones only 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 essential oil, BHT and eugenol) was placed in a cuvette, and 1 ml of 0.004% ethanolic solution of DPPH was added. Absorbance measurements commenced immediately. The decrease in absorbance at 517 nm was determined by UVVIS PerkinElmer Lambda EZ 201 spectrophotometer after 2 h for all samples. Ethanol was used to zero the spectrophotometer. The absorbance of the DPPH radical without the antioxidant, i.e. the control, was measured daily. Special care was taken to minimize the loss of free radical activity of the DPPH radical stock solution (Blois, 1958). All determinations were performed in triplicate. The percent inhibition of the DPPH radical by the samples was calculated according to the formula of Yen & Duh (1994): % Inhibition AC0 AAt =AC0 100 where AC(0) is the absorbance of the control at t = 0 min and 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 power FRAP is a simple direct test for measuring of antioxidant capacity. This method was initially developed to assay plasma antioxidant capacity, but can be used for plant extracts too. The total antioxidant potential of the sample was determined using a ferric reducing ability (FRAP) assay (Benzie & Strain, 1996) as a measure of antioxidant power. This assay measures the change in absorbance at 593 nm owing to the formation of a blue colored Fe2+-tripyridyltriazine compound from colorless oxidized Fe3+ form by the action of electron donating antioxidants. The working FRAP reagent was prepared by mixing 10 parts of 300 mmol/l acetate buer, pH 3.6, with 1 part of 10 mmol/l TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/l hydrochloride acid and with 1 volume of 20 mmol/l ferric chloride. Freshly prepared FRAP reagent (1.5 ml) was warmed to 37 C and a reagent blank reading was taken at 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 FRAP reagent. Final dilution of the sample in the reaction mixture was 1:34. The sample was incubated at 37 C throughout the monitoring period. The change in absorbance between the nal reading (4-min reading) and the M1 read-

ing was selected for the calculation of FRAP values. Standard curve was prepared using dierent concentrations (0.15 mmol/l) of FeCl2 4H2O. All solutions were used on the day of preparation. In the FRAP assay, the antioxidant eciency of the antioxidant under the test was calculated with reference to the reaction signal given by a Fe2+ solution of known concentration. The results were corrected for dilution and expressed in mmol Fe2+/l. All determinations 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 plant material was 0.14 mg/g. As shown in Table 1, the GC MS analysis of the aglycones revealed twenty-three compounds, representing 96.3% of the total aglycone fraction. Aliphatic alcohols and acids, terpene compounds, derivative of phenylpropanes and derivative of norisoprenoid were identied. The main aglycones were phenylpropanoids eugenol (44.0%) and chavicol (29.5%). Other quantitatively important aglycones were benzyl alcohol (5.7%), vanillin (2.9%), 2-phenyl ethanol (2.7%) and not identied isomer of 3,7-dimethyl-1,5-octadiene-3,7-diol (2.4%). The obtained results show only some qualitative similarity with those reported by Lang & Ho rster (1977). They found only four compounds in aglycone fraction of morphological undierentiated callus and cell-suspension cultures with thymol and linalool as major ones. This is probably due to dierence in the isolation of glycosides. The aglycones such as aliphatic alcohols, 2-phenylethanol, benzyl alcohol, eugenol, linalool, geraniol, nerol and a-terpineol can, more or less, be considered common in aglycone fraction of Lamiaceae family (Stahl-Biskup et al., 1993) and the eugenol was found to be the main aglycone in most plants of this 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 higher than that of the aglycones. Among 33 compounds identied in basil essential oil, representing 97.0% of the total oil (Table 2), monoterpene compounds, sesquiterpene compounds and derivative of phenylpropanoid were identied. The major compound was monoterpene alcohol linalool (28.6%). The second most important compound was phenylpropanoid estragole with the peak area of 21.7%. Other important 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%). This means that this essential oil represents a true methyl cinnamate chemotype (Guenter, 1965). All this supports the assumption that if alcohols and phenols are the main components of the essential oil, the corresponding aglycones can also be detected (Stahl-Biskup & Holthuijzen, 1995). Comparing the chemical composition of the essential oil

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O. Politeo et al. / Food Chemistry 101 (2007) 379385 Table 2 Chemical composition of basil essential oil No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Identied compound b-Pinene Limonene 1,8-Cineole Camphor Linalool Bornyl acetate Terpinen-4-ol a-Bergamotene Caryophyllene Aloaromadendrene Estragole a-Terpineol Germacrene D a-Humulene Carvone b-Cubebene b-Burbonene b-Elemene c-Cadinene Calamenene a-Amorphene b-Farnesene D-Cadinene a-Bisabolene (Z)-Methyl cinnamate Methyl eugenol (E)-Methyl cinnamate Spatulenol Eugenol Carvacrol a-Cadinol Torreyol Chavicol Total Peak area (%) 0.1 0.1 4.0 0.5 28.6 0.5 0.7 2.2 0.3 0.1 21.7 1.0 0.3 0.2 0.4 0.5 t 0.3 0.2 0.2 1.0 0.2 0.1 0.1 1.6 3.1 14.3 0.8 5.9 t 7.1 0.2 0.7 97.0 RIa HP-20M 1180 1185 1477 1518 1545 1563 1564 1632 1653 1673 1685 1694 1716 1710 1724 1900 1959 2019 2066 2105 2118 2120 2173 RIa HP-101 949 1005 1006 1109 1092 1252 1154 1407 1385 1450 1177 1176 1444 1417 1207 1059 1354 1364 1426 1483 1479 1452 1486 1506 1281 1378 1364 1368 1814 1614 b

Table 1 Chemical composition of free volatile aglycones isolated from basil No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Identied compound Hex-2-en-4-in-1-ol 3-Hexen-1-ol 1-Okten-3-ol Linalool Hotrienol Lavandulol a-Terpineol 3,7-Dimethyl-1, 5-octadiene-3,7-diolc 2-Butenoic acid 2-Hydroxymethyl benzoate Nerol Geraniol Benzyl alcohol 2-Phenyl ethanol 3,7-Dimethyl-1, 5-octadiene-3,7-diolc 2,2 0 -Oxybisdiacetate ethanol Eugenol Chavicol Phytol Vanillin 3-Hydroxy-b-damascone 4-Hydroxy-3, 5-dimethoxybenzaldehyde Hexadecanoic acid Total Peak area (%) 0.2 1.4 0.5 0.8 0.3 0.7 0.4 0.4 0.1 0.2 0.8 0.4 5.7 2.7 2.4 1.7 44.0 29.5 0.3 2.9 0.4 0.2 0.3 96.3 RIa HP-20M 1256 1351 1412 1510 1570 1634 1650 1676 1709 1715 1751 1803 1819 1844 1903 1937 2116 b b b RIa HP-101 838 963 1084 1157 1172 1253 1258 1096 1149 1615 1303 1385 1501 1627 1746 1972

, not identied. a Retention indices relative to C8C22 n-alkanes on polar HP-20M and apolar HP-101 column. b Retention times is outside of retention times of homologous series of C8C22 n-alkanes (identied by MS). c Correct isomer (E or Z) is not identied. Identication is performed by MS.

and aglycones, four compounds were established to be common: eugenol, chavicol, linalool and a-terpineol. Our results show moderate qualitative correlation in the chemical composition of essential oil and free volatile aglycones of 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 antioxidant capacity of basil volatile aglycones and essential oil in comparison with known synthetic antioxidant BHT and pure eugenol. The decrease in absorbance at room temperature was measured every 15 min until the reaction reached steady state or until absorbance declined less than 10%. Typical proles of the percent inhibition of the DPPH radical by essential oil, volatile aglycones, BHT and pure eugenol in the presence of stock solution concentration of 20 g/l are shown in Fig. 1. The percent inhibition of the DPPH radical as a function of the concentration for essential oil, corresponding mass of eugenol in essential oil and pure

, not identied. t, trace (<0.1%). a Retention indices relative to C8C22 n-alkanes on polar HP-20M and apolar HP-101 column. b Retention times is outside of retention times of homologous series of C8C22 n-alkanes (identied by MS).

eugenol is shown in Fig. 2a, and that for volatile aglycones, corresponding mass of eugenol in volatile aglycones and pure eugenol is shown in Fig. 2b. The decrease in concentration of the aglycones produced reduction in their capacities. The same behavior was observed for the essential oil as well as BHT and pure eugenol. Antioxidant capacities in series of concentrations of each of volatile aglycones, essential oils and pure compounds were used to calculate the eective relative concentration EC50. The amount of sample, necessary to decrease the absorbance of DPPH by 50% (EC50), was calculated graphically (% of inhibition was plotted against the logarithm of antioxidant concentration in reaction system). The data given in Table 3 show that eugenol possess the best radical scavenging capacity (EC50 = 0.096 g/l). The basil essential oil and known synthetic antioxidant BHT showed similar capacities

O. Politeo et al. / Food Chemistry 101 (2007) 379385

100

383

75

essential oil volatile aglycones BHT

AI (%)

50

25

eugenol

0 0 30 60 90 120

time (min)
Fig. 1. Typical prole of the percent inhibition of the DPPH radical in the presence of 20 g/l of basil volatile aglycones, basil essential oil, BHT and pure eugenol.

100 eugenol 75 essential oil equiv. mass of eugenol in essent. oil

AI (%)

50

25

0 0.001

0.01

0.1

10

100

(a)

log conc. (g/l)

100

eugenol volatile aglycones equiv. mass of eugenol in volatile aglycones

75

AI (%)

50

25

0 0.001

0.01

0.1

10

100

(b)

log conc. (g/l)

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.

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

nol (EC50 = 0.099 g/l), while EC50 for corresponding mass of eugenol in volatile fraction was 1.950 g/l. FRAP assay is quick and simple to perform, and the reaction is reproducible and linearly related to the molar concentration of the antioxidant present. It is based on comparison

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O. Politeo et al. / Food Chemistry 101 (2007) 379385

Table 3 Radical scavenging of basil volatile aglycones, basil essential oil, BHT and pure eugenol determined with DPPH method Antioxidant Volatile aglycones Essential oil BHT Eugenol Equivalent mass of eugenol in essential oil Equivalent mass of eugenol in volatile aglycones
a

EC50a 3.338 1.378 0.908 0.096 0.099 1.950

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

of the total amount of antioxidant to the reducing capacity of the sample. Absorbance changes are linear over a wide concentration range of antioxidant mixture. The reducing power (FRAP) of the liberated volatile aglycones and the essential oil, as well as BHT and pure eugenol, as determined by FRAP assay, is shown in Fig. 3. As in the previous method, decrease in concentration caused a reduction in the reducing power for all of the applied samples. The highest reducing power (FRAP) shows samples of pure eugenol. The reducing power of the volatile aglycones is comparable, but less than the reducing power of essential oil and BHT. A hierarchy in the reducing capacity of samples could be observed as well: eugenol > BHT > basil essential oil > basil volatile aglycones. Among the compounds identied in the essential oil and volatile aglycones from basil, eugenol was considered the main contributor of the antioxidant capacity. The antioxidant capacity of eugenol (Dorman, Surai, & Deans, 2000; Nagababu & Lakshmaiah, 1992; Satoh, Ida, Sakagami, Tanaka, & Fusisawa, 1998) has been reported earlier. Surprisingly, in spite of much higher percentage of eugenol in volatile aglycones (44.0%) than in essential oil (5.9%), the essential oil shows higher antioxidant capacity. The comparison of the plots in Fig. 2a shows that the antioxidant capacity of pure eugenol (EC50 = 0.096 g/l) is more potent

than that of the total essential oil (EC50 = 1.378 g/l). But the comparison of the plot for pure eugenol with the plot corresponding to equivalent mass of eugenol in total essential oil (EC50 = 0.099 g/l) shows very similar antioxidant capacities for both in all concentration range. This nding clearly suggests that the antioxidant capacity of total essential oil is due only or mainly to the presence of eugenol (5.9%) in its chemical composition and that other constituents do not have signicant eect on eugenol capacity. The plots presented in Fig. 2b show that the antioxidant capacity of total aglycones (EC50 = 3.338 g/l) is also mainly due to the presence of eugenol (44%) in its composition (EC50 = 1.950 g/l), but it is obvious that other aglycones antagonize antioxidant capacity compared to pure eugenol capacity (EC50 = 0.096 g/l). It could be explained with smaller antioxidant capacity of volatile aglycones compared to total essential oil. However, the DPPH results show that glycosidically bound basil aglycones possess a good free radical scavenging capacity comparable with basil essential oil and well-known antioxidant butylated hydroxytoluene (BHT), but less than pure eugenol. The results obtained by FRAP method show that these compounds were some less eective antioxidants than essential oil and BHT. The observed dierences can be explained by dierent solvent polarity in the two assays. It is known that substrate polarity does not aect to DPPH scavenging capacity (Koleva, van Beek, Linssen, de Groot, & Evstatieva, 2002). Glycosidically bound volatile compounds could be interesting as hidden potential of antioxidant compounds in basil or in other plants. Since volatile compounds can be released from nonvolatile glycoside precursors by enzymatic or chemical pathways during manufacturing process, these compounds can be considered as potential precursors of antioxidant substances in plant material and may contribute to the total antioxidant capacity of plants. This conrms the

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

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potential use of herbs and spices in the food industry to increase the shelf life of foodstus. The antioxidant properties of glycosidically bound volatile compounds from other plants 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, Project 0011-003. References
Adams, R. P. (1995). Identication of essential oil components by gas chromatography and mass spectroscopy. Carol Stream, IL: Allured Publ. Baratta, M. T., Dorman, H. J. D, Deans, S. G., Figueiredo, A. C., Barroso, J. G., & Ruberto, G. (1998). Antimicrobial and antioxidant properties of some commercial essential oils. Flavour and Fragrance Journal, 13, 235244. Baratta, M. T., Dorman, H. J. D., & Deans, S. G. (1998). Chemical composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano and coriander essential oil. Journal of Essential Oil Research, 10, 618627. Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power the FRAP assay. Analytical Biochemistry, 239, 7076. Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 181, 11991200. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft & Technologie, 28, 2530. Chaterje, A., Sukul, N. C., Laskal, S., & Ghoshmajumdar, S. (1982). Nematicidal principles from two species of Lamiaceae. Journal of Nematology, 14, 118120. Deshpande, R. S., & Tipnis, H. P. (1997). Insecticidal activity of Ocimum basilicum L. Pesticides, 11, 112. Dorman, H. J. D., Surai, P., & Deans, S. G. (2000). In vitro antioxidant activity of a number of plant essential oils and phytoconstituents. Journal of Essential Oil Research, 12, 241248. Guenter, E. (1965). The essential oil (Vol. III). New York: Van Nostrand, pp. 399415. Gunata, Y. Z., Bayonove, C. L., Baumes, R. L., & Cordonnier, R. E. (1985). The aroma of grapes. I. Extraction and determination of free and glycosidically bound fractions of some grape aroma components. Journal of Chromatography, 331, 8390. Koleva, I. I., van Beek, T. A., Linssen, J. P. H., de Groot, A., & Evstatieva, L. N. (2002). Screening of plant extracts for antioxidant capacity: a comparative study on three testing methods. Phytochemical Analysis, 13, 817.

Lang, E., & Horster, H. (1977). An Zucker gebundene regulare Mono terpene Teil II Untersuchungen zur Olbidung und Akkumulation atherischer Ole in Ocimum basilicum Zellkulturen. Planta Medica, 31, 112118. Lee, S. J., Umano, K., Shibamoto, T., & Lee, K.-G. (2005). Identication of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chemistry, 91, 131137. Milos, M., Mastelic, J., & Jerkovic, I. (2000). Chemical composition and antioxidant eect of glycosidically bound volatile compounds from oregano (Origanum vulgare L. ssp. hirtum). Food Chemistry, 71, 7983. Nagababu, E., & Lakshmaiah, N. (1992). Inhibitory eect of eugenol on non-enzymatic lipid peroxidation in rat liver mitochondria. Biochemical Pharmacology, 43, 23932400. Namiki, M. (1990). Antioxidants/antimutagens in food. Critical Reviews in Food Science & Nutrition, 29, 273300. Radonic, A., & Milos, M. (2003). Chemical composition and antioxidant test of free and glycosidically bound volatile compounds of savory (Satureja montana L. subsp. montana) from Croatia. Nahrung/Food, 47, 236237. Reuveni, R. A., Fleisher, A., & Putievsky, E. (1984). Fungistatic activity of essential oils from Ocimum basilicum chemotypes. Phytopathologische Zeitschrift Journal of Phytopathology, 110, 2022. Satoh, K., Ida, Y., Sakagami, H., Tanaka, T., & Fusisawa, S. (1998). Eect of antioxidants on radical intensity and cytotoxic activity of eugenol. Anticancer Research, 18, 15491552. Schwab, W., Mahr, C., & Schreier, P. (1989). Studies on the enzymatic hydrolysis of aroma components from Carica papaya fruit. Journal of Agricultural and Food Chemistry, 37, 10091012. Simon, J. E., Morales, M. R., Phippen, W. B., Vieira, R. F., & Hao, Z. (1999). Perspectives on new corps and new uses. In J. Janick (Ed.), A source of aroma compounds and a popular culinary and ornamental herb (pp. 499505). Alexandria, VA: ASHS Press. Stahl-Biskup, E., Intert, F., Holthuijzen, J., Stengle, M., & Schulz, G. (1993). Glycosidically bound volatiles. A review 19861991. Flavour and Fragrance Journal, 8, 6180. Stahl-Biskup, E., & Holthuijzen, J. (1995). Essential oil and glycosidically bound volatiles of lemon-scented thyme, Thymus citriodorus (Pers.) Schreb. Flavour and Fragrance Journal, 10, 225229. Tomaino, A., Cimino, F., Zimbalatti, V., Venuti, V., Sulfaro, V., De Pasquale, A., et al. (2005). Inuence of heating on antioxidant activity and the chemical composition of some spice essential oils. Food Chemistry, 89, 549554. Van Den Dool, H., & Kratz, P. D. (1963). A generalization of the retention index system including linear temperature programmed gas liquid partition chromatography. Journal of Chromatography, 11, 463471. Wannissorn, B., Jarikasem, S., Siriwangchai, T., & Thubthimthed, S. (2005). Antibacterial properties of essential oils from Thai medicinal plants. Fitoterapia, 76, 233236. Yen, G. C., & Duh, P. D. (1994). Scavenging eect of methanolic extracts of peanut hulls on free radical and active-oxygen species. Journal of Agriculture and Food Chemistry, 42, 629632.

Food Chemistry 115 (2009) 650656

Contents lists available at ScienceDirect

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

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

Jungmin Lee a,*, Carolyn F. Scagel b
a b

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

a b s t r a c t
This is the rst report to identify the presence of chicoric acid (cichoric acid; also known as dicaffeoyltartaric acid, which is a caffeic acid derivatized with tartaric acid) in basil leaves. Rosmarinic acid, chicoric acid and caftaric acid (in the order of most abundant to least; all derivatives of caffeic acid) were identied in fresh basil leaves. Rosmarinic acid was the main phenolic compound found in both leaves and stems. Chicoric 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 and leaves. 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 by HPLC/DAD/ESIMS/MS and co-chromatographed with purchased standard. The inuence of inoculation with an arbuscular mycorrhizal fungus (AMF), Glomus intraradices, on plant phenolic composition was studied on two basil cultivars,Genovese Italian and Purple Petra. Inoculation with AMF increased total anthocyanin concentration of Purple Petra but did not alter polyphenolic content or prole of leaves and stems, of either cultivar, compared to non-inoculated plants. In the US diet, basil presents a more accessible source of chicoric acid than does Echinacea purpurea, in which it is the major phenolic compound. Published by Elsevier Ltd.

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

Keywords: Phenolics Cichoric acid Caffeic acid derivatives Dicaffeoyltartaric acid Lamiaceae Glomus intraradices

1. Introduction Culinary herbs have been reported to possess antioxidant activities (Yanishlieva, Marinova, & Pokorny, 2006) suggesting that they might have potential human health benets. Basil (family Lamiaceae) is a popular herb in the US and Mediterranean diets. Basils importance as a culinary herb, its historic usage, essential oil composition and phenolics have been well reviewed by Makri and Kintzios (2008). Basil has shown antioxidant and antimicrobial activities 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 avonol-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 acid derivatives (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,

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

2007), but complete phenolic proles of basil have not been reported. Of the caffeic acid derivatives in basil, the present study is the rst to identify the presence of chicoric and caftaric acids in basil, respectively the second and third major phenolic acids present in basil leaves. Chicoric acid is the dominant phenolic reported in Echinacea purpurea (Molgaard, Johnsen, Christensen, & Cornett, 2003; Perry, Burgess, & Glennie, 2001) and has been found in all parts (ower heads, leaves, stems and root) of the E. purpurea plant ( Molgaard et al., 2003). Echinacea (family Asteraceae) has been reported to have potential antioxidant, anti-inammatory, antiviral and immunostimulating properties, arising from the naturally occurring alkamides, caffeic acid derivatives, polysaccharides and glycoproteins (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 Echinacea species possible health benets, its effectiveness was no better than that 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 herbal extracts have been very popular (US annual sales estimated at $100200 million during the years 20002006; Tilburt, Emanuel, & Miller, 2008) but, for US consumers, basil represents a more readily available and inexpensive source of these compounds.

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Phenolic production in a plant can be affected by biotic and abiotic factors (Hussain et al., 2008; Lee & Martin, 2009; Toussaint et al., 2007; Yao, Zhu, & Zeng, 2007). Colonization by arbuscular mycorrhizal fungi (AMF) is known to improve plant nutrient uptake and use (Clark & Zeto, 2000), and stress tolerance (Smith & Read, 1997). AMF colonization can also alter or enhance phenolic production within the host plant (Toussaint et al., 2007; Ganz, Kailis, & Abbott, 2002). The relationship between AMF colonization and the phenolic compounds produced in plants is still not well understood (Toussaint, 2007; Yao et al., 2007). The objectives of this study were to better identify the phenolic compounds found in the aerial portions of the fresh basil plant, and to determine the impact of AMF on plant phenolics within two cultivars. The polyphenolic contents of basil samples were determined using a blanched methanol extraction procedure, followed by HPLC/DAD analysis. The characterization of the polyphenolics found in the basil extracts was performed by HPLC/DAD/ESIMS/MS. 2. Materials and methods 2.1. Plant materials Common sweet basil and Thai basil (Ocimum basilicum, specic cultivar names unknown) were purchased from a local market in Nampa, ID, USA. Edible portions (mainly leaves) were separated from stems, and both fractions were then stored frozen at 80 C prior to extraction. Purchased sweet basil will be referred to as sweet basil, for clarication hereafter, to distinguish it from the other (sweet) basil samples of the AMF portion of this study. Two basil cultivars (Genovese Italian and Purple Petra) were used to examine the effect of inoculation with AMF on plant phenolic production. Echinacea whole plant herbal supplement extract was purchased from a local shop (Nampa, ID, USA) and analyzed by HPLC. The Echinacea herbal extract contained E. purpurea, according to the manufacturers label. All chemicals for polyphenolic extraction and HPLC analysis were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless indicated otherwise. Solvents and chemicals for this investigation were of analytical and high performance liquid chromatography (HPLC) grade. Puried chicoric acid standard was purchased from Indone Chemical Company, Inc. (Hillsborough, NJ, USA). 2.2. AMF inoculum The AMF, Glomus intraradices, originally obtained from Native Plants Incorporated (Salt Lake City, UT, USA), was propagated in pot cultures on roots of bunching onion (Allium cepa L. White Lisbon) grown in a 1:1 mixture of Willamette Valley alluvial silt loam and river sand (8 mg kg1 available phosphorus, pH 6.3) for ve 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 g1 of soil substrate) in the inoculum used in this study were estimated using the MPN (most probable number) method (Woomer, 1994). 2.3. Plant culture and AMF inoculation Surface-sterilized (30% sodium hypochlorite for 10 min, followed by a sterile water rinse) basil seeds were sown in 4 in. pots (Gage Dura Pot #GDP400) containing a steam-pasteurized (60 C for 30 min) 3:1 mixture of Willamette Valley alluvial silt loam and vermiculite (Horticultural Vermiculite, SunGro Horticulture, Bellevue, WA, USA). Available P in the growing substrate was 3 mg kg1, and pH was 6.2. One-half of the plants were inoculated

with AMF by hand-mixing 52 g of the AMF inoculum into the growing substrate in each pot before sowing seeds (AMF treatment). The same quantity of sterile (121 C, 15 min) inoculum was mixed into the growing substrate for the remainder of the plants before sowing seeds (control). Containers were placed in a glass house and watered as needed. Supplemental light ($720 lmol PAR m2 s1) was provided for 16 h d1 by high-pressure multi-vapour lamps. Day/night temperatures were controlled at $24/15 C during the experiment. After cotyledons had fully expanded, plants were fertilized weekly with 50 ml of a liquid fertilizer (Peters Professional, Scotts Company, Maysville, OH, USA) containing 380 mg N l1, 150 mg P l1, 380 mg P l1, 64 mg S l1, 100 mg Ca l1, 36 mg Mg l1, 4.6 mg Fe l1, 18 mg Cl l1, 0.55 mg Mn l1, 0.37 mg B l1, 0.024 mg Zn l1, 0.06 mg Cu l1, and 0.006 mg Mo l1. Plants were pruned back, to three nodes, six weeks after germination and owers that began to develop nine weeks after germination were removed from plants. 2.4. Plant harvest and colonization assessment All plants were destructively harvested 16 weeks after germination and separated into leaves, stems, and roots. Substrate was removed from roots by washing and samples of roots were taken for assessing AMF colonization. Samples of leaves and stems were taken for phenolic analyses (described below). The samples of fresh roots were cleared and stained, using a modied procedure of Phillips and Hayman (1970), in which lacto-phenol was replaced with lacto-glycerin, and assessed for AMF colonization. AMF colonization was measured on $1 cm sections of root samples, using the Biermann and Linderman (1980) method. 2.5. Sample preparation and extraction for polyphenolic analysis Frozen samples were liquid nitrogen-powdered and extracted with acidied methanol (0.1% formic acid, v/v) as described in Lee and Finn (2007) with the following modication: An IKA M20 Universal mill (IKA works Inc., Wilmington, NC, USA) was used to grind frozen samples. Frozen powder (5 g) with the rst methanol addition was blanched in a boiling water bath for 5 min, then immediately chilled in an ice bath for 10 min. This chilled mixture was centrifuged and the pellet was re-extracted, two additional times, with acidied methanol, as described in Lee and Finn (2007). Methanol was evaporated with a RapidVap Vacuum Evaporation System (Labconco Corp., Kansas City, MO, USA) at 40 C, and re-dissolved in water to a nal volume of 25 ml. Aqueous extracts were stored at 80 C prior to further analyses. To examine the importance of inactivating native enzymes in phenolic retention, sets of sweet basil leaves were powdered and then extracted, with and without the initial blanching step (in triplicates). 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, and expressed as mg cyanidin-3-glucoside 100 g1 tissue. TP absorbance was taken at 765 nm and was expressed as mg gallic acid 100 g1 tissue. A SpectraMax M2 microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA) was used. Both measurements were conducted in duplicate. 2.7. HPLC/DAD and HPLC/DAD/ESIMS/MS analyses Basil and Echinacea herbal extracts were analyzed as described in Lee and Finn (2007). Briey, solid phase C18 (Sep-Pak Plus;

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Waters Corp. Milford, MA, USA) clean-up, prior to HPLC analysis, was conducted (Lee & Finn, 2007). HPLC/DAD (HP1100; Agilent Technologies, Inc., Palo Alto, CA, USA) and HPLC/DAD/ESIMS/MS (MS was an XCT ion trap mass spectrometer, Agilent Technologies, Inc.) system and conditions were performed, using a previously published method (Lee & Finn, 2007). ESI was operated in negative ionization mode. Phenolic acids were quantied as caffeic acid at 320 nm. Flavonol-glycoside was quantied as quercetin-rutinoside at 370 nm. Both classes of phenolics were quantied, based on external standards. Phenolic compounds were identied, based on UVvisible (UVvis) spectra, retention time, molecular ions and fragmentation ions. When available, authentic standards were used to aid the identication of phenolics (namely, caffeic acid, chicoric acid, quercetin-rutinoside, and rosmarinic acid). A sum of the individual phenolics (determined by HPLC) will be referred to as total polyphenolics, to distinguish these values from TP in this paper. Phenolics other than anthocyanins will be referred to as polyphenolics 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 moment correlation coefcient (r) was used to assess the relationship between the TP obtained via spectrophotometer and total polyphenolics by HPLC (a = 0.05). 3. Results and discussion 3.1. Identication and quantication of phenolics from basil Three major phenolic compounds were found in purchased fresh sweet and Thai basil leaf samples (Tables 13), and in samples of Genovese Italian and Purple Petra (Table 4). Most of the basil phenolics were derivatives of caffeic acid. The dominant phenolic in all samples, regardless of plant tissue or cultivar (Tables 2 4), was rosmarinic acid (peak 11; Table 1 and Fig. 1A). Rosmarinic acid (caffeic acid dimer) has been found as the dominant phenolic in basil by numerous other researchers ( Javanmardi et al., 2002; Jayasinghe et al., 2003; Nguyen & Niemeyer, 2008). Rosmarinic acid is also the main phenolic acid in many other herbs of the family Lamiaceae, namely, oregano, sage, thyme, rosemary, spearmint, and marjoram (Fecka & Turek, 2008; Kivilompolo & Hyotylainen, 2007). The second most dominant phenolic, and previously unidentied in basil leaves, was chicoric acid (peak 7; Table 1 and

Fig. 1A). Identity of chicoric acid in basil leaves (Fig. 1A) was conrmed by comparison with a purchased pure standard (Fig. 1C) and E. purpurea herbal extracts (Fig. 1B), by evaluating retention time, UVvis spectra (Fig. 2), and MS information (Table 1). The UVvis spectra of chicoric acid from the three samples (sweet basil leaves, E. purpurea herbal extract, and pure standard) are overlaid in Fig. 2. Chicoric acid was present in all basil leaves examined (Tables 2 and 4), with Thai basil leaf samples having the highest level of chicoric acid (88.5 mg 100 g1 fresh weight; Table 2). Making direct comparisons between chicoric acid concentrations in basil leaves to concentrations reported in E. purpurea is complicated by other ndings being expressed as mg g1 of dry weight or ml of extract (Bergeron, Gafner, Batcha, & Angerhofer, 2002; Molgaard et al., 2003; Perry et al., 2001). A small level of chicoric acid was found in Thai basil stems (Table 2). Chicoric acid was not detected in stems of sweet basil, Genovese Italian, or Purple Petra. The third major phenolic identied was caftaric acid (caffeoyltartaric acid). This is the rst report identifying caftaric acid in basil leaves, though tartaric acid has been reported (Leal et al., 2008). Identication of caftaric acid (peak 2 in Table 1 and Fig. 1A) was conrmed by comparing retention time, UVvis spectra, molecular ion, and fragmented ions to caftaric acid from Pinot noir grapes (Lee & Martin, 2009). The basil caftaric acid peak was also compared to the caftaric acid peak of commercially-available E. purpurea herbal extracts (Fig. 1B), since they have been reported to contain caftaric acid (Bergeron et al., 2002; Perry et al., 2001). Caftaric acid was not detected in any stem samples from basil (Tables 2 and 4). We suspect that the potency of fraction VI from basil leaves, described by Jayasinghe et al. (2003), as having the most antioxidant activity, was due to chicoric acid. Though unidentied, Jayasinghe et al. (2003) suspected the dominant peak in fraction VI to be a caffeoyl derivative. Results presented by Nguyen and Niemeyer (2008) showed two other major 330 nm absorbers (present in their basil samples with much higher peak areas than caffeic acid), which the authors did not identify. We suspect that these unidentied peaks were caftaric acid and chicoric acid. Jayasinghe et al. (2003) reported the presence of numerous phenolic compounds in basil leaves ($20 compounds). We did not nd similar 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 data show some variation in phenolic composition between the basil cultivars. Peaks 1, 4, 6, and 9 were present in extracts from sweet basil leaves but not Thai basil leaf samples (Table 2). Similarly, peaks 2, 4, 6, 8, and 10 were present in extracts from leaves of Genovese Italian but not Purple Petra. Additionally, the

Table 1 Phenolics found in market-purchased basil leaves (Ocimum basilicum) from an unnamed cultivar of sweet basil. Of the basil cultivars examined in this study, this cultivar contained the most diverse assortment of compounds. ESI was operated in negative mode. Peaksa 1 2 3 4 5 6 7 8 9 10 11 Compounds Caffeic acid derivative Caftaric acid Cinnamyl malic acid Feruloyl tartaric acid Caffeic acid Caffeic acid derivative Chicoric acid Lithospermic acid Quercetin-rutinoside Cinnamic acid derivative Rosmarinic acid Molecular ions [M-H] 359 311 295 324 179 179 472 536 609 359 359 Fragmented ions (m/z) 197, 179 [caffeic acid-H] 179 [caffeic acid-H]-, 149 [tartaric acid-H] 163 [M-H-malic acid] = [coumaric acid-H], 131 [malic acid-H], 113, 119 282, 193 [ferulic acid-H], 149 [tartaric acid-H]135 149, 135 309 [M-H-tartaric acid], 291 [M-H-caffeic acid], 179 [caffeic acid-H] 491 301 [M-H-rutinoside] = [quercetin-H] 161 161

kmax (nm)b 335 330, 315, 330, 320, 330, 330, 330, 360 335, 330, 300s 295s 300s 290s 300s 300s 300s 285s 290s

a Peaks listed in the order of elution. Peak 2 was identied with the aid of Pinot noir grape extracts (Lee & Martin, 2009). Peaks 5, 7, 9, and 11 were co-chromatographed with purchased standards. All other peaks (1, 3, 4, 6, 8, and 10) were tentatively identied. b s: shoulder. Peaks 18 and 1011 were quantied as caffeic acid. Peak 9 was quantied as quercetin-rutinoside.

J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650656 Table 2 Phenolic composition of the market-purchased basil (Ocimum basilicum) leaves and stems from two unnamed cultivars of sweet and Thai basil. Componentsa Sweet basil Leaves TACY TP Polyphenolics Peak 2: caftaric acid Peak 7: chicoric acid Peak 11: rosmarinic acid Other minor compounds Total polyphenolics ndb 523 16.5 51.8 112 28.3 208 (15)c (1.18) (0.65) (1.86) (0.23) (3.50) Stems nd 244 nd nd 31.9 3.98 35.9 (4) Thai basil Leaves 2 605 1.93 88.5 128 17.6 236 (0) (6) (0.08) (2.03) (1.50) (1.44) (2.41) Stems 13 231 nd 0.30 40.3 9.41 50.0

653

(0) (1)

(4.74) (0.77) (5.51)

(0.04) (1.05) (0.06) (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 of cyanidin-glucoside 100 g1 tissue). TP: total phenolics (mg gallic acid 100 g1 tissue). Polyphenolics: polyphenolic compounds as determined by HPLC (mg 100 g1 of tissue). 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 3 Comparison of phenolic composition of market-purchased basil (Ocimum basilicum) leaves from an unnamed sweet basil cultivar after extraction with (blanched) and without (non-blanched) an initial blanching step before phenolic analyses by spectrophotometer and HPLC. Componenta TP Polyphenolics Peak 2: caftaric acid Peak 7: chicoric acid Peak 11: rosmarinic acid Other minor compounds Total polyphenolics Blanched extracts 523a (15)b 16.5a (1.18) 51.8a (0.65) 112a (1.86) 28.3a (0.23) 208a (3.50) Non-blanched extracts 478b (1) 14.6a (2.45) 46.9b (1.34) 89.6b (1.71) 24.6b (0.68) 176b (5.78)

a All values expressed on a fresh weight basis. TP: total phenolics (mg gallic acid 100 g1 tissue). Polyphenolics: phenolic compounds, as determined by HPLC (mg 100 g1 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 by different lower-case letters within a row are signicantly different (p 6 0.05, t-test).

Jayasinghe et al. (2003) results were based on extractions from dried basil leaves, compared to fresh basil samples analyzed in our study. As fresh leaves contain more water, concentrations of phenolics would be lower when expressed on a fresh weight basis, and a decrease in concentration may have contributed towards fewer compounds being identied in our study.

E. purpurea has been reported to be a major human dietary source of chicoric acid (Nusslein, Kurzmann, Bauer, & Kreis, 2000), but our data from basil and the reports of chicoric acid in iceberg 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 is among the Lamiaceae. Olah, Radu, Mogosan, Hanganu, and Gocan (2003) found chicoric acid in a herb, known as Cats whiskers (Orthosiphon aristatus) in the Lamiaceae. Chicoric acid might be present in other food sources, but has so far been unidentied due to its rapid degradation by native enzymes, and the difculty in obtaining a puried standard for comparison (Nusslein et al., 2000; Perry et al., 2001). Identication of chicoric acid in other food sources may be important because it has been reported to have stronger antioxidant activity than has rosmarinic acid (Dalby-Brown et al., 2005; Jayasinghe et al., 2003). Several minor peaks were identied from leaf and stem samples (Table 1 and Fig. 1A). All minor peaks were at concentrations of less than 6.6 mg 100 g1. Quercetin-rutinoside was a minor peak of the sweet 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). Lithospermic 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 4 Summary 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 Control Leaves TACY TP Polyphenolics Peak 2: caftaric acid Peak 7: chicoric acid Peak 11: rosmarinic acid Other minor compounds Total polyphenolics Fresh Weight (g per plant) ndb 644ac (79) 3.34a (1.30) 24.8a (3.63) 117a (19.4) 14.7a (2.77) 160a (25.4) 20.2a (2.7) Stems nd 376a (48) nd nd 72.4a 4.47a 76.8a 16.6a AMF Leaves nd 489a (63) 1.94a (0.45) 19.4a (1.84) 80.5a (8.78) 10.7a (0.88) 112a (11.7) 15.7a (1.1) Stems nd 335a (31) nd nd 60.3a 4.33a 64.6a 15.4a Purple Petra Control Leaves 65a (2) 325a (21) 0.44a (0.08) 11.42a (1.49) 35.2a (2.93) 3.46a (0.42) 50.5a (4.38) 22.9a (2.7) Stems 41a (5) 172a (12) nd nd 29.2a (3.86) 2.66a (0.43) 31.9a (3.90) 9.8a (0.6) AMF Leaves 88b (7) 390a (27) 0.36a 10.9a 39.4a 3.23a 53.9a 21.8a (0.11) (1.99) (3.36) (0.19) (5.12) (1.1) Stems 48a (5) 190a (25) nd nd 36.6a (5.32) 2.96a (0.14) 39.6a (5.31) 9.5a (1.4)

(9.25) (0.19) (9.24) (1.8)

(2.56) (0.64) (2.42) (0.4)

a All phenolic concentrations are expressed on a fresh weight basis. TACY: total anthocyanins (mg of cyanidin-glucoside 100 g1 tissue). TP: total phenolics (mg gallic acid 100 g1 tissue). Polyphenolics: polyphenolic compounds, as determined by HPLC/DAD (mg 100 g1 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 than anthocyanins 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 signicantly different (p 6 0.05, one-way ANOVA).

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A
7 2 1 Absorbance (mAU) 3 4 5 6 8 9

11

10

B
2

10

20

30

40 Time (min)

50

60

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

basil Echinacea purpurea chicoric acid

leaves, as expected from visual observations (Thai basil leaves had no visual purple colour and the petiole had a slight purple hue) (Table 2). Thai basil leaves had higher levels of TP than sweet basil leaves. 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 rosmarinic acid in basil leaves, leading to a potential underestimation of basil phenolic content by HPLC. The results from our studies indicate that, if only caffeic and rosmarinic acids were measured, approximately 2550% of the other individual phenolics in leaves and 4 18% of those in stems, would not be accounted for, based on mg 100 g1 of fresh weight.

Absorbance (mAU)

240

260

280 300 320 340 Wavelength (nm)

360

380

3.2. Effect of blanching prior to chemical extraction of sweet basil leaves During preliminary research (data not shown), we observed browning of basil tissue during sample preparation for phenolic analyses, similar to that described by others (Dogan, Turan, Dogan, Arslan, & Alkan, 2005); therefore we decided to evaluate the effect of an initial blanching step prior to chemical extraction of basil tissue. The TP value for blanched extracts was 9.0% higher than that of non-blanched extracts (Table 3). Blanched extracts had higher levels of chicoric acid (9.6%), rosmarinic acid (19.9%), other minor compounds (13.1%) and total polyphenolics by HPLC (15.7%) than did non-blanched extracts. Although not statistically signicant, caftaric acid was higher in blanched extracts, as well. These observed differences between blanched and non-blanched extracts might be greater in studies utilizing procedures with extended extraction times that do not include liquid nitrogen powdering or organic solvent. Extended extraction time, as well as higher temperatures, favours native enzymatic degradation of phenolics and could contribute toward large differences in phenolic concentrations. Perry et al. (2001) showed a greater than 50% loss in phenolics when Echinacea was initially extracted with water only, which is more favourable for native enzyme activity than is organic solvent extraction.

Fig. 2. Superimposed UVvis spectra chicoric acid peaks from market-purchased basil (Ocimum basilicum) leaves from an unnamed sweet basil cultivar, commercially-available Echinacea purpurea extract, and a pure chicoric acid standard.

1996). 5-Caffeoylquinic acid has been reported in basil (Jayasinghe et al., 2003), but 5-caffeoylquinic acid and its isomers (isomerized as described by Nagels, Van Dongen, DeBrucker, & DePooter, 1980 that produced 1-caffeoylquinic acid, 3-caffeolylquinic acid, and 4caffeoylquinic acid) were not found in these samples. Chromatograms extracted from a total ion current (TIC) MS scan chromatogram, in the negative ion mode at m/z 353, revealed weak chromatographic peaks, but they were not at the corresponding retention times of the caffeoylqunic acid isomers (Lee & Finn, 2007; Nagels et al., 1980). Other cinnamic acid derivatives were also present in minor quantities (Table 1 and Fig. 1A). Peaks other than caftaric, chicoric, and rosmarinic acids were combined and listed as other minor compounds (Tables 24). All basil stem fractions had lower phenolic concentrations and less variation in phenolic composition than did the corresponding cultivars leaf sample. No anthocyanins were detected in sweet basil leaves or stems (Table 2). Thai basil stems had higher concentrations of TACY than

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An initial blanching step, prior to extraction or processing, has often been included with samples known to turn brown rapidly, e.g. potatoes (Rodriguez-Saona, Giusti, & Wrolstad, 1998). Blanching 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 that blanching facilitated phenolic extraction compared to nonblanched 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 enzymes in analytical preparations. Susceptibility of basil phenolics to oxidation can be minimized, using an initial blanching step to maximize phenolic extraction and retention prior to analysis. 3.3. Inuence of analysis method on TP values The TP and total polyphenolic values we obtained from basil, using different methods (spectrophotometer vs. HPLC), were signicantly correlated (market-purchased basil: r = 0.991, p 6 0.05; AMF-study basil: r = 0.934, p 6 0.05). TP values from spectrophotometric analyses tended to be higher than the total polyphenolic values determined by HPLC/DAD, as previously observed (Lee & Finn, 2007; Lee & Martin, 2009). Higher TP values from spectrophotometric analysis are partially due to the interference of compounds found in plant samples (Waterhouse, 2002). 3.4. Effects of AMF on phenolic composition of basil Plants inoculated with AMF had high (>67%) root colonization and non-inoculated plants showed no sign of AMF colonization. Inoculation of basil with AMF had no effect on fresh weight of leaves or stems (Table 4), or dry weight of leaves, stems, or roots (data not shown). Leaves of Purple Petra plants inoculated with AMF had signicantly higher TACY than had non-inoculated plants, although the same trend was observed in Purple Petra stems, this difference was not statistically signicant (Table 4). Inoculation of basil with AMF had no inuence on TP, individual polyphenolic compounds, or total polyphenolics (Table 4). Copetta, Lingua, and Berta (2006) tested three AMFs (G. mosseae BEG 12, Gigaspora margarita BEG 34, and Gi. rosea BEG 9) and demonstrated AMF altered essential oil composition of Genovese basil, depending on the AMF isolated used for inoculation. The authors suggested that the increase in essential oil production in Gi. rosea inoculated plants may have been linked to higher plant biomass (Copetta et al., 2006). In our study, the isolate of AMF and phosphorus (P) concentration in the fertilizer used in this study were selected to grow plants with similar fresh weights, thus eliminating the effects of differences in composition due to biomass. Therefore increased TACY between inoculated and non-inoculated Purple Petra 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 the accumulation of anthocyanins in the leaves; therefore, there is a higher probability that non-mycorrhizal plants would have higher anthocyanin concentrations than would mycorrhizal plants. Toler, Morton, and Cumming (2005), however, reported that mycorrhizal sorghum had higher concentrations of anthocyanins than had nonmycorrhizal plants, but only when plants were under stress from elevated concentrations of copper (Cu) in the growing substrate. Several authors have speculated that many of the physiological advantages of mycorrhiza are only measurable when plants are growing under physiologically stressful conditions (Smith & Read, 1997). In our study, inoculated and non-inoculated plants contained similar amounts of nutrients (data not shown) and were provided with similar amounts of water. Therefore, increased TACY

in inoculated Purple Petra plants were probably not a result of increased water or nutrient stress. Using the same AMF species, Toussaint et al. (2007) reported that inoculation of Genova basil resulted in no signicant differences in concentrations of rosmarinic acid or caffeic acid in shoots (combination of stems and leaves), compared to non-inoculated plants when oven-dried samples from 7-week-old plants were analyzed using HPLC. Interestingly, they also reported variation in responses of basil to different isolates of AMF. Plants inoculated with G. caledonium or G. mosseae had higher concentrations of caffeic acid in shoots than had non-inoculated plants, and the response was independent of the effects of AMF on plant P nutrition. In our study, lack of response in basil phenolics to AMF inoculation may be a result of the isolate of AMF used in the study and 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 reported) inoculated olive plants had lower phenolic content than had non-AMF-inoculated plants, in both leaves and roots. Basil root phenolic composition was not the focus of this study. In the future, it would be interesting to determine whether root phenolics were altered due to AMF colonization, as reported by Toussaint et al. (2007) for rosmarinic acid and caffeic acid when basil plants were inoculated with specic AMF isolates. The effects of AMF inoculation on variation in basil phenolics due to cultivar, AMF isolate and colonization, and plant age will be explored in the future, as interest in this research has increased and there have been few studies in this eld (Toussaint et al., 2007). Plant secondary metabolites actively participate in plantmicrobe interactions, such as those in AMF-plant symbioses (Zhi-Lin, Chuan-Chao, & Lian-Quing, 2007). Enhancement of secondary metabolite accumulation in plants is of great importance in production of culinary herbs with antioxidant activity. The relationship between AMF and phenolic content alteration, within various plants, has been reviewed by Yao et al. (2007) and Toussaint (2007) and both agree that numerous factors need clarication in order to better dene the relationship between AMF and its host plants secondary metabolite production.

4. Conclusions To the best of our knowledge, this is the rst paper to identify the presence of chicoric acid and caftaric acid in basil. The presence of chicoric acid has now been reported in plants of the family Asteraceae (Echinacea, iceberg lettuce, and chicory) and Lamiaceae (basil and Cats whiskers). This is the rst report of chicoric acid in basil, and only the second to nd chicoric acid in plants of the Lamiaceae family. The response of basil phenolics to inoculation with AMF appears to depend on cultivar, polyphenolic class, plant tissue, and may also depend on AMF isolate. Regarding the potential health benets of agricultural foodstuffs, this study provides relevant information related to the dietary availability of chicoric acid. Chicoric acid is much more readily available from common and inexpensive basil leaves than from E. purpurea extracts, which was traditionally considered to be the main source of chicoric acid. Acknowledgements We thank Chris Rennaker, Jesse Mitchell, Anne Davis, and Rose Jepson of the USDA-ARS for technical assistance. This work was funded by USDA-ARS CRIS numbers 5358-21000-041-00D and 5358-12210-003-00D.

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J. Lee, C.F. Scagel / Food Chemistry 115 (2009) 650656 Lee, J., Durst, R. W., & Wrolstad, R. E. (2002). Impact of juice processing on blueberry anthocyanins and polyphenolics: Comparison of two pretreatments. Journal of Food Science, 67, 16601667. Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: Collaborative study. Journal of AOAC International, 88, 12691278. Lee, J., & Finn, C. E. (2007). Anthocyanins and other polyphenolics in American elderberry (Sambucus canadensis) and European elderberry (S. nigra) cultivars. Journal of the Science of Food and Agriculture, 87, 26652675. Lee, J., & Martin, R. R. (2009). Inuence of grapevine leafroll associated viruses (GLRaV-2 and -3) on the fruit composition of Oregon Vitis vinifera L. Cv. Pinot noir: Phenolics. Food Chemistry, 112, 889896. Makri, O., & Kintzios, S. (2008). Ocimum sp. (basil): Botany, cultivation, pharmaceutical properties, and biotechnology. Journal of Herbs Spices and Medicinal Plants, 13, 123150. Molgaard, P., Johnsen, S., Christensen, P., & Cornett, C. (2003). HPLC method validated for the simultaneous analysis of cichoric acid and alkamides in Echinacea purpurea plants and products. Journal of Agricultural and Food Chemistry, 51, 69226933. Nagels, L., Van Dongen, W., DeBrucker, J., & DePooter, H. (1980). High performance liquid chromatographic separation of naturally occurring esters of phenolic acids. Journal of Chromatography, 187, 181187. Nguyen, P. M., & Niemeyer, E. D. (2008). Effects of nitrogen fertilization on the phenolic composition and antioxidant properties of basil (Ocimum basilicum L.). Journal of Agricultural and Food Chemistry, 56, 86858691. Nusslein, B., Kurzmann, M., Bauer, R., & Kreis, W. (2000). Enzymatic degradation of chicoric acid in Echinacea purpurea preparations. Journal of Natural Products, 63, 16151618. Olah, N., Radu, L., Mogosan, C., Hanganu, D., & Gocan, S. (2003). Phytochemical and pharmacological studies on Orthosiphon stamineus Benth. (Lamiaceae) hydroalcoholic extracts. Journal of Pharmaceutical and Biomedical Analysis, 33, 117123. Perry, N. B., Burgess, E. J., & Glennie, V. I. (2001). Echinacea standardization: Analytical methods for phenolic compounds and typical levels in medicinal species. Journal of the Science of Food and Agriculture, 49, 17021706. Phillips, J. M., & Hayman, D. S. (1970). Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society, 55, 158161. Rodriguez-Saona, L., Giusti, M. M., & Wrolstad, R. E. (1998). Anthocyanin pigment composition of red-eshed potatoes. Journal of Food Science, 63, 458465. Smith, S. E., & Read, D. J. (1997). Mycorrhizal symbiosis (2nd ed.). San Diego, Calif., USA: Academic Press. Tada, H., Murakami, Y., Omoto, T., Shimomura, K., & Ishimaru, K. (1996). Rosmarinic acid and related phenolics in hairy root cultures of Ocimum basilicum. Phytochemistry, 42, 431434. Tilburt, J. C., Emanuel, E. J., & Miller, F. G. (2008). Does the evidence make a difference in consumer behavior? Sales of supplements before and after publication of negative research results. Journal of General Internal Medicine, 23, 14951498. Toler, H. D., Morton, J. B., & Cumming, J. R. (2005). Growth and metal accumulation of mycorrhizal sorghum exposed to elevated copper and zinc. Water, Air, and Soil Pollution, 164, 155172. Toussaint, J. P. (2007). Investigating physiological changes in the aerial parts of AM plants: What do we know and where should we be heading? Mycorrhiza, 17, 349353. Toussaint, J. P., Smith, F. A., & Smith, S. E. (2007). Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza, 17, 291297. Waterhouse, A. L. (2002). Unit I1.1: Polyphenolics: Determination of total phenolics. In R. E. Wrolstad (Ed.), Current protocols in food analytical chemistry (pp. 14). New York: John Wiley & Sons. Woomer, P. L. (1994). Most probable number counts. In R. W. Weaver (Ed.), Methods of soil analysis, part 2 (pp. 5979). Madison, Wis., USA: Soil Sci. Soc. Am., Inc.,. Yanishlieva, N. V., Marinova, E., & Pokorny, J. (2006). Natural antioxidants from herbs and spices. European Journal of Lipid Science and Technology, 108, 776 793. Yao, Q., Zhu, H. H., & Zeng, R. S. (2007). Role of phenolic compounds in plant defense: Induced by arbuscular mycorrhizal fungi. Allelopathy Journal, 20, 1 14. Zhi-Lin, Y., Chuan-Chao, D., & Lian-Quing, C. (2007). Regulation and accumulation of secondary metabolites in plant-fungus symbiotic system. African Journal of Biotechnology, 6, 12661271.

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 lietuvos moksl akademija, 2010 lietuvos moksl akademijos leidykla, 2010

chemija. 2010. vol. 21. No. 1. P. 5962

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 Department of Horticultural Sciences, Faculty of Agriculture, Urmian University, Urmia 5715944931, Iran Research Service Laboratory, Faculty of Chemistry, University of Tabriz, Tabriz, Tabriz 51666, Iran

The hydrodistilled essential oil from aerial parts of cultivated Ocimum basilicum L. plants from Northwest Iran was analyzed by gas chromatography / mass spectrometry. Forty seven components were identified, comprising 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 components 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--farnesene (1.1%) and -amorphene (1.1%) as their main ones. -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 percent 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 monoterpenes 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 1545 cm in height [1, 2]. This plant has obovate serrated opposite leaves and fragrant white or violet compound terminate flowers [2, 3]. Sweet basil is a cosmopolitan 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 applications in perfume, food, cosmetic and pharmaceutical industries [4]. Medicinally, this plant and its essential oil have long
* corresponding author. e-mail: hassanpouraghdam@gmail.com

been used as immunostimulant [5], sedative, hypnotic, local anesthetic [6], anticonvulsant, antitussive, diuretic [2], carminative, galactogogue, stomachic, spasmodic [7], vermifuge [8] and platelet anti-aggregant [9]. Furthermore, different biological 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, dysentery, 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]

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Mohammad Bagher Hassanpouraghdam, Abbas Hassani, Mohammad Safi Shalamzari

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 components, 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 chemotypes with major components greater than 50%, namely methyl chavicol, linalool, geraniol, linalool / methyl cinnamate, linalool / geraniol, methyl cinnamate / linalool and eugenol / 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 -cubebene were reported as the main components 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 -elemene as the main constituents of basil oil from Hungary. Meanwhile, linalool, (Z) cinnamic acid methyl ester, estragol, eugenol, 1,8-cineol, bergamotene, methyl cinnamate, -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, 1521]. However, only limited studies have been conducted so far on the compositional analysis of O. basilicum L. from Northwest Iran. The aim of the present study was to characterize for the first time the volatile oil composition of an endemiccultivated O. basilicum herb from Northwest Iran. EXPERIMENTAL Plant material sampling and preparation. The flowering above-ground parts of endemic purple green-leaved cultivated O. basilicum L. plants from Maragheh district in Northwest 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 representatives of the local native population were dried at room temperature (about 30 C) for 45 days until constant weight. The air-dried plant material was mixed and pulverized to obtain 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 carried out in triplicate. Gas chromatography / mass spectrometry. The analysis of the oil was carried out using a GC (Agilent Technologies 6890N) connected to a mass-selective detector (MSD, Agilent 5973B) equipped with an non-polar Agilent HP-5MS (5%-phenyl methyl polysiloxane) capillary column (30 m 0.25 mm i. d. and 0.25 m 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 until 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 l of the oil. The MS operating parameters were as follows: ionization potential, 70 eV; interface temperature, 200 C; and acquisition mass range, 50800. Identification and quantification of constituents. The relative percentage amounts of the essential oil components were evaluated from the total peak area (TIC) by the apparatus software. The identification of components in the volatile oil was based on a comparison of their mass spectra and retention 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 provided 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, retention 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 identified 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 monoterpenes (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%), isoneomenthol (7.5%), menthol (6.1%), pulegone (3.7%) and linalool (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%)

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

61

were the main subclass of 15 carbon sesquiterpenoidal compounds with trans-caryophyllene (2.2%), germacrene D (1.4%), trans--farnesene (1.1%) and -amorphene (1.1%) as the most abundant components. -Cadinol (2.9%), an oxygenated sesquiterpene, had the highest amount of its
Tab l e 1 . Essential oil composition of Ocimum basilicum L. from Iran
Compound
-Pinene Sabinene -Pinene -Myrcene 3-Octanol -Phellandrene p-Cymene Limonene 1,8-Cineole (Z)--Ocimene -Terpinene Fenchone Linalool cis-Rose oxide Camphor Menthone Menthol Iso-neomenthol Estragol Pulegone Chavicol Piperitone Isopulegol acetate Menthyl acetate Carvacrol -Cubebene Eugenol -Copaene -Bourbonene -Cubebene -Elemene Methyl eugenol trans-Caryophyllene trans--Bergamotene -Guaiene trans--Farnesene Germacrene D -Amorphene (E)--Ionone Bicyclogermacrene cis-Calamene Spathulenol Caryophyllene oxide Muurolol -Eudesmol -Cadinol Phytol Total

RI
0 939 0 975 0 979 0 991 0 991 1 003 1 025 1 029 1 031 1 037 1 060 1 078 1 097 1 108 1 146 1 153 1 172 1 187 1 196 1 237 1 250 1 253 1 278 1 295 1 299 1 351 1 359 1 377 1 388 1 388 1 391 1 404 1 419 1 435 1 440 1 457 1 485 1 485 1 489 1 500 1 540 1 578 1 583 1 646 1 651 1 654 1 943

Molecular formula
C10H16 C10H16 C10H16 C10H16 C8H18O C10H16 C10H14 C10H16 C10H18O C10H16 C10H16 C10H16O C10H18O C10H18O C10H16O C10H18O C10H20O C10H20O C10H12O2 C10H12O2 C9H10O C10H16O C12H20O2 C12H22O2 C10H14O C15H24 C10H12O2 C15H24 C15H24 C15H24 C15H24 C11H14O2 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C13H20O C15H24 C15H24 C15H24O C15H24O C15H26O C15H26O C15H26O C20H40O

%
0.1 0.1 0.3 0.1 0.1 0.1 0.1 1.5 0.2 0.1 0.2 0.3 1.7 0.2 0.3 33.1 6.1 7.5 21.5 3.7 0.1 0.3 0.3 5.6 0.2 0.1 0.1 0.5 0.1 0.3 0.5 1 2.2 0.7 0.1 1.1 1.4 1.1 0.1 0.5 0.2 0.4 0.4 0.1 0.2 2.9 0.1 97.9

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, -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 [49, 1221]. Taking into account the chemical 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 menthone, menthol, isoneomenthol and pulegone since these oxygenated monoterpenes are characteristic of mint plants and their high amounts in basil oil are unfamiliar. To our knowledge, 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 Rutaceae plants. On the other hand, low amounts of methyl eugenol and linalool weaken the chemo-similarity of the study plant with most of the above-cited literature [49, 1221]. In total, it is noteworthy that, although O. basilicum from different localities has been thoroughly investigated with regard 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 variations from diverse localities seem to be due to the divergent climatological and geographical conditions (light quality and quantity, soil characteristics, water and nutrient availability,
Table 2. Main classes, subclasses and chemical groups of Ocimum basilicum L. essential oil constituents from Iran
Class, subclass and chemical group of compound
Monoterpenoids Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpenoids Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total identified Chemical groups Alcohols Ketones Acetates Methylated compounds Oxides

%
77.8 2.6 75.3 12.8 8.8 4 7.3 97.9 42 37.8 5.9 1.1 0.8

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

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Mohammad Bagher Hassanpouraghdam, Abbas Hassani, Mohammad Safi Shalamzari

6. 7. 8. 9. 10. 11. m. ismail, Pharm. Biol., 44(8), 619 (2006). S. e. Sajjadi, Daru, 14(3), 128 (2006). m. Ozcan, j. c. chalchat, Czech J. Food Sci., 20(6), 223 (2002). j. W. Zhang, S. K. li, W. j. Wu, Molecules, 14, 273 (2009). X. chang, P. G. alderson, c. j. Wright, Environ. Exp. Bot., 63, 216 (2008). 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 Netherlands (1988). a. h. N. abduelrahman, e. a. alhussein, N. a. i. Osman, a. h. Nour, Int. J. Chem. Technol., 1(1), 1 (2009). v. D. Zheljazkov, a. N. callahan, c. l. cantrell, J. Agric. Food Chem., 56(1), 241 (2007). m. marotti, R. Piccaglia, e. Giovanelli, J. Agric. Food Chem., 44(12), 3926 (1996). e. Werker, e. Putievsky, U. Ravid, N. Dudai, i. Katzir, Ann. Bot., 71, 43 (1993). O. Politeo, m. jukic, m. milos, Croat. Chem. Acta, 79(4), 545 (2006). S. m. Keita, c. vincent, j. P. Schmit, a. Belanger, Flavour Frag. J., 15(5), 339 (2000). a. a. Kasali, a. O. eshilokun, S. adeola, P. Winterhalter, h. Knapp, B. Bonnlander, Flavour Frag. J., 20(1), 45 (2004). a. j. hussein, F. anwar, S. T. h. Sherazi, R. Prybylski, Food Chem., 108(3), 986 (2008). j. c. chalchat, m. m. Ozcan, Food Chem., 110(2), 501 (2008). S. R. vani, S. F. cheng, c. h. chuah, Am. J. Appl. Sci., 6(3), 523 (2009). R. P. adams, Identification of Essential Oil Components by Gas Chromatography / Quadrupole Mass Spectroscopy, allured Publishing corp., carol Stream, il, USa (2004).

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. basilicum L. plants studied in the present experiment might be a unique chemotype of this plant owing to its distinct volatile oil composition. However, this plea requires comparative studies based on detailed phytochemical assays. CONCLUSIONS In brief, the chemical composition of the essential oil of cultivated O. basilicum L. plant from Northwest Iran was characterized 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

12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22.

References
1. 2. a. Zarghari, Medicinal Plants (in Persian), Tehran University Publications, iran (1997). v. mozaffarian, A Dictionary of Iranian Plant Names (in Persian), Farhang moaaser Publishing company, iran (2004). a. Ghahreman, Plant Systematics: Cormophytes of Iran (in Persian), iran University Press, iran (1993). a. Koba, P. W. Poutouli, c. Raynaud, j. P. chaumont, K. Sanda, Bangladesh J. Pharmacol., 4, 1 (2009). 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).

Mohammad Bagher Hassanpouraghdam, Abbas Hassani, Mohammad Safi Shalamzari MENTONO IR ESTRAGOLIO TURINTIS ETERINIS ALIEJUS I IAURS VAKAR IRANE KULTIVUOJAMO OCIMUM BASILICUM L. Santrauka Eterinis aliejus, hidrodistiliavimo bdu iskirtas i iaurs vakar Irane kultivuojam Ocimum basilicum L. augal, buvo tiriamas duj chromatografijos-masi spektrometrijos metodu. Buvo identifikuoti 47 komponentai, kartu sudarantys 97,9 % eterinio aliejaus. Tarp komponent rasta 77,8 % monoterpenoid ir 12,8 % seskviterpenoid. Apskritai tirtasis eterinis aliejus yra apibdinamas kaip naujo mentonoestragolio tipo aliejus, kuris gali bti panaudotas maisto ir farmacijos pramonje.

3. 4. 5.

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Gonzlez-Ziga, Juan Antonio; Gonzlez-Snchez, Hctor Manuel; GonzlezPalomares, Salvador; Rosales-Reyes, Tbata; Andrade-Gonzlez, Isaac Microextraccin en fase slida de compuestos voltiles en albahaca (Ocimum basilicum L.) Acta Universitaria, vol. 21, nm. 1, enero-abril, 2011, pp. 17-22 Universidad de Guanajuato Guanajuato, Mxico
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Microextraccin en fase slida de compuestos voltiles en albahaca (Ocimum basilicum L.)

Juan Antonio Gonzlez-Ziga*, Hctor Manuel Gonzlez-Snchez**, Salvador GonzlezPalomares**, Tbata Rosales-Reyes**, e Isaac Andrade-Gonzlez***
RESUMEN
Los compuestos voltiles de la albahaca (Ocimum basilicum L.) fueron extrados mediante la microextraccin en fase slida (SPME) y analizados con cromatografa de gases-espectrometra de masas (GC-MS). Se evaluaron dos fibras, Polidimetilsiloxano/Divinilbenceno (PDMS/DVB, 65 m) y Carbowax/Divinilbenceno (CW/DVB, 65 m), para comparar la extraccin de componentes. Entre los 25 compuestos voltiles recuperados en la albahaca, se identificaron fenilpropanoides, monoterpenos, sesquiterpenos, steres, y aldehdos. Hubieron diferencias significativas (P < 0,05) entre las fibras analizadas. La comparacin de las dos fibras mostr que la extraccin con la fibra CW/DVB es aparentemente mejor tanto en el nmero de componentes aislados como en la concentracin total de los compuestos. Cuantitativamente, el componente ms importante fue el cinamato de metilo, seguido por el linalol.

ABSTRACT
Volatile compounds of basil (Ocimum basilicum L.) were extracted with solid phase microextraction (SPME) and analyzed with gas chromatography-mass spectrometry (GC-MS). Two SPME fiber coatings, Polydimethylsiloxane/Divinylbenzene (PDMS/DVB, 65 m) and Carbowax/Divinylbenzene (CW/DVB, 65 m) were evaluated in order to compare the extraction of components. Among the 25 volatile compounds detected were phenylpropanoids, monoterpenes, 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. Quantitatively, the most important component was methyl cinnamate, followed by linalool.

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

INTRODUCCIN La albahaca (Ocimum basilicum L., Lamiaceae) es una hierba anual, tiene gran aprecio por los consumidores debido a sus propiedades medicinales, as como tambin por su aroma y sabor caracterstico (Koba et al., 2009). En Mxico, el cultivo de esta planta se distribuye en casi todo el pas y es ampliamente usada en desrdenes 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 accin sobre el sistema digestivo y el sistema neurovegetativo (Arvy y Gallouin, 2007). La albahaca tambin tiene una diversidad de usos en alimentos, cosmticos, 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).
Palabras clave: SPME; voltiles; albahaca; cinamato de metilo. Keywords: SPME; volatile; basil; methyl cinnamate.

El gnero Ocimum, colectivamente llamado albahaca, crece en varias regiones del mundo (Simon et al., 1999) e incluye un gran nmero de especies, subespecies y variedades que son producto de una abundante polinizacin cruzada (Lawrence, 1988). Por estas razones, se ha investigado extensamente la composicin qumica del aceite esencial de la albahaca en diferentes pases (Fleisher, 1981; Lachowicz et al., 1996; Via y Murillo, 2003; Lee

*Unidad de Ciencias de los Alimentos. Instituto Tecnolgico Superior de La Huerta (ITSH). Av. Rafael Palomera No. 161, col. El Maguey, C.P. 48850. La Huerta, Jalisco, Mxico. Correo electrnico: chava1142@hotmail.com **Laboratorio de Biotecnologa. Universidad de Guadalajara (UdG). Centro Universitario de la Costa Sur (CUCSur). Av. Independencia Nacional No. 151. C.P. 48900. Autln de Navarro, Jalisco, Mxico. Correo electrnico: chava1142@yahoo.com.mx ***Planta Piloto de Procesos Agroindustriales. Instituto Tecnolgico de Tlajomulco, Jalisco (ITTJ). Km. 10 carr. San Miguel Cuyutln. C.P. 45640. Tlajomulco de Ziga, Jalisco, Mxico.

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et al., 2005; Koba et al., 2009), encontrndose que sta vara mucho segn el origen geogrfico de la planta (Arvy y Gallouin, 2007), el estado de desarrollo vegetativo (Fleisher, 1981), las condiciones agronmicas en su produccin (Miele et al., 2001), y las variedades cultivadas (Lachowicz et al., 1997; Via y Murillo, 2003). Sin embargo, hay pocos datos disponibles acerca de la caracterizacin qumica de las especies del gnero Ocimum cultivadas en Mxico. Los aceites esenciales contienen compuestos aromticos muy voltiles, los cuales son responsables de los olores y sabores caractersticos de las plantas (Padrini y Lucheroni, 1997; Gonzlez-Palomares y Vzquez-Garca, 2008). En la albahaca, el carcter aromtico de cada variedad se origina principalmente por una mezcla compleja de monoterpenos, sesquiterpenos, fenilpropanoides (Fleisher, 1981; Lachowicz et al., 1997; Miele et al., 2001; Wossa et al., 2008; Koba et al., 2009), cidos orgnicos (Argueta y Cano, 1994), alcoholes, aldehdos, cetonas y steres (Lee et al., 2005). Varios compuestos aromticos, tales como metil-chavicol (estragol), cinamato de metilo, metileugenol, eugenol, linalol y geraniol, han sido reportados como los mayores componentes de los aceites de O. basilicum (Sajjadi, 2006; Wossa et al., 2008). Estas diferencias en la composicin qumica, permitieron la clasificacin de la albahaca en quimiotipos [conocidos por los nombres segn el origen geogrfico (Simon et al., 1999)] con base en los componentes predominantes (Lawrence, 1988) o a los compuestos detectados en una cantidad superior al 20 % (Miele et al., 2001; Wossa et al., 2008). Actualmente se han aadido nuevos quimiotipos y subtipos a la clasificacin establecida por Lawrence en 1988 (Wossa et al., 2008). El anlisis por cromatografa de gases acoplada a espectrometra de masas (GC-MS) ha permitido conocer la composicin qumica y la abundancia relativa de los principales componentes de los aceites de O. basilicum (Acosta et al., 2003). Para lo cual, previamente se realiza la extraccin de los compuestos voltiles a travs de mtodos como la microextraccin en fase slida (SPME), que es muy popular en la actualidad (Fan y Sokorai, 2002; Marn y Cspedes, 2007; Reineccius, 2007; Gonzlez-Palomares et al., 2009, Gonzlez-Palomares et al., 2010a). Las virtudes de la SPME son ampliamente aclamadas e incluyen que es relativamente econmica, rpida, no utiliza disolventes en la preparacin de la muestra y es razonablemente sensible a la recuperacin de compuestos voltiles (Pawliszyn, 1997; Beaulieu y Lea, 2006; Marn y Cspedes, 2007; Reineccius, 2007). En esta tcnica, una fibra revestida con uno o

ms polmeros de extraccin remueve por adsorcin los analitos de la muestra (e.g. los compuestos aromticos), y luego es insertada directamente dentro del sistema GC-MS para la desorcin trmica y el anlisis (Marn y Cspedes, 2007; Reineccius, 2007; GonzlezPalomares et al., 2009). La combinacin de SPME y GC-MS ha sido exitosamente aplicada en la extraccin de compuestos orgnicos voltiles y semi-voltiles de diversas muestras (Vas y Vkey, 2004; Gonzlez-Palomares et al., 2010a). La escasez de informacin cientfica acerca de la composicin qumica de la albahaca cultivada en Jalisco, Mxico, gener como objetivo del trabajo: determinar los componentes aromticos ms abundantes de la albahaca (Ocimum basilicum L.) procedente de La Huerta, Jalisco, mediante la evaluacin de dos fibras de extraccin de compuestos voltiles por microextraccin en fase slida y cromatografa de gases- espectrometra de masas. La informacin generada servir como antecedente bsico para trabajos futuros dirigidos a la bsqueda de los compuestos voltiles de plantas popularmente empleadas en Jalisco, Mxico, como es el caso de la albahaca. MATERIALES Y MTODOS Muestras de albahaca: Las hojas y tallos frescos de albahaca (Ocimum basilicum L.), con 80 % de humedad, se colectaron de un campo de produccin de La Huerta, Jalisco, Mxico. El material vegetal cosechado provino de plantas en poca de floracin. En esta etapa fenolgica es cuando la albahaca tiene un mayor contenido de aceite esencial (Fleisher, 1981). Microextraccin en fase slida (SPME): Se emplearon dos fibras de extraccin, polidimetilsiloxano/divinilbenceno (PDMS/DVB, 65 m) y carbowax/ divinilbenceno (CW/DVB, 65 m). Las fibras se acondicionaron considerando las instrucciones provistas por el fabricante: 30 min a 260 C para PDMS/DVB y 30 min a 250 C para CW/DVB. Despus del acondicionamiento, se corrieron las fibras blanco para confirmar la limpieza del sistema GC-MS. La jeringa, fibras y viales para SPME se obtuvieron de la Compaa Supelco (Bellefonte, PA, USA). Las muestras de albahaca se homogeneizaron a temperatura ambiente durante 20 segundos. Los compuestos voltiles de las muestras homogenizadas fueron extrados por el mtodo de SPME (Cuevas-Glory et al., 2008), con modificaciones (Gonzlez-Palomares et al., 2009; Gonzlez-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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(Barnsted E-pure) a un vial de 40 mL, el cual se sell hermticamente por medio de una septa PTFE-silicn. El vial se incub a 70 C en un termobao con agitacin durante 30 min. Transcurrido este tiempo, la fibra para SPME se insert en el espacio de cabeza del vial, mantenindose la temperatura y la agitacin durante 40 min. La incubacin con temperatura y agitacin sirvi para promover el equilibrio entre los analitos en el espacio de cabeza del vial, la muestra y el polmero de la fibra, y as obtener una mayor concentracin de compuestos voltiles en la fibra. Al terminar el tiempo de extraccin, se retir del vial la fibra con los compuestos voltiles adsorbidos y se insert en el puerto de inyeccin de un cromatgrafo de gases con un tiempo de desorcin de 5 min. Este proceso se realiz con cinco repeticiones en las mismas condiciones. Cromatografa de gases-espectrometra de masas (GC-MS): Los compuestos voltiles 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 capilar polar Supelcowax-10 (Supelco, Bellefonte, PA, USA) de 30 m de largo x 0,25 m de dimetro interno y con fase estacionaria de polietilenglicol. Las condiciones empleadas durante el anlisis de las muestras fueron: temperatura del inyector y del detector de 190 C y 240 C, respectivamente. Se estableci una temperatura inicial del horno de 40 C, mantenida por 5 min hasta llegar a una temperatura final de 250 C con incrementos de 5 C por minuto. El gas acarreador fue helio grado cromatogrfico (INFRA S.A.), con un flujo de 0,8 mL/min (Gonzlez-Palomares et al., 2009; Gonzlez-Palomares et al., 2010b). Identificacin y cuantificacin de compuestos voltiles: Los compuestos voltiles de albahaca se identificaron por comparacin 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 cuantificacin se realiz con base en el porcentaje de rea de cada pico del cromatograma correspondiente a cada compuesto voltil de la albahaca (Gonzlez-Palomares et al., 2009; Gonzlez-Palomares et al., 2010b). Anlisis estadstico: Se evaluaron las fibras de extraccin utilizadas durante la SPME, con base en el nmero y concentracin de compuestos voltiles aislados. Las concentraciones totales de compuestos voltiles de la albahaca se obtuvieron del promedio de las cinco repeticiones (n = 5) realizadas en la microextraccin con las fibras PDMS/DVB y CW/DVB. Los datos generados fueron sujetos a anlisis estadstico

a travs de la prueba de t de Student y considerados significativos con P < 0,05 (Gonzlez-Palomares et al., 2010b). RESULTADOS Y DISCUSIN En albahaca (Ocimum basilicum L.) procedente de La Huerta, Jalisco, Mxico, se identificaron y cuantificaron 25 compuestos voltiles va GC-MS. En el aislamiento de los componentes aromticos mediante SPME, se emplearon dos fibras: PDMS/DVB y CW/ DVB. Del total de los compuestos extrados, 18 fueron comunes para ambas fibras (tabla 1).
Tabla 1. Compuestos voltiles de albahaca identificados mediante dos fibras de extraccin a travs de SPME/GC-MS.
Compuesto Concentracin (% de rea) PDMS/DVB:
Fenilpropanoides Cinamato de etilo Cinamato de metilo Linalol Neral -Pineno -Pineno -Mirceno p-Cimeno -Terpineno

CW/DVB: 4,11 15,00 10,50 0,90 0,63 0,44 0,32 0,45 2,00 1,70 0,51 1,10 2,15 1,41 2,05 1,85 2,21 1,35 0,99 1,95 0,81 0,90 1,54 0,70 1,89 25

3,11 8,50
Monoterpenos

6,00 -----1,60
Sesquiterpenos

-Cariofileno -Cadineno

0,93 -steres

Acetato de isoamilo Acetato de hexilo Acetato de etilo Acetato de octilo Acetato de bencilo 2-metilbutirato de etilo Miristato de etilo Benzoato de etilo Hexanoato de etilo Nonanal Hexanal Decanal (E)-2-Octenal (E)-2-Nonenal Total de compuestos:

0,23 0,92 1,10 1,17 1,19 1,25 1,21 0,97 -Aldehdos

0,94 0,91 0,45 0,47 0,59 18

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 extraccin, tanto en el nmero de componentes identificados (18 con la fibra PDMS/DVB y 25 con la fibra CW/DVB) como en la concentracin total: 31,54 % con la fibra PDMS/ DVB y 57,46 % con la fibra CW/DVB. Adems, con la fibra PDMS/DVB se aislaron menos terpenos que con la fibra CW/DVB. Estas discrepancias pueden deberse a la composicin de cada fibra y a la afinidad de los compuestos de la muestra a la matriz adsorbente. En el tequila, la fibra PDMS/DVB permiti muy baja cuantificacin de terpenos, la cual mejor al adicionar 100 % de NaCl, con temperatura y tiempo de extraccin de 25 C y 30 min, respectivamente (Pea-lvarez 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 permiti la clasificacin de los compuestos voltiles del caf con base en el origen geogrfico. Klimnkov et al., (2008) obtuvieron perfiles de compuestos voltiles de albahaca bastante similares entre las fibras PDMS/DVB y CW/DVB. En el actual estudio, los componentes extrados de la albahaca se agruparon con base en sus caractersticas estructurales en fenilpropanoides, monoterpenos, sesquiterpenos, steres y aldehdos. Los compuestos voltiles pertenecientes a los tres primeros grupos coincidieron con los reportados en otros trabajos de investigacin (Lachowicz et al., 1996; zcan y Chalchat, 2002; Via y Murillo, 2003; Lee et al., 2005; Chang et al., 2009). Estos autores han tabulado en la albahaca ms compuestos, sin embargo, debido a que fueron aislados por hidrodestilacin o por destilacin con vapor, posibles artefactos o productos de degradacin estn en algunas de estas listas. De otros estudios, que utilizaron SPME, correspondieron los compuestos ms abundantes (Reyes et al., 2007) y algunos mono y sesquiterpenos (Klimnkov et al., 2008). Con las dos fibras de extraccin utilizadas, el cinamato de metilo (fenilpropanoide), el linalol (monoterpeno), y el cinamato de etilo (fenilpropanoide) fueron los compuestos de mayor concentracin. 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; Via y Murillo, 2003; Reyes et al., 2007), o dentro de los mayores constituyentes del aroma (Lee et al., 2005; Muoz et al., 2007; Politeo et al., 2007; Klimnkov et al., 2008) en diferentes variedades de O. basilicum. Lee et al., (2005) tambin detectaron al cinamato de etilo aunque en baja concentracin. De acuerdo con Lawrence (1988),

el componente predominante es el que determina el quimiotipo de la albahaca. En la presente investigacin, el cinamato de metilo fue el principal componente, seguido por el linalol (razn 1,5:1). En algunas variedades de O. basilicum se han encontrado proporciones iguales o similares entre estos compuestos (Lachowicz et al., 1997; Via y Murillo, 2003) y se estableci que eran del quimiotipo cinamato de metilo, subtipo cinamato de metilo> linalol (Via y Murillo, 2003). Posiblemente dicho quimiotipo y subtipo sean tambin los que corresponden a la albahaca utilizada en este trabajo. No obstante, se requieren investigaciones posteriores para confirmar tal informacin. En las hojas de la albahaca se encuentran grandes cantidades de fenilpropanoides, monoterpenos y sesquiterpenos, as como metabolitos derivados de los cidos grasos. Estos grupos de compuestos, individualmente y en combinacin, imparten un distintivo aroma y sabor (Gang et al., 2001). Los cidos grasos insaturados y poliinsaturados son precursores de un gran nmero de compuestos voltiles que son importantes para definir el carcter aromtico, entre ellos, los steres y los aldehdos (Christensen et al., 2007). Los steres y los aldehdos han sido escasamente reportados en albahaca, sin embargo, en este estudio el mayor nmero de los compuestos extrados corresponden a estos dos grupos. En la albahaca se han registrado un amplio rango de aromas: a limn, rosa, alcanfor, licor, amaderado y afrutado (Simon et al., 1999). Los compuestos encontrados en esta investigacin proporcionan diversas notas: los fenilpropanoides y los steres, aromas afrutados (Christensen et al., 2007); los monoterpenos y sesquiterpenos proveen olores frescos, florales, a limn, dulces, herbceos, afrutados y amaderados (Tamura et al., 2001), y los aldehdos, notas frescas, verdes, ctricas, florales, jabonosas y oleosas (Rouseff y Perez-Cacho, 2007). Los compuestos voltiles se forman de los constituyentes mayores de las plantas a travs de varias rutas bioqumicas (Christensen et al., 2007). Los fenilpropanoides se biosintetizan a partir del cido cinmico a travs de la ruta del siquimato. Los monoterpenos y sesquiterpenos tienen su origen biosinttico en la va del cido mevalnico, tambin conocida como ruta del mevalonato (Via y Murillo, 2003; Wossa et al., 2008). Los steres y aldehdos resultan de la degradacin de los cidos grasos insaturados y poliinsaturados (Christensen et al., 2007). La composicin qumica de la albahaca puede verse influenciada por la variedad, las condiciones agroclimatolgicas (Lachowicz et al., 1997; Via y Murillo,

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2003), el estatus nutricional de las plantas (zcan y Chalchat, 2002; Sajjadi, 2006), el estado de desarrollo vegetativo (Fleisher, 1981), y la parte de la planta analizada (Muoz et al., 2007; Klimnkov et al., 2008). Las variedades de Ocimum basilicum L., exhiben distinto carcter aromtico, caractersticas morfolgicas, composicin qumica en los aceites esenciales, y quimiotipos (Simon et al., 1999). Varios mtodos analticos han sido desarrollados para determinar los constituyentes voltiles de los aceites esenciales, aunque algunos de ellos pueden ocasionar cambios qumicos en los compuestos o prdida de la mayora de los voltiles (Klimnkov et al., 2008). An cuando la extraccin de los compuestos voltiles se realice mediante el mismo mtodo, hay factores que pueden variar los resultados. Particularmente, en la SPME, el tipo y espesor del recubrimiento de la fibra usada, la adicin de un electrolito (e.g. una sal), las condiciones de extraccin (tiempo y temperatura), y la temperatura del puerto de inyeccin para la desorcin de los analitos de la fibra (Vas y Vkey, 2004). Las variaciones encontradas en el aislamiento de compuestos voltiles de la albahaca, entre este proyecto y otras investigaciones pueden deberse a la variedad de las plantas, al mtodo y las condiciones de extraccin utilizadas. Es importante recordar que ningn mtodo para aislar el aroma de una planta da una identificacin completa de los compuestos aromticos presentes en ella (Reineccius, 2007; Gonzlez-Palomares y Vzquez-Garca, 2008). La composicin del aroma es una mezcla compleja de sustancias que aunque lleva en s misma la huella del vegetal del que procede, cada planta tiene su esencia particular, la cual es nica e irreproducible (Padrini y Lucheroni, 1997; GonzlezPalomares et al., 2009). La sensibilidad de la SPME usada en este trabajo fue comparable con mtodos convencionales como la hidrodestilacin o la destilacin con vapor en cuanto a la extraccin de los fenilpropanoides, monoterpenos y sesquiterpenos. Segn 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 composicin qumica de la albahaca con respecto a reportes previos, principalmente estuvieron dadas por la variedad, el recubrimiento de la fibra, la adicin de sal, el tiempo y la temperatura de extraccin. Con el fin de ampliar y complementar la informacin obtenida de la albahaca, se justifican investigaciones posteriores de los compuestos voltiles y as dirigir la informacin obtenida hacia posibles aplicaciones.

CONCLUSIONES El perfil qumico de Ocimum basilicum L., procedente de La Huerta, Jalisco, Mxico est constituido principalmente por fenilpropanoides y monoterpenos, cuyos principales compuestos representantes son el cinamato de metilo y el linalol, respectivamente. La tcnica de SPME/GC-MS representa un mtodo apropiado para la extraccin de compuestos voltiles de la albahaca. En las condiciones analticas empleadas, la fibra CW/DVB present una mejor eficiencia de adsorcin de compuestos voltiles. AGRADECIMIENTOS Al Consejo Estatal de Ciencia y Tecnologa de Jalisco (COECYTJAL), por el apoyo econmico para realizar esta investigacin. A la doctora Lya Esther Saudo Guerra, doctora Martha Vergara Fregoso, maestra Ruth Catalina Perales Ponce y a la maestra Martha Daniela Concepcin Garca Moreno, autoridades de la Direccin General de Investigacin de la Secretara de Educacin Jalisco (SEJ), por las sugerencias en el desarrollo del trabajo. REFERENCIAS
Acosta, M., Gonzlez, M., Araque, M., Velazco, M., Khourt, N., Rojas, L., y Usubillaga, A. (2003). Composicin qumica de los aceites esenciales de Ocimum basilicum L. var basilicum, O. basilicum L. var purpurenscens, O. gratissimum L., y O. tenuiflorum L., y su efecto antimicrobiano sobre bacterias multirresistentes de origen nosocomial. Rev. Fac. Farm. 45(1):19-24. Argueta, V.A., y Cano, A.L. (1994). Atlas de las Plantas de la Medicina Tradicional Mexicana. Instituto Nacional Indigenista. Mxico. Pp: 86-88. Arvy, M.P., y Gallouin, F. (2007). Especias, aromatizantes y condimentos. MundiPrensa Libros. Madrid. Pp: 36-37. Beaulieu, J.C., y Lea J.M. (2006). Characterization and semiquantitative analysis of volatiles in seedless watermelon varieties using Solid-Phase Microextraction. J. Agric. Food Chem. 54:7789-7793. Chang, X., Alderson, P.G., y Wright, C.J. (2009). Enhanced UV-B radiation alters basil (Ocimum basilicum L.) growth and stimulates the synthesis of volatile oils. J. Hortic. For. 12:27-31. Christensen, L.P., Edelenbos, M., y Kreutzmann, S. (2007). Fruits and vegetables of moderate clime. In: Flavours and Fragances. Chemistry, Bioprocessing and Sustainability. Berger, R.G. (Ed.). Springer. Germany. Pp: 135-181. Costa, A.M.F., Parreira, C., y Vilas-Boas, L. (2001). Comparison of two SPME fibers for differentiation of coffee by analysis of volatile compounds. Chromatographia. 54(9-10):647-652. Cuevas-Glory, L.F., Ortiz-Vzquez, E., Centurin-Yah, A., Pino J.A., y Sauri-Duch, E. (2008). Desarrollo de un mtodo por microextraccin en fase slida para el anlisis de la fraccin voltil de la miel de abeja de Yucatn. Tc. Pecu. Mx. 46(4):387-395.

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Fan, X., and Sokorai, K.J.B. (2002). Changes in volatile compounds of -irradiated fresh cilantro leaves during cold storage. J. Agric. Food Chem. (50):7622-7626. Fleisher, A. (1981). Essential oils from two varieties of Ocimum basilicum L., grown in Israel. J. Sci. Food Agric. 32:1119-1122. Gang, D.R., Wang, J., Dudareva, N., Nam, K.H., Simon, J.E., Lewinsohn, E., and Pichersky, E. (2001). An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol. 125:539-555. Gonzlez-Palomares, S., Estarrn-Espinosa, M., Gmez-Leyva, J.F., and Andrade-Gonzlez, I. (2009). Effect of the Temperature on the Spray Drying of Roselle Extracts (Hibiscus sabdariffa L.). J. Plant Foods for Human Nutrition. 64(1):62-67. Gonzlez-Palomares, S., Estrada-Dichi, A., Del Val-Daz, R., Rosales-Reyes, T., Andrade-Gonzlez, I., y Hernndez-Estrada, A. (2010a). Determinacin de compuestos voltiles en noni (Morinda citrifolia L.) mediante microextraccin en fase slida y cromatografa de gases. Memoria del VI Congreso Internacional y XVII Congreso Nacional de Ingeniera Bioqumica. Colegio Mexicano de Ingenieros Bioqumicos, A.C. Acapulco, Guerrero, Mxico. Pp: 1-6. Gonzlez-Palomares, S., Rivera-Cambero, L.H., y Rosales-Reyes, T. (2010b). Anlisis de compuestos voltiles en cilantro (Coriandrum sativum L.). Revista Acta Universitaria. Universidad de Guanajuato. 20(1):19-24. Gonzlez-Palomares, S., y Vzquez-Garca, E.S. (2008). Caracterizacin de compuestos aromticos en fruta de noni (Morinda citrifolia L.). Boletn de ConCIENCIA y Tecnologa. Secretara de Educacin Jalisco (SEJ). (3):21-26. Klimnkov, E., Holadov, K., Hajlov, J., ajka, T., Poustka, J., and Koudela, M. (2008). Aroma profiles of five basil (Ocimum basilicum L.) cultivars grown under conventional and organic conditions. Food Chem. 107:464-472. Koba, K., Poutouli, P.W., Raynaud, C., Chaumont, J.P., and Sanda, K. (2009). Chemical composition and antimicrobial properties of different basil essential oils chemotypes from Togo. Bangladesh J. Pharmacol. 4:1-8. Lachowicz, K.J., Jones G.P., Briggs, D.R., Bienvenu, F.E., Palmer, M.V., Ting, S.S.T., and Hunter, M. (1996). Characteristics of essential oil from basil (Ocimum basilicum L.) grown in Australia. J. Agric. Food Chem. 44:877-881. Lachowicz, K.J., Jones, G.P., Briggs, D.R., Bienvenu, F.E., Palmer, M.V., Mishra, V., and Hunter, M.M. (1997). Characteristics of plants and plant extracts from five varieties of basil (Ocimum basilicum L.) grown in Australia. J. Agric. Food Chem. 45(7):2660-2665. Lawrence, B.M. (1988). A further examination of the variation of Ocimum basilicum L., In Flavors and Fragances: A world perspective. Lawrence, B.M., Mookerjee, B.D., and Willis, B.J. (Eds.). Elsevier Science Publishers B.V. Amsterdam. Pp: 61-169. Lee, S.J., Umano, K., Shibamoto, T., and Lee, K.G. (2005). Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem. 91:131-137. Marn, L.J.C., y Cspedes, C.L. (2007). Compuestos voltiles de plantas. Origen, emisin, efectos, anlisis y aplicaciones al agro. Revista Fitotcnia Mexicana. Sociedad Mexicana de Fitogentica. 30(4):327-351. Miele, M., Dondero, R., Ciarallo, G., and Mazzei, M. (2001). Methyleugenol in Ocimum basilicum L. Cv. Genovese Gigante. J. Agric. Food Chem. 49:517-521.

Muoz, A., Patio, J.G., Crdenas, C.Y., Reyes, J.A., Martnez, J.R., Stashenko, E.E. (2007). Composicin qumica de extractos obtenidos por destilacin extraccin simultnea con solvente de hojas e inflorescencias de nueve especies y/o variedades de albahacas (Ocimum spp). Scientia Et Technica. XIII(033):197-199. Murillo, E., Fernndez, K., Sierra, D.M., y Via, A. (2004). Caracterizacin fsico-qumica del aceite esencial de albahaca. II. Rev. Colomb. Quim. 33(2):139-148. zcan, M., and Chalchat, J.C. (2002). Essential oil composition of Ocimum basilicum L. and Ocimum minimum L. in Turkey. Czech. J. Food Sci. 20(6):223-228. Padrini, F., y Lucheroni, M.T. (1997). Aceites esenciales. De Vecchi, S.A. Barcelona. Pp: 8. Pawliszyn, J. (1997). Solid-Phase Microextraction. Theory and practice. WileyVCH Inc., New York, USA. 247p. Pea-lvarez, A., Capella, S., Jurez R., and Labastida, C. (2006). Determination of terpenes in tequila by solid phase microextraction-gas chromatographymass spectrometry. J. Chromatogr. A. 1134:291-297. Politeo, O., Jukic, M., and Milos, M. (2007). Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chem. 101:379-385. Reineccius, G.A. (2007). Flavour-Isolation Techniques. In: Flavours and Fragances. Chemistry, Bioprocessing and Sustainability. Berger, R.G. (Ed.). Springer. Germany. Pp: 409-426. Reyes, J.A., Patio, J.G., Martnez, J.R., y Stashenko, E.E. (2007). Caracterizacin de los metabolitos secundarios de dos especies de Ocimum (Fam. Labiatae), en funcin del mtodo de extraccin. Scientia et Technica. 33:121-123. Rouseff, R., and Perez-Cacho, P.R. (2007). Citrus Flavour. In: Flavours and Fragances. Chemistry, Bioprocessing and Sustainability. Berger, R.G. (Ed.). Springer. Germany. Pp: 117-134. Sajjadi, S.E. (2006). Analysis of the essential oils of two cultivated basil (Ocimum basilicum L.) from Iran. DARU. 14(3):128-130. Salazar, L. (2003). Catlogo de la coleccin del Jardn Etnobotnico. En: Jardn Etnobotnico, Museo de medicina tradicional y herbolaria. Cuernavaca, Morelos. Parrilla, L.A. (ed.). Instituto Nacional de Antropologa e Historia. Mxico. Pp: 69. Simon, J.E., Morales, M.R., Phippen, W.B, Vieira, R.F., Hao, Z. (1999). Basil: A source of aroma compounds and a popular culinary and ornamental herb. In: Perspectives on new crops and new uses. Janick, J. (Ed.). ASHS Press, Alexandria, VA. Pp: 499-505. Tamura, H., Boonbumrung, S., Yoshizawa, T., and Varanyanond, W. (2001). The volatile constituents in the peel and pulp of a green thai mango, Khieo Sawoei cultivar (Mangifera indica L.). Food Sci. Technol. Res. 7(1):72-77. Vas, G., and Vkey, K. (2004). Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis. J. Mass Spectrom. 39:233-254. Via, A., and Murillo, E. (2003). Essential oil composition from twelve varieties of basil (Ocimum spp) grown in Colombia. J. Braz. Chem. Soc. 14(5):744-749. Wossa, S.W., Rali, T., and Leach, D.N. (2008). Volatile chemical constituents of three Ocimum species (Lamiaceae) from Papua New Guinea. SPJNS. 26:25-27.

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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
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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--cadinol (5.9%) and trans--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 o f 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 clevengertype 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 HewlettPackard 6890 gas chromatograph fitted with a fused silica HP-5MS capillary column (30 m 0.25 mm; film thickness 0.25 m). The oven temperature was programmed from 60-280C at 4C/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, 200C.

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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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--bergamotene 1437 5.2 0.8 13 -humulene 1455 0.5 1.1 14 germacrene-D 1482 1.8 15 bicyclogermacrene 1496 0.9 16 germacrene-A 1504 0.7 17 -cadinene 1514 1.8 18 trans--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--cadinol 1643 5.9 24 -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 HP5MS 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--cadinol (5.9%), trans--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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8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20. 21.

REFERENCES Evans WC. Trease and Evans pharmacognosy. London: W.B. Saunders Company; 1996. p. 48. Bauer K, Garbe D, Surburg H. Common fragrance and flavor materials. 3rd edition, Weinheim: WileyVCH; 1997. p. 171. Chiej R. The Macdonald encyclopedia of medicinal plants. London: Macdonald and Co (Publishers) Ltd.; 1988. p. 207. Duke JA. CRC handbook of medicinal herbs. Boca Raton: CRC Press; 1989. p. 333. 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. 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. 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. Phippen WB, Simon JE. Anthocyanins in basil (Ocimum basilicum L.). J Agr Food Chem 1998; 46: 17341738. 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. 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. 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. British pharmacopoeia. Vol. 2, London: HMSO; 1988. p. A137-A138. Adams RP. Identification of essential oil components by gas chromatography /mass spectroscopy. Illinois: Allured Publ. Corp.; 1995. p. 69-351. Swigar AA, Silverstein RM. Monoterpenes. Infrared, mass, 1H-NMR, 13C-NMR spectra and kovats indices. Wisconsin: Aldrich Chemical Company Inc.; 1981. p. 1-121. 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. 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. 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. 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. 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: 4547. 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. Sanda K, Koba K, Nambo P, Gaset A. Chamical investigation of Ocimum species growing in Togo. Flav Fragr J 1998; 13: 226-232.

Volatile Chemical Constituents of three Ocimum species (Lamiaceae) from Papua New Guinea
Stewart W Wossa*1, Topul Rali2 and David N Leach3
2

University of Goroka, P. O. Box 1078, Goroka, Papua New Guinea. Chemistry Department, University of Papua New Guinea, P. O. Box 320, University, Papua New Guinea 3 Centre 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 crosspollination 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 broadspectrum 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.

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10.1071/SP08003

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

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 8hour 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 m 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: Table 1

initial temperature: 50oC; initial time 1.0 min; program rate 4oC/min; final temperature 300oC; 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--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 %).

Retention Index (RI) and percentage composition of the components of the Ocimum basilicum, O. tacilium and O. canum Percentage Composition (% Area) Chemical Constituents 1,8-cineole linalool estragole (methyl chavicol) octyl acetate nerol neral cis-isocitral geranial trans-isocitral neryl acetate eugenol -caryophyllene -farnescene cis--bisabolene bicyclosesquiphellandrene cis--bisabolene -cadinene 3-methoxy cinnamaldehyde Retention Index (RI) 1058 1110 1227 1234 1242 1261 1265 1288 1292 1367 1393 1464 1466 1565 1555 1565 1558 1629 O. basilicum 6.0 0.7 3.3 36.1 0.7 44.5 1.3 0.7 1.4 1.4 3.8 3.8 O. tacilium 0.4 96.6 0.4 0.8 0.8 1.6 O. canum 5.6 27.2 35.3 2.6 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

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Volatile Chemical Constituents of three Ocimum species: Wossa et al. 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--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. Republic of Guinea. Flavour and Fragrance Journal. 15(5), 339 341. Lachowicz, K.J.; Jones, G.P. and Briggs, D.R. (1998). The synergistic preservative effects of the essential oils of sweet basil (Ocimum basilicum L.) against acid-tolerant food microflora. Letters in Applied Microbiology. 26, 209 214. Lawrence, B.M. (1988). A further examination of the variation of Ocimum basilicum L. In: Flavors and Fragrances: A World Perspective. Lawrence, B.M., Mookherjee, B.D. and Willis, B.J. (Eds), Elselvier Science, Amsterdam, The Netherlands. Lawrence B.M. (1992). Chemical components of Labiatae oils and their exploitation. In: Advances in Labiatae Science. Harley, R.M. and Reynolds, T. (Eds), Royal Botanical Gardens: Kew, UK. Pp 399 436. Lawrence, B.M. (1993). Labiatae oils mother natures chemical factory. In: Essential Oils. Allured Publishing, Carol Stream, IL., pp 188 206. Mondello, L.; Zappia, G.; Cotroneo, A.; Bonaccorsi, I.; Chowdhury, J.U.; Mohammed Yusuf, M. and Dugo, G. (2002). Studies on the essential 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. Flavour and Fragrance Journal. 17(5), 335 340. Ntezurubanza, L.; Scheffer, J.J.C. and Looman, A. (1985). Composition of the essential oil of Ocimum canum growing in Rwanda. Pharmacy World & Science. 7(6), 273 276. Ozcan, M. and Chalchat, J-C. (2002). Essential oil composition of Ocimum basilicum L. and Ocimum minimum L. in Turkey. Czeckoslavakia Journal of Food Science. 20, 223 228. Sinha, G.K. and Gulati, B.C. (1990). Antibacterial and antifungal study of some essential oils and some of their constituents. Indian Perfumer. 34, 126 129. Sino, D.; Alam, K.; Tamate, J. and Rali, T. (1992). A preliminary study on the lemongrass oil from Papua New Guinea. Science in New Guinea, 18(3), 133 134. Svoboda, K.P.; Kyle, S.K.; Hampson, J.B.; Ruzickova, G. and Brocklehurst, S. (2003). Antimycotic activity of essential oils: the possibility of using new bioactive products derived from plants. In: Plant-derived antimycotics: Current trends and future prospects. Rai, M.K. (Ed), Binghampton, New York, USA. The Hawthorn Press Inc. pp 198 224. Vasudaran, P.; Kashyap, S. and Sharma, S. (1999). Bioactive botanicals from basil (Ocimum spp.). Journal of Science and Industrial Research. 58, 332 338. Wossa, S.W.; Rali, T. and Leach, D.N. (2004). Analysis of the essential oil compositions of some selected spices of Papua New Guinea. Papua New Guinea Journal of Agriculture, Forestry and Fisheries. 47(1), 17 21. Yusuf, M.; Begum, J.; Mondello, L. and Stagnod Alcontres, L. (1998). Studies on the essential oil bearing plants of Bangldesh. Part VI. Composition of the oil of Ocimum gratissimum L. Flavour and Fragrance Journal. 13(3), 163 166.

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.

5 REFERENCE
Adams, R.P. (1995). Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Pub. Corp., Carol Stream, IL. Carlton, R.R.; Waterman, P.G.; Gray, A.I. and Deans, S.G. (1992). The antifungal activity of the leaf gland volatile oil of sweet gale (Myrica gale) (Myricaceae). Chemoecology, 3, 55 59. Grayer, R.J.; Kite, G.C.; Goldstone, F.J.; Bryan, S.E.; Paton, A. and Putievsky, E. (1996). Intraspecific taxonomy and essential oil chemotypes in sweet basil, Ocimum basilicum. Phytochemistry. 43, 1033 1039. Holm, Y. (1999). Bioactivity of basil. In: Basil the genus Ocimum. Medicinal and Aromatic Plants Industrial Profiles. Vol. 10, UK, Harwood Academic Publishers, pp 113 135. Keita, S.M.; Vincent, C.; Schmit, J-P. and Belanger, A. (2000). Essential oil composition of Ocimum basilicum L., Ocimum gratissimum L. and Ocimum suave L. in the

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