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TESIS DOCTORAL
CARACTERIZACIÓN NUTRICIONAL Y AGRONÓMICA, ANÁLISIS DE LA ACTIVIDAD
BIOLÓGICA Y SELECCIÓN DE CRUCÍFERAS PARA USO ALIMENTARIO
Trabajo realizado en el Instituto de Investigación y Formación Agraria, Pesquera y
Alimentaria (IFAPA) - Centro Alameda del Obispo de Córdoba y en el Departamento de
Genética de la Universidad de Córdoba para optar al grado de Doctor por la licenciada en
Biología:
Myriam Magdalena Villatoro Pulido
Dirigido por:
Dra. Mercedes Del Río Celestino
Dr. Rafael Font Villa
Investigadora del IFAPA- Centro la
Mojonera, Almería
Investigador del IFAPA- Centro la
Mojonera, Almería
TITULO: Caracterización nutricional y agronómica, análisis de la actividad
biológica y selección de crucíferas para uso alimentario
AUTOR: Myriam Magdalena Villatoro Pulido
© Edita: Servicio de Publicaciones de la Universidad de Córdoba. 2011
Campus de Rabanales
Ctra. Nacional IV, Km. 396 A
14071 Córdoba
www.uco.es/publicaciones
[email protected]
ISBN-13: 978-84-694-5933-1
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Dra. Mercedes Del Río Celestino, Investigadora del Área de Mejora y Biotecnología de Cultivos
del IFAPA- Centro la Mojonera, Almería,
Dr. Rafael Font Villa, Investigador del Área de Tecnología, Postcosecha e Industria
Agroalimentaria del IFAPA- Centro la Mojonera, Almería, y
Dra. Ángeles Alonso Moraga, Catedrática del Departamento de Genética de la Universidad de
Córdoba, tutora de esta Tesis,
INFORMAN:
Que el trabajo titulado “CARACTERIZACIÓN NUTRICIONAL Y AGRONÓMICA, ANÁLISIS DE
LA ACTIVIDAD BIOLÓGICA Y SELECCIÓN DE CRUCÍFERAS PARA USO ALIMENTARIO”,
realizado por Myriam Magdalena Villatoro Pulido, bajo la dirección de los doctores Mercedes Del
Río Celestino y Rafael Font Villa, puede ser presentado para su exposición y defensa como Tesis
Doctoral en el Departamento de Genética de la Universidad de Córdoba.
Considerando que se encuentra concluida, dan el VºBº para su presentación y lectura.
Fdo.: Dra. Mercedes Del Río Celestino
Córdoba, Mayo de 2011.
Fdo.: Dra. Ángeles Alonso Moraga
Córdoba, Mayo de 2011.
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Fdo.: Dr. Rafael Font Villa
Córdoba, Mayo de 2011.
Esta investigación ha sido financiada por el Proyecto P06-AGR-02230, titulado “Selección
y Caracterización Agronómica y Nutricional de Crucíferas para Uso Alimentario e Industrial” de la
Consejería de Innovación y Desarrollo Tecnológico de la Junta de Andalucía. Además el capítulo
VII ha estado financiado por el Proyecto C03-070, titulado “Caracterización de la acumulación y
toxicidad de metales pesados y/o arsénico en las partes comestibles de variedades hortícolas de
crucíferas cultivadas en suelos” de la Consejería de Agricultura y Pesca de la Junta de Andalucía.
La doctoranda agradece al Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA) por la concesión de la beca predoctoral para la realización de esta tesis.
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Lo que sabemos es una gota de agua; lo que ignoramos es el océano.
Sir Isaac Newton (1642-1727)
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AGRADECIMIENTOS
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Podría llenar páginas y páginas de agradecimientos para todas aquellas personas que han
compartido estos años de tesis y que han colaborado este trabajo. Para empezar quisiera
agradecer a mis directores de tesis, Mercedes del Río y Rafael Font la oportunidad de haber
podido realizar la tesis con ellos. Gracias por todo lo que he aprendido con vosotros, gracias por
apoyarme siempre a hacer estancias, ponencias…Estos años no siempre han sido fáciles, con
vosotros he podido ver la parte más dura y difícil de la investigación en España, aunque también
la parte positiva cuando detrás hay un trabajo y un esfuerzo.
Gracias a Angelines Alonso por…todo, sin ella nada de esto no hubiera sido posible.
Gracias a Andrés Muñoz por toda su ayuda con el tratamiento estadístico, y por estar siempre
dispuesto con una sonrisa. A Jouad Anter por poder contar siempre con él como compañero y
amigo. Gracias a Marisol Paredes, a Fernando Calahorro y a Zahira Fernández por los buenos
momentos y la risa durante las comidas. Gracias a
vosotros porque entre todos habéis
conseguido lo que cualquier persona soñaría para su trabajo: el confundir la llave de su casa con
la de la puerta del laboratorio, creo que esto es más de lo dice todo.
Quisiera agradecer al IFAPA de Córdoba, a la Universidad de Córdoba, al IFAPA de
Almería, al Instituto de Investigación Alimentaria (IFR) de Norwich y al Departamento de Química
Farmaceútica de la Facultad de Farmacia de Génova por la colaboración prestada, así como a los
miembros que han colaborado. Me gustaría destacar en especial al Dr. Richard Mithen, la Dra.
María Traka, el Dr. Michele Forina y la Dra. Carla Armanino.
Especial mención a todos y cada uno de los colaboradores de los capítulos que conforman
este trabajo, sin vosotros estos resultados no serían posibles. Además quisiera agradecer su
apoyo al Dr. Antonio de Haro Bailón y a la Dra. Mª Dolores Luque de Castro.
Gracias a los miembros del tribunal por su buena disposición y su colaboración.
La lista de personas por nombrar sería interminable, amigos, compañeros, profesores y
tantas personas que han trabajado durante estos últimos años dando ánimos y consejo.
Por último y no menos importante quiero agradecer a mi familia su apoyo constante, su
comprensión en los momentos difíciles, su inestimable ayuda y por haberme hecho ver lo que es
realmente importante en la vida. Gracias a mis padres Paco y Lourdes, a mis hermanos Javier,
Juan Carlos y Eduardo. Por supuesto gracias a mi futuro esposo Alberto, te ha tocado compartir
conmigo la recta final y más difícil de este proyecto que termina, para comenzar el nuestro en
común. Sin tu paciencia y maravillosos consejos no se cómo lo hubiera podido terminar.
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Nota: el trabajo de esta tesis doctoral se presenta en parte en Inglés, ya que los capítulos que la
conforman han sido publicados o enviados a diferentes revistas de investigación y se han editado
los trabajos originales. Por esta misma razón las referencias de cada capítulo aparecen al final del
mismo tal y como establecen las normas de cada una de las revistas.
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ÍNDICE
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RESUMEN
INTRODUCCIÓN
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1. Concepto de alimento funcional
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2. Las Crucíferas: un grupo vegetal con fitoquímicos que le confieren
propiedades funcionales
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3. La rúcula
3.1. Clasificación taxonómica y procedencia.
3.2. Referencias históricas de la rúcula.
3.3. Fuentes de variabilidad genética
3.4. Bancos de germoplasma de rúcula
3.5. Situación actual en la mejora genética de la rúcula
4. El rábano
4.1. Procedencia, referencias históricas y usos
5. Fitoquímicos presentes en Crucíferas
5.1. Los glucosinolatos
5.2. Los isotiocianatos
5.3. Los compuestos fenólicos
5.4. Los carotenoides
5.5. Los carbohidratos
6. Los minerales
7. Acumulación de metales pesados en especies de crucíferas
8. Caracterización de los recursos genéticos
8.1. Caracterización agro-morfológica de Eruca
8.2. Caracterización organoléptica de Eruca
8.3. Caracterización nutricional y funcional de Eruca
8.3.1. Aproximación al papel fitoquímico de Eruca
8.3.2. La Espectroscopía por reflectancia en el infrarrojo cercano (NIRS) como
herramienta analítica en la caracterización del perfil de minerales en especies
vegetales.
8.3.3. Actividad biológica de las líneas de crucíferas.
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OBJETIVOS DE LA TESIS
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CAPÍTULO I: Diversidad fenotípica en rúcula (Eruca vesicaria subsp. sativa y
Eruca vesicaria subsp. vesicaria).
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Ensayo de inhibición del crecimiento tumoral
Estudio de la capacidad inductora de apoptosis
Estudio de la proteína p21
Modelo SMART de ensayo genotoxicológico y antigenotoxicológico in vivo
Supervivencia
JUSTIFICACIÓN DEL TRABAJO
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CAPÍTULO II: Características agromorfológicas, composición química y análisis
sensorial en hojas de rúcula (Eruca vesicaria subsp.sativa y Eruca vesicaria
subsp. vesicaria) y Erucastrum de una colección mundial.
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CAPÍTULO III: Aproximación al perfil fitoquímico de rúcula (Eruca sativa (Mill.)
Thell)
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CAPÍTULO IV- Caracterización y predicción por espectroscopía de reflectancia
en el infrarrojo cercano (NIRS) de la composición mineral de rúcola (Eruca
vesicaria subsp.sativa y Eruca vesicaria subsp. vesicaria).
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CAPÍTULO V: Análisis de la actividad biológica in vitro de extractos de rúcula,
Eruca vesicaria subsp. sativa (Mill.) Thell y sulforrafano.
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CAPÍTULO VI: Actividad in vivo de extractos de rúcula (Eruca vesicaria subsp.
sativa) y sulforrafano.
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CAPÍTULO VII: Actividad biológica del rábano, una crucífera, con contenido en
metales y metaloides
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DISCUSIÓN GENERAL
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CONCLUSIONES
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REFERENCIAS
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RESUMEN
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La necesidad de valor añadido en la agricultura andaluza unida a la casi inexistente Mejora
Genética en algunos géneros de hortícolas de hoja como la rúcula (Eruca), el alto precio de
mercado de estos vegetales que lo elevan a un producto gourmet, y los beneficios nutricionales
de las crucíferas en la salud humana, hacen de la rúcula un producto clave para un programa de
Mejora. En este trabajo se han caracterizado de forma multidisciplinar, entradas pertenecientes a
distintas especies y subespecies de Eruca (Eruca stenocarpa, Eruca vesicaria subsp. longirostris,
Eruca vesicaria subsp. vesicaria y Eruca vesicaria subsp. sativa) comparándolos con variedades
testigo comerciales.
Las líneas de trabajo y los resultados obtenidos han sido:
Caracterización agro-morfológica. La base inicial fue el Descriptor de Eruca recomendado por el
IPGRI. A partir del análisis de 15 caracteres agro-morfológicos en una colección constituida por 52
entradas se encontró una gran diversidad, y significativas características morfológicas que
distinguieron las entradas entre sí y también entre las especies y subespecies. Algunos de los
atributos morfológicos son considerados como marcas de calidad por agricultores (rendimiento,
días a floración, actitud de crecimiento de la planta) y consumidores (color, longitud, lobulación y
pubescencia de la hoja), por lo que podrían ser de gran interés comercial.
Caracterización sensorial. Con base en las normas internacionales (ISO, 2008) relativas al
análisis sensorial, se ha desarrollado un vocabulario específico que contribuirá a la caracterización
cualitativa de rúcola. El panel sensorial generó 27 descriptores simples clasificados en tres
diferentes grupos (7 para apariencia, 14 para sabor y 6 para textura). Con el propósito de
relacionar los atributos sensoriales con el contenido en glucosinolatos, que juegan un papel
decisivo en las propiedades organolépticas (olor, sabor amargo), se cuantificó el contenido de
estos compuestos. Los glucosinolatos mayoritarios fueron la glucorrafanina y la glucosativina. La
variabilidad cuantitativa de glucorrafanina encontrada entre las entradas de Eruca vesicaria
subesp. vesicaria
nos indica que es posible utilizar este material como base para la mejora
genética de la especie. La consecuencia de elevar los niveles de glucorafanina aumentaría el
interés nutracéutico que puede llegar a tener esta especie, ya que de éste glucosinolato se forma
sulforrafano (isotiocianato de la glucorrafanina), con interesantes propiedades anticarcinogénicas.
Desde el punto de vista de la calidad nutricional, se llevo a cabo el estudio del contenido
en minerales en hojas de Eruca, encontrándose una gran variabilidad entre las entradas para
todos los minerales (Cu, Fe, Zn, Mn, Mg, Na, y K) excepto para el contenido en Ca. Asimismo, el
estudio indicó que las entradas de Eruca fueron una buena fuente de minerales, particularmente
calcio, manganeso, hierro y potasio, lo que sugiere que su consumo diario puede contribuir en un
alto porcentaje a los requerimientos diarios de estos minerales a una persona. En relación al
contenido en minerales, se ha evaluado el potencial de la Espectroscopía por reflectancia en el
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infrarrojo cercano (NIRS) para la determinación de la concentración de minerales en rúcola. La
conclusión general es que la técnica NIRS puede ser usada en programas de mejora para
predecir el contenido en minerales en este cultivo (contenido total, Na, K, Fe, Mg y Zn) siendo las
correspondientes a Na y K las de mayor capacidad predictiva.
Con el objeto de estudiar el potencial de Eruca como alimento funcional con actividad
tumoricida, apoptótica, antimutagénica y antidegenerativa se llevaron a cabo análisis de la
actividad biológica in vivo e in vitro con extractos de dicha especie y con el isotiocianato
sulforrafano. Para ello, preciamente, se caracterizó el perfil fitoquímico (isotiocianatos,
compuestos fenólicos, carotenoides) de cuatro entradas que diferían en su contenido en
glucosinolatos, especialmente para su contenido en glucorrafanina. Los resultados indicaron que
la actividad in vitro de Eruca frente a líneas tumorales estaba relacionada con el contenido en
isotiocianatos así como por la interacción con otros compuestos fitoquímicos como fenoles y
carotenoides. Todas las concentraciones de sulforrafano y de extractos de rúcola ensayadas
fueron antigenotóxicas en el Test SMART de D. melanogaster, lo que indica una actividad
protectora del daño genético.
El problema de las crucíferas en uso alimentario es su tendencia a la acumulación de
metales(oides), por lo que además se hace necesario el análisis de la evaluación del estrés
oxidativo y mutagénico en modelos in vitro e in vivo para confirmar el potencial nocivo que los
metal(oides) acumulados en los tejidos pueda ejercer. En este sentido, se ha estudiado la
modulación de la genotoxicidad y citotoxicidad por el rábano. Los resultados obtenidos a partir del
Test SMART de Drosophila melanogaster y los ensayos de citotoxidad con células tumorales
demostraron que las plantas de rábano desarrolladas en suelos contaminados con metal(oides)
resultaron genotóxicas y menos citotóxicas que los rábanos desarrollados en suelos no
contaminados. Se ha sugerido que los principales agentes moduladores de la actividad genotóxica
de las plantas desarrolladas en suelos contaminados con metal(oides) provendrían de la
interacción entre los metal(oides) y los isotiocianatos.
Este trabajo demostró el valor de las entradas de Eruca caracterizadas como recurso
genético de gran potencial, por su alta variabilidad y calidad sensorial y nutritiva, cuyo uso en
futuros proyectos de mejora podría dar la oportunidad de ofertar al público más exigente la calidad
que está demandando.
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INTRODUCCIÓN
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1. Concepto de alimento funcional
Una dieta equilibrada debe garantizar una salud óptima, reduciendo los riesgos de
enfermedades carenciales y de enfermedades crónicas-degenerativas, sin dejar de ser
satisfactoria para el paladar (Gonzalvo-Heras et al., 2006). Nuestro estilo de vida actual ha ido
cambiando los hábitos de consumo alimentario, exigiendo una mayor calidad en los productos
naturales. Debido a esta demanda, surge la creación de nuevos alimentos aplicando nuevos
conocimientos científicos y tecnológicos que permitan desarrollar productos destinados a mejorar
la salud.
La definición de alimento del International Life Science Institute (ILSI) en 2004, establece
que éste puede ser considerado funcional si en su forma natural o procesada contiene un
componente, nutriente o no nutriente, con efecto selectivo sobre una o varias funciones del
organismo, con un efecto añadido por encima de su valor nutricional y cuyos efectos positivos
justifican que pueda reivindicarse su carácter funcional o incluso saludable, si se ha demostrado
de forma satisfactoria que posee un efecto beneficioso sobre una o varias funciones específicas
en el organismo, más allá de los efectos nutricionales habituales y que es relevante para la mejora
de la salud, el bienestar y la reducción del riesgo de enfermar (ILSI, 2004). Los alimentos
funcionales, en la medida que implican nuevos nutrientes o proporciones diferentes de los
mismos, pueden considerarse nuevos alimentos, según la clasificación establecida por la Unión
Europea y por el Comité Científico de la Alimentación Humana (Knudsen, 1999).
Algunos ejemplos de alimentos funcionales son los enriquecidos con determinadas
vitaminas, minerales, fibra alimenticia o ácidos grasos y los alimentos a los que se les ha añadido
sustancias biológicamente activas, como los fitoquímicos y otros antioxidantes. Actualmente, en
España, ya están disponibles en el mercado unos 200 productos de este tipo, sobre todo pan,
cereales, lácteos, zumos y sal; ello, gracias a que la industria alimentaria, puede proveer
alimentos con composición físico-química controlada o modificada, según el objetivo que se desea
obtener con su ingesta, basada en el principio del beneficio para la salud. Esto nos abre grandes
perspectivas tanto en investigación respecto a la prevención de ciertas enfermedades, y el
impacto de diversos nutrientes en patologías como el cáncer, enfermedades cardiovasculares y
neurológicas, como para la industria farmacéutica en la utilización de
desarrollo de nuevos fármacos.
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fitoquímicos para el
2. Las Crucíferas: un grupo vegetal con fitoquímicos que le confieren propiedades
funcionales
La familia Cruciferae (= Brassicaceae) presenta 390 géneros y 3.000 especies (Herbario
virtual del Mediterráneo Occidental), de los cuales 108 géneros tienen especies silvestres en
Europa. Esta familia botánica comprende especies originadas en zonas de clima templado, que
están adaptadas a desarrollarse y crecer en zonas con temperaturas moderadas, lo que ha
llevado a su cultivo en gran parte de los países de Europa. Son plantas herbáceas con hojas
alternas, simples, enteras o divididas a veces en roseta basal. Las inflorescencias se presentan en
racimo con flores hermafroditas, corola con pétalos libres y fruto en silicua o silícula. Esta familia
es llamada así porque las flores de estas plantas poseen todas cuatro sépalos y cuatro pétalos,
colocados en forma de cruz. Casi todas las especies viven en campos y huertos, siendo muchas
de ellas plantas arvenses.
La familia Cruciferae comprende numerosas
especies que han sido utilizadas
tradicionalmente por el hombre como hortícolas, forrajeras, o como fuente de condimentos y
aceites. Es una familia de gran importancia económica, con especies que presentan un
aprovechamiento agrícola destacado: nabos, nabizas y grelos (Brassica rapa), mostaza negra
(Brassica nigra), berza, repollo col asa de cántaro (Brassica oleracea), mostaza índia (Brassica
juncea), colza (Brassica napus), mostaza etíope (Brassica carinata), o el rábano (Raphanus
sativus). Algunas de las especies presentan propiedades medicinales como la bolsa de pastor
(Capsella bursa-pastoris), la rúcula (Eruca vesicaria), o la mostaza silvestre (Sinapis arvensis) y la
mostaza blanca (Sinapis alba).
Además de los usos anteriormente citados para los miembros de las crucíferas, diferentes
estudios han puesto de manifiesto resultados prometedores en cuanto a su utilización para
producción de biodiesel (Cardone et al. 2003), biofumigación (Kirkegaard y Saward 1998), y
recuperación de suelos contaminados por metales pesados (Nanda-Kumar et al. 1995, Del Río et
al. 2000).
En las últimas décadas el consumo de estos vegetales ha experimentado un fuerte
aumento de producción y venta en los países industrializados, al reconocérseles importantes
efectos beneficiosos en la salud de las poblaciones que los consumen. Así, los vegetales de la
familia Cruciferae juegan un importante papel en la salud y nutrición humana debido a su
contenido en glucosinolatos, isotiocianatos (ITCs), carotenoides, compuestos fenólicos, y
minerales (Mithen et al., 2000; Podsedek, 2007). En especial, la mayoría de los estudios dirigen
su atención al estudio de los glucosinolatos, los cuales, según amplias evidencias en la literatura,
son los principales responsables de las cualidades organolépticas, nutritivas y medicinales de las
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crucíferas (Mithen, 2001). Los glucosinolatos y sus productos de degradación han demostrado
ser unos potentes inhibidores del crecimiento de ciertas células tumorales. Así, se ha comprobado
que ciertos compuestos como el sulforrafano, derivado de la glucorafanina, un glucosinolato
presente en las hojas de rúcula y en otras crucíferas como el brécol, tienen un marcado efecto
protector contra determinadas sustancias carcinogénicas (Juge et al., 2007; Singh et al., 2009;
Keum et al., 2009; Chambers et al., 2009; Traka et al., 2010).
3. La rúcula
3.1. Clasificación taxonómica y procedencia.
Bajo el término de “rúcula” se incluyen varias especies de plantas pertenecientes a la
familia Cruciferae, siendo las más comunes aquellas pertenecientes a los géneros Eruca y
Diplotaxis. Eruca es originaria de la cuenca del Mediterráneo y Asia occidental. Es una planta
anual y parcialmente alógama (2n = 2x = 22), e incluye las especies Eruca stenocarpa y Eruca
vesicaria (Padulosi 1997). Esta última a su vez presenta las subespecies Eruca vesicaria subsp.
vesicaria, Eruca vesicaria subsp. sativa (Miller) Thell., Eruca vesicaria subsp. pinnatifida, Eruca
vesicaria subsp. longirostris (Uechtr.) Maire (Gómez-Campo, 1993; Gómez-Campo, 1999).
Ambas especies y subespecies pueden encontrarse de forma silvestre, aunque solo E.
vesicaria subsp. sativa (Miller) Thell. ha sido domesticada y ocupa un área geográfica más amplia
en el mundo. Las subespecies vesicaria y pinnatifida son endémicas de España y Noroeste de
África. La subsp. longirostra (Uechtr.) Maire, aunque también ha sido descrita, un detallado
análisis morfométrico basado en las dimensiones del fruto no termina de confirmar este estatus
(Gómez-Campo, 2003).
La rúcula es un cultivo conocido desde hace siglos. Su denominación proviene del latín
“uro” que significa “quemo”, debido al sabor pungente que tiene. Es importante como cultivo de
hoja en diferentes países circunmediterráneos entre los que se encuentran Italia, Grecia, Turquía,
Egipto y Sudán (Pimpini y Enzo, 1997). En España, son varias las empresas del sector de la IV
gama que la producen desde hace algunos años, como Verdifresh, Florette y Primaflor, entre
otras. En concreto, en Andalucía es producida y comercializada como hortaliza de hoja por la
empresa Primaflor S.L. en la provincia de Almería, constituyendo un producto tipo gourmet. En
India y China, es cultivada por el aceite de sus semillas (Sun-Ju et al., 2005). Se ha asilvestrado
en América del Norte, sur de África y Australia. En el resto del mundo, el órgano de consumo lo
constituyen las hojas y tallos jóvenes, que se ingieren crudos en ensaladas (Stephens, 2006).
Además se considera como planta medicinal y puede ser empleada en control biológico de plagas
(Gómez-Campo, 1995; D’Antuono et al. 2009). Como otras crucíferas, contiene un amplio rango
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de fitoquímicos promotores de la salud, incluidos carotenoides, vitamina C, fibra, flavonoides y
glucosinolatos (Podsedek, 2007).
3.2. Referencias históricas de la rúcula.
Antiguamente griegos y romanos conocían la rúcula por sus propiedades medicinales y
afrodisíacas (Morales y Janick, 2002). En la antigua Roma era consagrada a Príapo, plantándose
a los pies de las estatuas de esta deidad consagrada al potencial procreador de los machos.
Dioscórides (s. V a.C.) en el libro De Materia Libre Quinque advierte de que comida cruda
estimula la lujuria y que las semillas tienen las mismas virtudes. Columela hace referencia a su
provocativo efecto, pero conoce muy bien su técnica de cultivo: «... y también la rúcula y la
albahaca permanecen en su sitio, sin moverse, tal como han sido sembradas y no requieren otro
cultivo que el de estercolarlas y desherbarlas». Los hispanorromanos comparaban su poder
afrodisíaco con el anafrodisíaco de las lechugas, mientras que hispanovisigodos como Isidoro de
Sevilla mantienen el uso y conocimiento de las virtudes de esta planta: «... Eruca es como si dijera
uruca (quemadora), porque tiene unas propiedades abrasadoras y consumida frecuentemente en
la comida, inflama el apetito venéreo».
Se cree que la marginación de esta planta como hortaliza en España ha podido estar muy
relacionada con su condena por sus propiedades afrodisíacas. Los agrónomos hispanoárabes
también hablaban de su cultivo, entre ellos Ibn al-Awwam (siglo XII), quien comenta el uso de la
planta como aromatizante de mostos y arropes, moliendo la semilla y cubriendo con ellas la
superficie de las orzas en las que éstos se conservan (Nuez y Hernández-Bermejo, 2009).
3.3. Fuentes de variabilidad genética
Como definió Vavilov (Vavilov, 1935), la Mejora Genética Vegetal es la evolución de las
plantas dirigida por el hombre. Se trata básicamente de una elección hecha por éste de las
mejores plantas dentro de una población con características variables. La variabilidad, también
llamada biodiversidad o agrodiversidad, es la materia prima con la que se desarrolla la mejora
genética de plantas (Nuez y Ruiz, 1999a y 1999b; Picó y Ruiz-Quián, 2000). Y es en la
caracterización de los recursos disponibles donde podemos encontrar la materia prima que
estamos necesitando para la mejora de la calidad (Pitrat, 2002).
Lo cierto es que hasta bien entrado el siglo XX, la humanidad poseía un conjunto muy
diverso de Recursos Fitogenéticos en forma de cultivos tradicionales repartidos por todo el planeta
(FAO, 2007; www.uicn.org). Sin embargo, la preocupación por su conservación no se hizo
evidente hasta después de la llamada “revolución verde” (alrededor de 1960). Así, sólo el
mantenimiento de los Recursos Fitogenéticos existentes, proporcionará la materia prima o
material genético que, debidamente utilizado, permitirá obtener nuevas y mejores variedades de
23
plantas (Hawkes, 1991; Swanson, 1996). Paradójicamente, el mejorador que desarrolla nuevas y
uniformes variedades para el mercado, aumentando aún más la homogeneidad, al mismo tiempo
es absolutamente dependiente de poseer un “pool” genético asequible, muy amplio y bien
conservado que le permita afrontar nuevos retos del futuro. Este “pool” genético, tan necesario
para la agricultura, se halla principalmente mantenido en distintos bancos de germoplasma, tanto
nacionales como internacionales.
3.4. Bancos de germoplasma de rúcula
Algunos Centros Públicos e Internacionales poseen importantes Bancos de Germoplasma.
En España, el CRF (Centro de Recursos Fitogenéticos) del INIA (www.inia.es/crf), tiene la
responsabilidad de conservar duplicados un banco base de todas las colecciones españolas de
semillas y ser centro nacional de documentación. Actualmente, su inventario nacional recoge
información de 52 entradas de Eruca vesicaria (Cav) DC. De ellas 46 corresponden a España (37
de Castilla-La Mancha, 5 de Comunidad de Madrid, 3 de Aragón y 1 de Castilla-León). Respecto a
Diplotaxis tenuifolia (L.) DC. el CRF conserva 2 entradas de las cuales sólo una procede de
España (Castilla-La Mancha).
El interés de la rúcula como hortaliza de hoja para uso alimentario (Padulosi 1995; SilvaDías, 1997) y la iniciativa del proyecto por parte del International Plant Genetic Resources Institute
(IPGRI) en Especies Mediterráneas Infrautilizadas como mejora de conservación, derivó en el
establecimiento de la Red de Recursos Genéticos de Rúcula (Rocket Genetic Resources Network)
(Gómez-Campo, 1995; Padulosi y Pignone, 1997). El Germplasm Institute (IdG) y el Volcani
Center de Israel, Bet Dagan, tomaron la responsabilidad de supervisar las instituciones que
poseían fuentes genéticas de rúcula en el mundo. Además, la creación de dicho proyecto en 1994
tenía como objetivo promover este cultivo mediterráneo infrautilizado, supervisar las instituciones
que albergan germoplasma y salvaguardar su diversidad proporcionando así una amplia base
genética para su explotación (Padulosi y Pignone, 1997). Hasta entonces el conocimiento
existente sobre la variabilidad de la especie era muy escaso. La mayoría de los individuos
pertenecían a la subespecie sativa y al menos un 50% del total del germoplasma procedía de
Italia estando el resto de Europa mal representada. Se cree que en las colecciones de Eruca hay
cierto grado de duplicación y que los programas de recolección no han seguido estrategias bien
definidas, ni han prestado todo el apoyo económico necesario. Además falta información de la
colección mantenida en el USDA (United States Department of Agriculture) acerca del origen real
de las muestras y de su variabilidad genética.
Aunque líneas nuevas son continuamente añadidas a la colección de germoplasma
silvestre de rúcula (Pita-Vilamil et al., 2002), el género Eruca, comparado con otros vegetales de
la familia Cruciferae se encuentra infrautilizado desde la perspectiva de la Mejora Vegetal, ya que
24
según el estatus de las colecciones se puede observar que la rúcula ha sido objeto de escasos
programas de evaluación. Por tanto, se hace necesario el análisis de las características biológicas
y agronómicas del germoplasma para optimizar su utilización (Chin, 1994).
El bajo nivel de
caracterización de las colecciones existentes en los bancos mundiales se debe de acuerdo a
Padulosi y Pignone (1997) a los siguientes factores:
Al comportamiento alógamo de Eruca,
A la mínima multiplicación del material realizada.
A que las semillas presentan una baja germinación, siendo las silicuas altamente dehiscentes.
A la posibilidad de contaminación con polen local.
A que las semillas contienen un alto porcentaje de aceite siendo más complicadas de guardar
en bancos de germoplasma (ya que la desecación y almacenaje son decisivos).
3.5. Situación actual en la mejora genética de la rúcula
Hasta ahora son escasos los trabajos de mejora genética realizados sobre esta especie a
pesar de la importancia que está alcanzando en toda Europa, incluido nuestro país, como verdura
de IV gama. Estudios preliminares han revelado la amplia variabilidad genética existente para su
uso en programas de mejora genética (Padulosi, 1997, Warwick et al., 2007) es por ello que ha
sido utilizada como recurso genético para la mejora de otras crucíferas (Zhang et al., 2008; Sikdar
et al. 1987).
Actualmente, la producción de rúcula proviene de variedades de polinización abierta
procedentes en su mayoría de países del Norte de Europa, las cuales bajo nuestras condiciones
ambientales presentan larga duración del ciclo de cultivo (lo que permite mayor número de
cortes), bajo rendimiento, alta sensibilidad a patógenos (principalmente a mildiu), baja resistencia
a subida a flor, y escasa mejora de la calidad organoléptica y nutricional.
Las zonas de producción principales de rúcula son las provincias de Almería, Murcia,
Navarra, Barcelona o Mallorca entre otras, siendo comercializada en nuestro país como verdura
de IV gama y exportada a países como Alemania, Reino Unido, Escandinavia y Francia (Gómez,
2002). Si tenemos en cuenta que el precio como producto de IV gama en los supermercados
alcanza los 20 euros/Kg, y que este precio podría superarse mediante el incremento del valor
añadido como producto funcional con propiedades beneficiosas para la salud (alto contenido en
25
glucosinolatos), queda fuera de toda duda el interés potencial del cultivo y la mejora de esta
especie, y explicaría el interés de empresas
y de centros de investigación extranjeros (Norwich
Research Park en Reino Unido, Univ. Vila Real en Portugal, University of Bologna en Italia,
Saskatoon Research Centre en Canadá, Universidad de Izmir en Turquía) en el desarrollo de
líneas con alto valor añadido.
4. El rábano (Raphanus sativus L.)
Esta planta pertenece también a la familia Cruciferae y presenta gran importancia
económica (Schubert, et al., 2011). Su raíz puede ser globosa, elipsoide o cilíndrica, con colores
rojo, blanco, o violeta (Ecocrop- FAO). La parte comestible consiste en el hipocotilo ensanchado.
4.1. Procedencia, referencias históricas y usos.
El rábano es uno de los primeros vegetales cultivados. Algunos investigadores consideran
a China como la posible cuna de esta hortícola. Se tiene la certeza de que los egipcios y
babilonios consumían este tubérculo hace más de 4.000 años por haberse encontrado
representado en paredes interiores de la pirámide de Keops, en la que inscripción jeroglífica en la
recoge cuánto rábano, cebolla y ajo eran consumidos por los trabajadores de la pirámide. Además
era utilizado por los egipcios para limpiar los intestinos en el embalsamamiento de las personas
con menor poder adquisitivo (MacKendrick y Howe, 1952). Durante el primer milenio a.C. griegos
y romanos convirtieron al rábano en un alimento muy apreciado, extendiendo su consumo por
toda Europa gracias a las provincias conquistadas por estos últimos. El Dr. Alain Touwaide del
Departamento de Botánica del Museo Nacional de Historia Natural, Institución del Smithsonian de
Washington, encontró unas pastillas con extractos de rábano en unos contenedores recuperados
de un banco mercante romano del año 130 a.C. (Barley, 2010).
Su nombre proviene del término latino “radix” o raíz. El rábano actualmente se utiliza
como condimento en ensaladas y otros platos. Las hojas son utilizadas con fines comerciales
como fuente de proteína vegetal y las semillas como fuente de aceites en fertilizantes, jabones y
con fines nutricionales.
Históricamente, los rábanos han sido utilizados como plantas medicinales para una gran
variedad de enfermedades como disfunción hepática y mala digestión (Gutierrez y Pérez, 2004;
Lugasi, et al., 2005; Shukla et al., 2010).
Recientemente varios estudios han demostrado que los rábanos o extractos de los mismos
presentan actividad antioxidante (Lugasi et al., 2005; Wang et al., 2010), antimutagénica
26
(Nakamura et al., 2001), activación de elementos de respuesta antioxidante (ARE) (Hanlon et al.,
2011) y efectos antiproliferativos (Papi et al., 2008; Yamasaki et al., 2009; Beevi et al., 2010), así
como inducción de enzimas de detoxificación (Lee y Lee, 2006; Hanlon et al., 2007). En los
estudios de su actividad biológica se han utilizado extractos o fitoquímicos específicos de esta
especie, entre los que se incluyen glucosinolatos e ITCs (Hanlon et al., 2007; Papi et al., 2008;
Ben Salah et al., 2009), compuestos fenólicos (Sgherri et al., 2003) y antocianinas (Liu et al.,
2008; Wang et al., 2010).
5. Fitoquímicos presentes en Crucíferas.
El contenido nutricional de las crucíferas es variable y depende de las condiciones
ambientales donde se desarrolle la planta, la edad de la misma, las propiedades del cultivo, y el
método de conservación, procesamiento y preparación. En general, las partes verdes poseen un
bajo contenido en agua, así como en ácidos grasos y carbohidratos, lo que las convierte en
productos de bajo nivel calórico. Son además una buena fuente de minerales, particularmente en
antioxidantes, vitamina C y un alto contenido en aminoácidos. Contienen además un gran número
de nutrientes y fitoquímicos a los que se les atribuye un potente efecto antioxidante y que se
describen a continuación (Juge et al., 2007).
5.1. Los glucosinolatos.
Los glucosinolatos son metabolitos secundarios sintetizados por las plantas como defensa
ante la depredación. Se encuentran en la familia Cruciferae y en algunas otras familias.
La
molécula de glucosinolato consiste en una unidad de β-tioglucósido, una oxima sulfonada y una
cadena lateral variable, derivada de un aminoácido (Fig. 1).
Figura 1. Esquema de una molécula de glucosinolato. R corresponde a la cadena lateral variable
que puede ser alifático (con grupos hidroxilo o con azufre), aromático o indólicos.
Las diferencias en la estructura química de la cadena lateral son las que determinan la
existencia de más de 120 glucosinolatos dentro de la familia Cruciferae, siendo responsables de
las diferentes propiedades bioquímicas de los mismos (Mithen, 2001).
27
Los glucosinolatos se encuentran en el líquido intersticial celular. Cuando se rompe este
tejido se ponen en contacto con tioglucosidasas glucohidrolasas, también llamadas mirosinasas
que se encuentran en los idioblastos. Estas enzimas hidrolizan los glucosinolatos produciendo
varios productos, tales como ITCs, nitrilos, tiocianatos, epitionitrilos y oxazolidinas (Bones y
Rossiter, 2006) (Fig. 2).
Figura 2. Esquema general de la hidrólisis de un glucosinolato.
El tipo y proporción de estos productos de hidrólisis depende de la especie vegetal
estudiada, la cadena lateral, el pH, iones metales y otros elementos proteicos (Bones y Rossiter,
2006). Cuando se incuban glucosinolatos y mirosinasa purificados a pH neutro, los únicos
productos formados son ITCs. Sin embargo, cuando un glucosinolato, como la glucorrafanina es
hidrolizado al romperse el tejido vegetal de brócoli, de forma parecida a como sucede en la
masticación, el producto preponderante en un 60 u 80% es nitrilo (sulforrafano nitrilo) más que
ITC. Un cofactor de la mirosinasa, la proteína epitioespecífica (ESP), se sabe que condiciona los
productos formados a partir de la hidrólisis catalizada por mirosinasa dirigiendo la formación a un
epitionitrilo o nitrilo (Matusheski et al., 2006). Así, una baja cocción, por ejemplo, de brócoli,
preserva la mirosinasa y desnaturaliza la ESP, resultando en una conversión de casi un 100% a
ITC (sulforrafano). Sin embargo, una alta cocción del tejido vegetal, desnaturaliza también la
enzima mirosinasa, y se ingieren los glucosinolatos intactos. No obstante, los glucosinolatos
también pueden ser convertidos a ITC por la mirosinasa presente en el colon por la actividad de la
tioglucosidasa de la flora intestinal (Juge et al., 2007).
Como ya se ha comentado anteriormente, los glucosinolatos, además de ser responsables
del sabor característico de las crucíferas tienen un gran potencial anticancerígeno. Algunos
estudios en células tumorales y en roedores ha mostrado que ciertos glucosinolatos pueden
actuar como agentes bloqueantes, los cuales detoxifican carcinógenos previamente a la
carcinogénesis, y como agentes supresores, los cuales previenen la proliferación celular y puede
inducir la apoptosis (Mithen, 2001). Sin embargo, se ha observado que algunos glucosinolatos se
pueden degradar en productos goitrogénicos (Mithen, 2001). Otros autores también han descrito
que los extractos derivados de crucíferas, y sus productos de degradación pueden ser
genotóxicos (Lamy et al., 2008).
28
Además de existir una gran variabilidad en número (hay especies que presentan un solo
glucosinolato en su composición, mientras que otras poseen más de 30 diferentes), existe también
una gran variabilidad en la concentración de dichos compuestos dentro de una misma especie, así
como entre las distintas partes de la planta (raíces, tallo, hojas y semillas), o en función del estado
fenológico y de los nutrientes disponibles (Bellostas et al., 2007).
Hasta la fecha hay publicados varios estudios sobre el contenido en glucosinolatos de Eruca,
estando varios de ellos centrados en el contenido en glucosativina, glucorrafanina y glucoerucina
principalmente (Bennett et al., 2002; Kim e Ishii, 2006; Bennett et al., 2006; Bennett et al., 2007;
Selma et al., 2010; Bennett et al., 2007).
5.2. Los isotiocianatos.
Los ITCs son los responsables del sabor amargo, amostazado y picante (Mandiki et al.
2000) de las semillas y partes verdes de las crucíferas. Numerosos artículos demuestran los
efectos beneficiosos de los ITCs tales como los efectos en las enzimas de biotransformación
implicadas en el metabolismo de carcinógenos (Lampe y Peterson, 2002), prevención del cáncer,
inhibición de enzimas de biotransformación de fase I e inducción de enzimas de fase II (Spitz et
al., 2000), entre otros. En realidad, se ha especulado que los ITCs, son los responsables de los
efectos protectores de las crucíferas (Mithen, 2001; Traka et al., 2010). Los ITCs más investigados
son el 1-ITC-4-(metilsulfinil)-butano o sulforrafano (SF), el 4-(metiltio)butil ITC o erucina (ER) y el
3-metilsulfinilpropil-ITC o iberina (IB) (Jadhav et al., 2007) que son formados a partir de la
hidrólisis de la glucorrafanina, glucoerucina y glucoiberina, respectivamente. La ER además puede
obtenerse a través de la reducción in vivo del sulforrafano (Melchini et al., 2009). La interconversión in vivo de estos dos glucosinolatos y su similitud estructural han sugerido una actividad
biológica también semejante.
A diferencia de la mayoría de los ITCs, el SF contribuye poco al sabor, y es el ITC más
hidrofílico. El SF es el ITC más extensamente estudiado para descifrar los mecanismos implicados
en los efectos beneficiosos relacionados con la salud. El SF ha demostrado tener un efecto
protector contra la tumorigénesis inducida por carcinógenos y se cree que los efectos
quimiopreventivos probablemente impliquen varios mecanismos, los cuales interaccionan juntos
para reducir el riesgo de carcinogénesis (Juge et al., 2007). Estos mecanismos incluyen:
1) Inhibición de enzimas citocromo P450 de fase I: los procarcinógenos una vez que entran en el
organismo pueden oxidarse, así como reducirse o hidrolizarse pasando a intermediarios
altamente reactivos que pueden unirse a macromoléculas como el ADN, ARN o proteínas. Este
evento es llamado metabolismo de fase I (Nelson et al., 1993). Se ha demostrado además que
29
el SF in vitro puede inhibir la formación de los aductos de ADN inducidos por carcinógenos
(Yang et al., 1994).
2) Inducción de enzimas del metabolismo de fase II: las enzimas de fase II convierten los
carcinógenos en metabolitos inactivos preparados para ser excretados por el cuerpo y
previniendo su reacción con el ADN. El SF ha recibido mucha atención al descubrirse que es la
sustancia natural más potente de inducción de enzimas de fase II en animales y humanos in
vivo e in vitro incluso a las dosis administradas en la dieta (Prochaska et al., 1992). Además se
ha observado que no solo es activo en células carcinógenas, sino también en células no
transformadas (McMahon et al., 2003).
3) Funciones antioxidantes a través del incremento de los niveles en el tejido de glutatión: el SF
no es un antioxidante directo o prooxidante, sino que actúa indirectamente para incrementar la
capacidad antioxidante de las células animales y su capacidad para sobrellevar el estrés
oxidativo actuando sobre el GSH, que es un tripéptido importante que mantiene el equilibrio de
oxidación-reducción y protege a la célula contra los radicales libres (Zhang, 2000; Zhang, 2001;
Callaway et al., 2004).
4) Propiedades inductoras de apoptosis: se han descrito marcas clave de la apoptosis, tales como
condensación de la cromatina, traslocación del la fosfatidilserina a través de la membrana
plasmática y fragmentación del ADN en tratamientos con SF en células tumorales (GametPayrastre et al., 2000; Choi et al., 2003; Gingras, et al., 2004; Jackson et al., 2004). Además se
ha visto una implicación de apoptosis mediada por caspasas inducidas por tratamiento con SF
(como la caspasa 9) (Chiao et al., 2002). En la apoptosis mediada por SF se puede citar
también una implicación de la mitocondria, la familia proteica bcl-2, de JNK/MAPK y de p53
(Juge et al., 2007).
5) Inducción del arresto del ciclo celular: hay evidencias de que el SF ejerce un mecanismo
anticarcinogénico por arresto del ciclo celular a diferentes etapas de su progresión, por
regulación de la inhibición de CDKs, la disrupción de microtúbulos y modificación de histonas
entre otras (Juge et al., 2007).
6) Propiedades antiinflamatorias: implicando iNOS, Cox-2 y TNF-α (Juge et al., 2007).
7) Inhibición de la angiogénesis: actuando sobre la proliferación de las células endoteliales (Juge
et al., 2007).
30
En contraposición a todos estos efectos, el sulforrafano nitrilo (SFN) ha demostrado ser
inefectivo como inductor de enzimas de detoxificación. Por tanto se ha propuesto que la selección
de entradas con bajos niveles de proteína epitioespecífica pueden proporcionar una mayor
conversión de SF a SFN, con el incremento además de la actividad anticarcinogénica (Matusheski
et al., 2006).
Mientras que los efectos del SF han sido ampliamente estudiados in vivo e in vitro, el papel
protector de la ER presenta menos datos experimentales (Lamy et al., 2008). Munday y Munday
(2004) publicaron el papel inductor de la ER en las enzimas de detoxificación de fase II en varios
tejidos de rata; mientras que Harris y Jeffery (2008) demostraron una inducción de enzimas de
detoxificación de fase II por ER y SF en líneas celulares de carcinoma humano por un mecanismo
común. Incluso se ha descrito actividad antigenotóxica por ER en células de hepatoma humano
(HepG2) (Lamy et al., 2008).
La IB, un sulfóxido análogo del SF, ha sido también propuesta como un agente
quimiopreventivo (Jadhav et al., 2007), tal y como han mostrado estudios de sus efectos en
animales de laboratorio (Munday y Munday, 2004), por la inducción de enzimas de fase II. La IB
incrementa las actividades de la glutation S-transferasa y quinona reductasa en epitelio de vejiga
de rata, demostrando efectos protectores contra la carcinogénesis por compuestos químicos
(Staack et al., 1998). La iberina sobreexpresa la tioredoxina reductasa 1 en células humanas MCF
cells sugiriendo un papel en el mantenimiento del estatus de redox (Wang et al., 2005). Sin
embargo, los efectos anticancerígenos de la IB en células tumorales aún no ha sido investigado
en detalle.
El contenido en ITCs está menos estudiado que el de GLS. Se ha publicado que el ITC
mayoritario en hojas de Eruca es la erucina (Blazevic y Mastelic, 2008). Melchini y colaboradores
(2009) analizaron el contenido también en erucina y sulforrafano en liofilizado de hojas de Eruca.
Otros autores han citado la sativina como el ITC mayoritario en rúcula (Bennett et al., 2002). Se
pueden además encontrar algunos trabajos más enfocados al análisis cualitativo del contenido de
ITCs en este vegetal (Jirovetz et al., 2002; Miyazawa et al., 2002).
5.3. Los compuestos fenólicos.
Otro grupo de compuestos de interés que se encuentra presente en las crucíferas es el
formado por los compuestos fenólicos, los cuales se encuentran presentes en vegetales y frutas
en altos niveles (Androtopoulos et al., 2010). Algunos de ellos han mostrado propiedades
saludables (Prasain et al., 2010). Los fenoles aparecieron como una adaptación evolutiva de las
plantas al pasar del medio acuático al terrestre, además son los encargados del color de las flores
31
y de ciertos sabores, así como de funciones esenciales para la supervivencia de plantas
vasculares (Buchanan et al., 2000).
Los polifenoles poseen estructuras químicas que favorecen funciones antioxidantes como
de captación de radicales y quelantes de metales. Algunos pueden proporcionar beneficios
fisiológicos en situaciones patológicas asociadas con la producción de radicales libres. Sin
embargo, los mecanismos que relacionan las características químicas antioxidantes con los
efectos beneficiosos están aún por vislumbrar (Fraga, 2007). Se sabe que los polifenoles pueden
acutar como antioxidantes in vivo (Halliwell et al., 2008). Se piensa que pueden contribuir a la
prevención de enfermedades cardiovasculares, cáncer, osteoporosis, así como en la prevención
de enfermedades neurodegenerativas y de diabetes mellitus, aunque el mecanismo de actuación
no se ha esclarecido (Scalbert et al., 2005; Halliwell et al., 2008).
Los flavonoides son una gran sub-familia de compuestos fenólicos sintetizados por las
plantas como metabolitos secundarios con una estructura química común (Beecher et al., 2003).
Son el grupo más usual de fenoles en la dieta humana y se clasifican en flavonas, flavonoles,
flavanonas, e isoflavonas. Se han descrito potentes efectos in vitro anticarcinogénicos y
antiaterogénicos, incluyendo protección antioxidante de ADN y de lipoproteínas de baja densidad,
modulación de la inflamación, inhibición de la agregación plaquetaria y modulación de la expresión
de adhesión a receptor (Andersen et al., 2006).
Los flavonoides son potentes captores de radicales libres con capacidad antioxidante,
analizada in vitro (Heijnen et al., 2001) e in vivo (Pietta, 2000). Se ha publicado que la modulación
de las rutas de señalización celular por los flavonoides pueden prevenir el cancer por la
estimulación de enzimas de detoxificación de fase II (Kong et al., 2001), preservando la regulación
normal del ciclo celular (Chen et al., 2004; Stewart et al., 2003), inhibiendo la proliferación y la
inducción de apoptosis (Ramos et al., 2007), inhibiendo la invasión tumoral y la angiogénesis
(Bagli et al., 2004) y disminuyendo la inflamación (O'Leary et al., 2004). Sin embargo no hay
evidencia de los efectos pro-oxidantes sistémicos de estos compuestos en humanos y hay pocas
evidencias de los efectos antioxidantes también in vivo. Tienen otros efectos biológicos incluyendo
la capacidad de inhibir ciclooxigenasas, lipooxigenasas, metaloproteinasas y NADPH oxidasas.
Estas acciones pueden ser más importantes
in vivo que los efectos antioxidantes, aunque
muchos de ellos han sido demostrados solo in vitro (Halliwell et al., 2008).
En la literatura se pueden encontrar algunos trabajos sobre el estudio del contenido en
compuestos fenólicos de Eruca (Weckerle et al., 2001; Bennett et al., 2002; Arrabi et al., 2004).
Entre los compuestos fenólicos encontrados se pueden citar kaempferol, isohamnerin y
quercetina.
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5.4. Los carotenoides.
Los carotenoides constituyen una de las clases más importantes de pigmentos vegetales y
son abundantes en frutas y vegetales (Edge et al. 1997), puediendo ser clasificados en dos
grupos: carotenos y xantofilas. Son compuestos poli-isoprenoides de 40 átomos de carbono que
forman una cadena que constituye la “espina dorsal” de la molécula, pudiendo presentar
estructuras cíclicas (anillos) en los extremos, algunas de las cuales se complementan con grupos
funcionales que contienen oxígeno. Los carotenoides que contienen exclusivamente carbono e
hidrógeno en su estructura se conocen como carotenos, mientras que los derivados oxigenados
de estos hidrocarburos se conocen como xantofilas (luteína, zeaxantina, violaxantina) (RodríguezBernaldo de Quirós y Costa, 2006). Los carotenoides son pigmentos accesorios que se
encuentran en estructuras fotosintéticas, localizándose en la membrana de los tilacoides y en la
de envoltura de los cloroplastos. Los principales carotenoides de cloroplastos de plantas
superiores y microalgas son α y β-caroteno, luteína, violaxantina, zeaxantina y neoxantina.
Estos compuestos han mostrado actividad como antioxidantes biológicos, protegiendo las
células y los tejidos de los efectos perjudiciales de radicales libres y del oxígeno singlete. Su
comportamiento antioxidante depende de la concentración y localización en las células diana, así
como de otros factores (Van den Berg et al., 2000). La luteína y la zeaxantina actúan como
protectores en la región macular de la retina humana (Snodderly 1995). Además se ha observado
un incremento de la función inmune (Bendich 1989), protección contra quemaduras solares
(Matthews-Roth 1990) e inhibición del desarrollo de ciertos tipos de cáncer (Nishino 1998).
5.5. Los carbohidratos.
Los carbohidratos se corresponden a la mayoría de los componentes sintetizados por los
organismos vivos. Muchos de los carbohidratos representan formas de acumular carbón y
energía, mientras que otros son componentes estructurales de las paredes celulares,
proporcionando soporte a la planta (Buchanan et al., 2000). Desde un punto de vista nutricional,
reduciendo azúcares tales como glucosa, maltosa y fructosa, éstos pueden reaccionar con
aminoácidos y proteínas, dando lugar a la formación de productos de reacción Maillard (MRPs en
inglés), que afectan negativamente a las propiedades organolépticas y nutritivas de las plantas.
Los azúcares pueden llevar a cabo actividades antioxidantes, antimicrobianas o citotóxicas
(Chevalier et al., 2001). Por esta razón, junto con la dificultad del análisis de las diversas formas
de MRPs, muchos estudios fitoquímicos en plantas incluyen el análisis de azúcares (Onwukaeme
et al., 2007).
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6. Los minerales
Los humanos requieren más de 22 elementos minerales para cubrir sus necesidades
fisiológicas alimentarias. Algunos de estos elementos son requeridos en elevadas cantidades
como son por ejemplo el Calcio (Ca) y el Fósforo (P). Sin embargo, otros como el Hierro (Fe), Zinc
(Zn) y Cobre (Cu), sólo son necesarios en pequeñas cantidades (Welch y Graham, 2004; White y
Broadley, 2005).
A pesar de ser estos los nutrientes esenciales requeridos en menor cantidad en la dieta
humana actual, se observa que existen deficiencias en dichos elementos. Así se estima que de 6
billones de personas, entre el 60-80% son deficientes en Fe, más del 30 % son deficientes en Zn,
y más del 15% son deficientes en Ca, Mg y Cu (White y Broadley, 2005).
La deficiencia en Fe es la más extendida en el mundo, estando ligada a muchas
enfermedades como la anemia y en muchas de las muertes de mujeres embarazadas y neonatos,
además de ser causa de abortos. Principalmente la encontramos en el sur de Asia y África,
aunque los grupos de población con más riesgo son aquellos cuya dieta esta basada
principalmente en productos vegetales con bajo contenido en este elemento y poco consumo de
carne (Ortiz-Monasterio et al., 2007; Yang et al., 2007).
La deficiencia en Zn, también está muy extendida por el mundo, siendo este un elemento
implicado en la expresión de los genes, desarrollo y replicación celular (Hambrigde, 2000). A este
micronutriente se le atribuye el incremento de padecer diarreas, neumonía y malaria, además de
asociarse su deficiencia con un incremento en la mortalidad en niños menores de 5 años. Las
regiones más afectadas por esta deficiencia son el sur de Asia y África subsahariana,
principalmente afectando a niños (Caulfield y Back, 2004).
Los miembros de la familia Cruciferae son fuente de los principales elementos minerales
esenciales para el ser humano (Miller, 1987; House, 1999). Así lo han demostrado estudios
realizados en hojas en especies de Brassica, como Brassica oleracea var. capitata (Glew et al.,
2005) y Brassica juncea (Elles et al., 2000), los cuáles indicaron su potencial uso como suplementos
nutricionales de minerales concentrados en forma de cápsulas o tabletas. En dichos trabajos las
concentraciones medias de Fe, Zn, Mn, Se, Cr, Ca, Mg y P variaron desde 2500 hasta 73000 µg g-1
ps, además dichos elementos se encontraron en una forma más soluble y metabólicamente más
disponible que en los suplementos minerales comerciales específicos.
Respecto al contenido en minerales en Eruca se han publicado algunos trabajos
como los de Kawashima y Valente-Soares (2003), Bozokalfa y colaboradores (2010), Cavarianni y
34
colaboradores (2008), indicándose que las hojas de esta especie poseen concentraciones
significativas de los minerales principales.
7. Acumulación de metales pesados en especies de crucíferas
Hay algunas plantas que acumulan metales tóxicos, los cuáles no tienen conocido
beneficio directo para la planta (Baker y Brooks, 1989; Raskin et al., 1994), y sin embargo afectan
negativamente su calidad nutricional. Las primeras plantas hiperacumuladoras de metales
pesados caracterizadas pertenecían a las familias Brassicaceae y Fabaceae, aunque hoy día se
conocen al menos 45 familias distintas que presentan especies capaces de acumular metales.
Esta capacidad para acumular metales ha sido utilizada para su empleo en la descontaminación
de suelos (Del Río et al., 2000). En este sentido, se han realizado ensayos de campo para
fitoextracción continua de metales pesados con distintas especies acumuladoras: Thlaspi
caerulescens para Cd (Brown et al., 1995); Brassica oleracea, Raphanus sativus, Thlaspi
caerulescens, Alyssum lesbiacum, Alyssum murale y Arabidopsis thaliana para Zn, Cd, Ni, Cu, Pb
y Cr, respectivamente (Nanda-Kumar et al., 1995; Felix, 1997; Máthé-Gaspar y Anton, 2002).
Particularmente han sido numerosos los trabajos realizados en Brassica juncea y Brassica
carinata, identificándose líneas que acumulan metales en sus tallos y hojas con concentraciones
que exceden el 2% de su peso seco (Banuelos et al., 1993; Nanda-Kumar et al., 1995; Salt et al.,
1995; Del Río et al., 2000, 2005).
Teniendo en cuenta que algunas de las acumulaciones más altas de metales pesados y
arsénico en plantas cultivadas se han obtenido en especies de Brassica especialmente en sus
raíces (Liu et al, 1992, Bernal et al., 1994; Santamaria et al., 1996, Ebbs et al.,1997; CarbonellBarrachina et al., 1999), y el consumo frecuente que de las distintas variedades de especies de
Crucíferas existe a nivel mundial, algunos investigadores han visto necesaria la caracterización
del nivel de acumulación de metales(oides), así como los efectos biológicos derivados de la
ingesta de estas especies cuando son afectadas por distintos niveles de contaminación metálica.
8. Caracterización de los recursos genéticos
Los recursos genéticos, además de ser una necesidad para evitar la vulnerabilidad
genética, son una oportunidad para encontrar en ellos aquellas características de calidad que el
consumidor está demandando. Por tanto, la información asociada a estos recursos resulta de vital
importancia de cara a su utilidad y aprovechamiento (González-Andrés, 2001). Además, este
conocimiento es esencial para explicar la actividad biológica de las entradas y para planear
35
estrategias para el diseño de variedades que incrementen la salud del consumidor de estos
vegetales.
La caracterización es el establecimiento de todos los caracteres posibles de un cultivo.
Entre estos enfoques podemos citar los siguientes:
- Caracterización agro-morfológica: estudia cualquier órgano de la planta desde el
punto de vista cualitativo y cuantitativo, así como sus datos fenológicos.
- Caracterización sensorial: estudia las reacciones humanas a aquellas características
de los alimentos que se perciben por los sentidos de la vista, el oído, el gusto, el olfato y el
tacto, mediante personas entrenadas o instrumental analítico.
- Caracterización nutricional y funcional: estudia los valores nutricionales y funcionales
existentes en el material vegetal, como el nivel de glucosinolatos, ITCs, carotenoides,
minerales, carbohidratos, etc.
8.1. Caracterización agro-morfológica de Eruca.
Con el fin de conseguir un consenso, IPGRI publicó una lista de descriptores para la
identificación de distintas especies vegetales, entre ellas rúcula (IPGRI, 1999). Todos los
descriptores publicados presentan una lista de caracteres que se refieren a aquellas
características de la planta en todos sus estadios suficientemente estables para ser válidos a la
hora de definir y diferenciar las distintas variedades. A grandes rasgos la caracterización
morfológica puede estar basada en caracteres cualitativos, cuantitativos y fenológicos. Dentro de
los cuantitativos, los que consisten en utilizar sistemas de medición para cuantificar determinados
parámetros, reciben el nombre de morfométricos.
Respecto a rúcula, Chandel y Bhandari (1989) describieron una alta variabilidad genética
para distintos caracteres como tipo de planta, patrón de ramificación, pigmentación, tamaño de
silicua y forma, color y tamaño de la semilla en poblaciones en India. Warwick et al. (2007)
indicaron un alto potencial en poblaciones de Eruca a partir del estudio de caracteres agronómicos
y de la calidad de la semilla en trabajos realizados en Canadá. Egea-Gilabert et al. (2009)
evaluando 3 entradas silvestres y 1 entrada cultivada de E. vesicaria encontraron una alta
variabilidad para la mayoría de los caracteres agronómicos y morfológicos estudiados.
Recientemente, Bozokalfa et al. 2010 encontraron variabilidad en genotipos de Eruca para
distintos caracteres agronómicos como altura de la planta, semillas por silicua, peso de la semilla
y anchura de la silicua. Sin embargo, aunque se han realizado amplios estudios sobre estimas de
36
la variabilidad genética, heredabilidad y correlaciones de caracteres agronómicos en especies de
Brassica, no existe una detallada caracterización en genotipos de Eruca.
8.2. Caracterización organoléptica de Eruca.
La mejora genética en plantas orientada a la mejora sensorial, tal y como se ha
mencionado, es un objetivo muy reciente, y aún más en productos hortícolas. Sólo en contados
casos, esta mejora se ha orientado hacia la mejora de la calidad nutricional, y sólo muy
ocasionalmente, en la búsqueda de productos con una mayor calidad sensorial (Bartoszewski et
al., 2003). En aquellos casos, los tests sensoriales fueron usados como una herramienta
complementaria para seleccionar el germoplasma más apropiado y se basaron en las
valoraciones de una o dos personas capaces de distinguir algún defecto en un producto
(Hampson et al., 2000). Hoy en día, los estudios sensoriales no están sólo centrados en la
búsqueda de defectos, sino también en encontrar respuestas a los requerimientos del consumidor
y un nivel más alto de satisfacción sensorial (Hampson et al., 2000; Wismer et al., 2005).
Actualmente, un adecuado programa de Mejora Genética debe contener técnicas sensoriales y
estudios de consumo para identificar, entre las diversas posibilidades, los productos con mayor
probabilidades de éxito en el mercado (Harker et al., 2003; Jaeger et al., 2005).
La calidad sensorial es un concepto difícil de definir, el cual cubre, no sólo los atributos
intrínsecos del producto, sino también la interacción entre el producto y el consumidor. Esta
interacción contiene numerosos factores relativos a las características del alimento (composición
química, estructura y propiedades físicas), las características del consumidor (genéticas,
fisiológicas, sociológicas) y el entorno (geografía, cultura, gastronomía, religión, educación,
hábitos familiares, moda, precio). Es además necesario establecer una relación entre la
composición físico-química del producto y sus atributos organolépticos, como color, textura, aroma
(componentes volátiles) y sabor (dulce, ácido, salado, agrio) y también entre las percepciones
sensoriales y la aceptabilidad final del consumidor. Por la complejidad de estas técnicas, su
consolidación en el entorno académico e industrial no apareció hasta los ochenta (Moskowitz,
1993; Costell, 2000).
Hasta el momento los avances conseguidos en la mejora de caracteres organolépticos se
deben principalmente a la identificación de variedades tradicionales superiores y a su posterior
selección y corrección de deficiencias agronómicas. La mejora a partir de materiales
convencionales se ha abordado en numerosas especies y por vías distintas (Oraguzie et al., 2003;
Causse et al., 2001; Bartoszewski et al., 2003).
Respecto a rúcula, hay estudios muy específicos orientados a la extracción de volátiles,
responsables del aroma. Así, a partir del uso de GC, GC-MS y olfatometría se han identificado
37
más de 70 compuestos volátiles, entre ellos algunos ITCs y numerosos compuestos derivados de
butano, hexano, octano y nonano fueron los responsables del peculiar aroma de esta especie
(Jirovetz et al. 2002; Miyazawa et al., 2002; Nielsen et at., 2008,).
Otros estudios se han centrado en la influencia que las operaciones de procesado,
cortado, lavado, envasado y conservación, tienen sobre la calidad microbiológica, nutricional y
sensorial en productos mínimamente procesados de rúcula (Koukounaras et al., 2007; MartínezSánchez et al., 2006).
Generalmente, trabajos anteriormente publicados que están basados en componentes
nutricionales no se basan en el panel sensorial como una herramienta de análisis, sino que han
limitado su investigación al análisis instrumental (Bennett et al., 2002; Bennett et al., 2006). Existe
sólo un estudio directamente orientado a correlacionar el contenido en glucosinolatos con
atributos sensoriales en las especies Diplotaxis and Eruca vesicaria. (D’Antuono et al., 2009)
Hasta ahora, no se ha establecido un protocolo de caracterización sensorial incluyendo los
parámetros que puedan ser fácilmente adaptables a cualquier variedad de rúcula, ni desarrollado
un vocabulario específico capaz de dar resultados específicos y fidedignos.
8.3. Caracterización nutricional y funcional de Eruca
8.3.1. Aproximación al papel fitoquímico de Eruca
El perfil de glucosinolatos, flavonoides y análisis de algún carotenoide y algún isotiocianato
se ha realizado previamente en rúcula (Bennett et al., 2002; Niizu y Rodríguez-Amaya, 2005;
Bennet et al., 2007; Melchini et al., 2009). Sin embargo, no se han realizado estudios completos
del patrón de conversión de glucosinolatos a isotiocianatos, no se han realizado análisis
cuantitativos de carotenoides como la zeaxantina y β-criptoxantina, ni tampoco se ha realizado
ningún estudio del contenido en azúcares en rúcula. Estudios previos, además, se han centrado
en algún grupo de estos fitoquímicos en particular, pero no se ha realizado ningún estudio del
contenido completo de fitoquímicos en rúcula. Dada la escasa mejora de calidad que ha sufrido la
rúcula, este conocimiento es esencial como herramienta en programas de Mejora Genética, la
selección de las líneas más interesantes para el consumo humano, así como para la creación de
líneas para el mercado como alimentos funcionales.
8.3.2. La Espectroscopía por reflectancia en el infrarrojo cercano (NIRS) como herramienta
analítica en la caracterización del perfil de minerales en especies vegetales.
Las metodologías analíticas convencionales para la determinación de componentes de
calidad, muestran un alto grado de precisión en la medida, pero al mismo tiempo presentan
grandes inconvenientes, como son el alto coste del análisis, lentitud de la operación, necesidad de
38
personal especializado, destrucción de la matriz analizada, y polución del medio ambiente, debido
al uso de reactivos químicos, entre otros.
Estos antecedentes han llevado a la búsqueda de tecnologías analíticas alternativas, que
aunque perdiendo precisión en la cuantificación del analito, permitan un muestreo rápido y a bajo
coste económico, redundando en una importante descarga analítica para el laboratorio. Es por ello
una técnica con posibilidades reales de ser empleada como método de muestreo rápido, sencillo y
no contaminante en el control de la calidad (Font et al. 2005; Font et al. 2006) y seguridad
alimentaria (Clark et al. 1989; Font et al. 2004). En este sentido, la Espectroscopía en el Infrarrojo
Cercano (NIRS), ha mostrado un alto potencial para la predicción de minerales, tanto en matrices
orgánicas como en inorgánicas (Clark et al., 1989; Nilsson et al., 1996; Halgerson et al., 2004;
Cozzolino y Moron 2004; Petisco et al., 2005), si bien, aún no se ha aplicado dicha tecnología en
rúcula.
8.3.3. Actividad biológica de las líneas de crucíferas.
Los efectos beneficiosos atribuibles a los compuestos fitoquímicos de las crucíferas, así
como aquellos adversos atribuibles a los metales(oides) incluidos en esta tesis, han de
considerarse en el contexto de la complejidad existente en el organismo que los consume. Por
ello, se hace necesaria una evaluación sobre modelos biológicos que se asemejen a las
condiciones fisiológicas humanas. Se han publicado numerosos trabajos sobre la capacidad de
diferentes especies de crucíferas de inhibir procesos de tumorogénesis in vitro (Verhoeven et al.,
1997; van Poppel et al., 1999; Lynn et al., 2001). Estudios enfocados al análisis de la expresión
global génica pueden aportar pocas pruebas del los mecanismos potenciales derivados de
experimentos in vitro e in vivo con objeto de explicar los datos epidemiológicos a favor de que el
consumo de crucíferas puede reducir el riesgo de padecer cáncer (Traka et al., 2008). No
obstante, en dicho trabajo se pudo observar una perturbación en las vías de señal implicadas en
la carcinogénesis e inflamación. Hasta ahora sólo se ha realizado un estudio utilizando el material
vegetal de Eruca para analizar los efectos antigenotóxicos (Lamy et al., 2008) y otro analizando el
posible efecto protector de los glucosinolatos y productos de degradación de Eruca.
Ensayo de inhibición del crecimiento tumoral
La utilización de cultivos celulares para el ensayo de las características tóxicas de una
sustancia química constituye una metodología barata, rápida y evita el sacrificio de animales de
sangre caliente. Estas células se caracterizan por proliferar continuamente en suspensión, aunque
tras un periodo de cultivo, pierden su capacidad de división y se diferencian. Durante muchos
años, diferentes líneas celulares han sido extensamente estudiada para esclarecer los
mecanismos de citotoxicidad y que inducen diferenciación y apoptosis y así poder controlar su
proliferación en los organismos vivos (Collins et al., 1978; Conte-Anazetti et al., 2003).
39
Estudio de la capacidad inductora de apoptosis
La muerte celular o apoptosis juega un papel crucial en el desarrollo y el mantenimiento
de la homeostasis y eliminación de células dañadas o que no son necesarias en un futuro. La
correlación entre la inducción de la apoptosis y la citotoxicidad es una estrategia quimiopreventora
interesante. El ensayo de inducción de apoptosis estudia el grado de fragmentación del ADN que
ocurre durante la apoptosis entre otros mecanismos, pudiendo haberse observado inducción de
apoptosis en algunas líneas celulares por el tratamiento con sulforrafano (Kerr et al., 1972;
Higuchi, 2003; Juge et al., 2007; Gasper et al., 2007; Qian et al., 2009).
Estudio de la proteína p21
La proteína p21 es una proteína inhibidora de CDK, la cual es esencial para el crecimiento
celular, diferenciación y apoptosis (Xiong et al., 1993). Se sabe que la inducción de p21 causa el
arresto en las etapas G1 y G2 del ciclo celular en algunas líneas celulares. Además, esta proteína
juega un papel importante en el arresto celular inducido por SFN regulado por la proteína
supresora de tumores p53 en respuesta al daño al ADN (Kim et al., 2010). Se sabe que la ER y el
SF pueden causar un incremento significativo en los niveles de p21 a altas concentraciones (15–
25 µM) en células A549 (Melchini et al., 2009).
Modelo SMART de ensayo genotoxicológico y antigenotoxicológico in vivo
La elección de un sistema in vivo adecuado para la detección de agentes geno y
antigenotóxicos es de gran importancia, ya que las biotransformaciones pueden estar sesgadas
según el sistema de activación exógeno que se utilice en ensayos in vitro. La capacidad de
metabolizar promutágenos a compuestos activos o inactivos no es exclusiva de mamíferos sino
que aparece en todos los taxa biológicos (inclusive en plantas), por ello se propone el uso de
Drosophila melanogaster como sistema in vivo de detección de anti y xenobióticos desde muy
antiguo (Muller, 1927).
El ensayo de mutaciones y recombinaciones somáticas en alas de Drosophila (Somatic
Mutation And Recombination Test o S.M.A.R.T.) se basa en la detección de
alteraciones
genéticas producidas en las células de discos imaginales alares de la larva, que pueden
evidenciarse fenotípicamente en el tejido adulto después de la expansión clonal y la metamorfosis,
y fue desarrollado por Graf y colaboradores en 1984. Este ensayo ha mostrado ser capaz de
detectar actividad genotóxica en compuestos de estructura química variada, tanto mutágenos
directos como promutágenos, con diferentes métodos de acción genotóxica, como agentes
alquilantes, intercalantes o formadores de aductos, tanto sólidos, como líquidos, gaseosos,
simples o mezclas complejas (Graf et al, 1984; Alonso-Moraga y Graf, 1989; Graf at al, 1994;
Osaba et al, 1999). Además, ante esta evidencia indirecta, los estudios bioquímicos dan cuenta de
40
la presencia en Drosophila de enzimas implicados en el metabolismo de xenobióticos (Baars,
1980). Hoy día se considera un test de detección de primera línea; si además se tiene en cuenta
su bajo coste, eficiencia, versatilidad y rapidez podemos considerar al test S.MA.R.T como muy
apropiado para usar en Toxicología Genética utilizando como modelo un eucariota in vivo que no
necesita de activación metabólica exógena para detectar actividad de promutágenos y de
antimutágenos.
Supervivencia
El envejecimiento es un proceso multifactorial asociado a un declive en las funciones
biológicas y a una mayor incidencia de diversas enfermedades, como el cáncer, enfermedades
neurodegenerativas y diabetes. Se sabe que muchas frutas y verduras, y sus extractos, con
promotores de la salud y previenen o retardan la aparición de enfermedades relacionadas con el
envejecimiento. Incluso estudios preclínicos han demostrado que la dieta de Drosophila
suplementada con ciertas compuestos pueden promover la longevidad y la esperanza media de
vida (Trotta et al., 2006; Mockett y Sohal, 2006; Li et al., 2008). El modelo animal de supervivencia
en Drosophila para investigar las propiedades promotoras de longevidad y calidad de vida por la
alimentación es muy apropiado en programas de detección de nuevas sustancias debido a su
reducido life span, a su facilidad de cultivo con dietas simples o complejas, y al conocimiento
completo de su genoma, habiendo desvelado éste que la mitad de sus genes son homólogos a la
especie humana (Boyd et al., 2011; Jones et al., 2011).
41
JUSTIFICACIÓN DEL TRABAJO
42
Desde instituciones públicas y privadas en España, y en Andalucía en particular, se viene
realizando en los últimos años una apuesta clara y decidida en investigación y transferencia
dirigida a incrementar el valor añadido de los productos hortofrutícolas producidos en nuestro
país. Esta apuesta viene de la mano de la necesidad de ganar competitividad y rentabilidad en el
sector, como una respuesta al aumento de la competencia procedente de terceros países del arco
mediterráneo, con los que no es posible competir en precio, así como a la exigencia de un
incremento de la calidad en los productos tradicionales por parte de los mercados internos y
externos.
Con el fin de dar respuesta a la problemática planteada, la cual arrastra una evidente
pérdida de competitividad real sufrida por el sector hortofrutícola español y andaluz, el Ministerio
de Ciencia y Tecnología, a través del Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA) y la Junta de Andalucía a través de la Consejería de Innovación, Ciencia y
Empresa (CICE), vienen apoyando líneas específicas de investigación para incrementar el valor
añadido de la producción agraria española y andaluza, como queda reflejado en el Plan
Estratégico de la Agroindustria Andaluza (PEAA) Horizonte 2013. El PEAA, consensuado y
rubricado en 2009 por la propia Junta de Andalucía, Confederación de Empresarios de Andalucía
(CEA) y representación de los sindicatos mayoritarios, enumera una serie de líneas de actuación
prioritarias con el objetivo principal de incrementar la competitividad de las empresas del sector
hortofrutícola. Entre estas líneas se encuentran: 1) necesidad de aportar mayor valor añadido en
la cadena comercial; 2) apuesta por la innovación y programas I+D+i y, 3) la diversificación de la
oferta hacia nuevas necesidades de los mercados.
Por su parte, el IFAPA a través de su Plan Sectorial 2010-2013 (PEI) fija una serie de
líneas estratégicas de investigación y directrices para su cumplimiento. Estas son, entre otras las
siguientes: 1) Aumentar el valor añadido de la producción en fresco mediante la mejora de la
calidad, incremento de vida útil e innovación en el diseño y conservación de nuevos productos
transformados a partir de hortalizas y, 2) Responder a demandas concretas del sector que
mejoren la comercialización de sus productos,
En base a los criterios anteriormente mencionados, esta tesis aborda la caracterización de un
cultivo minoritario e infrautilizado como es la rúcola, La amplia variabilidad existente en una
colección constituida por líneas pertenecientes a diferentes especies de Eruca, y procedentes de
diversos origenes geográficos representa una fuente de germoplasma con múltiples posibilidades
en el campo de la Mejora Genética Vegetal, La búsqueda del incremento de valor añadido para
esta especie se ha realizado no solo en base a sus características agronómicas y morfológicas
sino también desde el punto de vista de la calidad nutricional ó funcional. Por tanto, se ha
43
propuesto como primer objetivo en esta tesis la caracterización de esta colección para la selección
de líneas de Eruca con mejores características agronómicas y nutricionales, mayor capacidad
antimutagénica y tumoricida (atribuible a los glucosinolatos) y alto contenido en minerales para su
futuro uso en programas de mejora.
Sin embargo, algunas especies de estos vegetales como el rábano son acumuladoras de
metales pesados tóxicos, cualidad que puede resultar negativa para su aprovechamiento
agronómico y funcional.
44
OBJETIVOS DE LA TESIS
45
La actividad científica desarrollada en la presente tesis doctoral trató de alcanzar los
siguientes objetivos globales y específicos:
Objetivo global 1: Estudio de la variabilidad vegetal existente en partes verdes de Eruca spp.
para su uso futuro en programas de Mejora Vegetal.
Objetivo específico 1.1: Caracterización morfológica, agronómica, bromatológica y
nutricional de líneas de Eruca.
Objetivo específico 1.2: Estudio del potencial de la Espectroscopía por reflectancia en el
Infrarrojo Cercano (NIRS) para la caracterización mineralógica en Eruca.
Objetivo global 2: Determinación de la actividad biológica (genotoxicidad y citotoxicidad) de
órganos vegetativos en Crucíferas de interés alimentario.
Objetivo específico 2.1: Determinación de la capacidad tumoricida y apoptótica de líneas
de Eruca mediante el modelo de inhibición de crecimiento tumoral con células HL-60, PC3 y
PNT1A y su relación con el contenido en glucosinolatos.
Objetivo específico 2.2: Determinación de la capacidad anti/mutagénica y antidegenerativa
de líneas de Eruca mediante el sistema SMART (Test de Mutación y Recombinación
Somática) de Drosophila melanogaster y en ensayos de supervivencia.
Objetivo específico 2.3: Determinación de la actividad biológica (genotoxicidad y
citotoxicidad) de Raphanus sativus cultivados en suelos contaminados con metaloides
46
47
CAPÍTULO I
Diversidad fenotípica de rúcola (Eruca)
Artículo en preparación
Phenotypic diversity of rocket (Eruca)
Myriam Villatoro-Pulidoa, Andrés Muñoz-Serranob, Rafael Fontc, Mercedes Del RíoCelestinoc.
a
IFAPA-Centro Alameda del Obispo, Córdoba, Spain.
b
Departamento de Genetica, Universidad de Córdoba, Córdoba, Spain.
c
IFAPA-Centro la Mojonera, Almería, Spain.
48
Abstract
Rocket (Eruca spp.) is a cruciferous crop used extensively in leaf salads as a Fourth
Generation vegetable. Despite the increasing economical importance of this crop limited
information is available on genetic variability for phenotypic traits. This information could enable a
proper management of germplasm collections in plant breeding. We studied a total of 52
accessions in a field trial at Córdoba (Spain). Data were recorded for 15 qualitative and
quantitative characters. The vegetal material consisted on accessions belonging to Eruca
stenocarpa, Eruca vesicaria subsp. longirostris, Eruca vesicaria subsp. vesicaria and Eruca
vesicaria subsp. sativa from of different geographic origins. Three commercial cultivars were
included in the study as control. High variability can be observed in most of the traits, showing also
good qualities like small leaves, high chlorophyll content, high growth rate, late flowering and
absence of pubescence for some of the accessions. The information on diversity among the agromorphological traits will be helpful for breeders in constructing their breeding populations.
49
1. Introduction
Rocket is a crop with increasing economic potential during the past decade for its use in
salads, although cooked leaves, flowers, and more recently sprouts (seedlings) are also consumed
(Padulosi, 1995; Bennet et al., 2007). The leaves for consumption are collected during the
vegetative stage.
Despite different species are referred under the name of rocket, the most common ones are
those belonging to Eruca and Diplotaxis genera. The taxonomic limits of the genus Eruca have
been moved with time. One of the accepted classifications of Eruca attends to the terms of Eruca
sternocarpa, Eruca vesicaria (L.) Cav. subsp. sativa (Miller) Thell., subsp. vesicaria, subsp.
longirostris and subsp. pinnatifida (Gómez-Campo, 1993; Gómez-Campo, 1999; Jalas et al., 1996,
Warwick et al., 2007). Recently it has been also accepted that Eruca contains a single species
Eruca vesicaria (L.) Cav., which, in tum, includes other intraspecific taxa (Pignone and GómezCampo 2011). All of these subspecies can be found in the wild state, but cultivated and consumed
rocket corresponds mainly to E. vesicaria subsp. sativa and it occupies a wider geographical area
in the world. The subspecies pinnatifida (Desf.) Emberger & Maire, is only local in the West
Mediterranean area, and it is endemic to north-western Africa (Warwick et al., 2007). Although
another subspecies, longirostra (Uechtr.) Maire, from the West Mediterranean area has also been
described, a detailed morphometric analysis based on fruit dimensions does not confirm its distinct
status (Gómez-Campo 2003). Other ancient synonyms are Brassica eruca L. and Raphanus eruca
(L.) Crantz.
Rocket currently used as a common component of salads in several European and Near
Eastern countries (Esiyok 1997; Pimpini and Enzo 1997; Yaniv 1996). This crop is grown for its
seed oil in India and Pakistan (Bhandari and Chandel 1997). It is considered a medicinal plant and
it can be employed in biological control of crop pests (Padulosi 1995; D’Antuono et al. 2008).
However, the increasing importance of rocket is the result of consumer desire for ready-to-use and
healthy vegetable products. Rocket as other members of Cruciferae family contains a wide range
of health promoting phytonutrients including vitamin C, fiber, flavonoids and glucosinolates (Mithen
et al., 2000; Bennett et al., 2004; Podsedek, 2007).
The interest of rocket as a useful plant for 4th generation of vegetables (Padulosi 1995;
Silva-Días, 1997) and the initiative of the project of the International Plant Genetic Resources
Institute (IPGRI) on Underutilized Mediterranean Species to join efforts and share research
findings in order to promote better conservation and use of rocket, led to the establishment of the
Rocket Genetic Resources Network (Padulosi 1995; Padulosi and Pignone, 1997). The collection
of wild rocket germplasm is in progress and new accessions are constantly added to seed banks
50
(Pita-Vilamil et al., 2002). Nevertheless, the genus Eruca, compared to other Cruciferous
vegetables, is considerably underdeveloped from a plant breeding perspective. Until the date,
limited rocket cultivars are available and variety selection have undergone (Morales et al., 2006)
and there is no detailed agronomic and morphological evaluation in Eruca spp. genotypes
(Warwick et al., 2007; Egea-Gilabert et al., 2009).
In EEUU and some European countries, as Italy, breeding programmes are being
conducted to encourage rocket cultivation and consumption (Paludosi and Pignone, 1997; Pico
and Nuéz, 1999; Morales et al, 2006). The most of commercial cultivars cultivated in Spain from
North Europe, and some impediments limit their production due to are not well adapted to climatic
agroclimatic conditions.
The particular characteristics of some of the accessions available from seed banks
(intenational or local) could be used to increase the production and the diversity of products
available to consumers and to improve their general quality (Pico and Nuéz, 1999).
Therefore, the objective of this work was to study the morphological and agronomical traits
of rocket germplasm for management purposes, in order to select the accessions showing the
most interesting characteristics for a future breeding programme.
2. Material and Methods
2.1. Plant material and greenhouse experiments
Fifty-two accessions of Eruca were acquired from different European genebanks collections
and collected from different countries of the world (Table 1). The vegetal material consisted in: 1
accession of Eruca stenocarpa, 1 accession of Eruca vesicaria subsp. longirostris, 10 accessions
of Eruca vesicaria subsp. vesicaria and 40 accessions of Eruca vesicaria subsp. sativa. The
commercial accessions from vegetable seed companies (PEX-17, PEX-55 and PEX-56) were used
as control (Table 1). For the morpho-agronomic characterization, an experiment was carried out in
field conditions in Córdoba (South Spain) (37º 53´ N; 4º 47´ W), in a randomized block design,
with two replicates. The rows were 5 m length, spaced 1 m from each other, with a seeding rate of
80 seeds per meter. First and last plants of each row were considered as borders. Plants were
managed in a conventional cultivation system following the crop recommendations indicated by
Pimpini and Enzo (1996), including soil preparation, pest and disease control, and harvest.
51
52
53
2.2. Agronomical and morphological analysis of the accessions
All accessions were characterized for different agronomical and morphological traits from
seedling up to the harvest of the crop during 2009 (Table 2). Traits selected and measured were
based on Descriptors for Rocket (IPGRI, 1999). Twenty plants at five-leaf stage were used to study
group A traits, while ten plants at flowering and maturity stage were used to study group B and C
traits respectively in each plot/replication. The agronomical traits measured were the content of
fresh matter (FM), plant height (PH) growth rate (GR), days to first flowering (DFF), and plant
growth attitude (PGA).
The morphological traits measured were spliced into quantitative and qualitative traits. The
quantitative traits measured were the leaf petiole length (LPL), leaf length (LL), leaf width (LW),
and leaf length/width ratio (LL/W). Qualitative traits selected were the leaf colour (LC), leaf blade
shape (LBS), leaf margins lobation (LML), leaf lobation (LLo), leaf pubescence (LP), leaf apex
shape (LAS), leaf blade thickness (LBT), petiole and/or midvein enlargement (PME), leaf rough
(LR) and flower colour (FC). The character of LC was measured with a Konica Minolta SPAD-502
chlorophyll meter, which indicates the relative chlorophyll content.
2.3. Statistical analysis
Quantitative traits were expressed as means and standard deviation, while qualitative traits were
expressed as median and robust coefficient of variation (cvr). Individual analyses of variance were
performed for each trait and comparison of means among accessions, species and subspecies
were made using Duncan's multiple range test at the p=0.05 level. The square root transformation
was applied for integer data.
3. Results
3.1. Agronomical analysis of the rocket accessions
Table 3 shows the results of the agronomical analysis. The agronomical traits measured
were the fresh matter content (FM), plant height (PH), growth rate (GR), days to first flowering
(DFF), and plant growth attitude (PGA). The FM ranged from 56.31 to 287.93 g of fresh weight of
plant/m of row for PEX-52 accession and the control accession PEX-55 respectively. Plant height
(PH) was only recorded in half of the accessions. It ranged from 0.33 m to 1.55 m for PEX-7 (E.
vesicaria subsp. vesicaria) and PEX-63 (E. vesicaria subsp. sativa) respectively. This character
was significantly higher for sativa than for subsp. vesicaria. GR trait showed great variability. This
character represents the ability of an accession to compete with weeds and, like the character of
PH, E. sativa accessions were the most vigorous. With regard to the number of days to first
flowering (DFF), the earliest accessions took 82 days to flower, while the last accessions were the
54
vesicaria accessions PEX-7, PEX-52 and PEX-93, flowering at 124 days. Control accessions
showed lower DFF (99-106 days) than those accessions mentioned previously (Table 2). Plant
growth attitude (PGA) refers to the growth position of the plant (if it is erect, intermediate or
prostrate). Eruca stenocarpa and longirostris are both intermediate, while vesicaria and sativa
showed the three categories of the trait. The commercial accessions showed erect (PEX-17),
intermediate (PEX-56) and postrate (PEX-55) plant growth attitude.
Table 2. Agronomical and morphological traits recorded in the rocket accessions during 2009.
Trait designation
Code
Description and categories of the trait
A. Seedling stage
Fresh matter
FM
Leaf colour
LC
Average of fresh weight of plant measured in g/m of row
Measured with Konica Minolta Spad-502 chlorophyll
meter. Expressed as SPAD units.
1. Orbicular, 2. Elliptic, 3. Obovate, 4. Spathulate, 5.
Ovate, 6. Lanceolate, 7. Oblong.
1.Entire, 2. Crenate, 3. Dentate, 4. Serrate, 5. Doubly
dentate, 6. Undulate
Leaf blade shape
LBS
Leaf margins lobation
LML
Leaf lobation
LLo
0. Absent, 1. Accentuated, 2. Markedly present
Leaf pubescence
LP
Leaf apex shape
LAS
Leaf blade thickness
Petiole and/or midvein
enlargement
LBT
3. Rada, 5. Intermediate, 7. Dense
1. Largely acute, 2. Acute, 3. Rounded, 4. Broadly
rounded
3. Thin, 5. Intermediate, 7. Thick
PME
1. Narrow, 2. Enlarged
Growth rate
GR
Leaf petiole length
LPL
Leaf length
LL
Leaf width
LW
Leaf length/width ratio
Leaf rough
LL/W
LR
3. Slow, 5. Intermediate, 7. Fast
Length from the stem to the lamina base including lobes
of largest leaf. Measured in cm.
Length of largest leaf from the stem to the apex of leaf
blade including petiole. Measured in cm.
Lamina width across the widest portion of the same leaf
used for LL. Measured in cm.
Ratio of leaf blade length to leaf width derived by LL/LW
0. Smooth, 3. Intermediate, 7. Rough
B. Flowering stage
Days to first flowering
Flower Colour
DFF
FC
Number of days from seed sowing to the appearance of
first open flower
W. White; C. Cream; Y. Yellow
C. Maturity stage
Plant height
Plant growth attitude
PH
PGA
Height of main shoot from soil level to the tip of
inflorescence. Measured in m.
E. Erect; I. Intermediate; P. Prostrate.
55
3.2. Morphological analysis of the accessions
The results of morphological quantitative traits are shown in table 4. The morphological
traits were measured following the descriptors for rocket Eruca spp. from IPGRI (International
Plant Genetic Resources Institute, 1999). These quantitative traits were leaf petiole length (LPL),
leaf length (LL), leaf with (LW), and leaf length/width ratio (LL/W).
Analysis of variance revealed significant differences among the accessions for the LPL and
LL, indicting that there was a high degree of variability for these characters. LPL was statistically
significant for the species and subspecies and among the accessions. The subspecies vesicaria
were very different than the rest regarding to the length of petiole. LPL showed values between
1.79 cm (PEX-52) and 8.25 cm (PEX-64) and LL showed values between 8.84 cm (PEX-53) to
23.96 cm (PEX-66). Accessions belonging to vesicaria presented lower LL than the rest. LL was
statistically significant only among the accessions. Concerning to LW, the accessions showed
values ranging from 1.69 cm to 4.05 cm and this character was statistically different for the species
and subspecies and among accessions. The LL/W ratio showed values between 3.51 and 6.89.
Among the accessions with a low LL/W ratio we could found PEX-52 accession, PEX-53 accession
(both belonging to vesicaria) and PEX-17 control accession.
The results of morphological qualitative traits are shown in table 5. These traits selected
were leaf colour (LC), leaf blade shape (LBS), leaf margins lobation (LML), leaf lobation (LLo), leaf
pubescence (LP), leaf apex shape (LAS), leaf blade thickness (LBT), petiole and/or midvein
enlargement (PME), leaf rough (LR) and flower colour (FC).
The content of chlorophyll was measured as an indicator of plant health. LC was
statistically significant for the different accessions (p<0.05), but not among the different species
and subspecies (p>0.05). LC ranged from 31.27 to 48.82 SPAD units, for PEX-55 and PEX-52 (for
sativa and vesicaria respectively), meaning that the leaves in commercial accessions had a less
intense green colour than the rest of accessions.
Morphological traits like the aspect of the leaf were evaluated because of the importance
for the acceptance of the final product for the consumer. LBS was significantly different among
accessions. Eruca stenocarpa (PEX-4) was the only accession with obovate leaves with respect to
this character. Seventeen of the accessions had spathulate leaves, eight had ovate leaves (one of
them belonging to longirostris), while eleven were lanceolate and fifteen leaves were oblong. Only
spathulate, lanceolate or oblong leaves were found in Eruca vesicaria vesicaria, while subsp.
sativa presented a wide variability (except obovate). With respect to LML, accessions belonging to
E. stenocarpa and longirostris had entire leaves. Among the ten accessions from vesicaria
56
species, seven of them had entire leaves, one accession had crenate leaves, and two of the
accessions had doubly dentate leaves. Most of the accessions of sativa had entire leaves and four
of them had crenate leaves. This character was significant different among the four species and
also among accessions. Relating to LLo, analysis of variance revealed significant differences
among accessions for this trait. Stenocarpa accessions had lobes markedly present in leaves, and
longirostris had accentuated lobes. Most of the accessions of vesicaria had markedly present
lobes and two of them had accentuated lobes in leaves. Among accessions of subspecies sativa
two of them had leaves without lobes (PEX-1 and PEX-11), ten of the accessions had accentuated
lobes and the rest of them had lobes markedly present. This character was statistically significant
for the different accessions studied.
Regarding to leaf pubescence (LP), E. stenocarpa had leaves with intermediate
pubescence. Vesicaria was the only species that had rada, intermediate and dense leaves in
relation to the pilosity, while longirostris and sativa had no pilosity in the leaves. This character is
undesirable for commercialization. LP was significant different for all the subspecies and
accessions. Although there are five descriptors for the leaf apex shape (LAS) trait, the accessions
showed only acute or rounded apex shapes. Most of the accessions of vesicaria and sativa
subspecies showed rounded leaves also some acute apex shapes were observed. LAS was only
significant different among accessions. Leaf blade thickness (LBT) is another important trait for
consumption, being preferably to select thin or intermediate leaves. This character resulted
statistically significant among accessions. E. stenocarpa had thin leaves, while logirostris had
intermediate leaves, and vesicaria and sativa had also thin leaves. Most of the leaves had narrow
petiole enlargement (PME), but leaves of two accessions of vesicaria and ten accessions of sativa
had enlarged petiole. Control accessions showed both, thin leaves (LBT) and narrow petioles
(PME) for PEX-17 accession and intermediate petioles for PEX-55 and PEX-56.
With respect to LR, leaves can be smooth, intermediate or rough, being this last category of
trait avoided for consumption. Leaves of E. stenocarpa were smooth, while longirostris had rough
leaves. Vesicaria showed wide variability with respect to this trait and most of accessions of sativa
were smooth and only three of them were rough. Both characters are also important for
consumption. Leaf rough (LR) is a trait with commercial value. It showed significant differences
among subspecies and accessions, presenting vesicaria and sativa in one group, and stenocarpa
and longirostris in other two separate groups. Flower colour was also recorded (FC). Accessions of
subsp. longirostris had yellow flowers, while vesicaria had white or cream flowers. Subsp. sativa
had four accessions with cream flowers, and the rest of them were white or yellow in similar
proportion.
57
4. Discussion
Plant genetic resources can be used to produce alternatives to major crops, helping to
diversify and to amplify the offer. Similarly, the development of genetic breeding programmes in
some species could encourage their cultivation, contributing to the diversification of both
production and supply.
Wild materials often represent untapped resources, and their use can add value, even in
species for which commercial varieties are already available, as it would expand their genetic basis
(Nuez and Hernández-Bermejo, 1994). Such programs should start with a precise characterization
of existing accessions. The agronomical and morphological analyses described in the present
study were made for rocket germplasm management purposes, and so that parent material
showing the most interesting characteristics could be selected for a future breeding program.
The results of the present study (Table 3, 4 and 5) revealed a high phenotypic variability in
the accessions studied of Eruca. Some of the traits studied in this work were significantly different
among accessions (LL, LBS, LLo, LAS and LBT) or among accessions and species/subspecies
(LPL, LW, LML, LP and LR). This is in agreement with previous studies that have also reported a
wide genetic variation for qualitative and quantitative traits in rocket germplasm (Duhoon and
Koppar, 1998; Warwick et al., 2007; Egea-Gilabert et al., 2009; Bozokalfa et al., 2010). EgeaGilabert and collaborators (2009) found similar values for LC, LPL, LW, LML, LLo, LP and LAS as
our results, but we observed a great variation. LL character exhibited a higher range in our study
(ranging from 8.84 to 23.96 cm) compared to the results published by Egea-Gilabert et al. (2009)
(ranging from 6.6 to 7.9 cm). Bozokalfa et al. (2010) reported a wide variation for the traits leaf
width and length, petiole length and thickness, and plant height among others. They found values
of LPL ranging from 3.15 to 6.56 cm in contrast to our results ranging from 1.79 cm to 8.25 cm as
in the trait of LL (ranging from 14.48 to 24.24 cm) compared to our results (ranging from 8.84 cm to
23.96 cm). Bozokalfa et al. (2010) reported LW values (4.25 to 8.51 cm) higher than ours (ranging
from 1.69 cm to 4.05 cm). Egea-Gilabert et al. (2009) observed similar variation in accessions for
the LBS trait, but they reported only obovate, ovate and spathulate shapes.
There are certain characteristics that have a great value in rocket such as high fresh
matter, intense green colour leaves, marked leaf lobation, small leaf and short petiole, late
flowering and absence of leaf pubescence.
The agronomic behaviour of the PEX-53 (vesicaria), PEX-14, PEX-58 and PEX-61 (sativa)
accessions grown under field conditions was good, with a fresh matter similar to the PEX-55
58
commercial accession, although the fresh matter of the other PEX-17 and PEX-56 commercial
accessions was significantly lower than that of the accessions mentioned previously (Table 3).
Concerning to leaf and petiole length, some accessions (PEX-52 and PEX-53) showed also
small leaves as the commercial accession (PEX-17) (Table 4). Furthermore, leaf lobation is also
present in a great number of accessions (Table 5). PEX-7, PEX-9 and PEX-52 accessions
presenting marked leaf lobation. In addition, the intensity of leaf colour, measured as chlorophyll
content (Table 4), was significantly higher in some accessions, PEX-52 accession presenting the
highest colour intensity (Table 5). Regarding to leaf pubescence vesicaria was the only species
that had rada, intermediate and dense leaves, while accessions belonging to longirostris and
sativa had no pilosity in the leaves.
Eruca is a fast-growing crop that flowers under long days and high temperature (Morales et
al., 2006). The accessions evaluated in this work took more days to flower (82-124 days) than to
other authors. Egea-Gilabert et al. (2009) reported flowering dates ranged from 34 to 58 days and
Warwick et al. (2007) in accessions of rocket evaluated in Canada reported dates ranged from 60
to 88 days. Accessions in our study flowered later also than the “Adagio” cultivar obtained by
Morales et al. (2006). Moreover, they measured the flowering date as the day that 50% of the
plants of that cultivar flowered compared to our measure as the day that flower the first plant of
each accession. However, it is important to bear in mind that the time of flowering also depends on
sowing time, and the reduced space that the plants have to grow could bring the flowering time
forward (Egea-Gilabert et al., 2009).
All these parameters could make some accessions good candidates to act as parent
material in a future breeding programme, favouring rocket germplasm conservation and
management.
5. Conclusions
This work showed genetic variability for the 52 rocket accessions studied considering the
morphological and agronomical traits. The study showed good qualities like high growth rate and
plant height, late flowering, erect growth attitude, absence of pubescence, thin leaves and smooth
leaves. This information is crucial to favour the germplasm conservation and management and the
best way of undertaking a breeding programme. In this way, it might be possible to select the
accessions that show the most interesting characteristics for marketing a quality product like the
accessions of Eruca vesicaria subsp. sativa PEX-14, PEX-58, PEX-61 and accessions of Eruca
vesicaria subsp. vesicaria PEX-7, PEX-9, PEX-52 and PEX-53.
59
Acknowledgements
This research was supported by the Consejería de Innovación, Ciencia y Empresa (Junta
de Andalucía), Project P06-AGR-02230, for which the authors are deeply indebted. We give
special thanks to the to the United States Department of Agriculture (USDA), to Dr. César GómezCampos and to the germplasm banks for providing seeds used in this work. Myriam VillatoroPulido was supported by Instituto de Investigación y Tecnología Agraria y Alimentaria (INIA)
contract.
60
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63
Table 3. Results of agronomic traits recorded in the accessions of rocket.
Accession
FM
LC
PH
GR
DFF
PEX-4
PEX-89
PEX-6
PEX-7
PEX-9
PEX-10
PEX-48
PEX-51
PEX-52
PEX-53
PEX-92
136.59±32.28de
134.93±1.54de
234.94±36.98fg
127.31±8.61cd
112.27±10.01cd
216.15±1.02fg
96.42±6.19bc
145.35±0.63df
56.31±4.13a
282.75±1.55gh
125.61±8.61cd
39.92±4.99bh
35.46±3.52fk
38.32±3.91cj
34.79±5.72gk
37.97±8.45cj
35.06±3.10gk
39.27±4.93bi
44.55±5.99ab
48.82±4.78a
36.98±4.11bf
34.97±5.24gk
1.03±0.15bc
1.05±0.20bc
1.01±0.10bc
0.33±0.03a
0.43±0.23a
1.17±0.07cd
1.22±0.08cd
1.20±0.00cd
0.81±0.10ab
0.64±0.38ab
0.90±0.11bc
5 (0.0%)b
5 (0.0%)b
5 (0.00%)b
5 (0.0%)b
5 (0.0%)b
3 (0.0%)a
5 (0.0%)b
5 (0.0%)b
5 (0.0%)b
5 (0.0%)b
5 (0.0%)b
100±0.66b
82±1.28a
99±0.94b
124±0.75c
PEX-93
PEX-1
PEX-11
PEX-14
PEX-15
PEX-17
PEX-55
PEX-56
PEX-58
PEX-59
PEX-60
PEX-61
PEX-62
122.14±2.08cd
127.14±2.08cd
145.41±1.10df
240.57±7.17fg
102.34±1.5bc1
92.23±6.39bc
287.93±1.55gh
93.72±14.72bc
384.36±5.47h
234.28±33.48fg
142.97±11.04df
239.56±6.92fh
189.20±44.90ef
36.56±6.01ek
35.23± 4.08gk
38.70±3.08cj
40.03±3.66bh
43.51±5.74bc
37.20±5.23dj
31.27±3.45ik
36.05±3.94ek
35.43±3.81fk
35.82±5.63fk
38.47±4.79cj
36.88±3.25dk
34.99±4.51gk
nd
1.05±0.14bc
1.20±0.00cd
1.10±0.00cd
1.20±0.00cd
0.93±0.08bc
1.28±0.17de
1.33±0.06de
1.10±0.00cd
1.16±0.14cd
1.03±0.06bc
1.16±0.15de
1.40±0.00de
5 (0.0%)b
5 (10.3%)b
5 (0.0%)b
7 (0.0%)c
7 (0.0%)c
3 (0.0%)a
5 (0.0%)b
5 (0.0%)b
7 (0.0%)c
3 (0.0%)a
3 (0.0%)a
3 (0.0%)a
7 (0.0%)c
124±0.60c
99±0.58b
PEX-63
PEX-64
PEX-65
145.82±8.27df
122.13±3.46de
112.49±12.71cd
34.93±5.79gk
35.02±9.05gk
37.53±3.26dj
1.55±0.28e
1.15±0.21cd
1.33±0.06de
7 (0.0%)c
7 (0.0%)c
5 (0.0%)b
PEX-66
117.31±5.74cd
33.72±4.83ik
nd
PEX-67
140.25±9.12df
37.48±5.53dj
40.61±3.47bg
34.93±2.93cj
41.76±3.71be
33.8±6.66ik
38.91±8.15cj
34.6±4.19gk
36.9±4.90dk
35.18±5.85gk
37.31±3.34dj
33.22±4.51ik
PEX-68
89.33±14.53ab
PEX-69
76.50±15.35a
PEX-70
151.45±2.47df
PEX-71
75.58±0.94a
PEX-72
126.14±22.46de
PEX-73
106.01±13.25cd
PEX-74
150.14±28.32df
PEX-75
72.11±27.81a
PEX-76
174.94±34.60ef
PEX-77
172.50±26.73ef
Table 3. Continued.
122±0.66c
99±1.28b
104±0.89b
101±0.55b
124±0.86c
109±0.62b
97±0.54b
99±1.35b
101±0.87b
106±0.57b
106±0.27b
100±0.47b
99±0.52b
101±0.43b
104±0.43b
104±0.57b
104±0.66b
101±1.28b
DFF PGA
I
100
82
99
124
122
99
104
101
124
109
97
I
P
E
E
124
99
99
101
106
106
100
99
101
104
104
104
101
I
P
E
E
E
P
P
P
P
E
P
I
P
E
E
E
P
I
105±0.39b
99
99
105
P
I
3 (0.0%)a
99±0.52b
99
E
nd
3 (0.0%)a
104±0.43b
104
I
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
5 (0.0%)b
5 (0.0%)b
7 (0.0%)c
7 (0.0%)c
3 (0.0%)a
5 (0.0%)b
5 (0.0%)b
7 (0.0%)c
7 (0.0%)c
3 (0.0%)a
101±0.43b
100±0.57b
101
100
99
82
101
82
99
82
96
82
P
99±0.89b
99±0.55b
99±0.66b
82±1.28a
101±0.89b
82±0.91a
99±0.89b
82±0.91a
96±0.52b
82±0.43a
Accession
FM
LC
PH
GR
DFF
PGA
PEX-78
PEX-79
PEX-80
200.68±41.25fg
148.24±0.32df
81.67±0.27ab
42.52±6.06bd
38.41±2.71cj
38.27±4.38cj
nd
nd
nd
5 (0.0%)b
7 (0.0%)c
7 (0.0%)c
103±0.43b
92±0.57b
82±0.66a
I
64
P
E
E
I
E
P
P
E
P
E
P
I
P
PEX-81
PEX-82
PEX-83
PEX-85
PEX-86
PEX-87
PEX-88
PEX-90
PEX-91
PEX-113
124.08±38.65cd
93.14±4.21bc
159.91±18.00ef
138.77±4.26df
236.11±86.49fg
185.92±0.27ef
140.85±23.97df
128.71±11.97de
125.61±8.61cd
63.92±15.30a
37.34±5.21dj
36.62±5.78ek
36.21±6.97ek
37.49±6.83dj
36.30±5.04ek
36.86±4.92dk
33.19±3.39ik
33.60±6.46ik
34.35±4.82hk
34.41±4.83hk
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
5 (0.0%)b
5 (0.0%)b
7 (0.0%)c
7 (0.0%)c
7 (0.0%)c
5 (0.0%)b
7 (0.0%)c
5 (0.0%)b
5 (0.0%)b
5 (0.0%)b
82±1.28a
82±0.89a
82±0.91a
82±0.66a
82±1.28a
96±0.89b
82±0.43a
95±0.57b
103±0.66b
96±1.28b
P
I
P
I
P
I
I
P
I
E
A: accession; FM: fresh matter (expressed as g/m of row); LC: Leaf color (expressed as SPAD
units); PH: Plant height (expressed as means and standard deviation); GR: Growth rate,
expressed as median and robust coefficient of variation (cvr); DFF: Days to first flowering; nd: not
detected.
Table 4. Results of morphologic quantitative traits (expressed as means and standard deviation)
recorded in the accessions of rocket.
A
LPL
LL
LW
LL/W
PEX-4
PEX-89
PEX-6
PEX-7
PEX-9
PEX-10
PEX-48
PEX-51
PEX-52
PEX-53
PEX-92
PEX-93
PEX-1
PEX-11
PEX-14
PEX-15
PEX-17
PEX-55
PEX-56
5.38±1.17a
5.34±2.24a
5.10±1.04b
2.88±1.03b
2.42±1.37b
5.50±1.16b
4.39±1.80b
4.57±1.23b
1.79±0.52b
2.19±0.82b
6.95±1.01b
6.30±0.71b
6.19±0.96a
3.91±1.28a
5.85±0.82a
7.11±1.54a
3.23±0.83a
6.67±1.41a
3.87±0.88a
14.05±1.67ef
13.30±2.24ef
15.58±5.67de
10.55±1.32fg
10.19±1.60fg
14.89±1.67de
13.21±2.54eg
12.73±1.50eg
10.33±1.84fg
8.84±3.34fg
14.94±1.45de
15.43±1.64de
13.14±1.44eg
11.89±1.08fg
14.01±1.57ef
17.36±3.12b
9.26±2.37bg
14.44±2.01de
11.00±1.04fg
2.63±0.52b
3.37±1.05a
2.93±0.48b
2.01±0.34b
1.69±0.28b
2.69±0.61b
2.70±0.92b
2.81±0.59b
2.98±0.62b
2.30±0.95b
3.20±0.39b
3.59±0.74b
2.57±0.34a
2.40±0.41a
2.74±0.52a
4.05±1.04a
2.68±0.63a
2.80±0.93a
2.77±0.25a
5.47±1.04cd
4.17±0.96ab
5.37±1.87cd
5.32±0.64cd
6.13±1.16d
5.75±1.29cd
5.21±1.39cd
4.72±1.21ab
3.51±0.49a
3.60±1.44a
4.69±0.35ab
4.42±0.77ab
5.15±0.53cd
5.09±1.01cd
5.29±1.15cd
4.39±0.81ab
3.59±1.03a
3.72±0.64a
3.98±0.36ab
A
LPL
LL
LW
LL/W
PEX-58
PEX-59
PEX-60
PEX-61
PEX-62
PEX-63
PEX-64
8.01±1.39a
4.80±1.47a
4.26±1.11a
5.01±0.94a
6.65±1.32a
7.71±0.59a
8.25±0.42a
16.61±1.04bc
12.65±1.92fg
11.21±2.06fg
12.92±1.25eg
16.72±1.96b
15.69±1.01de
17.83±1.21b
3.50±0.40a
3.02±0.45a
2.79±0.61a
2.85±0.41a
3.46±0.55a
3.16±0.39a
3.86±0.52a
4.79±0.53ab
4.24±0.78ab
4.08±0.65ab
4.59±0.54ab
4.91±0.76bc
5.01±0.53bc
4.68±0.67ab
Table 4. Continued.
65
PEX-65
PEX-66
PEX-67
PEX-68
PEX-69
PEX-70
PEX-71
PEX-72
PEX-73
PEX-74
PEX-75
PEX-76
PEX-77
PEX-78
PEX-79
PEX-80
PEX-81
PEX-82
PEX-83
PEX-85
PEX-86
PEX-87
PEX-88
PEX-90
PEX-91
PEX-113
6.05±1.41a
4.71±1.55a
4.61±1.19a
5.73±1.47a
5.48±2.58a
6.10±2.10a
5.25±1.32a
4.96±0.96a
4.65±1.32a
7.55±1.07a
6.56±1.24a
5.23±0.82a
6.83±1.93a
4.93±1.27a
5.44±1.55a
4.27±0.44a
4.85±1.21a
4.74±1.44a
4.90±1.41a
5.01±0.97a
6.20±1.32a
5.49±1.58a
5.28±0.64a
5.64±0.59a
6.07±0.87a
6.14±2.29a
15.82±2.04bd
23.96±3.55a
13.13±2.46eg
14.52±2.62de
12.74±4.83eg
16.70±2.83bc
13.52±3.10ef
11.95±1.75fg
12.53±2.89fg
16.70±1.92bc
15.03±3.16de
15.02±2.32de
16.98±2.22b
13.53±1.68ef
14.66±1.67de
11.18±1.17fg
15.52±7.09de
11.76±1.29fg
12.70±1.40fg
14.71±2.42de
14.81±2.15de
14.34±2.45de
12.94±2.08eg
13.50±2.94ef
14.37±1.63de
15.24±2.67de
3.17±0.43a
3.17±0.43a
3.02±0.57a
3.28±0.66a
2.74±1.28a
3.93±0.89a
3.08±0.91a
2.80±0.65a
3.03±0.67a
3.96±1.32a
3.28±0.58a
3.40±0.41a
3.50±0.88a
2.87±0.45a
3.30±0.86a
2.77±0.62a
3.27±0.54a
2.77±0.60a
3.18±0.58a
4.04±1.35a
3.28±0.57a
3.25±0.57a
2.65±0.27a
3.11±0.47a
3.25±0.43a
4.05±0.76a
5.05±0.82bc
6.89±0.86d
4.41±0.79ab
4.47±0.55ab
4.45±1.84ab
4.34±0.76ab
4.50±0.67ab
4.37±0.69ab
4.23±0.94ab
4.50±1.05ab
4.61±0.70ab
4.72±0.55ab
5.05±0.97bc
4.80±0.92bc
4.60±0.77ab
4.15±0.66ab
4.78±2.04ab
4.37±0.77ab
4.07±0.57ab
3.84±0.79ab
4.59±0.75ab
4.48±0.83ab
4.89±0.65bc
4.45±1.18ab
4.46±0.65ab
3.86±0.86ab
A: accession; LPL: Leaf petiole length; LL: Leaf length; LW: Leaf width; LL/W: Leaf length/width
ratio. Duncan's Multiple Range Test. Means with the same letter are not significantly different. LPL
and LW show the Duncan’s Multiple Range Test among groups and LL show the letters among
accession.
66
67
68
69
CAPÍTULO II
Características agromorfológicas, composición química y análisis
sensorial en hojas de rúcola (Eruca vesicaria subsp.sativa y Eruca
vesicaria subsp. vesicaria) y Erucastrum de una colección mundial
Artículo en preparación
Agro-morphological characteristics, chemical composition and sensory analysis in rocket
(Eruca vesicaria subsp. sativa and Eruca vesicaria subsp. vesicaria) and Erucastrum leaves
from a world collection.
Myriam Villatoro-Pulidoa, Rafael Fontb, Pilar Ruíz Pérez-Cachoc, Sara Obregón-Canod, Antonio De
Haro-Bailónd, , Mercedes Del Río-Celestinob.
a
IFAPA-Centro Alameda del Obispo, Córdoba, Spain.
b
IFAPA-Centro la Mojonera, Almería, Spain.
c
Departamento de Bromatología y Tecnología de Alimentos, Universidad de Córdoba, Córdoba,
Spain.
d
Instituto de Agricultura Sostenible, Córdoba, Spain.
Abstract
Background: Rocket and other minor species of Crucifers as Erucastrum are rich sources of
bioactive compounds with chemoprotective properties. In this work we present preliminary data
about the natural existing variability of Eruca and Erucastrum accessions. The objectives of our
research were a) to determine the variability among accessions of rocket (Eruca vesicaria subsp.
sativa and Eruca vesicaria subsp. vesicaria) and Erucastrum assessing their agro-morphological
characters, glucosinolate content and sensory attributes in leaves to investigate their potential in
the agro-food industry; b) to develop a specific lexicon for sensory analysis of rocket and
Erucastrum. Ten randomised accessions selected from a worldwide collection were studied under
field conditions for 17 different agro-morphological characters, in Cordoba, Spain during 2008 and
2009.
Results: The accessions displayed a great variability in the agro-morphological traits, sensory
attributes and glucosinolate content. The sensory panel generated 27 simple descriptors classified
70
in three different groups (for appearance, flavour and texture) to characterize qualitatively the
rocket accessions. Accessions displayed a great variability in the agro-morphological traits,
sensory attributes and glucosinolate content.
Conclusions: This variability provides an interesting and valuable material for further breeding
programs in order to generate lines that are key for a commercial objective.
Keywords: Eruca, Erucastrum, glucosinolates, sensory analysis, morphological analysis.
1. Introduction
It is estimated that of the 7000 edible species of vegetables around the world only a small
fraction, amounting more or less 150 species, are in fact being commercialized1. Among the plant
species with a scarce representation in the food market are those belonging to the genera Eruca
and Erucastrum.
A greater attention to rocket (Eruca Miller and Diplotaxis DC. genera) and to other neglected
species as those belonging to Erucastrum genus could represent an important step towards both
agricultural and diet diversification which ultimately contribute to improving our quality of life.
The Eruca species were widely consumed and mentioned in scripts about culinary habits in
the ancient Rome because of the peculiar taste of its leaves. The taxonomic position and ranking
of the taxa named Eruca vesicaria (L.) Cav. and E. sativa Miller are controversial. Treated as two
separate species by Greuter et al.2, E. vesicaria and E. sativa have been more recently united as
subspecies under the older, accepted name of E. vesicaria3-5. For the remainder of this study,
these two taxa will be referred to as E. vesicaria subsp. sativa and subsp. vesicaria.
Eruca has a wide distribution as a weed in cornfields, flax fields and on waste ground, along
roadsides and under sun-exposed and dry environments mainly in the Mediterranean and Asia.
Eruca is grown as a vegetable and as a cold weather oilseed crop to produce oil, called “jamba oil”
71
in India6. Leaves are eaten raw in salads or cooked in various culinary preparations, and are grown
or gathered from wild plants in Egypt (where it is very popular and the production can be
considered the largest in the world), Northern African countries, Italy, Turkey, Greece, Spain,
Sudan, Ethiopia, Somalia, Jordan, Israel, Slovenia, Japan, Brazil, Argentina, the USA, Australia,
the Caucasis area, and in several countries of Northern Europe. Variation of taste and pungency is
wide, depending on the species, its genetic diversity and the environment. Among the different
species of rocket existing in Europe, subspecies of Eruca are regarded as a delicacy. But rocket is
also considered a medicinal plant, with strong aphrodisiac effect, depurative properties, vitamin C
and mineral contents. More recently, rocket has been shown as a source of glucoraphanin (4methylsulfinylbutyl glucosinolate). This glucosinolate is present in seeds and leaves ant it is one of
the responsible molecules for many of the antioxidant and anticarcinogenic properties exhibited by
this vegetable7. Actually, vegetables of the Cruciferae family have an important role in human
nutrition due to their content in glucosinolates, isothiocyanates, carotenoids, phenolic compounds,
and minerals8,9.
Erucastrum nasturtiifolium (Poiret) O. Schulz is an annual or biannual herbaceous plant
distributed in the NW of the Mediterranean and sub-Mediterranean regions, which shows some
variability in its life-history traits. This species colonizes several contrasting habitats that differ in
disturbance type and resource availability10. Erucastrum has been used for hybridization in crop
breeding as other Cruciferous species11,12.
Estimates of genetic diversity and relationships between germplasm collections are very
useful for facilitating efficient germplasm collection and management. Many tools are now available
for studying variability and the relationships among accessions. However, morphological, sensory
and agronomic characterization is the first step in the description and classification of the
germplasm13. In this work we present preliminary data about sensory, morphological and
agronomic traits of Eruca and Erucastrum to investigate their potential in the agro-food industry.
Until the date little work has been performed to characterize rocket. D’Antouno and collaborators 14,
tried to correlate the glucosinolate content and sensory attributes of some species of Diplotaxis
and Eruca vesicaria. They found that low glucosinolate content is related to higher acceptance of
intake. The objectives of our research were a) to determine the variability among accessions of
rocket (Eruca vesicaria subsp. sativa and Eruca vesicaria subsp. vesicaria) and Erucastrum from a
worldwide collection assessing their agro-morphological characters, glucosinolate content and
sensory attributes; b) to develop a specific lexicon for sensory analysis of rocket and Erucastrum.
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2. Material and methods
2.1. Plant material and greenhouse experiments
Nine accessions of Eruca and one accession of Erucastrum were acquired from different
European genebanks collections and commercial seed companies (Table 1).
Seeds were
germinated in Petri dishes for 48 h under a minimum temperature of 25ºC. Pots were placed in
greenhouse under natural light, temperature of 27/18 ºC (day/night), and a relative humidity of
50/70% (day/night). When plants reached adequate height (8-12 cm), they were transferred to soil.
These accessions were grown in Cordoba, Spain (37º 53´ N; 4º 47´ W) during 2008 and 2009
under the semiarid conditions of Andalusia. A randomized complete block design with two
replications was used. Plants were collected for chemical and sensory analyses.
Table 1. List of rocket (Eruca vesicaria subsp. sativa and Eruca vesicaria subsp. vesicaria) and
Erucastrum accessions used in this work.
Code
Gender
Subspecies
Institution or Company
Botanischer Garten der
Universitat, Karlsruhe
(Germany)
PEX-1
Eruca
sativa
PEX-6
Eruca
vesicaria
INIA Madrid (Spain)
PEX-8
PEX-10
Erucastrum
Eruca
nasturtiifolium
vesicaria
PEX-11
Eruca
sativa
PEX-14
Eruca
sativa
PEX-15
Eruca
sativa
PEX-17
Eruca
sativa
PEX-48
PEX-56
Eruca
Eruca
vesicaria
sativa
INIA Madrid (Spain)
INIA Madrid (Spain)
Dipartimento di Scienze
Botaniche, Palermo (Italy)
Faculté des Sciences
Agronomiques, Gembloux
(Belgium)
Jardin Botanique, Ville de
Limoges (France)
Tozer Seeds Ltd., Cobham,
Surrey (United Kingdom),
(Variety Sky)
INIA Madrid (Spain)
Rocalba, Gerona (Spain)
Origin
Germany
Iran (Persepolis
Ruins)
Spain (Zaragoza)
Spain (Zaragoza)
Italy (Palermo)
Begium
France
United Kingdom
Spain (Navarra)
Spain
2.2. Agronomical and morphological analysis of the accessions
All accessions were characterized for different agronomical and morphological traits from
seedling up to the harvest of the crop during 2009 (Table 2). Traits selection and measurement
techniques were based on Descriptors for Rocket15. Group A traits were studied at the 5 leaf stage
using 20 plants per accession randomly selected. Group B traits were recorded on 10 plants per
accession in each plot/replication, while group C characters were recorded at maturity. For
characters such as fresh matter (FM, average fresh weight of plant measured as g m -1) and
number of leaves/plant (NL, average of number of leaves per plant) 10 plants per plot per
73
replication were used in plants at the optimum time for consumption. The character of leaf colour
(LF) was measured with a Konica Minolta SPAD-502 chlorophyll meter. This apparatus performs
field measurements of the relative chlorophyll content without damaging the leaf (expressed as
SPAD units).
Table 2. Morphological traits recorded in the rocket accessions during 2009.
Trait designation
Code
Description and categories of the trait
A. Seedling stage
Fresh matter
Number of leaves
Leaf colour
FM
NL
LC
Leaf blade shape
LBS
Leaf margins lobation
LML
Leaf lobation
Leaf pubescence
Leaf apex shape
Leaf blade thickness
Petiole and/or
midvein enlargement
Growth rate
LLo
LP
LAS
LBT
Average of fresh weight of plant measured in g m-1
Average of number of leaves per plant
Measured with Konica Minolta Spad-502 chlorophyll meter
1. Orbicular, 2. Elliptic, 3. Obovate, 4. Spathulate, 5. Ovate,
6. Lanceolate, 7. Oblong.
1.Entire, 2. Crenate, 3. Dentate, 4. Serrate, 5. Doubly
dentate, 6. Undulate
0. Absent, 1. Accentuated, 2. Markedly present
3. Rada, 5. Intermediate, 7. Dense
1. Largely acute, 2. Acute, 3. Rounded, 4. Broadly rounded
3. Thin, 5. Intermediate, 7. Thick
PME
1. Narrow, 2. Enlarged
GR
Leaf petiole length
LPL
Leaf length
LL-s
Leaf width
LW-s
Leaf length/width ratio
LL/W
3. Slow, 5. Intermediate, 7. Fast
Length from the stem to the lamina base including lobes of
largest leaf. Measured in cm.
Length of largest leaf from the stem to the apex of leaf blade
including petiole. Measured in cm.
Lamina width across the widest portion of the same leaf
used for LL. Measured in cm.
Ratio of leaf blade length to leaf width derived by LL/LW
B. Flowering stage
Days to first flowering
DFF
Number of days from seed sowing to the appearance of first
open flower
PH
Height of main shoot from soil level to the tip of inflorescence.
Measured in m.
C. Maturity stage
Plant height
2.3. Statistical analysis
Analyses of variance were performed for each trait and comparison of means among
accessions was made concerning each trait using Fisher´s protected least significant difference
(LSD) at P≤0.05. Quantitative traits were expressed as means and standard deviation, while
qualitative traits were expressed as median and robust coefficient of variation (cvr).
2.4. Sample pre-treatment and storing
Leaves from 10 plants per accession were collected once they were ready for human
74
consumption and washed with tap water. The accessions used for sensory analysis were directly
evaluated. The accessions assigned for glucosinolate analysis were weighed to assess their
biomass, stored at -20 ºC, and freeze-dried until analysis with the different methodologies as
indicated below.
2.5. Glucosinolates analysis by liquid chromatography with ultraviolet photometric detection
Glucosinolate analyses were performed in 2008 and 2009. Freeze-dried rocket leaves
(100mg) were grounded using the Janke and Kunkel mill (model A10, IKA-Labortechnik) for 20
sec, and a two-step glucosinolate extraction was carried out using a water bath at 75 °C to
inactivate myrosinase. The obtained flour was heated for 15 min in 2.5 mL of 70% aqueous
methanol and 200 µL of 10 mM sinigrin as an external standard (Sinigrin hydrate, 85440 Fluka) in
the first step. A second extraction was done after centrifugation (5 min, 5 x 103 g) using 2 mL of
70% aqueous methanol. The combined glucosinolate extracts (1 ml) were pipetted onto the top of
an ion-exchange column containing Sephadex DEAE-A25 (1 ml, 40-125 µm bead size, 30000 Da
exclusion limit). Desulfation was performed by addition of purified sulfatase (75 µl, EC 3.1.6.1, type
H-1 from Helix pomatia) (Sigma-Aldrich) solution. Desulfated glucosinolates were eluted with 2.5
mL (0.5 mL x 5) of Milli-Q (Millipore) ultrapure water and analysed with a 600 HPLC instrument
(Waters) equipped with a 486 UV tunable absorbance detector (Waters) set at a wavelength of 229
nm. Separation was carried out using a Lichrospher 100 RP-18 in Lichrocart column (125 mm x 4
mm i.d., 5 µm particle size, Merck). The HPLC chromatogram was compared to the desulphoglucosinolate profile provided by three certified reference standards recommended by the E.U. and
the ISO16. The content of glucosinolates was quantified using glucotropaolin as external standard,
and expressed as micromoles per gram of dry weight, after considering the relative response
factors of the individual glucosinolates, according to the ISO norm 17. The total glucosinolate
content was computed as the sum of all of the individual glucosinolates present in the sample.
2.6. Sensory analysis
2.6.1. Rocket samples used in the generation of vocabulary
Ten accessions of rocket leaves were evaluated, eight of them from genebanks and two
commercial companies of rocket from two different harvests in order to extend the generation of
vocabulary. Accessions PEX-1, PEX-6, PEX-8, PEX-10, PEX-11, PEX-14, PEX-15 and PEX-48
were analysed in 2008 and PEX-6, PEX-8, PEX-11, PEX-14, PEX-17 and PEX-56 in 2009.
Two leaves of each sample were placed in a Petri dish immediately before tasting and were
served at room temperature. Leaves from the different accessions of Eruca were collected from
two-three plants (experimental unit) and the commercial brands were taken directly from the
packet (experimental unit).
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2.6.2. Assessors
Ten trained panellists (7 female, 3 male) from the University of Córdoba (Córdoba, Spain),
aged (25-50 years), evaluated the accessions of rocket. These panellists had prior experience in
the sensory evaluation of many products18-20. Testing was carried out in the sensory laboratory
located at the University of Córdoba (Córdoba, Spain), equipped with a round table for training
sessions and individual booths according to the international standards 18. All analyses were
conducted in the morning (10.00-12.00 h).
2.6.3. Descriptive analysis
The Unguided Free Selection technique19,22,23 was used to develop a preliminary sensory
language (appearance, flavour and texture attributes) in accordance with the international
standards24. The lexicon was developed over 4 sessions of 1 to 1.5 hours in tasting booths during
2008 and 2009 (6 h) and 3 opening sessions of 1.5 hour each in 2009 (4.5 h). The assessors
generated individually the sensory terms individually in the tasting booths during 2008 and 2009.
Four to five samples, labelled with 3-digited random numbers were served, 1 at a time, over a
session. The tasting procedure was established as follow: (1) to take one leave, break it and rub it
with the hands and then sniff intensely (orthonasal odor); (2) to assess the aroma (retronasal
odour), taste and trigeminal attributes; (3) to take the second leave and assess the appearance
and (4) to bite the second leave and assess the texture attributes. Panellists were asked to
describe the samples before, during and after tasting23.
3. Results and discussion
3.1. Agronomical and morphological analysis of the accessions
The results of quantitative traits analyses are shown in Table 3. There are certain
characteristics that have a great interest depending on: (a) consumer taste, such as intense green
colour leaves, marked leaf lobation; (b) retail in baby leaf form, which means small leaf and short
petiole; (c) cultivation practices that concern the growers, such as days to flowering to identify
sources for late flowering, yield, number of leaf in the rosette at harvest time, etc. The agronomical
traits analysed were fresh matter (FM), leaf colour (LC), plant height (PH), growth rate (GR), and
days to first flowering (DFF); the rest of analysed characters were morphological traits. Significant
differences were observed in terms of biomass production (FM), PEX-6, PEX-10 and PEX-14
accessions, showed the highest biomass, with values ranging from 216.2 to 240.6 g of fresh
weight of plant m
-1
of row. The fresh matter was significantly lower in commercial accessions
(PEX-17 and PEX-56). There was variation in the number of leaves (NL) ranging form 5.6 to 11
leaves. This character in our work was higher than other accessions evaluated in Spain by EgeaGilabert and collaborators25 (ranging from 6.6 to 8.3 leaves).
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LC measures the chlorophyll in leaf that is an indicator of plant health. It ranged from 35.2 to
43.9 SPAD units. The intensity of leaf colour (Table 3) was significantly higher in Erucastrum
(PEX-8) and subsp. vesicaria (PEX-48), which presented the highest colour intensity. Significant
differences were found for leaves length (LL) and petioles (LPL). Leaf length ranged from 9.3 cm to
17.4 cm, and petiole lengths ranged from 3.2 cm to 7.1 cm. The petiole length was significantly
shorter in commercial accessions (PEX-17 and PEX-56) and in PEX-8, PEX-11 and PEX-48
accessions. The leaf width (LW) and the ratio leaf length/ leaf width (LL/LW) were both variable.
The days to first flowering (DFF) ranged from 99 to 106 days. As the plants are harvested before
shooting and flowering, DFF is recommended to be as high as possible. All the accessions
evaluated in this study took more time to flower (99–106 days) than other
accessions evaluated in Spain by Egea-Gilabert and collaborators25 (34-58 days) and in Canada
by Warwick and coleagues26 but agree to those studied in Israel27 (60–88 days). In our trial the
commercial accession (PEX-17) took 106 days to flower, showed no significant differences
compared with the other accessions, which is adequate to start a breeding programme. Attending
to Erucastrum nasturtiifolium (PEX-8 accession), our results contrasts with those obtained by
Chamorro and Sans10 who found a late flowering in wild populations of this species in the northern
of Spain (133 days).
The results of qualitative traits are shown in Table 4. Morphological traits, such as leaf blade
shape (LBS), leaf lobation (LLo), leaf pubescence (LP) and leaf blade thickness (LBT) were
evaluated because of their importance from the consumers’ point of view in terms of acceptance of
the final product. The results of LBS were as follow; six of the accessions had spathulate leaves
(PEX-1, PEX-10, PEX-11, PEX-14, PEX-15 and PEX-56), while the other accessions were
lanceolate (PEX-6, PEX-8 and PEX-48) or oblong (PEX-17). Egea-Gilabert and collatorators25
observed similar variation in accessions for this trait, but they reported obovate, ovate and
spathulate shapes. Regarding LML, most of the leaves were entire, and four of the accessions
showed crenate leaves (PEX-1, PEX-8, PEX-10, and PEX-11). Leaf lobation was present in
accessions PEX-8, PEX-10, PEX-14 and PEX-56; and it was markedly present in accessions PEX6, PEX-15, PEX-17 and PEX-48. This character was only absent in two of the accessions (PEX-1
and PEX-11). All accessions showed absence of leaf pubescence (LP), except the commercial
accession PEX-17.
There was variation for the character leaf apex shape (LAS) (acute and rounded). All the
accessions included in this study showed narrow petioles (PME) except the commercial accession
PEX-56 that presented enlarged petioles. Pubescence and blade thickness of the leaves are
characters of commercial importance for consumption. Concerning to leaf blade thickness (LBT),
the commercial accessions PEX-17 showed thin leaves as PEX-1, PEX-10, PEX-11, and PEX-14.
However, the commercial accession PEX-56 presented intermediate leaves.
78
The character of GR represents the ability of an accession to compete with weeds. PEX-14
and PEX-15 were the most vigorous accessions. The Duncan test showed statistically significant
differences (p<0.05) among accessions for the traits compared, but not between both subspecies.
Most of the accessions with highest biomass are those belonging to subsp. vesicaria (PEX-6, PEX10), and to genus Erucastrum (PEX-8). Both are not used for human consumption due to the low
domestication of the subspecies vesicaria and Erucastrum nasturtiifolium; nevertheless, these
accessions could be interesting from an applied breeding point of view. The accession belonging
to subsp. sativa with highest biomass is PEX-14. This accession had late flowering (101 days), it
had absence of pilosity or PME, and it had fast growth rate.
3.2. Glucosinolate composition of the accessions
Table 5 shows the concentrations of the individual and total glucosinolates of the accessions
evaluated during 2008 and 2009. Among the aliphatic glucosinolates (GLS), glucoerucin (GER)
and glucoraphanin (GRA) were the most abundant, followed by glucoiberverin (GIV), gluconapin
(GNA), progoitrin (PRO), gluconapoleiferin (GNL) and glucobrassicanapin (GBN). Gluconasturtiin
was the only aromatic GLS detected. Other GLS present were the indole GLS, where glucosativin
(4-Mercapto) was the predominant indole followed by 4-hidroxyglucobrassicin (4-OH GBS), 4metoxyglucobrassicin (4-OM GBS) and neoglucobrassicin (NGBS) (Table 5).
The influence of sowing year on GLS concentration and profile in accessions was high. Total
glucosinolate concentration in leaves harvested in 2008 was higher than the concentration in 2009,
which is in agreement with findings by other researchers teams in cruciferous28-31. Generally, the
highest concentrations of total GLS occurred when crops were harvested during periods of high
temperatures and long day length29. In our study, the lower temperatures were registered during
2009. With respect to the contents of individual GLS, glucosativin and glucoraphanin were affected
by environmental conditions, so higher content were detected in 2008 than in 2009, except in the
case of glucoerucin, which increased.
The accessions showed a total content of glucosinolates ranging from 14.0 to 39.95 µ moles
of glucosinolates g-1 of dry weight (dw). The accessions of E. vesicaria subsp. vesicaria (PEX-6
and PEX- 10), and the genus Erucastrum (PEX-8) were the accessions with the highest total
content of glucosinolates (31.80, 39.95 and 37.69 µ moles of glucosinolates g -1 dw respectively) in
2008. These accessions could be adequate candidates for future breeding programs. The PEX-17
and PEX-56 commercial accessions showed lower mean values of total GLS (14 and 20 µmoles of
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80
81
glucosinolates g-1
dw respectively) and specially, glucoraphanin
(GRA<5 µ moles of
glucosinolates g-1 dw) in 2008.
The PEX-8, PEX-10 and PEX-11 accessions showed the highest content of GRA (>15 µ
moles of GRA g-1 dw) during 2008, which have been widely reported to possess cancer preventive
activity7. The total GLS and GRA contents of rocket leaves in the current study were higher than
those found in other studies7,32.
Scarce studies have been carried out on seeds of Erucastrum species. In a preliminary
study, Daxenbichler et al.33 reported the presence of glucoraphenin (4-methylsulphinyl-3-butenyl)
and gluconapin (3-butenyl) in seeds of Erucastrum nasturtiifolium and Erucastrum laevigatum,
respectively. Furthermore, Agerbirk and collaborators34 have reported that predominant leaf
glucosinolate content from Erucastrum canariense was sinigrin, in contrast to the results of the
present study.
3.3. Sensory analysis of the accessions
The preliminary lexicon for the rocket leaves is presented in Table 6 (appearance and texture
attributes) and Table 7 (flavour: odour/aroma, basic taste and trigeminal attributes).
The analytical panel generated 27 simple descriptors classified in three different groups: 7 for
appearance (hue, intensity of colour, leaf size, leaf shape, leaf margins, leaf petiole length and
visual texture) 6 for texture (tender, crunchy, moist, fibrous, palate coating and prickly in hands)
and 14 for flavour (7 for odour/aroma: green/grass/clover, radish, tomatoes, green onion,
artichoke, lemon, almond; 4 for basic tastes: sweet, acid, salty and bitter and 3 for trigeminal
sensations: pungent, astringent and burning).
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83
84
85
From the overall appearance results, it showed that samples could be differentiate by their
aspect. Thus, accessions PEX-1, PEX-11, PEX-14, PEX-48 and PEX-56 had leaves with a green
colour while PEX-6, PEX-8, PEX-17 showed yellowish-green leaves. In addition, some of the
accessions had purple venation (PEX-8, PEX-11 and PEX-14). This result does not correspond
completely to the measure of the chlorophyll performed in the morphological analyses. This
difference can be due to the smaller size of sample in the sensory analyses in contrast to the
morphological analyses and to the variability of the accession.
Most of the samples showed small size, elongated shape of leaf and long petiole except the
accessions PEX-56 that presented medium size, PEX-6, which had rounded shape, and PEX-11
that had small petiole. This correlates with the morphological analyses. However, there were many
differences among the samples for the leaf margin sensory attribute. Thus, the accessions PEX-11
and PEX-56 showed entire leaves, the PEX-6 had undulated margin, the PEX-1, PEX-8, PEX-14
had dentate leaves while the PEX-17 and PEX-48 had lobulated margins. Finally, only the
accessions PEX-6 and PEX-17 (as in morphological analysis) presented pubescence on their
leaves. PEX-6 comes from Persepolis ruins and it is an Eruca vessicaria subsp. vesicaria that is
not normally used for human consumption. However PEX-17 is a commercial line of Eruca
vesicaria subsp. sativa and this character is undesirable for commercialization.
With regard to the texture attributes, all the accessions were tender, crunchy and moist
except the accession PEX-17 that showed a dry and not crunchy texture. In addition, the samples
PEX-6, PEX-11 and PEX-48 presented fibres adhering on/in the teeth during mastication. Finally,
in the accessions PEX-8 and PEX-48 the panel perceived a coating, which remained in the palate
after swallowing rocket leaves.
Finally, flavour results showed that the majority of the samples were described with
green/grass/clover, radish, lemon peel and fruit nuts (almond) odour, and aroma terms.
In
addition, all accessions of the subspecies vesicaria (PEX 6, PEX-10 and PEX-48) showed green
onion odour/aroma except the PEX-8 that had tomatoes odour/aroma notes. Finally, the accession
PEX-15 had an artichoke odour. All samples are characterized as sweet except the accession
PEX-14 that is described as salty; the accessions PEX-11 and PEX-14 were bitter and PEX-1 was
acid. In mouth, all samples are characterized as pungent: the accessions PEX-1, PEX-6 and PEX10 are described as pungent at the initial masticatory phase; the samples PEX-15 and PEX-48
showed a constant pungent sensation during all the masticatory phase; the accessions PEX-8 and
PEX-11 increased their pungent intensities during the masticatory phase while the accessions
PEX-14, PEX-17 and PEX-56 were pungent at the end of the masticatory phase. It is remarkable
the differences between 2008 and 2009. Accessions grew in 2008 were more mature than in 2009,
because all the accessions had radish, and clover notes but only accessions from 2008 had also
86
tomato or artichoke notes. This can be due to environmental conditions like rain and temperature.
In a previous work performed by D’Antuono et al.14, they pointed out the relationship of the
glucosinolate content of rocket and sensory perception. In our work it is not possible to attribute
that a certain glucosinolate or the total content of them can be related to a determinate flavour.
Padilla et al. 35 reported that the bitterest varieties of Brassica rapa had higher glucosinolates and
gluconapin content than the less bitter varieties. Some authors have also reported a relation
between the bitter taste and glucosinolate degradation products36,37. Therefore, in a previous
study31 the researchers could not find correlation between the characteristic flavour of cabbages
and the glucosinolate content, and concluded that the flavour was probably due to other
phytochemicals or the synergism among them, not just of hydrolysis products derived from
glucosinolates. All these compounds are synthetized by plants as defence against predation, but
they have been also reported to have healthy properties. Enhancing the phytochemical content of
plant foods is a good option for disease prevention. However these bioactive compounds can
produce a rejection of the consumer because of the unpleasant flavour. The food industry has
been removing these compounds during decades. Thus food designers face the dilemma of
increasing the content of healthy related phytochemicals and the incompatibility with consumer
acceptance25.
4. Conclussions
This work has shown the variability of the accessions of rocket and Erucastrum for
agronomic, morphological and sensory attributes. This variability provides an interesting and
valuable material for further breeding programs in order to generate lines of interest for the agrofood industry. In general, a high variation was observed for most of the 12 morphological and 5
agronomical traits showing significant differences. Some accessions showed good qualities, such
as high fresh matter, small leaves, high chlorophyll content, absent pilosity, late flowering, and high
glucosinolate content. Thus, PEX-14 accession showed the highest biomass, had late flowering
(101 days), absence of pilosity, and it had fast growth rate. Regarding the glucosinolate content,
PEX-6, PEX-8 and PEX-10 could be good candidates for future breeding purposes because of
their high total glucosinolate content. In addition, the presence of high glucoraphanin content in
some accessions (PEX-8, PEX-10 and PEX-11) should be studied more exhaustively since this
aliphatic glucosinolate is the precursor of sulforaphane, a potent anti-cancer isothiocyanate.
This is the first work that creates a preliminary special lexicon for rocket sensory analysis.
The analytical panel generated 27 simple descriptors: 7 for appearance, 6 for texture and 14 for
flavour. This lexicon has been developed from 9 rocket accessions and 1 accession of the genus
Erucastrum. It was not possible in this study to correlate a determined flavour to the glucosinolate
content or to the presence of a certain glucosinolate, suggesting that other phytochemicals may be
87
involved in the characteristic flavour of this Cruciferous species.
Acknowledgements
This research was supported by the Consejería de Innovación, Ciencia y Empresa (Junta de
Andalucía), Project P06-AGR-02230, for which the authors are deeply indebted. We give special
thanks to the UCO sensory panel for their volunteer participation and to Dr. César Gómez-Campos
and to the germplasm banks for providing vegetal material for this work. Myriam Villatoro was
supported by Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)
contract.
88
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91
92
CAPÍTULO III
Aproximación al perfil fitoquímico de rúcola (Eruca sativa (Mill.) Thell)
Artículo en preparación:
An approach to the phytochemical profile of rocket (Eruca sativa (Mill.) Thell)
Myriam Villatoro-Pulido1, Feliciano Priego-Capote2, Beatriz Álvarez-Sánchez2, Shikha Saha3, Mark
Philo4, Sara Obregón-Cano5, Antonio De Haro.Bailón5, Rafael Font6, Mercedes Del Río-Celestino6
1
IFAPA Centro-Alameda del Obispo, Department of Plant Breeding and Biotechnology, Córdoba,
Spain.
2
Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales,
University of Córdoba, Córdoba, Spain.
3
Phytochemicals and Health Programme, Institute of Food Research, Norwich Research Park,
NR4 7UA Norwich, United Kingdom.
4
Metabolomics and Mass Spectrometry, Institute of Food Research, Norwich Research Park, NR4
7UA Norwich, United Kingdom.
5
Department of Plant Breeding, Institute of Sustainable Agriculture (IAS-CSIC), Alameda del
Obispo s/n, 14080 Córdoba, Spain.
6
IFAPA Centro La Mojonera, Department of Plant Breeding and Biotechnology, La Mojonera,
Almería, Spain.
93
Abstract
The purpose of this study was to determine the profile of different families of compounds with
nutraceutical and organoleptical properties in leaves or four rocket accessions (Eruca vesicaria
subsp. sativa). The target families were glucosinolates, isothiocyanates, phenolic compounds,
carotenoids and carbohydrates. The four accessions were named according to the total content of
glucosinolates that ranged from 14.02 to 28.24 µmol/ g of dry weight. Glucoraphanin represented
up to 52% of the total glucosinolate content in leaves (high glucosinolate content 1 accession).
Accessions showed differences in the hydrolysis of glucoraphanin and formation of the
isothiocyanate, sulforaphane. Data showed no correlation between both compounds in leaves,
which suggested differences in the myrosinase activity within accessions. In addition leaves of
rocket had variable phenolic profiles represented by quercetin-3-glucoside, rutin, traces of
myricetin, quercetin and phenolic acids such as ferulic and p-coumaric acids. The total carotenoid
content ranged from 16.2 to 275 µg/g of dry weight revealing a high variability. Lutein was the main
carotenoid ranging from 8.3 to 124.3 µg/g dw. The low glucosinolate content 2 accession is a good
candidate for future breeding programs because of its pattern of healthy beneficial related
compounds. However, further research is essential to evaluate the biological activity of these four
accessions, assessing the possible non-desirable effect before planning strategies to design
functional food and improving consumer’s health.
Keywords: Eruca sativa, rocket, glucosinolate, isothyocianate, polyphenol, carotenoid, sugar.
Abbreviations: GL: glucosinolate; ITC: isothiocyanate.
94
1. Introduction
Many studies associate a highly significant cancer risk reduction with increasing Cruciferae
consumption (Juge et al., 2007). The term “rocket” refers mainly to Eruca and Diplotaxis genera
within the Cruciferae family. Eruca sativa contains a wide range of health-promoting
phytochemicals including glucosinolates (GLs) (and their degradation products), phenolic
compounds, and carotenoids among others (Bones and Rossiter, 2006; Bennett et al., 2002; Niizu
and Rodríguez-Amaya 2005).
When GLs are exposed to myrosinase (thioglucohydrolase), during tissue damage, etc.,
glucose and an unstable intermediate are formed. This intermediate degrades to produce a sulfate
ion, and a variety of products including isothiocyanates (ITCs), nitriles and, to a lesser extent,
thiocyanates, epithionitriles and oxazolidines. The kind and proportion of these hydrolysis products
depend on the plant species studied, on the GLs itself (as side chain substitution), and reaction
conditions like pH, metal ions or epithiospecifier protein (Bones, & Rossiter, 2006; Bennet et al.,
2007). It has been speculated that the ITCs, hydrolysis products of GLs, are responsible for the
protective effects of Brassica vegetables (Mithen, 2001).
Other family of compounds with nutraceutical properties are phenols, which are the most
abundant antioxidants in diet and they can act as antioxidants in vivo (Halliwell, 2008).
Considerable evidence indicates also that some of the protective effects of phenols on fruits and
vegetables may be due to flavonoids (Clifford, & Brown, 2006).
Carotenoids constitute one of the most important classes of plant pigments. Their antioxidant
behaviour depends on the concentration and localization in the actual target cells, tissues or
cellular compartments, as well as on other factors (Van den Berg et al., 2000).
Until date no data on sugar fractions alditols and saccharides in Eruca sativa have been
published yet despite their contribution to organoleptical properties. Additionally, it is known that
nonstructural carbohydrates, among other solutes, act as osmoregulators and osmoprotectors of
the tolerance response to abiotic stresses (Gómez-González et al., 2010).
The present work is part of an ongoing breeding program to obtain varieties of rocket with
potential health benefits. The material for this work has been acquired from different European
genebanks. After a previous screening of the material attending to the GLs profile we have
selected four accessions with breeding interest covering a wide range of this compound. The
accessions are named attending to the total content of GLs: Low Glucosinolate Content (LGC1
and LGC2), and High Glucosinolate Content (HGC1 and HGC2). The objective has been to
95
characterize different bioactive fractions, apart from GLs, such as ITCs, phenolic compounds,
carotenoids and carbohydrates of these four accessions of rocket to be considered as evaluation
parameters in breeding programs and before planning strategies to design functional foods for
improving consumer’s health.
2. Material and methods
2.1. Plant material and greenhouse experiments
Four lines of rocket differing in the concentration and pattern of GLs were selected for this
study. These accessions are part of a germplasm collection located at the IFAPA- La Mojonera,
Almería (south Spain). Seeds of Eruca sativa LGC1 (commercial variety Sky), LGC2, HGC1 and
HGC2 were obtained from Tozer Seeds Lyd (Cobham, Surrey, U.K.), Faculté des Sciences
Agronomiques of Gembloux, Belgium, Dipartimento di Scienze Botaniche of Palermo, Italy and
Botanischer Garten der Universitat of Karlsruhe, Germany, respectively. Seeds were germinated in
Petri dishes for 48 h at 25 ºC. Pots were placed in greenhouse under natural light at 27/18 ºC
(day/night) and a relative humidity of 50/70% (day/night). When the plants reached 8–12 cm, they
were transferred to a field in Córdoba, Spain (37"51'42'N, 04'48'00'W; 220m asl). The experiment
was designed as a randomized complete block consisting of rows of 5 meters length with three
replicates each.
2.2. Sample pre-treatment and storage
Leaves from 10 randomly selected plants per replicate were harvested eight weeks after
transplanting and on the same day. They were washed, weighed to assess their biomass, and
placed in Ziploc-type freezer bags at –20 ºC for post-harvest storage. The samples were freezedried up and ground using a pestle and mortar.
2.3. GLs analysis by liquid chromatography with ultraviolet photometric detection (LC-UV)
GL composition was determined by a LC-UV method, according to Font and collaborators
(2005). 100 mg of freeze-dried sample was heated at 75ºC for 15 min in 2.5 mL of 70:30
methanol–water and 200 µL of 10 mM sinigrin as an external standard (Sinigrin hydrate, 85440
Fluka) according to the ISO norm (ISO 9167-1, 1992). A second extraction was applied after
centrifugation (5 min, 5 x 103g) using 2 mL of 70:30 methanol-water. The combined GLs extracts
were pipetted (1mL) onto the top of an ion-exchange column containing 1 mL of Sephadex DEAEA25 (40-125 µm bead size, 30000 Da exclusion limit). Desulfation was carried out by addition of 75
µL of purified sulfatase (EC 3.1.6.1, type H-1 from Helix pomatia) (Sigma-Aldrich) solution.
Desulfated GLs were eluted with 2.5 mL of Milli-Q (Millipore) ultrapure water and analyzed with a
600 HPLC instrument (Waters) equipped with a model 486 UV tunable absorbance detector
(Waters) fixed at a wavelength of 229 nm. Separation was carried out using a Lichrospher 100 RP-
96
18 in Lichrocart column (125 mm x 4 mm i.d., 5 µm particle size, Merck).
The HPLC
chromatogram was compared to the desulfo-GL profile provided by three certified reference
materials recommended by the U.E. and ISO (CRMs 366, 190 and 367) (Commission of the
European Communities, report EUR 13339 EN, 1-75) (Wathelet, Wagstaffe, & Boenke, 1991).
2.4. Sulforaphane, iberine and sulforaphane nitrile determination by liquid chromatography and
mass spectrometry detection (LC–MS)
Freeze-dried leaves (40 mg) were hydrolyzed in 1 mL phosphate saline buffer (PBS),
incubated at 37°C for 2 h, and then centrifuged (at 13,000 g, 30 min at 4ºC) to obtain ITCs from
GLs. Supernatant was directly analyzed using liquid chromatography with mass spectrometric
detection with 1100 Agilent LC system (Agilent Technologies, Waldbronn, Germany) equipped with
a diode array detector and a single quadrupole mass spectrometry detector. A linear gradient from
0.1% formic acid in H2O (mobile phase A) to 0.1% formic acid in CH3CN (mobile phase B) as
mobile phases with flow rate 0.3 mL/min was used. The chromatographic column was a
Phenomenex Luna C-18 (150 mm × 4.6 mm i.d., 3 µm particle size). The gradient started at 0%
solution B increasing over 30 min to 30% B and, finally, re-equilibration to 0% B for 10 min.
Sulforaphane and iberin were monitored spectrophotometrically at 229 nm, and also using
selected ion monitoring (SIM) targeted on m/z 178.0 and m/z 164.3 for ITCs sulforaphane and
iberin, respectively. The standards (S8044 and I0416 LKT Laboratories, Inc., USA, for
sulforaphane and iberin, respectively) were correlated with the ITCs in order to quantify them after
identification based on retention time and mass spectrum. Quantification was carried out by
comparison to external standard calibration curves (linear regression coefficients >0.99).
Sulforaphane nitrile was monitored using the same LC-MS method by selected ion monitoring
(SIM) in positive mode monitoring the ion at m/z 146.0.
2.5. Erucin determination by gas chromatography/mass spectrometry analysis (GC-MS)
The solution of hydrolysed ITCs in PBS (0.5 mL) was added to the same volume of
dichloromethane (CH3Cl2) for extraction of erucin. The organic phase was isolated and centrifuged
at 13,000g for 30 min at 4 °C. The erucin content was measured by gas chromatography-mass
spectrometry (GC–MS) with identification based on comparison to GC retention time and mass
spectrum provided by erucin standard (E6880-LKT Laboratories, Inc., USA). GC-MS analysis was
performed using a Trace GC Ultra™ (Thermo Scientific) operated in selected ion monitoring (SIM)
mode with positive ionization by electron impact (EI+). Separation was carried out using a ZB-5mS
(Phenomenex®, Netherlands), 30 m × 0.25 mm × 0.25 µm capillary column. The injection volume
was 1 µL in splitless mode with a splitless time of 45 s and injector temperature of 250 °C. The
97
oven temperature program was linear with a ramp from 40 °C min to 250 °C at 10 °C/min. The
source and transfer line temperatures were 200 and 250 ºC, respectively. The ions monitored for
erucin identification (ER) were m/z 146, 161, 61 and 115.
2.6. Determination of the total phenolic fraction
The concentration of total phenolic compounds was estimated by a modified version of the
Folin–Ciocalteu method (Singleton, & Rossi, 1965), using gallic acid as standard, for which a
calibration curve was run with solutions of 50, 100, 200, 300, 400, 500 and 600 mg/L of this
compound. A 0.06 mL aliquot of extract 1.58 mL of distilled water, 0.1 mL of Folin–Ciocalteu
reagent and 0.3 mL of Na2CO3 (20% w/v) were mixed and heated at 50 ºC for 5 min. After 30 min,
the absorbance was measured at 765 nm against a blank similarly prepared, but containing 70:30
ethanol–water mixture (pH 3.2) instead of extract. Sodium carbonate (Panreac), Folin–Ciocalteu
reagent (FCR) and gallic acid (both from Sigma–Aldrich) were used to determine the total phenol
fraction. The absorbance was measured with a ThermoSpectronic UV–visible Spectrometer
(Thermo Fisher Scientific, USA).
2.7. Analysis of the phenolic fraction by liquid chromatography tandem mass spectrometry (LCMS/MS)
Freeze-dried shoots (200mg) were agitated overnight in 30 mL of 70:30 ethanol–water
mixture at pH 3.2 fixed with formic acid (Heimler et al., 2007). Prior to LC–MS analysis, 100 µL of
extract was evaporated to dryness and reconstituted in 100 µL of initial mobile phase for injection
of 10 µL in the chromatograph. Liquid chromatography analysis was performed with an Agilent
(Palo Alto, CA, USA) 1200 Series LC system coupled to an Agilent 6410 triple quadrupole (QqQ)
mass analyzer. The data were processed using a MassHunter Workstation Software from Agilent
for qualitative and quantitative analysis. An Inertsil ODS-2 C18 analytical column (4.0 mm i.d.×250
mm; 5 µm particle size, GL Sciences Inc., Tokyo, Japan) was used for chromatographic
separation. Separation of the phenolic compounds was performed in 71 min, being the mobile
phases A and B 0.4% aqueous formic acid and 50:50 (v/v) acetonitrile–methanol, respectively. The
flow rate and the column oven temperature were set at 1 mL/min and 35 ºC, respectively. The
chromatographic method was as follows: the initial mobile phase was set at 4% of B, which was
increased to 50% in 40 min and, then, to 60% B in 5 min.
Finally, it was gradually changed to 100% mobile phase B in 3 min and maintained for 17
min. A re-equilibration step of 6 min was programmed after each chromatographic run. Analyses
were carried out in selected reaction monitoring (SRM) negative ionization mode with nitrogen as
drying and nebulizing gas. The operating conditions of the ESI–QqQ, were: flow rate and
temperature of drying gas 10 mL/min and 325 ºC, respectively, nebulizer pressure 40 psi, capillary
98
99
voltage 2700 V and dwell time 200 ms. The quantification transition and fragmentation conditions
for each phenolic compound are shown in Table 1.
The panel of phenolic compounds was composed of benzoic acid derivatives
(protocatechuic, vanillic and syringic acids), methyl and ethyl esters from gallic acid, cinnamic
acids (p-coumaric, ferulic and caffeic acids), stilbene (trans-resveratrol), the flavonols (kaempferol3-O-rutinoside, quercetin, quercetin 3-ß-D glucoside and myricetin) and the flavone glycoside rutin
hydrate, which were purchased from Sigma–Aldrich (St. Louis, MO, USA). The flavonols ((+)catechin, (−)-epicatechin and procianidins B1, B2 and A2) were from Extrasynthese (Genay
Cedex, France).
2.8. Analysis of the carotenoid content
Carotenoids were extracted using a modification of the method described by Tadmor and
collaborators (2000). 400 mg of sample were rehydrated with 5 mL ethanol containing 1 mg/mL
butylated hydroxytoluene (BHT) using a Polytron homogenizer. One mL of a 40% (w/v) KOH
methanolic solution was added to each tube, and the samples were saponified for 10 min at 85 ºC,
cooled in an ice bath, then, 2 mL of ice-cold water was added. The suspensions were extracted
twice with 2 mL of hexane by vigorous vortexing followed by a 2000g centrifugation for 10 min at
room temperature. The upper hexane layers were pooled and evaporated to dryness and
resuspended. The carotenoids were dissolved before injection in 800 µL of an acetonitrile–
methanol–dichloromethane (45:20:35 v/v) solution, filtered through a 0.22 µm PTFE syringe filter
(Millipore) directly to sample vials, and 10 µL were injected into the chromatograph. The analyses
were carried out on an HPLC apparatus equipped with binary pump, in-line vacuum degasser,
autosampler injector, a Waters Symmetry C18 column (4.6 mm x 150 mm, 5 µm) and a dual λ
absorbance detector (model 2487), controlled by Breeze workstation.
The initial mobile phase consisted of acetonitrile–methanol (97:3, v/v) containing 0.05%
(v/v) triethylamine. A linear gradient of dichloromethane from 0 to 10% in 20 min at the expense of
acetonitrile was used, and then, the dichloromethane was kept constant at 10% until run
completion. The flow rate was 1.0 mL/min and the column temperature was 30º C. A λ absorbance
detector was used to detect coloured carotenoids at 450 nm. The compounds were identified by
comparison of retention times, coinjection with known standards, and comparison of their UVvisible spectra with authentic standards (β-carotene, β-cryptoxanthin, lutein and zeaxanthin).
Quantification was carried out by external standardization. Full standard curves were made
in triplicate with five different concentrations for each carotenoid. The curves, which passed
through or very near to the origin, were linear and bracketed the concentrations expected in the
samples.
100
2.9. Analysis of the sugar fraction by gas chromatography with mass spectrometry detection
200 mg of freeze-dried shoots was extracted by overnight agitation in 30 mL of 70:30
ethanol–water mixture at pH 3.2 fixed with formic acid as in the determination of the phenolic
fraction (Heimler et al., 2007). A 150 µL aliquot of this extract was evaporated to dryness and
reconstituted in 150 µL of derivatization solution, which consisted of 50-µL pyridine from Merck
(Darmstadt, Germany), 98-µL N,O−Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 2 µL
trimethylchlorosilane (TMCS) from Sigma–Aldrich. The reaction mixture was vortexed at room
temperature for 1 h, before the GC–MS analysis, which was carried out with a Varian CP 3800 gas
chromatograph coupled to a Saturn 2200 ion trap mass spectrometer (Sugar Land, TX, USA)
equipped with a FactorFour capillary column (VF-5 ms 30 mx0.25 mm, 0.25 µm) from Varian (Palo
Alto, USA). Thus, after derivatization, 1 µL of the analytical sample was injected into the
chromatograph. The injector temperature was fixed at 280 °C, and the injection was in the
split/splitless mode. Helium at a constant flow-rate of 1.3 mL/min was used as carrier gas. The
oven temperature program was as follows: initial temperature 65 °C (held for 2 min), increased at
6 °C/min to 300 °C (held for 30 min). The ion-trap mass spectrometer was operated in the electron
impact ionization (EI) positive mode, for which the instrumental parameters were set at the
following values: filament emission current 80 µA; transfer line, ion trap and manifold temperatures
were kept at 280, 200 and 50 °C, respectively. The MS–MS process was carried out by collisioninduced dissociation (CID) in non-resonant excitation mode. Table 2 shows the optimal MS–MS
parameters for each compound. Carbohydrate standards D-(-)-arabinose, D-(+)-mannose, D-(-)fructose, D-(-)-galactose, D-(+)-glucose, D-(+)-sucrose, and D-(+)-melazitose (the latter used as
internal standard) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3. Results and discussion
3.1. GL content in leaves of rocket
The biosynthesis of GLs consists of three main stages: amino acid elongation, synthesis of
the GL from the amino acid and chain modifications. The methionine derived GLs form
homomethionine if the elongation occurs in carbon 3. By contrast, if elongation is produced in
carbon 4, the product formed is dihomomethionine. Homomethionine forms the GL glucoiberverin
that can be oxidized to form glucoiberin. The content of iberin, the ITC formed from the hydrolysis
of glucoiberin, has been determined (Table 3). Therefore, glucoiberin should have also been
detected in the GL analysis. The GL obtained from dihomomethionine is the glucoerucin, which is
oxidized to form glucoraphanin. In fact, glucoraphanin was the GL with highest concentration in
101
102
HGC1 and HGC2 accessions (14.90 and 12.64 µ mol of GL/g dw, respectively). Upon alkilation,
glucoraphanin forms gluconapin, which was not detected in LGC1 and HGC1 accessions. The
level of gluconapin was low (0.34 µ mol of GLs/g dw) in LGC2 and HGC2. Hydroxylation of
gluconapin forms progoitrin, which was found at half of content of its precursor in accessions LGC2
and HGC2 (0.17 and 0.14 µ mol of GLs/g dw), and was not detected in accessions LGC1 and
HGC1. Concerning aromatic GLs, gluconasturtin was the only one found in rocket accessions,
which ranged from 0.24 to 0.82 µ mol/g dw.
Regarding to tryptophan-derived GLs, the compounds detected are indolyl group
derivatives. The levels of glucobrassicin, 4-hydroxyglucobrassicin, 4-metoxiglucobrassicin and
neoglucobrassicin were quite low (ranging from 0.03 to 0.16 µ mol of GLs/g of vegetal tissue). By
contrast, glucosativin was the indolyl GL that exhibited the highest content with a maximum mean
value of 11.40 µ mol/g for HGC1 accession as it has been reported in previous works (Bennet et
al., 2002).
The accession LGC1, LGC2, HGC1, and HGC2, showed a total content of GLs of 14.02,
19.4, 28.24 and 27.65 µ moles of GLs/g of freeze-dried vegetal tissue, respectively. The content of
glucoerucin and glucobrassicin is similar to that reported by Kim and Ishii (2006), who published a
content of 3.28 and 0.04 µ mols/g dw for glucoerucin and glucobrassicin respectively. Nevertheless
the level of glucoraphanin (1.25 µ mols/g dw) and total GL content (11 µ mols/g dw) is higher in our
work. In a previous work, Bennet and collaborators (2006) reported higher values for glucoerucin
(ranging from 1.96 to 9.78 µ mols/g dw) and glucosativin (ranging from 15.34 to 30.67 µ mols/g
dw) in contrast to our values (ranging from 0.14 to 4.03 µ mols/g dw, and from 8.10 to 11.40 µ
mols/g dw for glucoerucin and glucosativin respectively). Bennet and collaborators (2007) reported
higher values of total GLs (55.4 µ mols/g dw), glucoerucin (32.0 µ mols/g dw), glucosativin (31.3 µ
mols/g dw), but lower values of glucoraphanin (6.1 µ mols/g dw) and glucobrassicanapin (0.2 µ
mols/g dw) than our work.
3.2. Isothyocianate content in leaves of rocket
The ITCs detected were 1-ITC-4-(methylsulfinyl)-butane (sulforaphane), sulforaphane-nitrile,
3-methylsulphinylpropyl-ITC (iberin) and 4-(methylthio)butyl ITC (erucin) (Table 3). Sulforaphane is
derived from glucoraphanin and erucin is obtained from hydrolysis of glucoerucin, but also through
in vivo reduction of the ITC sulforaphane (Melchini et al., 2009). The in vivo inter conversion of
these two ITCs and their structural similarity has suggested a similar biological activity. Iberin is
formed from glucoiberin, which was not detected in these accessions. Accessions showed
differences in the hydrolysis of glucoraphanin and formation of sulforaphane ranging from 4.12%
(LGC1 accession) to 97.35% (LGC2 accession). Pearson’s correlation of glucoraphanin and
sulforaphane was not significant in leaves of rocket (-0.38, P >0.05), which suggested differences
103
in the hydrolysis activity of related enzymes within accessions.
Sulforaphane mean content ranged from 0.15 to 5.90 µg/g dw (Table 3) and sulforaphane
nitrile was found ranging from 0.15 (HGC1 accession) to 0.97 (LGC2 accession) µg/g dw. This
compound has been found to be ineffective as an inducer of some detoxification enzymes. The
selection of accessions with low levels of the epithiospecifier protein might provide higher
conversion of sulforaphane than sulforaphane nitrile with improved potency as anticarcinogenic
food (Matusheski et al., 2006). LGC2 accession showed the maximum level for iberin (1.55 µmol /g
dw). Erucin could be quantified only in the LGC2 accession (0.01 µmol /g dw). Our data are in
concordance to that reported by Melchini et al. (2009), who reported values of 3.46 µ mol of
sulforaphane /g dw and 0.05 µ mol of erucin/g dw. Nevertheless, it has been previously published
that erucin is the major ITC in rocket leaves (Blazevic y Matelic, 2008), while other authors have
stated that the most abundant ITC in rocket is sativin (Bennett et al., 2002).
3.3. Phenolic compounds in leaves of rocket
Quercetin derived compounds such as quercetin-3-β-glucoside (isoquercetin) or rutin were
the most abundant flavonoids in the rocket accessions (Table 4). HGC1 accession showed the
maximum mean values for quercetin-3-β-glucoside (1680.0 µg/g dw). Rutin was found ranging
from 12.00 to 27.00 µg/g dw in accessions of rocket (Table 4).
There are previous data on the phenolic and flavonoid content of rocket species (Weckerle
et al., 2001; Bennett et al., 2002; Arrabi et al., 2002). Weckerle and collaborators reported
quercetin disinapoyl tri-O-glycoside in leaves of rocket, in contrast to our study and Bennett and
collaborators (Bennett, Rosa, Mellon, & Kroon, 2002) suggested that the leaves analysed in the
study of Werkerle and collaborators were not E. sativa or they were an Italian ecotype with a very
different leaf flavonoid profile to common commercially available E. sativa. Bennet and
collaborators (2006), also showed that kaempferol di-O-glycoside was the major flavonoid in young
leaves of Eruca. Selma and collaborators (Selma et al., 2010) reported alsokaempferol,
isohamnerin and quercetin in leaves of Eruca sativa. Jin and colleagues (2009) reported also that
the predominant flavonoids in rocket leaves were quercetin, kaempferol, and isorhamnetin. These
discrepancies in phenolic contents between studies and varieties could be the result of multiple
factors,
including
methodology
(all
reports
used
different
approaches
for
extraction,
chromatography, and quantification), sample characteristics and conditions, including variables
such as growth (Brown et al., 2002).
Differences were found among the accessions of rocket in their mean flavonol contents.
Quercetin was detected only in HGC1 accession (13.50 µg/g dw). This flavonol is a recognized
supplement that could increase the nutraceutical value of rocket. Myricetin was detected only in
HGC1 and LGC2 accessions (3.00 µg/g dw) (Table 4). Based on Hertog et al. (1995), a flavonol
104
105
106
content > 300 µg/g dw in food can be considered high. Therefore, the flavonol content was low in
accessions of rocket (<20 µg/g dw).
Apart from flavonoids, ferulic acid, a phenolic acid (ranging from 30.60 to 48.00 µg/g dw)
was detected in the rocket accessions. The mean content of total phenolic ranged from 4474.5 to
32700 µg/g dw (Table 4). Our results showed variability between accessions and demonstrated
that leaves of rocket, especially LGC1 and HGC2 accessions are an excellent source of phenolic
compounds. Both accessions reached higher values than those found in others sprouting species
such as broccoli (27962.5 µg/g dw approximately) (Perez-Balibrea et al. 2001) and even richer
than the commercial broccoli florets (Vallejo et al., 2002). Nevertheless LGC2 and HGC1 were the
accessions that had more qualitative variability regarding to the phenols studied.
3.4. Carotenoid content in leaves of rocket
Lutein was the principal carotenoid, followed by β-cryptoxanthin, β-carotene, zeaxanthin and
violaxanthin (Table 5). Neoxanthin was found in lower proportions (less than 1% of total carotenoid
content). The total carotenoid content ranged from a minimum mean value of 19.51 µg/g dw (LGC1
accession) to a maximum mean value of 263.91 µg/g dw (LGC2 accession). HGC1 and LGC1
accessions showed the maximum and minimum mean values for lutein (124.30 and 8.30 µg/g dw),
respectively. LGC2 exhibited the highest β-carotene concentration with a mean content of 20.71
µg/g dw. All the rocket accessions had zeaxanthin and β-cryptoxanthin, and this is the first report
on quantitative analysis of both carotenoids. β-Cryptoxanthin has only one-half of the provitamin A
activity of β-carotene, but in many accessions of rocket (LGC1, LGC2 and HGC1), a higher
concentration of the former than that of β-carotene has been found. Of the rocket accessions
analyzed, LGC2 and HGC1 had the highest concentrations of neoxanthin, violaxanthin, lutein,
zeaxanthin and β-cryptoxanthin. The LGC1 accession presented the lowest levels of these main
carotenoids, whereas HGC2 had intermediate concentrations.
Table 5. Carotenoid content (mean±standard deviation) isolated from rocket leaves.
Carotenoid
Compound
Neoxantin
Violaxanthin
Xantophylls
Lutein
Zeaxanthin
β-Cryptoxanthin
Carotene
β-Carotene
Total Content of Carotenoids
a
LGC1
a
nd
0.20±0.01
8.30±0.10
1.91±0.01
8.01±0.04
0.90±0.03
19.51±0.41
LGC2
2.20±0.00
8.20±0.10
115.20±0.40
24.10±0.20
93.70±0.10
20.71±0.20
263.91±0.30
b
HGC1
0.80±0.01
13.00±0.20
124.30±0.40
25.81±0.03
81.71±0.21
14.62±0.10
260.31±0.50
HGC2
0.30±0.01
2.50±0.02
55.60±0.20
12.81±0.01
1.70±0.10
14.90±0.10
87.91±0.20
nd: non-detected.
107
b
Concentrations, expressed as µg/g dw, are means±standard error of at least three independent
extractions and analyses of ten plants.
In previous works (Ramos and Rodríguez- Amaya, 1987; Kimura and Rodríguez-Amaya,
2003; Niizu and Rodríguez-Amaya 2005) lutein and β-carotene were reported as the most
abundant carotenoids with mean contents of 50 µg/g for lutein and 30 µg/g for β-carotene. The
contents of lutein in our work are higher than those found in previous works, but lower than the
values found for β-carotene.
It has been suggested that 6 mg of lutein per day may reduce the risk of age related macular
degeneration in a percentage of 43% (Seddon, 1994). This concentration is equivalent to
consuming ~7 kg tomatoes, 2 salad bowls of spinach, or, as revealed by this study, ~ 320 g of
fresh leaves of rocket. Although lutein is not a provitamin A, it is a more effective antioxidant than
many other carotenoids. It inhibits in vitro lipid oxidation in a more efficient manner than βcarotene, α-carotene or lycopene.
3.5. Sugars in leaves of rocket
Glucose, the primary photosynthetic product, was the predominant sugar in leaves (Table
6). In fact, this sugar represents >70% of the total soluble carbohydrates in leaves of rocket. This is
not surprising as glucose represents the major transport sugar in rocket and contributes
significantly to osmotic adjustment. Sucrose, fructose, galactose arabinose and mannose were
found at low concentration (Table 6). The capability of supporting prolonged water deficiency is
known for rocket (Ashraf, 1994). The activity of these substances is related to their ability to raise
the osmotic potential of the cell. Similar results were also reported in Eruca by Ashraf (1994), who
found that salt tolerant populations had significantly higher soluble sugars in their leaves that salt
sensitive populations at varying salt levels of the growth medium.
Table 6. Carbohydrate (mean±standard deviation) isolated from rocket leaves.
Classification
Elemental
composition
C5H10O5
Analyte
Monosaccharide
C6H12O6
Arabinose
Mannose
Fructose
Glucose
Galactose
Disaccharide
C12H22O11
Sucrose
a
LGC1
HGC1
HGC2
a
nd
0.15±0.11
0.01±0.03
nd
0.19±0.02 1.94±0.31
1.24±0.02
0.36±0.11
nd
5.48±0.22
nd
nd
31.19±0.10 38.55±0.65 35.87±1.01 38.46±0.04
1.44±0.13 2.14±0.04
1.63±0.21
1.86±0.10
2.06±0.05
nd: non-detected; concentrations expressed as mg/g.
108
LGC2
5.97±0.18
2.96±0.21
1.75±0.01
4. Conclusions
In summary, GLs and ITCs were detected in varying levels in leaves of four rocket
accessions. Glucoraphanin represented up to 52% (HGC1 accession) of the total GLs in the
leaves. Accessions showed differences in the degradation of glucoraphanin and formation of
sulforaphane up to 97.35% of yield (LGC2 accession). Total GLs and ITCs contents present in
leaves suggested differences in the myrosinase activity within accessions.
Concerning phenols and carotenoids, it is only possible to affirm that an adequate
consumption of them can prevent some diseases (Halliwell, 2008). However, it is not possible to
state that the level of phenols or the presence of a certain specific single phenol in the accessions
is enough to exert a protective action. It has been proposed that the synergistic effect among
phenols in samples with high content of them can enhance their antioxidant effect (Pignatelli et al.,
2006). It has also been reported that some phenols are capable of having anti/pro-oxidant effect
and that depends to a great extent on the system used and the relative amounts of the phenolic
(Makris and Rossiter, 2001).
The high levels of GLs, myrosinase activity, phenols and carotenoids in some of the
accessions (LGC2 accession) can be transferred through genetic engineering or conventional
breeding programs to commercial lines such as “Sky” to increase its potential benefit on human
health. However, further research is essential to evaluate the biological activity of these four
accessions, assessing the possible non-desirable effect before planning strategies for designing
functional food and improving consumer’s health.
Acknowledgments
This work was supported by the project P06-AGR-02230 (CICE, Junta de Andalucía) and
FEDER funds. Myriam Villatoro was supported by Instituto Nacional de Investigación y Tecnología
Agraria y Alimentaria (INIA) contract. Authors gratefully acknowledge the Institute of Food
Research , Norwich, U.K., for provision of technical materials and support. They thank Dr Richarch
Mithen, (Department of Natural Products and Health, IFR), Dr. Angeles Alonso-Moraga
(Department of Genetics, University of Córdoba, Spain) and Dr. Mª Dolores Luque de Castro,
(Department of Analytical Chemistry, University of Córdoba, Spain), for insightful advice in all
aspects of the work.
We also thank to Faculté des Sciences Agronomiques of Gembloux
(Belgium), Dipartimento di Scienze Botaniche of Palermo, Italy and Botanischer Garten der
Universitat of Karlsruhe, Germany, for the supply of vegetal material.
109
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112
113
CAPÍTULO IV
Caracterización y predicción por espectroscopía por reflectancia en el
infrarrojo cercano (NIRS) de la composición mineral de rúcola (Eruca
vesicaria subsp.sativa y Eruca vesicaria subsp. vesicaria).
Enviado a:
Journal of Science of Food and Agriculture
Characterization and prediction by near-infrared reflectance of mineral composition of rocket
(Eruca vesicaria subsp. sativa and Eruca vesicaria subsp. vesicaria)
Myriam Villatoro-Pulidoa, Rafael Moreno Rojasb, Andrés Muñoz-Serranoc, Vanessa Cardeñosaa,
Manuel Ángel Amaro Lópezb, Rafael Fontd, Mercedes Del Río-Celestinod.
a
IFAPA-Centro Alameda del Obispo, Córdoba, Spain.
b
Departamento de Bromatología y Tecnología de Alimentos, Universidad de Córdoba, Córdoba,
Spain.
c
Departamento de Genetica, Universidad de Córdoba, Córdoba, Spain.
d
IFAPA-Centro la Mojonera, Almería, Spain.
114
Abstract
Background: Minerals are essential for human nutrition and they are obtained from our diet.
Crucifer vegetables are a good source of these nutriments. The aim of this research was to
determine the genetic variability for mineral content and to evaluate the use of NIRS for prediction
of ashes and minerals among and within the species E. vesicaria subsp. sativa and subsp.
vesicaria (rocket). The minerals studied were: iron (Fe), copper (Cu), sodium (Na), potassium (K),
calcium (Ca), magnesium (Mg), manganese (Mn) and zinc (Zn).
Results: The maximum mean values obtained for all the accessions (mean±se) were: 23.55±0.15
mg ashes/100 g, 27.33±0.42 mg Fe/100 g, 1.81±0.04 mg Cu/100 g, 283.38±4.69 mg Na/100 g,
7158±101.71 mg K/100 g, 6461±121.97 mg Ca/100 g, 689±11.40 mg Mg/100 g, 10.16±0.12 mg
Mn/100 g, and 6.71±0.04 mg Zn/100 g of dry weight.
Conclusions: The statistical analysis showed significant differences for all the minerals for each
accession studied individually and for accessions grouped within countries except for the Ca. The
results indicated that NIRS can be used as a rapid screening method for determining total mineral,
Fe, Na, K, and Zn in rocket.
Keywords: Eruca, NIRS, minerals, iron, calcium, manganese.
Abbreviations: Ca: calcium; Cu: copper; Fe: iron; K: potassium; NIRS: Near-infrared reflectance
spectroscopy; Mg: magnesium; Mn: manganese; MPLS: modified partial least squares; Na:
sodium; RPD: ratio SD to SECV; SD: standard deviation; SEC: standard error in calibration; SECV:
standard error of cross-validation; SEP: standard error of prediction; SNV-DT: standard normal
variate and detrend transformations; Zn: zinc.
115
1. Introduction
Minerals are integral part of human and plant nutrition that support biological processes
during different stages of growth and development1. Vegetables of the Cruciferae family are widely
consumed and have a valuable role in human nutrition due to their content in glucosinolates,
carotenoids, vitamins, phenolic compounds, and minerals2-5 found in their tissues. The term
“rocket” refers to some species belonging mainly to the Eruca (Miller) and Diplotaxis (DC.) genera
within the Cruciferae family. The most recent classification attends to the terms of Eruca vesicaria
(L.) Cav. subsp. sativa (Miller) Thell., and subsp. vesicaria6. Recently, Bozokalfa and collaborators7
reported that E. vesicaria subsp. sativa is a mineral source for human nutrition. However, no
information is available about the genetic variability of mineral composition of Eruca vesicaria
subsp. vesicaria and the breeding potential for improvement of the mineral content.
Chemical determinations of different minerals in forages and leafy vegetables are
commonly performed by current techniques, which are time-consuming and expensive8. Nearinfrared reflectance spectroscopy (NIRS) has been widely used as a fast and cost-effective method
for determining forage nutritive value9. Although minerals theoretically do not absorb energy in the
near-infrared spectrum, some of the inorganic minerals in forages can be predicted by NIRS 10-12,
heavy metals in Brassica juncea, and arsenic content in Amaranthus blitoides by its association
with organic molecules13. Clark and colaborators10 reported that NIRS calibrations for the
macrominerals Ca, P, Mg, and K were useful in crested wheatgrass (Agropyron cristatum (L.)
Gaertn) and alfalfa (Medicago sativa L.). Halgerson and coleagues11 obtained similar results, in
which Ca, P, and K concentrations were accurately predicted in leaves and stems of alfalfa hay,
whereas Mg and S predictions were less consistent and Na prediction failed. Stoltz14 reported that
Ca, K, P, and Mg calibrations in alfalfa and white clover (Trifolium angustifolium L.) were
unsuccessful. Moreover in other studies it was difficult to obtain accurate NIRS predictions for
minerals15.
This study is part of an ongoing breeding program focused on obtaining varieties of rocket
with enhanced healthy properties for human nutrition. The knowledge of phytochemical characters
of the population has an important impact on the crop improvement as well as the conservation of
genetic resources. Our objectives were to determine the genetic variability for mineral content
among and within the subspecies E. vesicaria subsp. sativa and subsp. vesicaria; and to evaluate
the potential of NIRS to predict ashes, iron (Fe), copper (Cu), sodium (Na), potassium (K), calcium
(Ca), magnesium (Mg), manganese (Mn) and zinc (Zn) contents in rocket.
116
2. Material and methods
2.1. Plant material and greenhouse experiments
Table 1 shows the twenty-seven accessions of Eruca vesicaria analysed in this work.
Vegetal material has been acquired from the North Central Regional Plant Introduction Station
Ames, Iowa, USA, which collected the samples from researchers or worldwide gene banks from
Island of Sicily in Italy (Si), Italy (I), Turkey (T), India (In), Egypt (E), Iran (Ir), Poland (Po), Pakistan
(Pa), United Kingdom (UK), Canada (C), and an unknown country (U). We have distinguished
between Sicily and Italy because of the possible effects of isolation of the material being Sicily an
island.
Seeds were germinated in Petri dishes for 48 h under a minimum temperature of 25ºC.
Pots were placed in greenhouse under natural light, temperature of 27/18ºC (day/night) and a
relative humidity of 50/70% (day/night). When plants reached adequate height (8-12 cm), they
were transferred to soil. These accessions were grown in Cordoba, Spain (37º 53´ N; 4º 47´ W)
during 2007 under the semiarid conditions of Andalusia. A randomized complete block of design
seven replications was used.
2.2. Sample pre-treatment and storing
Leaves from 10 plants per accession (with seven replicates) were harvested together eight
weeks after transplanting and on the same day once they were ready for human consumption.
Then, they were washed with tap water, weighed to assess their biomass, and placed in Ziploctype freezer bags at –20 ºC for post-harvest storage. The samples were freeze-dried up to perform
the mineral analysis.
2.3. Analysis of mineral composition of the accessions
For the mineral composition analysis of the rocket, the dry mineralization method described
by Moreno Rojas and collaborators16 was used. Washed and homogenized samples (25 g) were
weighed into porcelain crucibles, previously dried in a furnace at 100 ºC to constant weight, from
which, and from the initial fresh weight, the moisture content was calculated. Once the samples
were dried, they were incinerated in a muffle furnace at 460 ºC for 15 h. The ash was bleached
after cooling by adding 2ml of 2N nitric acid, drying it on thermostatic hotplates and maintaining it
in a muffle furnace at 460 ºC for 1 h. Ash recovery was performed with 5 ml of 2N suprapur nitric
acid, making up to 15 ml with O.1N suprapur nitric acid. The determinations were carried out by
flame atomic absorption spectrophotometry, except for sodium and potassium, which were
analysed by flame atomic emission. For the determination of all the elements, except potassium, it
was necessary to dilute the samples 1/100, and, in the case of calcium and magnesium,
lanthanum chloride (LaCl3.7H2O) was added to make up a final concentration of 0.27% of the
117
sample, in order to prevent anionic interferences, which might modify the result of the
determinations. Elemental analyses were performed with a Perkin-Elmer model 2100 atomic
absorption spectrophotometer equipped with a Perkin-Elmer AS-50 autosampler, standard air–
acetylene flame and single element hollow cathode lamps and background correction with
deuterium lamp for manganese.
Table 1. List of accessions of rocket (Eruca vesicaria subsp. sativa and subsp. vesicaria) with the
USDA codex, botanical classification and country of origin.
Accession USDA Codex
27572
S1
Gender
Species
Sub-species Country of origin
Eruca
vesicaria
sativa
Sicily (Italy)
S2
27573
Eruca
vesicaria
sativa
Sicily (Italy)
S3
27574
Eruca
vesicaria
sativa
Sicily (Italy)
S4
27575
Eruca
vesicaria
sativa
Sicily (Italy)
S5
27576
Eruca
vesicaria
sativa
Sicily (Italy)
S6
27577
Eruca
vesicaria
sativa
Sicily (Italy)
S7
27578
Eruca
vesicaria
sativa
Sicily (Italy)
S8
27579
Eruca
vesicaria
sativa
Sicily (Italy)
S9
27580
Eruca
vesicaria
sativa
Sicily (Italy)
S10
27581
Eruca
vesicaria
sativa
Sicily (Italy)
S11
27583
Eruca
vesicaria
sativa
Italy
S12
27584
Eruca
vesicaria
sativa
Italy
S13
27585
Eruca
vesicaria
sativa
Italy
S14
27586
Eruca
vesicaria
sativa
Italy
S15
179279
Eruca
vesicaria
sativa
Turkey
S16
181054
Eruca
vesicaria
sativa
India
S17
216034
Eruca
vesicaria
sativa
India
S18
250021
Eruca
vesicaria
sativa
Egypt
S19
251497
Eruca
vesicaria
sativa
Iran
S20
311742
Eruca
vesicaria
sativa
Poland
S21
426699
Eruca
vesicaria
sativa
Pakistan
S22
426708
Eruca
vesicaria
sativa
Pakistan
S23
426712
Eruca
vesicaria
sativa
Pakistan
S24
633203
Eruca
vesicaria
sativa
Pakistan
S25
633202
Eruca
vesicaria
sativa
United Kingdom
S26
633218
Eruca
vesicaria
vesicaria
Canada
S27
633219
Eruca
vesicaria
vesicaria
Unknown
118
2.4. Optimization of the analysis procedure
The entire analytical procedure was tested for sensitivity, precision, accuracy and limit of
detection
16-20
in order to assess the degree of reliability. The sensitivity was defined as being the
concentration required of an element (in mg/l) to produce a 1% absorption signal, comparable to a
reading of 0.0044 absorption units. The precision of the method was established by the calculation
of between-assay variation coefficients from the data of ten independent analyses, including the
pre-treatment steps, carried out at different times on a commercial mushroom sample. In order to
check the accuracy of the method in the determination of Cu, Fe, Zn, Mn, Ca, Mg, Na, and K, five
samples of ‘‘Citrus Leaves’’ (Standard Reference Material, SRM1572), supplied by the National
Bureau of Standards (NBS), were analysed. The recovery value means of the entire mineral
elements considered are found to be within the interval of confidence (P<0.05) calculated for the
value certified.
2.5. NIRS analysis
Spectra of leaf ground samples from twenty-seven accessions of E. sativa were obtained in
a near infrared spectrophotometer (NIRSystems mod. 6500, Foss-NIRSystems, Inc., Silver Spring,
MD, USA) in the reflectance mode, acquiring their spectra over a wavelength range from 400 to
2500 nm (visible and near infrared regions). The absorbance values (log 1/R, where R is
reflectance) were registered at 2 nm intervals. Calibration equations for ashes, Fe, Cu, Na, K, Ca,
Mg, Mn and Zn were developed using the program GLOBAL v. 1.50 (WINISI II, Infrasoft
International, LLC, Port Matilda, PA, USA). Calibration equations were computed using the raw
optical data (log 1/R, where R is reflectance), or first or second derivatives of the log 1/R data, with
several combinations of derivative (gap) sizes and smoothing [i.e. (0, 0, 1, 1; derivative order,
segment of the derivative, first smooth, second smooth); (1, 4, 4, 1); (1, 10, 10, 1); (2, 5,5, 2); (2,
20, 20, 2)]. Wavelengths from 400 to 2500 nm every 8 nm, were used to perform the different
calibration equations. The regression method employed to correlate spectral information and
mineral content in the samples was modified partial least squares (MPLS). This regression method
constructs a number of factors as linear combinations of the original spectral data, performing a
regression on the factor scores to derive a prediction equation. The final objective of the
mathematical procedure is to reduce the high number of spectral data points (absorbance values
from 400 to 2500 nm every 2 nm, i.e. 1050 data) and to eliminate the correlation of absorbance
values presented by neighbouring wavelengths9. Standard normal variate and detrend
transformations (SNV-DT)21 were used to correct baseline offset due to scattering effects
(differences in particle size among samples). Cross-validation was performed on the calibration set
for determining the best number of terms to use in the equation, as well as to determine the ability
of each equation to predict on unknown samples22.
119
2.6. Statistical analysis
SAS statistical software23 was used to evaluate the mineral concentrations with the PROC
GLM and Duncan mean homogeneity test (P<0.05). Duncan test was used to determine the
significant differences between means of ashes and minerals of the accessions grouped in
countries and individually.
2.7. Average and SD spectra of rocket
The second derivative average spectrum of rocket plants used in this work was obtained to
identify and correlate the different absorption bands to specific absorbers. In the first step, the
original absorbance values at each wavelength (raw optical data from 400 to 2500 nm, every 2 nm)
were averaged, and the resulting average spectrum was standardized using the algorithms SNV
and DT. In a second step, the standardized spectrum was transformed into its second derivative
(2, 5, 5, 2).
The second order derivative transformation of the original spectrum resulted in a spectral
pattern display of absorption peaks pointing downward rather than upward. The SD (standard
deviation) spectrum shows the standard deviations of the absorbance values of the samples at
specific wavelengths (from 400 to 2500 nm, every 2 nm). It is a way of easily displaying those
spectral regions that are more highly variable in apparent absorption among samples and,
therefore, in concentration of a determined absorber. Together with the correlation plot, the SD
spectrum gives information about those wavelengths with high potential of being used in modelling
the MPLS factors for the parameter being studied.
2.8. Correlation plot of total mineral content vs. wavelength in rocket plants
The correlations of the total mineral content vs. wavelength for each plant sample were
obtained by using the whole set of samples, to identify those spectral regions more highly
correlated with the total mineral content in the tissues of rocket. Spectral data were standardized
by using SNV+DT
21
to interpret in a simpler way the correlation plot of spectral data vs. total
mineral content in the whole set of samples. This mathematical pre-treatment of the spectral data
eliminates the background of constant correlation due to any existing relationship between total
mineral content and particle size. In theory, areas matching absorption bands in the spectra of the
constituent being measured should have positive correlations in the correlation plot, while areas
corresponding to absorption bands in the spectra of other constituents could have positive,
negative or zero correlations depending on the inter-correlations between constituents24.
120
a
Fig. 1. Near infrared mean spectrum (a) and standard deviation (b) of rocket samples.
b
Wavelengths
Wavelength
3. Results
3.1. Characterization of ashes and minerals in rocket
Table 2 shows the mean concentrations (mg/100 g of dry weight) and standard errors of
content of ashes, Fe, Cu, Na, K, Ca, Mg, Mn, and Zn of the accessions grouped within countries.
The mean content of ashes and minerals of the countries (expressed in dry weight) were: ashes
ranging from 18.03 to 22.43 mg/100 g from Egypt and Pakistan; Fe ranging from 6.9 to 20.07
mg/100 g for the United Kingdom and Turkey; Cu ranging from 0.73 to 1.64 mg/100 g for Poland
and India; Na ranging from 84.48 to 243.95 mg/100 g for Turkey and Poland; K ranging from 2822
to 6557.2 mg/100 g for Egypt and Iran; Ca ranging from 3184.5 to 5005 mg/100 g for The United
Kingdom and Pakistan; Mg ranging from 240.7 to 484.18 mg/100 g for Egypt and Pakistan; Mn
ranging from 3.43 to 10.16 mg/100 g for The United Kingdom and Unknown; and Zn ranging from
3.27 to 6.71 mg/100 g for Egypt and Unknown respectively. This collection of accessions has been
grown under same conditions of soil and environment. Differences in the accumulation of minerals
have to be due to genetic differences. This variability is essential for breeding programs focus on
the selection of the most adequate lines.
121
122
Table 3 shows the mean concentrations (mg/100 g of dry weight) and standard errors for
the content of ashes and minerals for each accession. The minimum and maximum mean values
of minerals contented in each accession were: 18.03 mg/100 g and 23.55 mg/100 g of ashes for
S18 (Egypt) and S23 (Pakistan); 6.97 mg/100 g and 27.33 mg/100 g of Fe for S25 (U.K.) and S3
(Sicily); 0.591 mg/100 g and 1.81 mg/100 g of Cu for S10 (Italy) and S16 (India); 84.48 mg/100 g
and 283.38 mg/100 g of Na for S15 (Turkey) and S17 (India); 2863.5 mg/100 g and 6461 mg/100 g
of Ca for S14 (Italy) and S24 (Pakistan); 3.43 mg/100 g and 10.16 mg/100 g of Mn for S25 (U.K.)
and S27 (Unknown); 3.26 mg/100 g and 6.71 mg/100 g of Zn for S18 (Egypt) and S27 (Unknown);
2890.7 mg/100 g and 7158 mg/100 g of K for S27 (Unknown) and S11 (Italy); 240.75 mg/100 g
and 689 mg/100 g of Mg for S18 (Egypt) and S24 (Pakistan) respectively. Both analyses showed
significant statistically differences for the content of all the minerals except for the Ca. It is shown in
Table 3 that there is some material that can be used for breeding programs due to their high
content in some of the minerals like the accessions S3, S5, S9 (all of them from Sicily) or S22
(from Pakistan), among others.
The result of the cluster for the accessions grouped within countries and the pie graphs for
the major (Ca, Mg, and K) and minor (Na, Fe, Mn, Zn, and Cu) minerals of the countries for the
first distance of the cluster is presented on Fig. 2. It is worth to mention the nearness of India and
Pakistan in the cluster, which are geographically close one to each other and the distance of the
island of Sicily and the mainland of Italy. Also, it is interesting that the only two accessions of E.
vesicaria subsp. vesicaria belonging to the countries of Canada and Unknown are grouped
together. Each pie for group of countries shows the major or minor minerals expressed as 100%.
The pies for the major minerals show a content of K ranging from 40% to 58%, Ca ranging from
27% to 52%, Mg ranging from 3% to 31%, and Na ranging from 1% to 3%. It is worth to mention
the higher percentage of the content of K of the accessions from Iran, Poland and Sicily, the
content of Mg in Italy and Turkey, and the content of Ca in Canada and Unknown. Regarding to
the pies for the minor minerals it is important to point out the content of and Zn ranging from 14%
to 74%, and the iron ranging from 16% to 64%. The rest of minerals are in low and similar
concentration.
123
Table 3. Mean values and standard errors of ashes and mineral content (mg/100 g of dry weight)
of rocket accessions (Eruca vesicaria subsp. sativa and subsp. vesicaria).
Accession
Ashes
Fe
Cu
Na
Ca
Mn
Zn
K
Mg
S1 (Si)
20.92b
10.35e
0.68b
211.4b
3339.7a
4.46c
3.75f
5564.2c
349.00b
S2 (Si)
21.73b
12.85e
0.817b
216.7b
3601.7a
5.88c
4.98c
6167.8c
411.83b
S3 (Si)
19.29c
27.33a
1.43ab
182.8c
4285.0a
5.62c
4.15e
4045.5e
395.50b
S4 (Si)
20.73b
12.63e
0.638b
182.9c
3947.2a
5.94c
4.02f
6324.8c
510.00b
S5 (Si)
19.60c
21.84b
1.07ab
152.8c
4111.6a
5.43c
4.24e
4754.4d
351.00b
S6 (Si)
21.03b
10.65e
0.767b
168.1c
3696.7a
4.68c
3.98f
6658.2b
375.33b
S7 (Si)
19.23c
12.99d
0.850b
196.6b
4154.3a
6.26c
4.28e
6805.8b
327.50b
S8 (Si)
20.66b
12.74d
1.04ab
188.1c
4175.8a
6.81b
4.79d
6894.5b
358.67b
S9 (Si)
18.78c
13.16d
1.19ab
193.6b
4513.8a
6.76c
3.63g
6500.8b
322.17b
S10 (Si)
20.39b
13.21d
0.592b
165.8c
4004.0a
5.12c
4.19e
5759.7c
401.67b
S11 (I)
19.05c
17.73c
1.34ab
192.3b
3547.2a
6.10c
3.59g
7158.0a
352.83b
S12 (I)
19.46c
23.12b
1.58ab
176.7c
3801.5a
6.18c
3.88f
6034.0c
337.67b
S13 (I)
21.21b
8.26f
1.16ab
260.9b
3275.5a
6.32c
3.71g
3257.7f
382.50b
S14 (I)
20.74b
8.22f
0.832b
208.0b
2863.5a
6.25c
4.39e
3731.8f
380.83b
S15 (T)
19.50c
20.07b
1.05ab
84.5d
3489.8a
3.74d
3.68g
5218.5d
401.25b
S16 (In)
18.99c
15.12c
1.81a
130.1d
4323.0a
4.97c
3.99f
4207.2e
385.33b
S17 (In)
18.99c
8.48f
1.48ab
283.4a
4057.2a
4.96c
4.04f
5210.8d
359.40b
S18 (E)
18.03c
16.81d
1.14ab
220.2b
3478.0a
5.52c
3.27g
3756.0f
240.75b
S19 (Ir)
18.75c
7.64f
0.772b
201.3b
4011.7a
4.87c
4.15e
6557.2b
289.83b
S20 (Po)
18.22c
7.26f
0.733b
243.9b
4107.7a
4.30c
4.41e
6368.3c
300.33b
S21 (Pa)
23.32a
8.50f
0.883ab 254.9b
5540.5a
5.01c
4.54e
3149.3f
371.00b
S22 (Pa)
21.76b
13.73d
1.28ab
196.4b
4206.3a
4.92c
5.08c
4631.8d
477.83b
S23 (Pa)
23.55a
19.95b
1.51ab
154.3c
4297.5a
3.64d
3.88f
5908.8c
467.17b
S24 (Pa)
20.41b
13.44d
1.23ab
209.3b
6461.0a
4.59d
3.89f
4907.3d
689.00a
S25 (UK)
20.37b
6.97f
0.833 b
224.3b
3184.5a
3.43d
4.39d
3589.3f
371.50b
S26 (C)
21.99a
9.27f
0.852 b
210.9b
3636.3a
5.90c
5.40b
3015.3f
396.50b
S27 (U)
21.56b
10.91e
1.04ab
178.4c
3975.0a 10.16a 6.71a
2890.7f
314.83b
12.54
1.01
191.15
3949.84
5.34
4.21
4962.76
374.78
0.417
0.040
4.691
121.97
0.121
0.044
101.71
11.40
Mean
S.E.
20,25
0.153
Duncan's Multiple Range Test. Means with the same letter are not significantly different (p<0.05).
124
Fig. 2. Cluster of the accessions grouped within countries and pie graphs for the major and minor
minerals for the first five groups of countries.
125
3.2. Mean and SD spectra of the rocket plant samples
Figure 1 (1a and 1b) shows the mean and SD spectra SNV+DT [second derivative
treatment (2, 5, 5, 2)] of the freeze-dried samples of rocket used to conduct this work (n=190). The
(2, 5, 5, 2; SNV+DT) average spectrum showed absortion bands in the visible region of the
spectrum with a maximum at λ= 676 nm, which corresponds to electronic transitions in the red,
which has been assigned to chlorophyll25.
The NIR region of the spectrum (Fig. 1a) showed characteristic absorption bands at 1432
and 1916 nm related to O–H stretch second and first overtones of water, respectively; 1726, 2310
and 2348 nm related to C–H stretch first overtones and combination bands of lipids26; at 2058 nm
related to N–H stretch of amides24 and 2000 and 2274 nm related to O–H+C–O deformation, O–H
stretch plus deformation, and O–H+C–C stretch of starch24, respectively. The highest SDs of the
spectral data were found in the visible region of the spectrum (electronic transitions in the red) and
also in the regions from 1800 to 2000 nm and from 2100 to 2300 nm (Fig. 1b).
3.3. Calibration and validation
Range, mean and standard deviation for mineral contents in the calibration set of samples
are shown in Table 4. Total mineral content, and also the contents of Na, Fe, Ca and K showed a
wide range in composition. Results obtained in the calibration and cross-validation processes for
minerals are also shown in Table 4. For all the mineral studied in this work, the second derivative
transformation of the raw optical data, with a gap of 5 nm and 5 and 2 nm for the first and second
smooth, respectively, yielded the equations with the highest accuracy in the cross-validation.
Table 4. Mean, standard deviation (SD), range, calibration and cross-validation statistics for rocket
samples (Eruca vesicaria subsp. sativa and subsp. vesicaria) using SNVD and first and second
derivatives.
Minerals N a
Mean
Range
S.D. a R2 CAL b SEC c R2VALd
Ash
Total
181
185
19.1
8450.5
12- 25
3.1
0.54
3211.2 - 13454.5 2160.3 0.59
2.119 0.31
1378.8 0.52
2.81 1.10
1528.8 1.41
Fe
Cu
Na
K
Ca
Mg
Mn
Zn
180
177
185
175
170
175
170
175
24.3
0.8
138.8
4304
3638.2
5.4
5.14
3.814
6.6 – 95.7
0.06 - 2
13.4-318.9
1635 - 7416
1847 - 5228
1.58 - 13.1
0.94 - 11.39
1.60 - 6.95
7.67
0.23
39.5
684.6
590.9
1.84
1.52
0.59
13.9
0.35
45.32
779.7
957.1
2.22
2.13
0.83
16.5
0.3
67.9
1482
754.2
2.51
1.98
1.1
0.78
0.52
0.66
0.79
0.39
0.44
0.41
0.71
a
N: number of samples used to perform the calibration models.
b
R2 CAL: coefficient of determination in calibration.
126
0.57
0.26
0.56
0.73
0.22
0.27
0.21
0.40
SECVe RPDf
1.18
0.85
1.50
1.90
0.78
1.13
0.92
1.32
c
SEC: standard error in calibration.
d
R2 VAL: coefficient of determination in cross-validation.
e
SECV: standard error of cross-validation.
f
RPD: ratio SD to SECV
3.4. Correlation plot for total mineral content vs. wavelength
Areas of high positive (500-700 nm; 1100–1300 nm; 1400-1900 nm) or negative (1900–
2400 nm) correlations were Wavelengths
found between wavelength and total mineral content in rocket samples
around 500-700, 1100–1300, 1400 and 1900–2400 nm (Fig. 3). These regions had considerable
influence in the spectra due to the strong relationship between minerals and other constituents,
principally with O–H tones and with C–H combination tones (organic functional groups)
26,27
.
Fig. 3. Wavelength correlation of total mineral content in rocket samples using SNVD and second
derivative as treatment.
4. Discussion
4.1. Mineral content in accessions of rocket
The mean results of mineral content of this study (see table 3) for Fe, Cu, Na, Mn, and Zn
(12.54mg Fe/100g, 1.01mg Cu/100g, 191.15mg Na/100 g, 5.34mg Mn/100 g, and 4.21mg Zn/100
g of dry weight) are higher than the results published by Kawashima and Valente-Soares28 who
reported values of 6.66mg Fe/100 g, 0.66mg Cu/100 g, 26.66mg Na/100 g, 2mg Mn/100 g, and
2.66mg Zn/100 g respectively. Nevertheless Bozokalfa and collaborators7 reported higher values of
these minerals than those of this work (77.38mg Fe/100 g, 5.15mg Cu/100 g, 220mg Na/100 g,
30.85mg Mn/100 g, and 29.78mg Zn/100 g). The concentrations of K and Ca (4962.76mg K/100 g,
and 3949.84mg Ca/100 g) were higher than those contents reported by Kawashima and Valente-
127
Soares (2420mg K/100 g, and 653.33mg Ca/100 g), Bozokalfa et al. (3573.33mg K/100 g, and
713.33mg Ca/100 g), and Cavarianni et al. (2866.66mg K/100 g, and 1533.33mg Ca/100 g) 29. The
content of Mg was lower than the concentration reported by Bozokalfa (380 mg Mg/100 g), but
higher than those found in leaves of rocket by Kawashima and Valente-Soares (120mg/100 g) and
Cavarianni et al. (286.66mg/100 g). The concentrations of the different minerals found in the
accessions studied in this work (Table 3) show a wide variability. No significant differences were
found between E. vesicaria sativa and vesicaria regarding to the mineral content except in the
accession S27 for Zn and Mn. Nevertheless it would be necessary a higher number of accessions
to make a taxonomic differentiation in both subspecies.
Table 5 shows the Pearson correlation for the content of minerals. Significant positive
correlations (p< 0.05) were found between the pairs K and Fe, Mg and Ca, Mn and Cu, Mn and
Na, Na and Zn, and Zn and Mn; whereas the significant negative correlations (p< 0.05) were found
between the pairs Na and Fe, Zn and Fe, and Zn and K.
Some vegetables show a high content of minerals, but their bioavailability is low due to the
presence of phytate, which is a main inhibitor of Fe and Zn absorption30. Vegetables are also
another source of dietary Ca. However, Ca absorption from vegetables is generally considered low
because they contain substances, like phytate, oxalate, and dietary fibre components, which bind
Ca forming compounds not absorbable by plants
31
. It has been reported that oxalate salts are
poorly soluble at intestinal pH and oxalic acid is known to decrease Ca absorption in monogastric
animals. Although the effect of oxalate on Ca absorption in humans is less clear
30
. Studies have
shown that foods with high concentrations of phytic acid apart form oxalic acid may reduce Ca
availability. Nevertheless, Brassica vegetables are essentially phytate- and oxalate-free
vegetables; therefore dietary fibre components and organic acids are the constituents that could
influence mineral availability and the consequent absorption 31.
Minerals in diet are required for metabolic reactions, transmission of nerve impulses, rigid
bone formation and regulation of water and salt balance32. The daily requirements of an adult
person are as follows (mg/day): 9–18 Fe, 1.1 Cu, 3100 K, 7–9.5 Zn, 1300-1500 Na, 300–350 Mg,
1.8-2.3 Mn, 900–1000 Ca
33
. Based on our data, supposing that a person consume a course of
rocket salad of approximately 80 g/day (and taking into account a content of moisture of 85%), the
calculated content for all the minerals is below of the recommended values (0.66 mg of Fe, 0,05
mg of Cu, 265 mg of K, 0.22 mg of Zn, 10 mg of Na, 20 mg of Mg, 0.28 mg of Mn and 210 mg of
Ca). The mineral showing the highest content was Ca. The accession S24 (subsp. sativa,
Pakistan) showed a total amount of Ca of 512mg (for 80g of salad expressed in fresh weight) that
means the half part of the daily requirement for this mineral. Therefore, consumption of 80g of
rocket can provide 25% Fe, 11.5% Cu, 2% Na, 57% Ca, 46% Mn, 8% Zn, 20% K, 11% Mg of the
128
daily requirements. The low concentration of sodium (<2%) and the presence of a high amount of
K could suggest the utilization of rocket in an anti-hypertensive diet. In fact K from fruit and
vegetables can reduce blood pressure
34
. Therefore, rocket is a good source of minerals including
iron, potassium, magnesium, manganese, copper and calcium (Table 2) and it is shown that the
quality and concentrations of minerals found in this work in Eruca vesicaria subspecies are proper
for human consumption at nutritional levels.
4.2. NIRS analysis
The wavelength correlation plot for total mineral content showed high correlations in the
visible region related to absorptions by plant pigments (400–700 nm) and in the NIR region,
principally associated with OH overtones (Fig. 3) Others authors who used NIRS for predicting
minerals in forages and legumes reported similar absorption regions, although some differences at
specific wavelength absorptions were found. Because trace elements are found in different
complexes and the complexes appear to be different both within and among forages and legumes,
this will lead to differences in wavelengths selected
12,35
.
The SEC (standard error in calibration) and SECV (standard error of cross-validation)
obtained in this work (Table 4) for the different trace minerals agreed with those reported by other
authors in forages and legumes12,35,36. These authors reported similar results for Fe (R2: 0.74 and
SEC: 15), Zn (R2: 0.72 and SEC: 3.8), and K (R2: 0.82 and SEC: 3.47) and better results for Na
(R2: 0.83 and SEC: 0.7), Cu (R2: 0.82 and SEC: 0.84), Mn (R2: 0.74 and SEC: 50) and Ca (R2:
0.75 and SEC: 1.10).
Cross-validation resulted in coefficients of determination (1-VR) of 0.31, 0.52, 0.51, 0.26,
0.56, 0.73, 0.22, 0.44, 0.21, 0.40 for ashes, total mineral, Fe, Cu, Na, K, Ca, Mg, Mn and Zn
contents (Table 4, Fig. 3), indicating that the 31%, 52%, 51%, 26%, 56%, 73%, 22%, 44%, 21%,
40%of the variability present in the data was explained by the respective calibration equations.
Limited studies have been done to exploring the capabilities of NIRS to determine minerals in
cruciferous plants. The SECV obtained in the cross-validation were lower than their respective
SDs, indicating that NIRS is able to determine Ash, total mineral, Fe, Na, Mg, K and Zn
concentration change in the tissues of rocket.
When a cross-validation is performed in the calibration set, NIR prediction error is defined
as the standard error of cross-validation (SECV). Statistically, the SECV is the standard deviation
for the residuals due to differences between reference and NIR predicted values for samples used
in the calibration, using a specific calibration equation. For a comparison of the potential of the
prediction among the equations obtained, a standardization of the different SECVs is needed. In
this way, the RPD, defined as the SD to SECV ratio
37
was estimated for each equation. As it is
129
shown in Table 4, the prediction ability of the calibration equations obtained for minerals in Eruca
vesicaria were in the order K>Fe>Na>total mineral>Zn>Ash>Cu>Mn>Ca.
From SEP (standard error of prediction) and SD data reported in forages, legumes
12,35,36,38,39
and cruciferous
40
for ash and minerals, RPDs for tall fescue (Festuca arundinacea
Schreb.), crested wheatgrass (Agropyron cristatum and A. desertorum), alfalfa (Medicago sativa
L.), white clover (Trifolium repens L.), Red clover (Trifolium pratense L.), Persian clover (Trifolium
resupinatum L.); Bird’s-foot-trefoil (Lotus corniculatus ) and Indian mustard (Brassica juncea) were
calculated. Although sometimes differences could exist between SECV and SEP, in general terms
RPDs reported in this work in rocket are similar for K (RPD: 1.95) for forages
Indian mustard
40
38
, Zn (RPD: 1.34) for
and Cu (RPD:0.9) for crested wheatgrass. However, RPDs in rocket were lower
than those obtained for legumes for Ash, Na, Fe, Mn and Ca (RPD: 3.52, 2.12, 2.08, 2.38, 2.27,
respectively 12,39.
The different r2 values obtained in the cross-validation for the equations reported of Na and
K (Table 4, Fig. 4), were characteristic of equations that can be used for a good separation of the
samples in the validation set into high, medium and low Na and K contents
9
(Fig. 4).
Poor
calibrations were obtained for Ash, Cu Mn and Ca where coefficients of determination were smaller
probably due to the narrow concentration range of some of these elements and/or low
concentration of the associated organic compounds sensed by NIRS (Fig. 4). The total mineral
content, Fe, Mg and Zn equations showed 1-VR values characteristic of equations that could be a
useful tool for preliminary screening of Eruca vesicaria lines for mineral content in a breeding
programme.
5. Conclusions
There is a great variability in relation to the content of minerals in the accessions from
different geographic origins. Significant differences for all the minerals, except for the Ca were
found between the accessions. This is an important issue for breeding programs and safety food
studies and there are some interesting accessions, like S3, S5, S9 (all of them from Sicily) or S22
(from Pakistan) for the selection and improvement of mineral content in rocket. Rocket is a good
source of minerals because of its tendency of hyper-accumulation of some of them. This study
reflects the importance of rocket in the contribution of mineral content, especially in the contribution
of Fe, Mn, K, and Ca to human diet.
The results presented in this paper also show that it is possible to use NIRS technology for
determining mineral contents in ground samples of Eruca vesicaria plants for screening purposes.
130
The use of this technique represents an important reduction of the analysis time, at a low cost and
without using hazardous chemicals, and will be used in future research aiming to select the best
genotypes after the screening of thousands of plants in a breeding program of Eruca vesicaria.
Acknowledgements
The authors wish to express their thanks to the Consejería de Innovación, Ciencia y
Empresa (Junta de Andalucía), Project P06-AGR-02230, for the funding for this research, and also
to the United States Department of Agriculture (USDA) for providing the seeds used in this work.
Myriam Villatoro-Pulido was supported by Instituto de Investigación y Tecnología Agraria y
Alimentaria (INIA) contract.
131
Ash
Fig. 4. NIRS predicted data vs. chemical reference data for mineral in rocket (dry weight).
Total Mineral Content
Fe
Cu
Na
132
K
Fig. 4. Continued.
Ca
Mg
Mn
Zn
133
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137
CAPÍTULO V
Análisis de la actividad biológica in vitro de extractos de rúcola (Eruca
vesicaria subsp. sativa (Mill.) Thell) y sulforrafano.
Artículo en preparación
Analysis of in vitro biological activity of extracts of rocket, Eruca vesicaria subsp. sativa
(Mill.) Thell and sulforaphane
Myriam Villatoro-Pulido1, Maria Traka2, Jaouad Anter4, Zahira Fernández-Bédmar4, Rafael Font3,
Andrés Muñoz-Serrano4, Richard Mithen2, Ángeles Alonso-Moraga4, Mercedes Del Río- Celestino3
1
2
IFAPA Centro-Alameda del Obispo, Córdoba, Spain
Phytochemicals and Health Programme, Institute of Food Research, Norwich Research Park,
NR4 7UA Norwich, United Kingdom
3
IFAPA Centro La Mojonera, Almería, Spain
4
Genetics Department, Campus Universitario de Rabanales, University of Córdoba, Spain.
138
Abstract
Rocket, Eruca vesicaria subsp. sativa (Mill.) Thell., is a member of the Cruciferae family that
has recently gained popularity consumed as raw salad. The health benefits of consuming
cruciferous vegetables are considered to be due to the biological activity of glucosinolate
degradation products (isothiocyanates). However, it is conceivable that other phytochemicals
within crucifers may also have biological activity that could contribute to these healthy benefits. In
this work we investigate the in vitro activity of the isothiocyanate sulforaphane (SF), the in vitro
activity of the treatment with rocket extracts, and the relation with their phytochemical composition.
Three approximations have been used to analyse the effects of the SF and the Eruca vesicaria
subsp. sativa extracts: (i) effect on cell growth, (ii) effect on apoptotic induction activity, and (iii)
effect on expression on p21 protein, an essential protein for the cellular growth. Our results show
that the extracts affect differently to the normal (PNT1A), and tumoural cells (HL60 and PC3)
depending on the assay and the pattern of phytochemicals, especially on the isothiocyanate
content. Apoptotic induction activity in the treatments has been observed at different degree with
the accessions of Eruca vesicaria subsp. sativa and SF. The expression of p21 protein was not
enhanced when the cells were treated with the rocket extracts at the different studied
concentrations. This study helps to understand the capacity of some phytochemicals compounds
to affect the promotion and proliferation of tumoural cells as a key point of the cancer prevention.
Keywords:
carotenoids,
cytotoxicity,
Eruca
vesicaria
subsp.
sativa,
glucosinolates,
isothyocianates, polyphenols, rocket, sulforaphane.
Abbreviations: ER, erucin; GAPDH, glyceraldehyde-3-phosphatase dehydrogenase; GLs:
glucosinolates; HL-60, human leukaemia cells; ITCs, isothiocyanates; PBS, phoetal bovine serum;
PC3, human cancerous prostate cell line; PNT1A, Human post pubertal prostate; SF,
sulforaphane;
WST-1,
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate.
139
1. Introduction
The consumption of some vegetables such as crucifers has been related with a reduction in
the risk of cancer (Juge et al., 2007). The biological activity of isothiocyanates found in cruciferous
vegetables may provide an explanation for this correlation (Traka et al., 2010). The isothiocyanates
are derived from the hydrolysis of the glucosinolates, which are sulphur containing secondary
metabolites found primarily in the Cruciferae family (Mithen, 2001). Rocket, Eruca vesicaria subsp.
sativa (Mill.) Thell., is a member of the Cruciferae family. This vegetable is widely used mainly as
raw vegetable or as a spice for its peculiar taste. It contains health-promoting phytochemicals such
as glucosinolates, isothiocyanates, phenols, flavonoids and carotenoids (Mithen et al., 2000; Juge
et al., 2007). There is wide evidence of in vitro and in vivo activity of these compounds although
there are limited studies using the vegetal material itself (Lamy et al., 2008).
The isothiocyanates SF, erucin (ER) and iberin, a sulfoxide analogue of SF (Jadhav et al.,
2007), are thought to be some of the best isothiocyanates (ITCs) candidates for anticancer
therapy. SF has been proved to increase the antioxidant capacity of the cell related also to Phase
II detoxification enzymes, inhibits tumoural cell proliferation, and can act as apoptotic inductor
(Juge et al., 2007; Fimognari et al., 2007). Treatment of tumoural cells with ER and SF induces
Phase III detoxification system in human carcinoma cell lines through a common molecular
mechanism (Harris and Jeffery, 2008). Several phenols have shown healthy properties such as
antioxidant capacity in vivo and in vitro, induction of Phase II detoxification enzymes, inhibition of
proliferation and apoptosis among others (Androutsopoulos et al., 2010; Prasain et al., 2010;
Heijnen et al., 2001; Pietta, 2000; Kong et al., 2001; Ramos et al., 2007). Rutin has a powerful in
vitro antioxidant capacity against some antioxidant testing systems (Yang et al., 2008), as well as
anti-inflammatory and antitumoural (Calabró et al, 2005). Myricetin is highly effective scavenging
reactive species of oxigen (ROS), and exhibits cytoprotective effects against oxidative stress
(Shimmyo et al., 2008). Quercetin is a powerful antioxidant
in every system used (Makris and
Rossiter, 2001). Carotenoids have also shown antioxidant activity depending on the localisation
and concentration (Van den Berg et al., 2000). The carotenoid β-carotene and the xantophyll lutein
act both as antioxidants. β-carotene is the most potent provitamin A (Di Mascio et al., 1989) and
lutein is one of the carotenoid implicated in the reduction of the risk of cataract and macular
degeneration (Seddon et al., 1994).
The knowledge about the phytochemical content and profile that contributes to improve the
consumer’s health in the Cruciferae family can be essential for the selection of certain accessions
in Genetic Breeding programs. For that reason it is essential to study the in vivo and in vitro
behaviour of the extracts of these vegetables apart from the phytochemical compounds it-selves.
140
In this work we have studied the in vitro activity of some accessions of rocket previously
characterized for covering a wide range of glucosinolate, isothiocyanate, polyphenol, and
carotenoid content (see chapter 3). These accessions are named attending to the total content of
glucosinolates: Low Glucosinolate Content 1 (LGC1), Low Glucosinolate Content 2 (LGC2), High
Glucosinolate Content 1 (HGC1), and High Glucosinolate Content 2 (HGC2).
An interesting chemopreventive therapy strategy is the correlation between cytotoxicity and
apoptosis-inducing activity (Qian et al., 2009). The programmed cell death or apoptosis plays
important role in the development and maintenance of homeostasis and elimination of damaged or
no longer necessary cells. Apoptosis include DNA fragmentation and other morphological cell
features (Kerr et al., 1972; Higuchi, 2003; Juge et al., 2007; Gasper et al., 2007).
We have used three human cell lines for this work: 1) HL60 human leukaemia cells have
been used to assess the possible tumouricide effect of the plant material and SF, and the
apoptosis-inducing activity. HL60 tumour cells have been intensely used in literature to study the
control of proliferation (Collins et al., 1978; Fahey and Talalay, 1999; Conte-Annazetti et al., 2003).
2) A human cancerous prostate cell line, PC3 (Kaighn et al., 197), which is deficient in the tumour
suppressor gene PTEN, was used to analyse the viability and the effects of rocket extract on the
p21 protein compared to SF. The protein p21 is a CDK inhibitor protein that is essential for cellular
growth, differentiation and apoptosis (Xiong et al., 1993). 3) The cell line PNT1A, Human post
pubertal prostate, established by immortalisation of normal adult prostatic epithelial cells by
transfection with a plasmid containing SV40 genome (Cussenot et al., 1991), was used to assess
the viability of the normal cells treated with the plant extract.
The objectives of this paper were: a) to investigate the in vitro activity of the treatment
with Eruca vesicaria subsp. sativa extracts and sulforaphane with three human cell lines, and b) to
relate the phytochemical composition of the Eruca vesicaria subsp. sativa extracts with the
biological activity.
2. Material and methods
2.1. Plant material and greenhouse experiments
Seeds of Eruca vesicaria subsp. sativa LGC1 (cv. Sky), LGC2, HGC1 and HGC2 were
obtained from Tozer Seeds Lyd (Cobham, Surrey, U.K.); Faculté des Sciences Agronomiques of
Gembloux, Belgium; Dipartimento di Scienze Botaniche of Palermo, Italy; and Botanischer Garten
der Universitat of Karlsruhe, Germany, respectively. Seeds were germinated in Petri dishes at a
temperature of 25ºC for 48 h. Pots were placed under natural light, temperature of 27/18ºC
141
(day/night) and a relative humidity of 50/70% (day/night) in the greenhouse. When the plants
reached proper height (8-12 cm), they were transferred to soil.
2.2. Sample preparation
The accessions were collected once they were ready for human consumption. Then, they
were washed with tap water, weighed to assess their biomass, stored at -80º C and freeze-dried.
2.3. GLs analysis by liquid chromatography with ultraviolet photometric detection
Freeze-dried leaves of rocket (100 mg) were ground in a Janke and Kunkel (A10 mill, IKALabortechnik). The flour was heated at 75 °C to inactivate myrosinase (15 min, 2.5 mL of 70%
aqueous methanol). Sinigrin (200 µL, 10 mM) was added as an external standard (Sinigrin hydrate,
85440 Fluka). After centrifugation (5 min, 5 x 103g) glucosinolates were extracted with 2 mL of 70%
aqueous methanol. 1 mL of the GL extracts was pipetted onto the top of an ion-exchange column
with Sephadex DEAE-A25 (1 mL, 40-125 µm bead size, 30000 Da exclusion limit). Purified
sulfatase (75 µL) was added for desulfation (EC 3.1.6.1, type H-1 from Helix pomatia, SigmaAldrich). Desulfated GLs were eluted with Milli-Q (Millipore) ultrapure water (2.5 mL) and analyzed
with a 600 HPLC instrument (Waters) equipped with a 486 UV absorbance detector (Waters) at
229 nm. A Lichrospher 100 RP-18 in Lichrocart column (125 mm x 4 mm i.d., 5 µm particle size,
Merck) was used for separation and the HPLC chromatogram was compared to the desulpho-GL
profile provided by three certified reference materials recommended by U.E. and ISO (CRMs 366,
190 and 367) (Commission of the European Communities, report EUR 13339 EN, 1-75) (Wathelet
et al., 1991). The content of GLs was quantified using sinigrin according to the ISO norm (ISO
9167-1, 1992). The total GL content was computed as the sum of all the individual GLs present in
the sample.
2.4. SF determination by liquid chromatography and mass spectrometry detection (LC-MS)
Freeze-dried leaves (40mg) of E. vesicaria subsp. sativa Mill. were hydrolysed in
phosphate saline buffer (PBS), incubated during 2 hours and then centrifuged (13,000g, 30 min at
4 °C) to obtain ITCs from GLSs. Supernatant was analysed using liquid chromatography with mass
spectrometric detection with positive API-ES (LC/MS) with an 1100 Agilent LC system (Agilent
Technologies, Waldbronn, Germany) equipped with a diode array detector and a mass
spectrometric detector. SF was monitored using absorbance at 229 nm, and with a selected ion
monitoring (SIM) targeted on m/z 178.0. SF quantification was performed by comparing the mass
spectrum and the retention time (S8044 Laboratories, Inc., USA) basing on retention time and
mass spectrum. A gradient liquid chromatographic separation was performed on a C18- 3µm (150
x 4.6 mm) column, 0.1% formic acid in H2O and 0.1% formic acid in CH3CN as mobile phase (flow
rate 0.3 mL/min).
142
2.5. In vitro cytotoxicity assays
2.5.1. Cell cultures and incubation conditions
The human leukaemia cell line HL60 was supplied by Dr. José M. Villalba-Montoro
(Department of Cell Biology, Univ. Cordoba, Spain). The HL60 cell line was grown in suspension in
RPMI 1640 medium (Invitrogen) containing the antibiotics penicillin, streptomycin and amphotericin
(commercial mixture, A5955, antibiotic-antimycotic solution 100x stabilised, Sigma), 10% heatinactivated fetal bovine serum (FBS) (S01805, Linus) and L-glutamine (G7513, Sigma). Cultures
were incubated at 37 °C in a humidified atmosphere containing 5% CO2 (Shel Lab). In order to
maintain logarithmic growth, cultures were passed in 10ml bottles every 2-3 days. The PC3 human
prostate cancer cell line and the PNT1A Human post pubertal prostate normal cell line were
purchased from the American Type Culture Collection (Rockville, MD, USA) and the Health
Protection Agency Culture Collections respectively. PC3 and PNT1A cell lines were cultured as
monolayers in HAMS (F-12K Nutrient Mixture Kaighn’s Modification (1x) liquid, Invitrogen) and in
RPMI-1640 media (Invitrogen), respectively. Both media were supplemented with 10% heatedinactivated fetal bovine serum (FBS) (S01805, Linus), 2 mM L-glutamine (210551-040 Invitrogen),
penicillin (100 IU/ml, P3032, Sigma-Aldrich), and maintained in a humidified incubator at 37°C and
5% CO2.
2.5.2. Survival assay
Cell viability was carried out by the Trypan Blue dye (T8154, Sigma) exclusion assay. The
starting cell concentration was 105 cells/ml. The tumoural cell line was incubated in 2 ml well plates
with increasing concentrations of filtered lyophilized plants and the major isothiocyanate, SF,
whereas the negative controls had only culture medium. Three separate experiments were carried
out to calculate means for statistical analysis and plotting. Cells were counted adding an aliquot of
10 µl of Trypan Blue dye to 10 µl of the culture. After mixing it, cells were counted on a Neubauer
chamber under a light inverted microscope (AE30/31, Motic). Cells were counted after 72 h of
exposure to establish a growth curve. The concentration of tested compound causing 50%
inhibition of cell growth, IC50 value, was also estimated. Curves were plotted as survival
percentage with respect to controls at 72 hours of growth.
2.5.3. Cell viability assay
The effect of the accessions on the PC3 and PNT1A cells was evaluated using a WST-1
(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) cell proliferation kit
(Cat. No. 11 644 807 001, Roche Applied Science, Germany). Both cell lines were cultured in 96well plates in a final volume of 100 µl/well culture medium. Cells were cultivated until 70% of
confluence before adding the ITCs in PBS in various concentrations. Each dose was tested three
times. WST-1 reagent (10 µl/well) was added and incubated for 40 minutes. A scanning multiwell
microplate ELISA reader (ELx808, Ultra Microplate Reader; BIO-TEK Instruments, Inc., Winooski,
143
VT) was used to quantify the formazan dye produced by metabolically active cells measured at an
absorbance at 450 nm.
2.6. Apoptotic induction activity
Apoptosis is characterised by fragmentation of DNA at internucleosomal linker sites giving
bands of 180-200 bp and multiples (Higuchi, 2003). The HL-60 tumoural cell line was used with the
aim of analyse apoptotic induction. Tumoural cells were treated for 5 hours at the same
concentrations as the cytotoxicity test with the four Eruca accessions and SF. Cells were
centrifuged at 4000rpm during 5 minutes, washed with ice-cold PBS and pelleted. DNA was
extracted with a commercial kit (Dominion mbl, MBL 243). Total DNA was treated with RNAse
(Quiagen) during 30 minutes at 37ºC. A yielding of 1500ng of DNA was loaded on 2% agarose gel
followed by a 120 min and 50v electrophoresis. The oligonucleosomal DNA fragments were
visualized by staining with ethidium bromide and photographed under UV light.
2.7. Western blotting analysis
PC3 cells were treated when they were at a 70% of confluence. After 24 hours, cells were
lysed with RIPA buffer (100 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1% NP-40 and 50 mM Tris-HCl
[pH 7.2]) and then centrifuged. The supernatant fraction was measured for protein concentration
using a Bicinchoninic Acid Kit (BCA). Equivalent amount of proteins were separated by 10% SDSpolyacrilamide gel electrophoresis and transferred to nitrocellulose membranes. Transfer quality
was verified with Ponceau S staining solution. Membranes were blocked using blocking buffer (5%
non fat milk in TBS Tween 1%) for 1 h under shaking and incubated with the anti-p21 Waf1/Cip1
(Cat.No.#2946, Cell Signalling Technology, Inc.) at the dilutions and times recommended in the
manufacturer’s instructions. After treatment with appropriate HRP-conjugate secondary antibodies
(Cell Signaling Technology, Inc.), the blot was developed by Fluor s-Max Multi-Imager System
(Biorad Laboratories, Inc.). SuperSignal® West Pico Chemiluminescent Substrate (PIERCE) was
used according to the commercial recommendations. Quantitative analysis of protein gels was
performed by using Quantity One software (Biorad Labs). Glyceraldehyde-3-phosphatase
dehydrogenase (GAPDH) was detected as a loading control for the blot (Cat.No.4300, Ambion,
Inc.).
2.8. Statiscal analysis
One-way analysis of variance (ANOVA) applied to data of phytochemical contents was
used to detect differences between accessions. Statistical analysis was done by using the SPSS
Version 10.0 software (SPSS, 2000).
144
3. Results
3.1. Phytochemical composition of the rocket accessions
The phytochemical composition of the rocket accessions (Eruca vesicaria subsp. sativa)
(analysed in Chapter III) is summarised in Table 1 for glucosinolates, isothyocianates, polyphenols
and carotenoids content.
Table 1. Phytochemical composition of four accessions of rocket (Eruca vesicaria subsp. sativa)
leaves (see Chapter III).
Compound
LGC1
LGC2
HGC2
HGC1
0.14±0.00c(1)
3.64±0.00c
9.15±0.30a
14.02±0.30b
2.70±0.03b
6.06±0.03b
8.10±0.80a
19.40±1.04b
4.03±0.03a
12.64±0.20a
8.10±0.80a
27.65±0.30a
0.63±0.08c
14.90±1.46a
11.40±0.08a
28.24±1.61a
0.15±0.02b
0.75±0.09a
0.02±0.00c
nd(2)
5.90±0.33a
0.97±0.01a
1.55±0.12a
0.01±0.00
1.33±0.07b
0.34±0.01a
1.37±0.03a
nd
0.81±0.01b
0.15±0.00a
0.57±0.01b
nd
Polyphenols
Isoquercitrin
Rutin
Quercetin
Myricetin
Ferulic acid
Total content
1023±12.01b
nd
nd
nd
33.00±1.50a
32248.5a
774.0±12.01b
18.00±1.50b
nd
3.00±3.02a
30.60±3.02a
18750.1b
1621.5±9.01a
12.01±0.03b
nd
nd
nd
32700.2a
1680±15.00a
27.00±1.50a
13.50±15a
3.00±0.01a
48.00±4.50a
4474.5a
Carotenoids
β-Carotene
Lutein
Total content
0.90±0.03
8.30±0.10c
19.51±0.41c
14.90±0.10b
55.60±0.20b
87.91±0.20b
14.62±0.10b
124.30±0.40a
260.31±0.50a
Glucosinolates
Glucoerucin
Glucoraphanin
Glucosativin
Total content
Isothiocyanates
Sulforaphane
Sulforaphane- nitrile
Iberin
Erucin
(1)
20.71±0.20a
115.20±0.40a
263.91±0.30a
For each phytochemical component, means followed by a common letter are not significantly
different from each other using Duncan Multiple Range Test (P<0.05).
(2)
nd: non-detected. Content of glucosinolates and isothiocyanates expressed as µmol/g of dry
weight (dw). Content of polyphenols and carotenoids expressed as µg/g dw. Data expressed as
mean±standard deviation.
HGC1 and HGC2 accessions showed the highest glucoraphanin content and total content
of glucosinolates with approximately 14 and 28 µmol /g dry weight, respectively. Accessions
showed differences in the hydrolysis of glucoraphanin and formation of SF ranging from 4.12%
145
(LGC1 accession) to 97.35% (LGC2 accession). Pearson’s correlation of glucoraphanin and SF
was not significant in leaves of rocket (-0.38, P >0.05), which suggested differences in the
myrosinase activity within accessions. This leads to the conversion of glucosinolates to other
metabolites like nitriles, which are less potent in inhibiting cancer cell growth than the
corresponding isothiocyanates (Nastruzzi et al., 2000), and/or to epithionitriles, which there is no
available information in the literature regarding their biological activities (Wittstock et al., 2003).
The mean content of total phenolic ranged from 4474.5 to 32700 µg/g dw (Table 1). Our
results showed variability between accessions and demonstrated that leaves of rocket, specially
LGC1 and HGC2 accessions are an excellent source of phenolic compounds (32248.5 and
32700.2, respectively). Nevertheless LGC2 and HGC1 were the accessions, which had more
qualitative variability regarding to the phenols studied.
The total carotenoid content ranged from a minimum mean value of 19.51 µg/g dw (LGC1
accession) to a maximum mean value of 263.91 µg/g dw (LGC2 accession). HGC1 and LGC1
accessions showed the maximum and minimum mean values for lutein (124.30 and 8.30 µg/g dw),
respectively. LGC2 exhibited the highest β-carotene concentration with a mean content of 20.71
µg/g dw.
3.2. Effects of Eruca vesicaria subsp. sativa extracts on cell growth
Cell survival of the selected material was evaluated by the trypan blue exclusion assay after
72 h of treatment on HL60 cells. The accessions were assayed at the concentrations of 0.031,
0.062, 0.125, 0.25, 0.5, 1 and 2 mg/ml, but the HGC2 accession which was assayed at the
concentrations of 0.062, 0.125, 0.25, 0.5, 1, 2 and 4 mg/ml. SF was assayed at the concentrations
of 4.35, 8.7, 17.5, 25, 50 and 100 µM. The results (Fig. 1) are expressed as survival percentage
with respect to the controls. Both the SF and the LGC2 accession have been highly cytotoxic. The
results of the cytotoxic assays showed different IC50, being 0.4, 0.42, 1 mg/ml and 6.5 mM for
HGC2, LGC2, HGC1 accessions and SF respectively. In the case of the accession with the lowest
glucosinolate content (LGC1), the IC50 was not reached. The shapes of the curves were different
for each case. The SF curve showed the most negative slope, followed by the curves of LGC2 and
HGC2 accessions.
Cell viability was also measured with the WST-1 proliferation assay. The results for this
assay did not show significant differences for the four accessions of rocket at low concentrations
for 24h of treatment in PNT1A and PC3 cells viability (Fig. 1). We have also found that at low
concentrations, LGC2 accession of rocket had the same effect on the viability of PNT1A and PC3
cells.
146
Fig. 1. Effects of SF and extracts from four accessions of rocket (LGC1, LGC2, HGC1 and HGC2)
on viability of HL-60, PC3 and PNT1A cells. Cell viability was assessed by trypan blue exclusion
test and WST-1 assay. Data are expressed as percentages of control (mean±SD values from three
independent experiments).
3.3. Relation between viability of HL-60 cells and SF content
Viability of HL-60 cells and SF content in the four Eruca accessions at a concentration of
2mg/ml is plotted in figure 2. It shows a negative correlation between both parameters. The viability
decreased as the SF content increased in the samples.
147
Fig. 2. Correlation between viability of HL60 cell line and SF content for the four rocket accessions
at 2mg/ml.
3.4. Effects of Eruca vesicaria subsp. sativa extracts on apoptotic induction activity
The apoptosis-inducing activity of the crude extract of the four accessions of Eruca
vesicaria subsp. sativa and SF was investigated in the HL60 human promyelocytic leukaemia cell
line (Figure 3). This cell line was treated with the same concentrations as in the trypan blue
cytotoxicity assay for 5 h of treatment. SF induced internucleosomal DNA fragmentation at the
concentrations of 8, 16 y 32 µM. Some accessions induced also internucleosomal DNA
fragmentation but with less intensity.
Fig. 3. Nucleosomal DNA fragmentation. HL-60 leukemic cells were exposed to various
concentrations of the extracts of the accessions (A) LGC1, (B) LGC2, (C) HGC2, (D) HGC1, and
(E) SF for 5 hours. DNA was extracted from cells and was subjected to 2% agarose gel
electrophoresis at 50 V for 120 minutes. (A), (B), (C), and (D): lane M, DNA size markers; lane 1,
control; lane 2, 0.08 mg/ml; lane 3, 0.175 mg/ml; lane 4, 0.25 mg/ml; lane 5, 0.5 mg/ml; lane 6, 1
mg/ml; lane 7, 2 mg/ml. (E): lane M, DNA size markers; lane 1, control; lane 2, 4.35 µM; lane 3, 8.7
µM; lane 4, 17.5 µM; lane 5, 25 µM; lane 6, 50 µM; lane 7, 100 µM.
(A)
(B)
(C)
(D)
148
(E)
3.5. Effects of Eruca vesicaria subsp. sativa extracts on the expression of the p21 protein
LGC2 accession at a concentration of 2mg/ml did not affect the expression levels of p21
protein in PC3 cells (Fig. 4). The treatment with rocket extract was also compared with the SF at a
concentration of 25 µM that increased the expression of the protein as it has been reported in
literature (Dashwood and Ho, 2007; Melchini et al., 2009; Kim et al., 2010).
Fig 4. Effects of the treatment of PC3 cells with the LGC2 accession of rocket (2mg/ml) and SF (25
µM) on the expression of p21 protein. Western blotting analysis was performed to measure p21
protein levels in treated and untreated PC3 cells for 24 h and quantitative analysis of 21 protein
levels was performed using Quantity one software. Glyceraldehyde-3-phosphatase dehydrogenase
(GAPDH) was used as a loading control for the western blot.
149
4. Discussion
Prevention by dietary phytochemicals is an important approach in cancer management
(Surh, 2003). The dietary intake of cruciferous vegetables has been associated with a lower risk of
some types of cancers, and it is thought that the hydrolysis products of glucosinolates, the
isothiocyanates, and other phytochemicals like phenols and carotenoids are the responsible
molecules of this protective effect (Mithen et al., 2000; Juge et al., 2007). It has been extensively
reported in literature over the past 20 years in vivo and in vitro studies that propose
isothiocyanates as important chemopreventive agents and antitumour activity, although the
mechanisms of these activities is not fully clarified (Wu et al., 2009). In the past years the trend in
Genetic Breeding has been to increase the content of healthy related phytochemicals like
glucosinolates and isothiocyanates (Faulkner et al., 1998; Sarikamis et al., 2006). Nevertheless it
has been also proposed that concentrations required for the ITCs to exert protective activity are in
the low micromolar range (<30µmol/L) and higher concentrations can make disappear this
protective effect (Tang and Zhang, 2004). Therefore, it is essential to study the in vitro behaviour of
the extracts of these vegetables, with different profile of phytochemicals, apart from the
compounds it-selves. The information about the profile that contributes to improve the consumer’s
health is necessary for the selection of accessions in genetic breeding programs.
The results of trypan blue exclusion assay (Fig. 1) agree with those previously reported
concluding that SF is an effective agent against the proliferation of a variety of cancer cells in a
dose-dependent manner (Yao, et al., 2008, Shankar et al., 2008; Kim et al., 2010). The SF IC50
was significantly lower than those found for the treatments with the vegetal extracts, apart from the
accession with the lowest glucosinolate content (LGC1), in which this IC50 was never reached. In
Figure 2 it is observed as the content of SF can be related to the antiproliferative effect.
The viability results with the WST1 assay (Fig. 1) are not in agreement with those previously
reported, where both SF and ER showed a strong antiproliferative effect at higher concentrations
(Harris and Jeffery, 2008). It has been also reported that the dietary isothiocyanate SF inhibits the
growth of the cancerous PC3 prostate cells at lower concentrations than the non-cancerous
PNT1A cell line (Traka et al., 2010). It is known that some polyphenol compounds can causes
overestimation of the WST assays, leading to inconsistent results between cell growth and cell
viability (Maioli et al., 2009). Therefore the differences between trypan blue and WST assays could
be attributed to the fact that WST can be reduced by phenolics (Anter et al., 2011). This artefact
also explains the low differences between PC3 cells and PNT1A cells treated with the accession
LGC2.
SF induced internucleosomal DNA fragmentation (Fig. 3) at the concentrations ranging
from 8 to 32 µM. This agrees with previous studies, like the one performed by Yeh and Yen (2005)
150
in which results indicated that SF showed a strong growth-inhibitory effect, but with higher doses of
SF than in our study (30 to 100 mM). Nevertheless accessions did not show the same intensity of
fragmentation. This can be due to the time of exposure of the substances or to a non
fragmentation-associated apoptotic mechanism.
The p21 protein is a CDK inhibitor that is essential for cellular growth, differentiation and
apoptosis (Xiong et al., 1993). It is known that the induction of p21 molecule causes the arrest in
both G1 and G2 checkpoints in some cell lines and also that this protein plays an important role in
SF-induced cell cycle arrest regulated by tumour suppressor p53 in response to DNA damage. The
study performed by Kim et al. (2010) showed that SF induced the p21 expression and its
transactivation to induce G2/M phase arrest. It can be found in literature that ER and SF can cause
a significant increase in p21 levels at high concentrations (15–25 µM) in A549 human
adenocarcinoma lung epithelial cells (Melchini et al., 2009). Experimental data obtained from
prostate cell lines, included PC3, showed the increase of p21 protein expression by SF 15 µM
(Dashwood and Ho, 2007). Our results showed that the treatment with the with the accession with
the highest SF content (LGC2) at a concentration of 2mg/ml did not affect the expression levels of
p21 protein in PC3 cells (Fig. 4), in contrast to the expected results obtained elsewhere with the
treatment of SF, where has been observed an induction of p21. The fact that the extract does not
induce the expression of the protein can be due to the content of SF in the LGC2 accession. At this
concentration (12.5µM) the accession was not able to increase the protein level expression and
others concentrations of vegetal extracts should be assayed.
This study has shown that SF has been proved to be an important chemopreventive agent,
and LGC2 and HGC2 accessions, but especially the LGC2 showed the best in vitro behaviour with
a high antiproliferative effect. Our results have also indicated that the healthy protective effect of
rocket accessions can be due to the content in isothiocyanates and the phenol and carotenoid
content.
The LGC2 accession had low glucosinolates content (19.4 µmol /g dw) and the highest SF
and total carotenoid content among the accessions analysed, with 5.90 µmol /g dw and 264 µg /g
dw, respectively. On the other hand, the HGC2 accession had high glucosinolate content (27.6
µmol /g dw), low SF content (1.3 µmol /g dw) and the highest total polyphenol content with 32700.2
µg /g dw. Previous studies have indicated different mechanisms of action for different
phytochemicals (Lampe. 1999), which may help to explain the observed healthy benefits in this
work. Lutein, the major carotenoid found in Eruca has been shown to have anti-carcinogenic
activities in vitro (Mares-Perlman et al., 2002). Flavonols such as quercetin have been shown to
modulate DNA damage from genotoxins in vitro (Agullo et al., 1996) and have anti-proliferative
effects (Kuo 1996). The ferulic acid have been found to exert free radical scavenging activity,
151
protection against DNA breakage in mammalian cells and inhibition of phase I enzyme activity
(Ferguson et al., 2005). Therefore, the in vitro behaviour of an entire plant extract can be attributed
to the combination of its bioactive components. Our results agreed with previous works performed
in other species of the family Cruciferae like watercress (Nasturtium officinale R. Br.). In these
studies the antigenotoxic effect of watercress extract (reduction in damage to DNA), indicate that
phenethyl isothiocyanate was not identified as the only potentially active components (Kassie et
al., 2003; Boyd et al., 2006). Watercress is a rich source of a variety of phytochemicals inclluding
not only glucosinolate derivarives but flavonoids such as quercetin, hydroxycinnamic acids, and
carotenoids such as β-carotene and lutein.
We would suggest a selection criterion of rocket accessions taking into account the
conversion to isothiocyanate rather than to the content of total glucosinolates or a glucosinolate in
particular for a breeding program. This is due to the fact that the glucosinolate levels do not reflect
the amounts of the corresponding isothiocyanate that will be formed (Matusheski et al., 2006).
Furthermore the conversion of glucosinolates, to other metabolites than isothiocyanates, have
proven little or no healthy activities and they even may counteract the protective effect of
isothiocyanates (Nastruzzi et al., 2000, Wittstock et al., 2003). Along with GL/ITC conversion the
high content and wide variability of phenols and carotenoids have to be taken into account for
selection of accessions with added value for human health.
5. Conclusions
SF has been shown to be useful in chemoprevention. Our results suggest that the high
content of isothiocyanate in the rocket extracts exert the healthiest role with respect to the DNA
protection. The biological activity (cytotoxicity and apoptosis) of rocket is due to the ITC content
and the combined effects of phytochemicals that taken together may also account for this DNAprotective effect of rocket. Therefore, further studies should be conducted to define this cytotoxic
behaviour, and the mechanisms of action must be examined more closely.
A promising panorama is depicted concerning to the evaluation and genetic selection of
plant material with an appropriate pattern of nutraceutical compounds in order to obtain better
quality products for human nutrition.
Acknowledgements
The authors thank to the Consejería de Innovación, Ciencia y Empresa (Junta de
Andalucía, Spain) for funding the Project [P06-AGR-02230] and to Gloria Fernández, (IAS-CSIC,
152
Cordoba) for technical assistance in the analysis of plants. We also acknowledge to the Faculté
des Sciences Agronomiques of Gembloux, Belgium; Dipartimento di Scienze Botaniche of
Palermo, Italy and Botanischer Garten der Universitat of Karlsruhe, Germany for providing the
seed material for this work. Myriam Villatoro was supported by a Instituto Nacional de
Investigación y Tecnología Agraria y Alimentaria (INIA) contract.
153
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157
158
CAPÍTULO VI
Actividad in vivo de extractos de rúcola (Eruca vesicaria subsp. sativa
(Miller) Thell) y sulforrafano
Enviado a:
Food and Chemical Toxicology
In vivo biological activity of rocket extracts (Eruca vesicaria subsp. sativa (Miller) Thell) and
sulforaphane
M. Villatoro-Pulido1, R. Font2, S. Saha3, Obregón-Cano4, S., J. Anter5, Zahira Fernández-Bédmar5,
A. De Haro Bailón4, A. Alonso-Moraga5, M. Del Río- Celestino2
1
IFAPA, Centro-Alameda del Obispo, Córdoba, Spain
2
IFAPA, Centro La Mojonera, Almería, Spain
3
Phytochemicals and Health Programme, Institute of Food Research, Norwich Research Park,
NR4 TUA Norwich, United Kingdom
4
Department of Agronomy and Plant Breeding, Institute of Sustainable Agriculture, Spanish
Council for Scientific Research (CSIC), Alameda del Obispo s/n, 14080 Córdoba, Spain
5
Genetics Department, Campus Universitario de Rabanales, University of Córdoba, Spain.
159
Abstract
Eruca is thought to be an excellent source of antioxidants as phenolic compounds,
carotenoids, glucosinolates and their degradation products, like isothiocyanates. Sulforaphane is
one of the most potent antioxidants of Eruca isolated until the date. In this work we investigate: i)
the DNA protective activity of Eruca extracts and sulforaphane (under and without oxidative stress)
in Drosophila melanogaster; and ii) the influence on Drosophila melanogaster life span treated with
Eruca extracts and sulforaphane. Our results showed that among the Eruca extracts tested,
intermediate concentrations of the Es2 accession exhibited no genotoxic activity, antigenotoxic
activity and also enhanced the health span portion of the live span curves. Sulforaphane presented
a high antigenotoxic activity in the SMART test of D. melanogaster and intermediate
concentrations of this compound (3.75 µM) enhanced average life span. The results of this study
indicate the presence of potent antigenotoxic factors in rocket, which are being explored further for
their mechanism of action.
Keywords: Eruca vesicaria subsp. sativa, rocket, glucosinolates, isothyocianates, antigenotoxicity,
life span.
Abbreviations: GL: glucosinolate, ITC: isothiocyanate, SF: sulforaphane.
160
1. Introduction
Senescence is a multifaceted process caused by a gradual decline in physiological function
and an increased incidence of various diseases, including cancer, neurodegenerative diseases,
and diabetes (Fontana et al., 2010; Kenyon et al., 2010). A prime candidate of senescence has
been the damage caused by the reactive oxygen species (ROS), which are endogenous molecular
species generated primarily during respiration of the cell (Harman 1956; Kenyon, 2010). This
theory named as the free radical theory of ageing (Harman, 1956), states that the cumulative
damage by oxygen free radicals is the major driver of ageing. It has been also proposed that
increasing antioxidant defence should decrease steady-state levels of oxidative damage, which
would then increase life span (Lutsgarten et al., 2011). Evidence suggests also that transformed
cells use ROS signals to drive proliferation and other events required for tumour progression. This
confers a state of increased basal oxidative stress; making them vulnerable to chemotherapeutic
agents that further augment ROS generation or that weaken antioxidant defences of the cell
(Schumacker, 2006). It seems that anticancer therapy is frequently efficient in this early stages of
the disease (Benhar et al., 2002). Thus food consumption plays an important role modulating the
quality of live of an organism (Boyd et al., 2011). The strongest evidence that vegetables and fruits
are related to a potential reduction in cancer risk comes from epidemiological studies for
cruciferous vegetables (Gasper et al., 2007). Among vegetables, species of cruciferous like rocket
(Eruca vesicaria subsp.sativa) contain a range of health-promoting phytochemicals including
carotenoids, vitamin C, fibres, polyphenols, and glucosinolates (GLs). It has been speculated that
the isothiocyanates (ITCs) like sulforaphane (SF), obtained from hydrolysis of GLs, are in great
part responsible for the protective effects of cruciferous vegetables (Mithen, 200; Juge et al.,
2007). SF is the most investigated ITC in vivo and in vitro and it is derived from the GL
glucoraphanin.
The extensive knowledge of the genetics of Drosophila melanogaster and the long
experimental experience with this organism has made it useful in genetic toxicology (Graf et al.,
1984; Graf et al., 1998). The somatic mutation and recombination test (SMART) in wings of
Drosophila melanogaster is based on the loss of heterozygosity for two genetic markers that affect
the phenotype of wing hairs. There is a wide variety of compounds and complex mixtures that have
been assayed with the SMART test, such as food additives, beverages and insecticides (Yeh and
Yen, 2005, Romero-Jiménez et al., 2005; Villatoro-Pulido et al., 2009).
Anti-ageing and anti-degenerative assays can be carried out using different Drosophila
melanogaster strains in order to perform life span trials with specific chronic diets in controlled
environments (Fleming et al., 1992). In this work we describe a study designed to examine the
effects of rocket extract and SF supplementation in the diet on life span in Drosophila
161
melanogaster. This animal model is an excellent system to investigate the longevity-promoting
properties of compounds and nutraceutical extracts because it has a short life span, can be
cultured on simple diets, and has a rich genetic resource with a fully sequenced genome, and,
more importantly, over half of the fly genes have mammalian homologs (Boyd et al., 2011; Jones
et al., 2011). With the evidences of the protective role of cruciferous vegetables and the
compounds that they contain described previously we also have hypothesised that the treatment
with SF and/or Eruca extracts may enhance the Drosophila life span.
With these evidences of the protective effects of SF and cruciferous vegetables we have
focused on the concept of chemoprevention. DNA protection and enlarging life span assays in
animal models are two important in vivo insights for evaluation of the health-promoting role of
rocket and SF. The main objectives of this work were: (i) to analyse the glucoraphanin, SF and
total GL content of four accessions of Eruca sativa, (ii) to establish the genotoxic and antigenotoxic
activities of the Eruca material, using the in vivo Drosophila melanogaster SMART test, (iii) and to
examine the effects of Eruca sativa extracts and SF supplementation in the diet on the life span
and health span of Drosophila.
2. Material and methods
2.1. Plant material and greenhouse experiments
Seeds of Eruca vesicaria subsp. sativa Es1 (cv. Sky), Es2 , Es3 and Es4 were obtained
from Tozer Seeds Lyd (Cobham, Surrey, U.K.), Faculté des Sciences Agronomiques of Gembloux,
Belgium, Botanischer Garten der Universität of Karlsruhe, Germany, and Dipartimento di Scienze
Botaniche of Palermo, Italy, respectively. They were germinated in Petri dishes at a temperature of
25ºC for 48 h. Pots were placed under natural light, temperature of 27/18ºC (day/night) and a
relative humidity of 50/70% (day/night) in the greenhouse. When the plants reached proper height
(8-12 cm), they were transferred to soil.
2.2. Sample preparation
The accessions were harvested once they were ready for human consumption. They were
washed with tap water, weighed to assess their biomass, stored at -80º C and freeze-dried.
2.3. Glucosinolate analysis by liquid chromatography with ultraviolet photometric detection
Freeze-dried leaves of rocket (100 mg) were ground in a Janke and Kunkel (A10 mill, IKALabortechnik). The flour was heated at 75 °C to inactivate myrosinase (15 min, 2.5 mL of 70%
aqueous methanol). Sinigrin (200 µL, 10 mM) was added as an external standard (Sinigrin hydrate,
162
85440 Fluka). A second extraction was applied after centrifugation (5 min, 5 x 10 3g) with 2 mL of
70% aqueous methanol. 1 mL of the GL extracts was pipetted onto the top of an ion-exchange
column with Sephadex DEAE-A25 (1 mL, 40-125 µm bead size, 30000 Da exclusion limit). Purified
sulfatase (75 µL) was added for desulfation (EC 3.1.6.1, type H-1 from Helix pomatia, SigmaAldrich). Desulfated GLs were eluted with Milli-Q (Millipore) ultrapure water (2.5 mL) and analysed
with a 600 HPLC instrument (Waters) equipped with a 486 UV absorbance detector (Waters) at
229 nm. A Lichrospher 100 RP-18 in Lichrocart column (125 mm x 4 mm i.d., 5 µm particle size,
Merck) was used for separation and the HPLC chromatogram was compared to the desulpho-GL
profile provided by three certified reference materials recommended by U.E. and ISO (CRMs 366,
190 and 367) (Commission of the European Communities, report EUR 13339 EN, 1-75) (Wathelet
et al., 1991). The content of GLs was quantified using sinigrin according to the ISO norm (ISO
9167-1, 1992). The total GL content was computed as the sum of all the individual GLs present in
the sample.
2.4. Sulforaphane determination by liquid chromatography and mass spectrometry detection (LCMS)
Freeze-dried leaves (40mg) of E. vesicaria subsp. sativa were hydrolysed in phosphate
saline buffer (PBS), incubated during 2 hours and then centrifuged (13,000g, 30 min at 4 °C) to
obtain ITCs from GLSs. Supernatant was analysed using liquid chromatography with mass
spectrometric detection with positive API-ES (LC/MS) with an 1100 Agilent LC system (Agilent
Technologies, Waldbronn, Germany) equipped with a diode array detector and a mass
spectrometric detector. SF was monitored using absorbance at 229 nm, and with a selected ion
monitoring (SIM) targeted on m/z 178.0. SF quantification was performed by comparing the mass
spectrum and the retention time (S8044 Laboratories, Inc., USA) basing on retention time and
mass spectrum. A gradient liquid chromatographic separation was performed on a C18- 3µm (150
x 4.6 mm) column, 0.1% formic acid in H2O and 0.1% formic acid in CH3CN as mobile phase (flow
rate 0.3 mL/min).
2.5. Genotoxicity assays
2.5.1. Strains
Detailed genetic information of the mutations is provided by Lindsley and Zimm (Lindsley
and Zimm, 1992). Two Drosophila melanogaster strains were used containing genetics markers on
the left arm of chromosome 3: a) mwh/mwh, carrying the wing cell marker multiple wing hairs
(mwh) (Yan et al., 2008) and b) flr3/In(3LR)TM3, ri pp sep bx34e es BdS (flr3/TM3, BdS abbreviated).
Being the wing cell marker flare (flr3) (Ren et al., 2007) a zygotic recessive lethal, which is
maintained in the strain over the balancer chromosome TM3. All experimental flies were reared in
a humidified, temperature-controlled incubator at 25 ºC and 65% humidity.
163
2.5.2. Treatment procedure
Crosses
mwh/mwh males were mated to virgin females with the genotype flr3/TM3, Bd . An optimal
S
design requires the double of females than males. Flies are allowed to mate for 3 days to obtain an
optimal production of hybrid eggs on the fourth day after mating.
Treatments
Genotoxicity and antigenotoxicity tests were carried out as described by Graf et al. (Graf et
al., 1984; Graf et al., 1998). Flies are allowed to lay eggs for an 8-hour period. After 72 ± 4 hours,
the emergent transheterozygous larvae were washed with distilled water and transferred to
treatment vials. Lyophilized leaves of the accessions and SF (Sigma S6317) were dissolved in
distilled water at room temperature at decreasing concentrations. For SF treatments, the highest
concentration was selected supposing that 100% of the maximum value for the content of
glucoraphanin contended in 5mg/mL (highest concentration of rocket extract analysed) of vegetal
extract was hydrolysed to sulforaphane. Fresh solutions were added to 4 mL treatment vials with
0.85 g of Drosophila Instant Medium (Formula 4-24, Carolina Biological Supply, Burlington NC,
USA). Negative and positive controls were prepared with distilled water and hydrogen peroxide
0.12M
(Sigma,
cat.
number
H-1009)
as
genotoxine
(Romero-Jiménez
et
al.,
2005).
Antigenotoxicity tests were carried out by mixing the mutagen (hydrogen peroxide) with the
lyophilised samples in the same concentrations as genotoxicity tests concentrations. Groups of
100 larvae were fed in chronic treatments until pupation at 25 ± 1 °C. The emergent adult flies
were stored in a 70% ethanol solution.
2.5.3. Wing scoring
The wings of transheterozygous marker flies (mwh flr+/mwh+ flr3) were separated in sexes,
removed and mounted on slides using Faure´s solution. Both dorsal and ventral surfaces of the
wings were scored under a microscope with the 400x magnification. Wing hair mutations (clones)
were scored among a total of 24,000 monotricoma cells/wing. When positive results are obtained
in genotoxic treatments (positive control and some single treatments), the balancer wings
(mwh/TM3,BdS) were also mounted and analysed.
2.5.4. Data evaluation and statistical analysis
Wing spot data are spliced into small single spots of 1 or 2 mwh or flr3 cells, large single
spots with three or more mwh or flr3 cells, and twin spots with mwh and flr3 cells. The total number
of spots was also evaluated.
For evaluation of the genotoxic effects, the frequency of spots in the treated assay was
compared to negative controls. The statistical significance of spots frequency per wing was
164
assessed using a multi-decision procedure to determine whether a result was positive, negative or
inconclusive, based on two alternative hypotheses (Frei and Würgler, 1988).
In the balancer-heterozygous genotype (mwh/TM3,BdS), mwh spots are produced mainly
by somatic point mutation and chromosome aberrations, since mitotic recombination between the
balancer chromosome and its structurally normal homologue is a lethal event. To quantify the
recombinogenic activity of the mutagenic samples, the frequency of mwh clones on the marker
transhetorozygous wings (mwh single spots plus twin spots) was compared with the frequency of
mwh spots on the balancer transheterozygous wings. The difference in mwh clone frequency is a
direct measure of the proportion of recombination (Zimmering et al., 1990).
To evaluate the antigenotoxic effects of the selected material and compounds, the
percentage of inhibition of mutagenic events by lyophilised samples was calculated from the
control-corrected frequencies of total spots, as proposed by Abraham (1994). More detailed
information is provided by Villatoro-Pulido (Villatoro-Pulido et al., 2009).
Inhibition= (genotoxine alone-sample plus genotoxine)x100 /genotoxine alone
2.6. Life span experiments
The life span experiments were carried out following a modified method of Chavous et al
(2001). The same strains and synchronised crosses as the genotoxicity and antigenotoxicity tests
were used. Three-day old transheterozygous larvae were fed with SF and Es2 and Es4 accessions
at increasing concentrations and water as negative control. Adults were collected after one week
using light CO2 anaesthesia. Then they were transferred to 22 mg of Carolina medium
supplemented with 1 mL of a dilution of the same compound and concentration as they had before
hatching. To minimise any density effects on mortality, we separated two vials with ten males each
and another two with ten females each for each concentration. Deaths were scored and the
medium renewed twice a week.
2.6.1. Statistical analysis of life span
The Kaplan–Meier estimates of the survival function for each control and concentration are
plotted as survival curves. The statistical analyses and signification of the curves were assessed
by the SPSS 15.0 statistics software (SPSS Inc. Headquarters, Chicago, IL, USA) using the LogRank (Mantel-Cox) method.
165
3. Results
3.1. Glucosinolate and isothiocyanate composition of the rocket extracts
Accessions of rocket differed in the total content of GLs (Table 1), which ranged from 14.79
(Es1) to 28.24 (Es4) µmol /g of dry weight (dw). Glucoraphanin content ranged from 3.24 (Es1) to
14.90 (Es4) µmol /g of vegetal tissue, whereas SF content ranged from 0.14 (Es1) to 5.90 (Es2)
µmol /g dw. Pearson’s correlation of glucoraphanin and sulforaphane content was not significant in
leaves of rocket (-0.38, P >0.05). This suggests differences in the hydrolytic enzymes activities
among accessions as it has been proposed in broccoli (Matusheski et al., 2006).
Kim and Ishii (2006) reported levels of glucoraphanin (1.25 µ mols/g dw) and total GL
content (11 µ mols/g dw), which were lower than those we found (minimum mean values of
glucoraphanin of 3.24 µmol /g and 14.79 µmol /g of total glucosinolate content). Bennet and
collaborators (2006) reported values of glucoraphanin (6.1 µ mols/g dw) in the range of our work.
Our data about the isothiocyanate content are in concordance to those reported by Melchini et al.
(2009), who reported values of 3.46 µ mol of sulforaphane /g dw.
Table 1. Phytochemical composition of the accessions of rocket (Eruca vesicaria subsp. sativa)
leaves.
Accessions
Compound
Es1
Es2
Es3
Es4
Total content
14.79±1.20
20.10±1.12
27.2±0.98
30.52±0.90
Glucoraphanin
3.24±0.15
6.30±0.15
12.88±0.28
16.96±1.11
0.14±0.07
6.25±0.15
0.82±0.09
1.24±0.13
Glucosinolates
Isothiocyanate
Sulforaphane
Content of GLs and ITCs expressed as µmol /g of dw. Data expressed as mean±standard
deviation.
3.2. Genotoxicity and antigenotoxicity assays of treatments with rocket extracts and sulforaphane.
The Somatic Mutation And Recombination Test (SMART) was applied to assess the
genotoxicity and antigenotoxicity of the rocket accessions, which differed for the contents of GLs,
glucoraphanin and SF. Table 2 shows the results for the genotoxicity assays. The water-negative
and hydrogen peroxide positive controls for the proliferative imaginal discs of the wing in
Drosophila larvae gave the expected results. Hydrogen peroxide exhibited a total mutation rate
(0.375 mutant clones/wing), which duplicated the negative control rate (0.175), implying that the
166
Table 2. Genotoxicity in the Drosophila SMART Test of the treatments with rocket and
sulforaphane.
Frequency of spots per wing (number of spots) and diagnosis
Compounds
(1)
Number
Small spots
Large spots (more
Twin spots
Total spots
of wings
(1-2 cells)
than two cells)
m=5
m=2
m=2
m=5
H 2O
80
0.15 (12)
0.0125 (1)
0.0125 (1)
0.175 (14)
H2O2 (0.12 M)
40
0.35 (14) +
0 (0) -
0.025 (1) -
0.375 (15) + *
Rocket (mg/ml)
Es1
[0.675]
40
0.125 (5) i
0 (0) -
0.025 (1) i
0.15 (6) i
[1.25]
40
0.2 (8) i
0 (0) -
0 (0) -
0.2 (8) i
[2.5]
40
0.175 (7) i
0.075 (3) i
0 (0) -
0.25 (10) i
[5]
40
0.125 (5) i
0.1 (4) +
0.05 (2) i
0.275 (11) i
[0.625]
40
0.175 (7) i
0.025 (1) i
0.025 (1) i
0.225 (9) i
[1.25]
40
0.1 (4) -
0.025 (1) i
0 (0) -
0.125 (5) -
[2.5]
40
0.15 (6) i
0 (0) -
0.025 (1) i
0.175 (7) i
[5]
40
0.175 (7) i
0 (0) -
0 (0) -
0.175 (7) i
[0.625]
40
0.125 (5) i
0.05 (2) i
0 (0) -
0.175 (7) i
[1.25]
40
0.2 (8) i
0.025 (1) i
0.025 (1) i
0.25 (10) i
[2.5]
40
0.15 (6) i
0.05 (2) i
0 (0) -
0.2 (8) i
[5]
40
0.275 (11) i
0.05 (2) i
0 (0) -
0.325 (13) i
[0.675]
40
0.1 (4) -
0. (0) -
0.05 (2) i
0.15 (6) i
[1.25]
40
0.125 (5) i
0 (0) -
0.05 (2) i
0.175 (7) i
[2.5]
40
0.125 (5) i
0 (0) -
0 (0) -
0.125 (5) -
[5]
40
0.325 (13) +
0.075 (3) i
0.025 (1) i
0.425 (17) + *
[5] Serrate
40
0.075 (3) -
0 (0) -
0 (0) -
0.075 (3) -
Es2
Es3
Es4
SF (µM)
(1)
[12.6]
40
0.175 (7) i
0 (0) -
0.025 (1) i
0.2 (8) i
[6.3]
40
0.1 (4) -
0 (0) -
0 (0) -
0.1 (4) -
[3.15]
40
0.25 (10) i
0 (0) -
0 (0) -
0.25 (10) i
[1.575]
40
0.125 (5) i
0 (0) -
0 (0) -
0.125 (5) -
Statistical diagnoses according to Frei and Würgler (Frei and Würgler, 1988): + (positive), -
(negative) and i (inconclusive). Significance levels α = β = 0.05.
167
(2)
Genotoxic activity in balancer-heterozygous (mwh/TM3, BdS) larvae for genotoxic
concentrations.
Table 3. Antigenotoxicity in the Drosophila SMART Test of the treatments with rocket and
sulforaphane in combined treatments with hydrogen peroxide as genotoxine.
Frequency of spots per wing (number of spots) and diagnosis
Compounds
(1)
Number
Small spots
Large spots (more
Twin spots
Total spots
Inhibition
of wings
(1-2 cells)
than two cells)
m=5
m=2
Rate
m=2
m=5
H 2O
80
0.15 (12)
0.0125 (1)
0.0125 (1)
0.175 (14)
H2O2 (0.12 M)
40
0.35 (14)+
0 (0) -
0.025 (1)-
0.375 (15)+
Rocket (mg/mL) + 0.12 M H2O2
Es1
[0.675]
40
0.225 (9) i
0.025 (1) i
0.025 (1) i
0.275 (11) i
0.26
[1.25]
40
0.075 (3) -
0.025 (1) i
0 (0) -
0.1 (4) -
0.73
[2.5]
40
0.15 (6) i
0.05 (2) i
0 (0) -
0.2 (8) i
0.46
[5]
40
0.025 (1) -
0 (0) -
0 (0) -
0.025 (1) -
0.93
[0.625]
40
0.125 (5) i
0 (0) -
0.025 (1) i
0.15 (6) i
0.6
[1.25]
40
0.1 (4) -
0.05 (2) i
0 (0) -
0.15 (6) i
0.6
[2.5]
40
0.3 (12) i
0 (0) -
0 (0) -
0.3 (12) i
0.2
[5]
40
0.175 (7) i
0 (0) -
0.025 (1) i
0.2 (8) i
0.46
[0.625]
40
0.15 (6) i
0.025 (1) i
0 (0) -
0.175 (7) i
0.53
[1.25]
40
0.175 (7) i
0.025 (1) i
0.025 (1) i
0.225 (9) i
0.4
[2.5]
40
0.275 (11) i
0 (0) -
0 (0) -
0.275 (11) i
0.26
[5]
40
0.175 (7) i
0.025 (1) i
0 (0) -
0.2 (8) i
0.46
[0.675]
40
(6) i
0.025 (1) i
0 (0) -
0.175 (7) i
0.53
[1.25]
40
0.2 (8) i
0.025 (1) i
0.025 (1) i
0.25 (10) i
0.33
[2.5]
40
(5) i
0.025 (1) i
0 (0) -
0.15 (6) i
0.6
[5]
40
(12) i
0.025 (1) -
0 (0) -
0.325 (13) i
0.13
Es2
Es3
Es4
SF (µM)
168
[1.575]
40
0.125 (5) i
0 (0) -
0.025 (1) i
0.15 (6) i
0.6
[3.15]
40
0.075 (3) -
0.025 (1) i
0.025 (1) i
0.125 (5) -
0.66
[6.3]
40
0.15 (6) i
0.025 (1) i
0.025 (1) i
0.2 (8) i
0.46
[12.6]
40
0.175 (7) i
0 (0) -
0 (0) -
0.175 (7) i
0.53
(1) Statistical diagnoses according to Frei and Würgler (Frei and Würgler, 1988): +
(positive), - (negative) and i (inconclusive). Significance levels α = β = 0.05.
accuracy of the genotoxicity and antigenotoxicity assays was ensured (Romero-Jiménez et al.,
2005). 45 and 80 wings were evaluated for each concentration and the water control respectively.
Data were expressed as small, large, twin and total spots/wing scored. Negative results were
found for all the accessions and SF except for the Es4 accession at highest concentration (5
mg/mL), this accesion had the highest content of GLs and low content of SF (Table 1). Many of the
concentrations assayed exhibited even lower genotoxicity values than the water control. It can be
observed that there is recombinogenicity associated (82%) (Table 2) in the mutagenic
concentration when we look on the spots/ wing scored in balancer wings (Serrate phenotype). The
hydrogen peroxide is a recombinogenic genotoxine with an activity of 44% (Villatoro-Pulido et al.,
2009), which is nearly the half of recombinogenic potency of the higher concentration of the Es4
accession.
The antigenotoxicity against the oxidative mutagen hydrogen peroxide in the Drosophila
wing spot test is included in Table 3. The test showed that Eruca accessions and SF were able to
detoxify the genotoxic activity of hydrogen peroxide although no dose effect was observed. The
vegetal samples and SF exhibited a percentage of inhibition that ranged from 0.13 (Es4 accession
at a concentration of 5 mg/mL) to 0.93 (Es1 accession at a concentration of 5 mg/mL).
In order to study the exact relation of the GLs content of the accessions to its genotoxic
activity, we represented the total spots/wing as dependent variable with respect to the GLs content
corresponding to the assayed Eruca concentrations. A significant linear tendency (p≤ 0.004) was
observed and positive significant associations are also found for glucoraphanin contents (p≤ 0,001)
and total mutation rates.
3.3. Survival assay of treatments with rocket extracts and sulforaphane.
Consumption of diet rich in vegetables is thought to increase antioxidant defence and
therefore life span. To evaluate whether supplementing Drosophila diet with SF and Eruca extracts
could promote the survival of flies, we compared the life span of flies fed with Drosophila Instant
Medium to flies fed supplemented with different concentrations of SF extracts (Fig.1), wih different
concentrations of Es2 accession extracts (high SF: 6.25 µmol/g) (Fig. 2) and with different
concentrations of Es4 accession extracts (low SF: 1.24 µmol/g) (Fig. 3).
The control has lived a maximum of 124 days, which was higher than the treatment with
SF. The maximum survival time of flies treated with SF in days (Fig. 1) ranged from 90 days (for
the concentration of 1.87 µM) to 104 days (for the concentration of 3.75 µM). Figure 2 shows the
169
results of flies fed with the accession Es2, which lived from 92 days (for the concentration of 2.5
mg/mL) to 129 days (for the concentrations of 0.625 and 1.25 mg/mL). Nevertheless the highest
survival was found for the concentration of 1.25 mg/mL in the Es4 accession (Fig. 3). This
accession showed a minimum survival of 107 days for the concentration of 2.5 mg/mL and a
maximum of 133 days. The control life span was only exceeded by the concentrations of 0.625 and
1.25 mg/mL for Es2 accession and 1.25 mg/mL for Es4 accession.
Additional information on the life span data related to the quality of life can be obtained from
the highest part of the life span curves. We have compared the survival curves of living flies for
water control and the rest of substances at ≥75%. This part of the living flies entire life span was
considered the health span of a curve, which is characterized by low and more or less constant
age-specific mortality rate values (Soh et al., 2007). Log Rank (Mantel-Cox) analyses of the
corresponding health span curves of SF, Es2 and Es4 accessions have been performed and the
results are summarized in Fig. 4. All the SF concentrations increase significantly the health span of
Drosophila compared to the control, except for the highest concentration (15 µM), although a doseresponse was not observed. The lowest concentration in the Es2 accession extract (0.625 mg/mL)
and the concentrations of 0.625 and 2.5 mg/mL in the Es4 accession extract increase also
significantly the quality of life of the individuals comparing to the control.
Fig. 1. Survival curves of Drosophila melanogaster fed with different concentrations of
sulforaphane.
170
Fig. 2. Survival curves of Drosophila melanogaster fed with different concentrations of Es2
accession of rocket.
Fig. 3. Survival curves of Drosophila melanogaster fed with different concentrations of Es4
accession of rocket.
171
Fig. 4. Health span means at ≥75% survival flies (for control, sulforaphane, and Es2 and Es4
accessions) and significances of the Log Rank (Mantel-Cox) analysis of the curves compared
to the control treatment. Asterisks indicate higher values of significant (P≤0.05).
4. Discussion
Chemoprevention is basic for the reversion, inhibition and prevention of cancer, and,
optimally, requires the use of non- toxic agents that inhibit molecular steps during the carcinogenic
pathway (Fimognari et al., 2007). Studies to analyse global gene expression have found little
evidence to support potential mechanisms derived from in vitro and in vivo experiments to explain
the epidemiological data which show that consuming a cruciferous vegetable for one year may
reduce risk of cancer. Although they found evidence for the perturbation of signalling pathways
implicated in carcinogenesis and inflammation (Traka et al., 2008). To this date few studies have
been carried out using the plant material of Eruca to asses the antigenotoxic effects. Previous
works conducted with tumoural cells have not shown genotoxic effects even at high concentrations
and genotoxic effects derived from vegetables of the family Cruciferae and their ITCs using the
Salmonella assay and CHO cells (cancerous hamster ovary cells) have been reported (Lamy et al.,
2008). In a recent work, researchers (Jin et al., 2009) found evidence that the protective effect of
GLs and their hydrolysis products of Eruca vesicaria subsp. sativa on colon cancer cells HT-29
might be counteracted by other phytochemicals present in rocket leaves. Additionally, they stated
that a very small amount of GLs is required to initiate cell defence mechanisms against oxidative
stress, and therefore increased concentrations do not have an additive effect.
172
Furthermore, it has been sought that concentrations required for the ITCs to exert
protective activity are in the low micromolar range (<30µmol/L) and higher concentrations can
make disappear this protective effect (Tang and Zhang, 2004).
Recently, Vázquez-Gómez et al., (2010) reported that SF resulted mutagenic only in the
standard (ST) cross of the SMART test of Drosophila with a significant induction of small spots
frequency. These researchers, however, used a concentration of 140 µM of SF, while in our work
the highest concentration was 12.6 µM and did not resulted genotoxic. It is thought that SF acts
indirectly to increase the antioxidant capacity of the cell and does not work as a direct-acting
antioxidant or pro-oxidant (Juge et al., 2007). Our results are in agreement with those of Tang &
Zhang, (2004). In our work the only accession that showed genotoxicity was Es4 at a
concentration of 5 mg/mL. The recombinogenic activity–calculated as Zimmering et al., (1990)
recommended is 82% out of the total genotoxic activity induced in this work (0.425 mwh
spots/wing), which means a high level of the recombinogenic activity of the similarly high Eruca
sativa Es4 concentrations chronic treatments.This accession had the highest content of
glucoraphanin (16.96 µmol /g) and total content of GLs (30.52 µmol/g), although the yielding SF
content was low (1.24 µmol /g).
More than two hundred effects of hydrogen peroxide as genotoxine (Allen and Tresini,
2000) have been described, and there are several antigenotoxic studies that demonstrate its
genotoxic power –both mutagenic and recombinogenic (Romero- Jiménez et al., 2005, VillatoroPulido et al., 2009). All Eruca accessions were able to detoxify the genotoxine supplemented with
the treatments.
Our data show that the highest concentration (5 mg/mL) of Es4 accession extract is related
to higher mutation rates, and the rest of concentrations of the four Eruca sativa extracts are safe
with respect to the integrity of the DNA in proliferative somatic cells of Drosophila melanogaster
(Table 2 y 3).
The highest life span corresponded to the Es4 accession. We hypothesised that Es2
accession (with low GL: 22.10 µmol/g and high SF: 6.25 µmol/g content) should result healthier
than Es4 (with high GL: 30.52 µmol/g and low SF: 1.24 µmol/g) since that beneficial effects of
glucosinolates are attributed to isothiocyanates rather than to the glucosinolates themselves.
Cruciferous plants, including rocket have been shown to produce not only isothiocyanates but
other alternative bioactive breakdown products as nitriles and epithionitriles, which at higher
concentrations may initiate mutagenic, cytotoxic, and carcinogenic processes (Martin-Dietz et al.,
1991). The results of this study showed that Es4 accession was not toxic when was consumed
chronically, possibly the content of other phytochemicals (polyphenols, carotenoids) may
173
counteract the possible deleterious effect of this accession. Previous studies have indicate that
isothiocyanates were not the only potentially active components responsible of antigenotoxic
effects in other species of Cruciferous and flavonoids and carotenoids had a potent antioxidant
activity (Boyd et al., 2006; Gil et al., 2004; Kassie et al., 2003). The results for health span curves
with the treatment of 0.625 and 2.5 mg/mL concentrations of Es4 accession are in agreement with
those obtained for geno/antigenotoxicity in which the same concentrations of plant extracts show
the lowest mutation rates.
The Drosophila strains used in this work are not extended life mutants. Average life span
data of Drosophila melanogaster vary widely and are strongly dependent on the rearing conditions.
When we compare our results of average control life span to those appearing in previous works,
we found values lower than ours (between 33 and 100 days) (Trotta et al., 2006; Mockett and
Sohal, 2006; Li et al., 2008). Furthermore, assays of supplementation with different concentrations
of treatments like nectarine, cocoa, broccoli or lamotrigine, reported values also lower with normal
diet (like 10% sugar and 10% yeast extract), dietary restriction (2.5% sugar and 2.5% yeast
extract), or anoxia treatments (Li et al., 2008; Bahadorani and Hilliker, 2008; Avanesian et al.,
2010; Boyd et al., 2011).
5. Conclusions
Our results suggest that the high GL content accession at highest concentration were
genotoxic, while the rest of them exhibited genotoxicity values lower than the water control. All the
Eruca accessions for all the concentrations and SF were able to detoxify the genotoxic activity of
hydrogen peroxide with inhibition rates ranging from 0.13 to 0.93. Drosophila health span was
increased with all the concentrations of SF (except the highest) and with low and intermediate
concentrations (0.625 mg/ ml of Es2 and Es4, and 2.5 mg/ ml of Es4) of Eruca extract treatments.
These endpoints affecting the DNA integrity and antidegenerative activity of rocket can help to
understand the role and potential use of plants containing GLs. In addition, these results may
suggest that moderate consumption of Eruca vesicaria subsp. sativa and its derivative product, SF,
may have the potential to strengthen the antioxidant defence system and extend life span of
mammals due to the homology with Drosophila genome. As a consequence, we must state that
evaluation of biological properties and appropriate profile in GLs and their hydrolysis products is
needed for genetic breeding and selection of accessions in order to establish safe, health
promoting and most promising entries with added value of Eruca vesicaria subsp. sativa.
Acknowledgements
The authors thank to the Consejería de Innovación, Ciencia y Empresa (Junta de
Andalucía, Spain) for funding the Project P06-AGR-02230 and to Gloria Fernández, (IAS-CSIC,
174
Cordoba) for technical assistance in the analysis of plants. We gratefully acknowledge the Institute
of Food Research (IFR), Norwich, U.K., for provision of materials and technical support. We
acknowledge Dr. Richard Mithen for his insightful advice in analysis of accessions and Dr. Andrés
Muñoz-Serrano for advice in statistical analysis. Myriam Villatoro was supported by Instituto
Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) contract.
175
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180
CAPÍTULO VII
Actividad biológica del rábano, una crucífera, con contenido en metales
y metaloides
Publiado como:
Modulation of genotoxicity and cytotoxicity by radish grown in metal-contaminated soils
Myriam Villatoro-Pulido, Rafael Font, Maria Isabel De Haro-Bravo, Magdalena Romero-Jiménez,
Jaouad Anter, Antonio De Haro Bailón, Ángeles Alonso-Moraga, and Mercedes Del Río-Celestino
Mutagenesis vol. 24 no. 1 pp. 51–57, 2009
doi:10.1093/mutage/gen051
181
Abstract
Members of the Brassicaceae family are known for their anticarcinogenic and genetic
material protective effects. However, many of the species of this family accumulate high amounts
of metals, which is an undesirable feature. Radish (Raphanus sativus L.) has shown to accumulate
metals in roots to a higher extent than others members of Brassicaceae. The main objectives of
this work are (i) to study the distribution of the accumulated As, Pb and Cd in radish plants and (ii)
to establish the genotoxic, antigenotoxic and cytotoxic activities of the root and shoot of this
vegetable. Results indicate that (i) the shoots of radish accumulate higher concentrations of
metal(oid)s than roots; (ii) the shoots were genotoxic at the different concentrations studied, with
the root showing such genotoxic effect only at the highest concentration assayed; (iii) the
antigenotoxic potential of radish is reduced in plants with high metal content and (iv) the
tumouricide activities of the radish plants were negatively correlated to their metal(oid) contents.
An interaction between metal(oid)s and the isothiocyanates (hydrolysis products of the
glucosinolates) contained in the radish is suggested as the main modulator agents of the genotoxic
activity of the plants grown in contaminated soils with metal(oid)s.
182
1. Introduction
Several studies have indicated that vegetables, particularly leafy crops, grown in heavy
metals contaminated soils have higher concentrations of heavy metals than those grown in
uncontaminated soil (1). A major pathway of soil contamination is through atmospheric deposition
of heavy metals from point sources such as metaliferous mining, smelting, agricultural and
industrial activities (2). In addition, foliar uptake of atmospheric heavy metals emissions has also
been identified as an important pathway of metal contamination in vegetable crops (3).
Elements such as Pb, Cd and As are not eliminated and can accumulate in human vital
organs producing progressive toxicity. Arsenic (As) is one of the most important global
environmental toxicants. Chronic arsenic poisoning can cause serious health effects including
cancers, melanosis, hyperkeratosis, restrictive lung disease, peripheral vascular disease,
gangrene, diabetes mellitus, hypertension and ischaemic heart disease (4–7). Lead and cadmium
are among the most abundant heavy metals and are particularly toxic. Cadmium can impair renal
function, and some studies indicate a neoplastic effect (8). Lead is a well-known physiological and
neurological toxic affecting many biochemical processes and almost every organ and system in the
human body (9).
Typical uncontaminated agricultural soils contain 1–20 mg/kg of As in the soil (10), 2–300
mg/kg of Pb in the soil and 0.01–2 mg/kg of Cd in the soil (11). Generally, in unpolluted
environments, ordinary crops do not accumulate enough arsenic to be toxic to humans. However,
in arsenic contaminated soil, the uptake of arsenic by the plant tissue is significantly elevated,
particularly in vegetables and edible crops (12). Therefore, there is, concern regarding
accumulation of As in agricultural crops and vegetables grown in arsenic-affected areas.
Some members of the Brassicaceae family have been shown to accumulate from moderate
to high levels of Pb, Cr, Cd, Ni, Zn and Cu (13). Carbonell-Barrachina et al. (14) also reported that
radish (Raphanus sativus L.) plants grown on higher soil concentrations of As accumulated high
As concentration in roots and shoots.
Besides the logical cautions about the use of contaminated soils, there is a great concern
about As and heavy metal pollution in Spain due to an environmental accident in a pyrite mine
located in the city of Aznalcóllar, Sevilla (Southern Spain) (15,16). Arsenic, lead and cadmium from
these soils may accumulate in any of the agricultural species being grown in them and enter the
human food chain through their edible parts.
183
This pollutants are all potential carcinogens and therefore they are dangerous when present
in human diet. Studies of genotoxicity and antigenotoxicity can help to evaluate the risk/safety and
effectiveness of healthy food products (17). The somatic mutation and recombination test
(SMART) in wings of Drosophila melanogaster is a well-known eukaryotic assay based on the loss
of heterozygosity for two genetic markers affecting the phenotype of wing hairs (18). This wing
spot test is a versatile and reliable system to test complex mixtures for geno/antigenotoxicity. It
was shown to be suitable to carry out both genotoxicity and antigenotoxicity assays, thanks to the
capabilities of treated larvae to bioactivate metabolites either as single compound or as complex
mixtures depending on the form on which they are up taken (19,20). A wide variety of compounds
and complex mixtures have been assayed with this test, such as food additives, beverages and
insecticides (21,22).
In the case of arsenic, a well-known genotoxin and carcinogen, Rizki et al. (23) concluded
that inorganic arsenic was non-genotoxic in the SMART test for D. melanogaster. However, there
have been no studies to test complex mixtures such as edible vegetables grown in contaminated
soils that contain metals.
HL60 human leukaemia cells have been used in cytotoxicity assays in order to determine the
tumouricide activity of the plant. The HL60 line was isolated from peripheral blood leukocytes of a
36-year-old Caucasian patient suffering from promyelocytic leukaemia (24). This cell line has been
studied intensely for many years in order to clarify the mechanisms that induce the differentiation
of normal cells into tumoural cells, with a view to control this proliferation in living organisms (25).
In this way, the induction of differentiation and apoptosis in tumoural cells would be an efficient
anticancer therapy strategy.
The main objectives of this work are (i) to study the uptake and distribution of As, Pb and Cd
in radish plants and (ii) to establish the genotoxic, antigenotoxic and cytotoxic activities of this
vegetable’s roots and shoots.
2. Material and methods
2.1. Plant material and greenhouse experiments
The plant species studied was the variety of radish namely Middle East Giant of R. sativus L.
Pots were placed in the greenhouse under natural light, temperature of 27/18°C (day/night) and a
relative humidity of 50/70% (day/night). Seeds were germinated in Petri dishes for 48 h and when
the plants had reached adequate height (8–12 cm), they were transferred to plastic pots containing
3 kg of contaminated soil in order to study the uptake and accumulation of As, Pb and Cd.
184
The contaminated soil was obtained from the experimental area ‘El Vicario’ (37° 26‘21’’N,
6°13‘00’’W) within the Green Corridor, close to the pyrite mine of Aznalcóllar (26). The soil was
classified as Typic Haploxeralf. One week before planting, soil was mixed with commercial potting
mixture (1:1 vol). The commercial potting mixture was used as control. The soil was a sandy loam
soil (sand 50%, silt 33% and clay 17%) which chemical characteristics were pH = 6.0, C org =
35%, NT = 0.3% and organic matter = 60%.
In order to study the As, Pb and Cd accumulation, a complete random design was used for
40 days of exposure. Controls with unpolluted soil were also included. All treatments were
replicated 10 times.
2.2. Sample preparation and chemical analysis
The plants were separated into shoots and edible parts, washed with tap water, rinsed
several times with distilled water and weighed to assess their biomass. The arsenic concentration
was determined by FIA-HG-AAS (27), and heavy metal contents (Pb and Cd) were determined by
AAS with graphite chamber (Perkin Elmer Analyst 600 with an autosampler AS 800) (28). The
accuracy and precision of the analytical methods was assessed by carrying out analyses of the
Community Bureau of Reference reference sample CMR 279 (sea lettuce) (29). The values
obtained for the reference sample by FIA-HG-AAS and AAS were concordant with the certified
values (data not showed).
2.3. Genotoxicity assays
Strains Two Drosophila strains were used containing genetics markers on the left arm of
chromosome 3: mwh/mwh, carrying the wing cell marker multiple wing hairs (mwh) and
flr3/In(3LR)TM3, ri pp sep bx34e es BdS (flr3/TM3, BdS abbreviated). This wing cell marker flare
(flr3) is a zygotic recessive lethal, which is maintained in the strain over the balancer chromosome
TM3. More detailed genetic information is provided by Lindsley and Zimm (30).
2.4. Treatment procedure
2.4.1. Crosses
Virgin females with the genotype flr3/TM3, BdS were mated to mwh/ mwh males. An optimal
design requires 300 females and 150 males. Flies are allowed to mate for 3 days in order to obtain
an optimal production of hybrid eggs on the fourth day after mating.
2.4.2. Treatments
Genotoxicity tests were carried out as described by Graf et al. (18). Hybrid eggs were
collected over an 8-h period. After 72 ± 4 h later, the emergent larvae were washed from
185
remaining feeding medium using a 20% sodium chloride solution and transferred to treatment
vials. These vials contained 0.85 g of Drosophila Instant Medium (Formula 4-24, Carolina
Biological Supply, Burlington, NC), and different concentrations of lyophilized vegetable samples
wetted with distilled water. The negative controls were prepared with medium and water and
positive controls with medium and hydrogen peroxide as oxidative genotoxin (22). Antigenotoxicity
tests were performed by mixing the mutagen (hydrogen peroxide) with the lyophilized samples in
appropriate concentrations. Larvae were fed until pupation ( ~48 h) at 25 ± 1°C. After emergence,
adult flies were collected and stored in a 70% ethanol solution.
2.4.3. Wing scoring
Twenty pairs of wings of each control and concentrations of transheterozygous marker flies
(mwh flr þ/mwhþ flr3) were removed and mounted on slides using Faure’s solution. Female and
male wings were mounted separately. Both dorsal and ventral surfaces of the wings were analysed
under a photonic microscope with the x400 magnification. Wing hair mutations (spots) were scored
among a total of 24 000 monotricoma cells per wing. In the positive control and genotoxic single
treatments, balancer wings (mwh/TM3, BdS) were also mounted.
2.5. In vitro cytotoxicity assays
2.5.1. Cell culture and incubation conditions
The human leukaemia cell line HL60 (promyelocytic cells) was supplied by Dr Jose´ M.
Villalba-Montoro (Department of Cell Biology, University of Cordoba, Spain). HL-60 myeloid
leukaemia cells were grown in RPMI-1640 medium (Invitrogen, Verviers, Belgium) supplemented
with the antibiotics penicillin, streptomycin and amphotericin (commercial mixture, A5955,
antibiotic–antimycotic solution 100 x stabilized, Sigma, St. Louis, MO, USA), L-glutamine (G7513,
Sigma) and heat-inactivated foetal bovine serum (S01805, Linus), in a humidified atmosphere
containing 5% CO2 at 37°C (Shel Lab, Cornelius, OR, USA) (31). Cultures were passed every 2–3
days to maintain logarithmic growth. Cells were grown at a density of 105 cells/ml before beginning
the assay in 2-ml well plates. The HL-60 cells of the assays were incubated with increasing
concentrations of filters from lyophilized plants whereas the negative controls had only culture
medium. At least three independent repetitions of the assays were carried out to calculate means
for statistical analysis.
2.5.2. Survival assay
Cell viability was determined by the trypan blue dye (T8154, Sigma) exclusion test. Cells
were counted by adding an aliquot of 10 ll of the culture to 10 ll of the trypan blue dye. The mix of
cells and dye were put on a Neubauer chamber and counted under a light inverted microscope
(AE30/31, Motic). Aliquots were taken at 24, 36 48, 60 and 72 h of incubation. After each
incubation period, a growth curve was established and IC50 values (concentration of tested
186
compound causing 50% inhibition of cell growth) were estimated. Curves are expressed as
survival percentage with respect to controls at 72 h of growth.
2.6. Data evaluation and statistical analysis
Student t-test [applied to data of metal(oids) contents of soil and plants grown in control and
contaminated soils] was used to detect differences between soils and plants in their concentration
of the two heavy metals (Pb and Cd) and As. The test allowed us to see how and where the plants
concentrate the pollutants analysed. SPSS Version 10.0 software (32) was used to perform all
statistical analyses.
For each plant, we calculated the shoot/root metal concentration quotient (MS/MR) as a
measure to assess the metal(oid) uptake strategy of plants.
Wing spot data are broken down into three different categories: small single spots (S)
consisting of 1 or 2 mwh or flr3 cells, large single spots (L) with three or more mwh or flr3 cells and
twin spots (T) with mwh and flr3 cells. The total number of spots was evaluated.
For evaluation of the genotoxic effects, the frequency of spots in the treated assay was
compared to negative controls, using distilled water. The statistical significance of spots frequency
per wing was evaluated using a multi-decision procedure to determine whether a result was
positive, negative or inconclusive based on two alternative hypotheses (33).
In the balancer-heterozygous genotype (mwh/TM3, BdS), mwh spots are produced mainly by
somatic point mutation and chromosome aberrations since mitotic recombination between the
balancer chromosome and its structurally normal homologue is a lethal event. To quantify the
recombinogenic activity of the mutagenic samples, the frequency of mwh clones on the marker
transheterozygous wings (mwh single spots plus twin spots) was compared with the frequency of
mwh spots on the balancer transheterozygous wings. The difference in mwh clone frequency is a
direct measure of the proportion of recombination (20).
The percentage of inhibition of mutagenic events by lyophilized samples was calculated from
the control-corrected frequencies of total spots, as proposed by Abraham (34): [inhibition 5
(genotoxin alone-sample plus genotoxin) x 100/genotoxin alone].
187
3. Results
Availability and accumulation of metal(oids) Soil analysis Table I shows concentrations of
heavy metals (Pb and Cd) and arsenic of the contaminated soils used in this work. The mean
concentration of total and diethylene triamine pentaacetic acid (DPTA) extractable arsenic in the
contaminated soils (75 and 4 mg/kg, respectively) were significantly higher than control soil and
higher than the upper limit of the range from normal soils, as shown in Table I (10,11). Mean
concentration values of total and DPTA-extractable Pb (83 and 14 mg/kg) and Cd (0.4 and 0.08
mg/kg) in contaminated soil were significantly higher than control soil, although both Pb and Cd
concentrations in contaminated soils were within the range found in normal soils (10,11). Available
data in the literature show that the values of As concentration in contaminated soils (<20 mg/kg)
can be considered toxic for plant growth (2). Plant-available metal was very low (Table I) and was
within the ranges of normal soils.
Table 1. Total and DPTA extractable concentrations of Pb, Cd and As in soils.
Total
Pb (mg/ kg)
Cd (mg/ kg)
As (mg/ kg)
DPTA extractable
Control
Contaminated
Control
Contaminated
41.8±0.1
0.2±0.0
9.1±0.1
83.2±0.1*
0.4±0.0*
74.8±0.1**
4.2±0.1
0.01±0.0
0.10±0.0
14.1±0.2*
0.08±0.0*
3.8±0.1*
Normal ranges
in soils
2-300 (b)
0.01-2 (b)
0.1-20 (a)
Means were compared between the control and contaminated soils for Pb, Cd and As within each
total and DPTA extractable concentrations; ns=not significant; *, **, ***=significantly different at
P<0.05, P<0.01 and P<0.001, respectively.
3.1. Accumulation and distribution of Pb, Cd and As by radish plants
The metal(oid)s concentrations from plants grown in contaminated and uncontaminated soils
are shown in Table II. Due to the low bioavailability of metals in the soils used (Table I), the
accumulation of metals in the tissues of the plants studied was low (Table II). Significant
differences were found for Pb, Cd and As concentrations in roots and shoots from radish plants
from contaminated and uncontaminated sites (Table II).
When radish plants were grown in contaminated soil, a little portion of As, Pb and Cd
remained in the roots, with a major portion of the element translocating to the shoots (Table II).
Radish plants accumulated significantly higher concentration of metal(oid)s in shoots as compared
to the roots. The behaviour of the radish plants with respect to metal(oid)s was characterized in all
the cases by MS/MR (shoot/root metal concentration quotient >> 1).
188
Table 2. Accumulation of Pb, Cd and As in the different tissues of the radish.
Pb (mg/ kg)
Part
Cd (mg/ kg)
As (mg/ kg)
Control Contaminated Control Contaminated Control Contaminated
Shoot 0.8±0.4
1.6±0.7*
0.5±0.1
0.9±0.2*
0.7±0.0
3.9±0.8*
Root 0.6±0.5
0.2±0.1ns
0.2±0.0
0.4±0.0ns
0.2±0.2
1.2±0.2*
Comparisons between control and contaminated soils in parts of radish for Pb, Cd and As; ns=not
significant; *, **, ***=significantly different at P<0.05, P<0.01 and P<0.001, respectively.
3.2. Genotoxicity assays of R.sativus L.
The SMART was applied to discern the genotoxicity and possible antigenotoxicity of radish
plants grown in standard and metal-contaminated soils. The proliferative imaginal discs of the wing
in Drosophila larvae gave the expected results for internal water-negative control and concurrent
hydrogen peroxide-positive control. Hydrogen peroxide exhibited a total mutation rate (0.225
mutant clones per wing) which duplicates the control rate, implying that the accuracy of the
genotoxicity and antigenotoxicity assays was ensured (22).
Table III shows the results obtained with the genotoxicity assays. Data are expressed as
small single, large single, twin and total spots per wing scored in 40 transheterozygous wings for
each assayed concentration. The non-metal-treated roots were not genotoxic for any of the
concentrations (0.125 total spots per wing on average). The metal-treated roots showed genotoxic
results but only at the highest concentration (5 mg/ ml), and the metal-treated shoot parts were
genotoxic for all the assayed concentrations (5, 2.5 and 0.625 mg/ml).
In order to evaluate the recombinogenic potency of mutagenic concentrations, we also
looked at additional information on the spots per wing scored in balancer wings (Serrate
phenotype) where no recombinogenicity is accounted (Table III). Values of recombinogenicity with
respect to the total induced clones ranged from 16 to 50%, with the aerial part reaching the highest
values (50%) at the two highest concentrations. It is remarkable that the hydrogen peroxide used
as oxidative genotoxin gave a recombinogenic activity lower (44%) than the shoot of metal-treated
radish.
All the samples tested for genotoxicity were tested in parallel for antigenotoxicity against the
oxidative mutagen hydrogen peroxide in the Drosophila wing spot test. Hydrogen peroxide is a
well-known mutagen in D. melanogaster. Studies published by Romero-Jiménez et al. (22) and
Allen and Tresini (35) have described .200 effects of hydrogen peroxide on .100 genes, including
those of stress. The results of the antimutagenic effects against hydrogen peroxide are shown in
Table IV. The only sample that had genotoxic effect was the treated root at concentrations of 0.625
189
mg/ml. As expected, the antigenotoxic effects of non-contaminated roots were higher than those of
roots grown in contaminated soils.
Table 3. Genotoxicity in the Drosophila SMART by radish.
Frequency of spots per wing (number of spots) and
diagnosis (1)
Compounds
Numbe Small spots Large spots
r
(1-2 cells) (more than
of
m=2
two cells)
wings
m=5
Estimated
Twin spots Total spots
recombination
m=5
m=2
percentage
H 2O
40
0.05 (2)
0 (0)
0 (0)
0.05 (2)
H2O2 (0.12 M)
H2O2 (0.12 M) (S)(2)
40
40
0.175 (7) +
0.125 (5) i
0.025 (1) i
0 (0) i
0.025 (1) i
0.225 (9) +
0.125 (5) i
[0.625]
40
0.1 (4) i
0 (0) -
0 (0) -
0.1 (4) i
[2.5]
[5]
40
40
0.075 (3) i
0.1 (4) i
0 (0) 0.1 (4) i
0 (0) 0 (0) -
0.075 (3) i
0.2 (8) i
MTR
[0.625]
[2.5]
[5]
[5] (S)
40
0.125 (5) i
0.075 (3) I
0 (0) -
0.2 (8) I
40
40
40
0.15 (6) i
0.2 (8) i
0.15 (6)
0 (0) 0.025 (1) i
0.025 (1)
0 (0) 0 (0) -
0.15 (6) I
0.225 (9) +
0.175 (7)
MTS
[0.625]
[0.625] (S)
[2.5]
[2.5] (S)
[5]
[5] (S)
40
0.25 (10) +
0.05 (2) I
40
40
40
40
40
0.275 (11)
0.25 (10) +
0.075 (3)
0.2 (8) i
0.125 (5)
00 (0) 0.05 (2)
0.05 (2) i
0 (0) i
44
Raphanus sativus
L(mg/ml)
NMTR(3)
(1)
22
0.025 (1) i 0.325 (13) +
0 (0) 0 (0) -
0.275 (11) +
0.25 (10) +
0.125 (5)
0.25 (10) +
0.125 (5)
16
50
50
Statistical diagnoses according to Frei and Würgler (32): + (positive), - (negative) and i
(inconclusive). Significance levels = 0.05.
(2)
Genotoxic
activity
in
balancer-heterozygous
(mwh/TM3,BdS)
larvae
for
genotoxic
concentrations.
(3)
Non metal treated root, NMTR; metal treated root, MTR; and metal treated shoot, MTS.
190
For root of plants that grew in non-contaminated soils, the percentage of inhibition increased
with the doses (0.11, 0.22 and 0.33 for the doses 0.625, 2.5 and 5 mg/ml, respectively, which
means an average inhibition rate of 0.22). These results agree with data (data not shown, 36) from
other species of the same family, in which a strong antimutagenic activity against hydrogen
peroxide has been detected. Results obtained from the samples that grew in contaminated soil
were diverse: the highest concentrations were capable of inhibiting the damage induced by
hydrogen peroxide (0.11 inhibition average for treated root). Nevertheless, the lower
concentrations of treated roots could not inhibit the effect, but increased the mutagenic activity of
hydrogen peroxide.
Table 4. Antigenotoxicity in the Drosophila SMART by radish in combined treatments with the
genotoxin hydrogen peroxide.
Frequency of spots per wing (number of spots) and
diagnosis (1)
Compounds
Numbe Small spots Large spots
r
(1-2 cells) (more than
of
m=2
two cells)
wings
m=5
Twin spots Total spots
m=5
m=2
Inhibition
percentage
H 2O
40
0.05 (2)
0 (0)
0 (0)
0.05 (2)
H2O2 (0.12 M)
40
0.175 (7) +
0.025 (1) i
0.025 (1) i
0.225 (9) +
NMTR(2)[0.625]
[2.5]
[5]
40
40
40
0.125 (5) i
0.1 (4) 0.1 (4) +
0.025 (1) i
0.05 (2) i
0.025 (1) -
0.05 (2) i
0.025 (1) i
0.025(1) -
0.2 (8) i
0.175 (7) i
0.15 (6) i
0.11
0.22
0.33
MTR [0.625]
[2.5]
[5]
40
40
40
0.225 (9) +
0.075 (3) i
0.125 (5) i
0.05 (2) i
0.05 (2) I
0.05 (2) I
0.025 (1) i
0 (0) 0 (0) -
0.3 (12) +
0.125 (5) i
0.175 (7) i
- 0.33
0.44
0.22
MTS [0.625] MTS
[2.5]
[5]
40
40
40
0.175 (7) 0.125 (5) i
0.175 (7) i
0.025 (1) i
0 (0) 0 (0) -
0 (0) 0 (0) 0 (0) -
0.2 (8) i
0.125 (5) i
0.175 (7) i
0.11
0.44
0.22
Raphanus sativus
L(mg/ml) + 0.12 M
H 2O 2
(1)
Statistical diagnoses according to Frei and Würgler (32): + (positive), - (negative) and i
(inconclusive). Significance levels = 0.05.
(2)
Non metal treated root, NMTR; metal treated root, MTR; and metal treated shoot, MTS.
191
3.3. Cytotoxicity assays
Figure 1a, b and c show the results of cytotoxicity assays performed using lyophilized radish
material against exponential growing of HL-60 cancer cells. The curves express the survival
percentage with respect to controls growing after 72 h of treatment. The relative growth of the
tumour cells decreased as concentration increased in the three experiments. Nevertheless, shapes
and IC50s were different for each case. The lethal dose 50 were reached at a concentration of
0.65 mg/ml for the control sample (Figure 1a) whereas this dose was reached at a concentration of
5 mg/ml with sample of contaminated roots (Figure 1b). This value was 15 times higher than the
dose needed to inhibit tumour growth by control roots. As stated above, metals are related to
cancer promotion, and the moderately high content of As, Cd and Pb in the treated roots could
explain the high inhibitory concentrations needed. The IC50 was not reached with the samples
from contaminated shoots. Higher concentrations of the samples were tested (data not shown) and
the IC50 was never reached. In this case, two main factors could have influenced this unhealthy
result: the high amount of metals incorporated and the lack of glucosinolates in the aerial part of
the plant. It is needed to say that the radish shoot is not normally used as a food, but it is the root
that is the edible part.
4. Discussion
The accumulation in the tissues of the plants studied was low (17, 20 and 5% for Pb, Cd and
As, respectively) (Table II) due to the previous chemical treatments (soil amendments with calcium
carbonate and ferric oxides) used by regional authorities to fix metals in the soil of Aznalcóllar (37)
(Table I).
The concentrations of Pb, Cd and As in shoots of radish (1.6, 0.9 and 3.9 mg/kg dw,
respectively) were higher than those found in roots (edible part). These results contrast with those
from Marchiol et al. (38), which reported a higher concentration of Pb and Cd in the root system of
radish as compared to the shoots. Tlustos et al. (39) found that the distribution of arsenic among
plants was affected by the rate of As in soil. Plants grown on lower As soil accumulated more As in
the leaves than in the roots, whereas those grown on higher As soil had more As in the roots than
in the leaves. Carbonell-Barrachina et al. (14) also reported that radish plants grown in soils with
higher concentrations of As had a higher concentration of As in the roots than in the shoots.
192
Fig. 1. Relative growing of HL60 tumour cells at 72 h of treatments with non- metal-treated root (a),
metal-treated root (b) and metal-treated shoot (c) samples.
From the perspective of human and animal consumption, the Pb, Cd and As concentrations
in radish plants in our study were below the maximum concentrations allowed by the statutory limit
set for metal(oid)s content in vegetables (3, 0.5 and 10 mg/kg dw, respectively) (40,41).
The levels of As, Pb and Cd found in radish plants in this experiment could have been higher
considered the low bioavailability of As, Pb and Cd in the soils used. Further research is needed
using higher As, Pb and Cd content in soils to determine the plant ability to accumulate these
elements in their edible parts.
193
The results of genotoxicity observed for contaminated shoots and roots can be due to the
presence of metal(oid)s that are related to DNA damage and have a negative influence on the
protective role of radish. Nevertheless, although carcinogenicity of metals(oid)s is not always
related to genotoxicity results, we found a clear relation between metal content and genotoxicity in
our study.
In the case of arsenic, a well-known genotoxin and carcinogen, Rizki et al. (23) concluded
that inorganic arsenic is non-genotoxic in the SMART test for Drosophila. Recent evidence from
experimental studies in mammals indicates that methylated metabolites of arsenic are more
genotoxic than inorganic arsenic. These authors (23) hypothesized that inorganic arsenic is nongenotoxic in Drosophila because they are unable to biotransform arsenic to methylated forms. The
absence of biomethylation in Drosophila could explain the lack of genotoxicity for inorganic arsenic
and the genotoxicity of methylated arsenic in the SMART wing spot assay found by these authors.
Our experiment design solves this limitation of Drosophila. Radish grown in contaminated soils
incorporate and bioactivate arsenic inorganic species in bioavailable molecules that results
mutagenic for Drosophila. Our results open a new way to test complex single and complex
compounds in Drosophila by feeding larvae not with the inactive molecule but with the molecules
that are already bioactivated by plants in the same way and concentrations that would be
consumed by humans.
Cadmium, a potent immunotoxic metal, induces DNA strand breaks, sister chromatid
exchanges and chromosomal aberrations in human cells; on the other hand, lead is considered a
potential mutagen by inducing direct DNA damage, clastogenicity and inhibition of DNA synthesis
or interfering with DNA repair (42). The genotoxicity observed in Drosophila fed by contaminated
radish could be due to the sum of the three mutagenic activities of As, Cd and Pb which are
bioavailable and bioactivated.
The percentage of inhibition (Table IV) calculated by the algorithm of Abraham (34) for
antigenotoxicity assays gives the reduction ability of mutagenic effect of the plant assayed against
hydrogen peroxide. The synergism observed between hydrogen peroxide and metal(oid)s can
activate several genes which codify stress enzymes that detoxify the effects of hydrogen peroxide,
but only at the highest concentrations (43). The glucosinolate and isothiocyanate content of
Raphanus could behave as desmutagens by counteracting the effect of the reactive oxygen
species (ROS) generated by hydrogen peroxide and metals, but only when a certain threshold
concentration is reached.
194
When assayed using model systems in which both intragenic and multilocus mutations can
readily be detected, arsenic is, indeed, found to be a strong, dose-dependent mutagen which
induces mostly multilocus deletions. Furthermore, the roles of oxygen and nitrogen reactive
species in mediating the genotoxic response are presented in a systematic and logical fashion in
support of a working model. The data from the study of Hei and Filipic (44) suggest that
antioxidants may be a useful interventional treatment in reducing the deleterious effects of arsenic.
Cd leads to the enhanced production of ROS and exerts its effects on cellular structure and
mechanisms, and Pb may also generate ROS and cause oxidative damage to DNA. Pb can be
substituted for Zn in several proteins which function as transcriptional regulators, e.g. Zn finger
(42). Nevertheless, a negative synergism between the two types of doses of ROS (those produced
by metals and those produced by hydrogen peroxide) is observed in our experiences, probably
due to stress genes that become activated.
The cytotoxic activity of radish can be explained by the high content in glucosinolates and
isothiocyanates in the root part of the plant (45). Evidence supporting the relation between
metal(oid)s and cancer has been previously reported (5,8). In the case of the As, much of the
evidence suggests that As and most of its derivates are cancer promoters rather than carcinogens
in animal studies (46). The mechanisms of metal-induced carcinogenesis may involve induction of
lipid peroxidation and an increase in the levels of free radical within the cells, following Pb or Cd
exposure, suggesting that the induction of genotoxicity and carcinogenicity is achieved by indirect
interactions (e.g. oxidative stress) of these metals with DNA (42). Cd affects cell proliferation and
differentiation. This metal interferes with antioxidant defence mechanisms and stimulates the
production of ROS, which may act as signalling molecules in the induction of gene expression (46)
and suppression of apoptosis (42). The inhibition of DNA repair processes by Cd represents a
mechanism by which the genotoxicity of other agents is enhanced and may contribute to the
tumour initiation by this metal (47). Lead is considered as another potential human carcinogen.
Cruciferous vegetables are also known to concentrate Cd and Pb, and these metals are
considered to be potential human carcinogens (42).
The absence of genotoxicity of the non-contaminated root and the genotoxicity of
contaminated shoot and root has been demonstrated. The antigenotoxic effects of noncontaminated roots are higher than the effects of the shoots grown in contaminated soil. All the
samples showed tumouricide activity but with different rates of inhibition. The non-treated plants
had the highest anti-proliferative activity. The glucosinolates and its hydrolysis products could be
the principal modulator agents of the antigenotoxic and cytotoxic activities of the plants grown in
soils contaminated with metals.
195
It has already been mentioned that governmental agencies have set limits for Pb and Cd
concentrations above which horticultural crops of the family Brassicaceae is considered unsuitable
for human consumption (40). This regulation sets the maximum limit for Pb and Cd in vegetables
at 0.30 and 0.20 mg/ kg wet weight, respectively, and 1 mg/kg wet weight for As (41). For edible
part of radish analysed in the present study, total Pb, Cd and As concentrations were 0.02, 0.04
and 0.12 mg/kg wet weight (90% of water content) which did not exceed limits.
Also, the European Environment and Health Information System of the World Health
Organization (WHO) has established regulatory guidelines regarding dietary Pb, Cd and As intake.
It recommends a provisional tolerable weekly intake (PTWI) of 25, 7 and 15 μg/kg body wt of total
Pb, Cd and As, respectively (48). To estimate the ‘degree’ of Pb, Cd and As intake through radish,
our results were interpreted in terms of the WHO PTWI. Using the means of radish, weekly
consumption of the Spanish population of 183 g (49), mean Pb, Cd and As concentrations in
radish and human body weight (70 kg), weekly intake calculated were 5.20 x 10-2 , 14.9 x 10- 2
and 31.5 x 10-2 μg/kg body wt for Pb, Cd and As, respectively.
On the basis of the results obtained in this work, we can conclude that it is necessary to
perform more studies to review the criteria used to set the maximum limits allowed by law
regarding different metals. This statement is based on the fact that in spite of the estimated weekly
intake of total Pb, Cd and As does not exceed the safety limits allowed by the WHO current
legislation, this consumption of metals caused more genotoxic effects and less citotoxicity than the
radish cultivated in non-contaminated soil. Both, SMART and cytotoxicity tests, offer a rapid and
cost-effective first-pass screening capable to assess toxicity when conventional toxicology data are
limited or lacking.
Funding
Consejería de Agricultura y Pesca (Junta de Andalucía, Spain) (C03-070).
Acknowledgements
The authors thank Gloria Fernández (IAS-CSIC, Córdoba) for technical assistance in the analysis
of plants.
Conflict of interest statement: None declared.
196
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201
DISCUSIÓN GENERAL
202
En el presente trabajo se ha abordado la caracterización agronómica y nutricional de
crucíferas de uso alimentario, así como el análisis de la actividad biológica y su selección. Uno de
los objetivos generales del presente trabajo ha sido contribuir al conocimiento de los recursos
fitogenéticos como herramienta para la mejora de rúcola. Se ha caracterizado la variabilidad agromorfológica de entradas de rúcola de diferentes orígenes geográficos incluidas variedades
comerciales como referencia. La utilización de material vegetal no domesticado puede representar
una importante fuente de recursos, ya que puede aportar valor añadido a variedades comerciales
ya disponibles (Nuez y Hernández-Bermejo, 2009). El material vegetal ha consistido en diversas
entradas de Eruca stenocarpa, Eruca vesicaria subsp. longirostris, Eruca vesicaria subsp.
vesicaria y Eruca vesicaria subsp. sativa seleccionadas de varios bancos de germoplasma. Se
analizaron un total de 15 caracteres agro-morfológicos en 52 entradas de rúcola. Estos análisis
agro-morfológicos han sido previamente diseñados para el estudio del manejo de germoplasma
de rúcola, con objeto de poder seleccionar el material vegetal que muestre características de
interés para ser utilizados como parentales en programas de mejora. Los resultados han mostrado
una alta diversidad en el germoplasma de rúcola, observándose gran variabilidad en la mayoría de
los caracteres evaluados (Tablas 3, 4 y 5, capítulo I). La variabilidad encontrada ha sido puesta de
manifiesto en estudios previos (Duhoon y Koppar, 1989; Egea-Gilabert et al., 2009; Bozokalfa et
al., 2010).
Algunos de los caracteres estudiados en este trabajo fueron estadísticamente significativos
entre las entradas como longitud, forma, lobulación y grosor de la hoja y forma del ápice de la
hoja. Otros caracteres además, fueron significativos entre las entradas, especies y subespecies
como la longitud del peciolo, lobulación del margen de la hoja, ancho, pubescencia y rugosidad de
la hoja.
Todas las entradas de Eruca estudiadas presentaron mayor número de días a floración
que líneas de Eruca previamente estudiadas en otros trabajos (Yaniv, et al., 1998; Warwick et al.,
2007; Egea-Gilabert, et al., 2009), incluso más que cultivares comerciales de floración tardía
(Morales et al., 2006). El carácter agronómico días a floración tiene gran importancia económica
debido a que permitiría un mayor número de cortes de hoja, si bien es conocido que factores
como el momento de siega, el espacio por unidad de siembra (Egea-Gilabert et al., 2009) y el
tiempo de siembra (Padulosi y Pignone, 1997) pueden influir sobre el valor promedio de este
carácter. También en el caso de la longitud de hoja hemos obtenido valores superiores a otros
autores (Egea-Gilabert, et al., 2009) En esta Tesís se ha encontrado mayor variabilidad para
algunos de los caracteres morfológicos estudiados (longitud del peciolo, longitud, forma y color de
la hoja) que los descritos en trabajos previos (Egea-Gilabert et al., 2009; Bozokalfa et al., 2010).
El comportamiento agronómico de las entradas PEX-53 (vesicaria), PEX-14, PEX-58 y
203
PEX-61 (sativa) presentaron similar peso fresco que la variedad comercial PEX-55, y fueron
significativamente más altas que las variedades comerciales PEX-16 y PEX-56 (Table 3). Así
mismo, entradas (PEX-7, PEX-52 PEX-93) pertenecientes a la subp. vesicaria, presentaron una
floración más tardía cuando se compararon con las tres variedades comerciales.
Respecto a otros caracteres morfológicos fue posible seleccionar las entradas PEX-14,
PEX-58, PEX-61 (sativa) y PEX-7, PEX-9, PEX-52 y PEX-53 (vesicaria), las cuáles mostraron
características interesantes (hoja de pequeño tamaño, alto contenido en clorofila, ausencia de
pubescencia en sativa y alta lobulación de las hojas en vesicaria) desde el punto del consumidor.
Para obtener entradas de Eruca con valor aplicable en Mejora Vegetal que puedan competir
adaptándose a la lucha de los mercados nos hemos planteado, no sólo la selección de líneas con
un deseable comportamiento agronómico robusto, sino que posean un valor alimenticio que
atraiga al consumidor. La rúcola es preferida en consumo crudo frente a otros vegetales por
diversas características sensoriales: su aspecto (color, vellosidad y forma de las hojas), por su
textura, aromas en boca y por sus características sensaciones trigeminales. Estas últimas
características sensoriales vienen dadas en última instancia por la hidrólisis de los glucosinolatos
(mediado por la enzima mirosinasa) a isotiocianatos y otros metabolitos. En el capítulo II se han
mostrado datos preliminares acerca del contenido en glucosinolatos y los atributos sensoriales,
así como el desarrollo de un léxico específico para el análisis sensorial de un total de 10 entradas
de rúcola y Erucastrum.
Ciertas características sensoriales como las de fase visual arrojaron diferencias entre las
entradas (color púrpura en venación, tamaño y forma de la hoja) y entre las valoraciones en
campo y las realizadas en el panel de cata (margen de la hoja o pubescencia), estas últimas
debidas posiblemente al muestreo necesariamente reducido en cata. Respecto al resto de fases
sensoriales, se ha encontrado una variedad de descriptores como césped, rábano, piel de limón y
almendra, que arrojan diferencias entre entradas. Sin embargo, hemos detectado un patrón de
diferencias para ciertas notas (coles, rábano, trébol, tomate y alcachofa ente otros) que arrojan
resultados estadísticamente diferentes debido exclusivamente al efecto fijo de la cosecha (2008
versus 2009).
En un intento por relacionar las diversas variables sensoriales con el contenido en
glucosinolatos, que es la característica diferencial de las crucíferas que estudiamos, se cuantificó
el contenido de glucosinolatos. Entre los glucosinolatos alifáticos, los mayoritarios fueron la
glucoerucina y glucorrafanina, este último con valores de hasta 17.9 µ moles of glucosinolates/ g
de tejido en peso seco (en la subespecie vesicaria), y en el caso de la especie Erucastrum (PEX8) llego a alcanzar 23.1 µ moles of glucosinolates/ g. Es importante resaltar la presencia de este
204
glucosinolato por sus propiedades anticancerígenas (Farham et al., 2004; Sun-Ju y Gensho,
2006). El glucosinolato indólico mayoritario fue la glucosativina que probablemente es derivado de
una desmetilación de la glucoerucina (Bennet et al., 2006), lo que explicaría los bajos niveles de
glucoerucina que muestran las hojas; y el único glucosinolato aromático detectado fue la
gluconasturtina.
La variabilidad cuantitativa de glucorafanina encontrada en entradas de Eruca vesicaria
subesp. vesicaria
nos indica que es posible utilizar este material como base para la mejora
genética de la especie. La consecuencia de elevar los niveles de glucorafanina aumentarían el
interés nutracético que puede llegar a tener ésta especie, ya que de éste glucosinolato se forma
sulforrafano (isotiocianato de la glucorafanina), con interesantes propiedades anticarcinogénicas.
El contenido total en glucosinolatos y de glucorrafanina fue más alto para la mayoría de las
entradas estudias al encontrado en las hojas de las dos variedades de rúcola utilizadas como
control (PEX-17 y PEX-56), así como en estudios previos (Bennett et al., 2006; Kim e Ishii, 2006).
Se ha podido observar la influencia en el año de recolección, ya que la concentración de
glucosinolatos en 2008 resultó más alta que las determinadas en 2009. Este hecho se ha
relacionado con las temperaturas más altas registradas durante el año 2008. La influencia
ambiental sobre el contenido en glucosinolatos ha sido ampliamente descrita en estudios previos
en especies de crucíferas de hoja (Rosa et al., 1996; Charron et al., 2005; Velasco et al., 2007;
Cartea et al., 2008).
Desafortunadamente
no
se
ha
podido
relacionar
un
determinado
contenido
de
glucosinolatos con ninguna de las características sensoriales, a diferencia de otros trabajos
previos (D’Antuono et al., 2009; Padilla et al., 2007; Fenwick et al., 1983), por lo que se puede
sugerir que el sabor puede ser debido a otros fitoquímicos o al sinergismo entre ellos.
A pesar de los efectos beneficiosos de los isotiocianatos, ciertos productos de degradación
de los glucosinolatos pueden presentar en algunos casos un sabor no deseable, produciendo un
rechazo por parte del consumidor. Nos encontramos ante el dilema de continuar mejorando para
la disminución de estos compuestos para evitar posibles sabores desagradables o incrementar el
contenido de fitoquímicos con la consiguiente incompatibilidad con la aceptación por parte del
consumidor.
En el capítulo III se ha abordado la caracterización para el contenido en glucosinolatos,
isotiocianatos, fenoles, carotenoides y carbohidratos de cuatro entradas de rúcola previamente
rastreadas en el banco de germoplasma para alto y bajo contenido en glucosinolatos totales. Los
resultados han mostrado un contenido en glucosinolatos totales de 14.0, 19.4,
27.6 y 28.2
205
µmoles / g (peso seco) de tejido liofilizado para las muestras denominadas LGC1, LGC2, HGC1 y
HGC2 respectivamente. Se ha analizado además el contenido de 13 de los glucosinolatos
contenidos en las muestras de rúcola.
En cuanto al rendimiento en isotiocianatos, el sulforrafano fue el mayoritario, seguido de la
erucina e iberina, siendo la entrada LGC2 la que presentó más variabilidad y el valor más alto en
isotiocianatos. El contenido de sulforrafano nitrilo también fue determinado. Las entradas
mostraron diferencias significativas en la hidrólisis de glucorrafanina y formación de sulforrafano
con un porcentaje de conversión entre 4.12 y 97.35% para las entradas LGC1 y LGC2
respectivamente. Puesto que el sulforrafano nitrilo no es capaz de inducir las enzimas de
detoxificación a diferencia del sulforrafano sería conveniente estudiar la selección de entradas con
bajos niveles o ausencia de la proteína epitioespecífica (cofactor responsable de la generación de
epitionitrilos y tiocianatos orgánicos) y altos niveles de mirosinasa (Matusheski et al., 2006) y
comprobar que estas líneas exhiben alto rendimiento de sulforrafano, con lo que se incrementaría
el valor añadido de las mismas. Nuestros resultados contrastan con otros donde se cita a la
erucina (Blazevic y Mastelic, 2008) o la sativina (Bennett et al., 2002) como los isotiocianatos
mayoritarios en Eruca.
Los fenoles más abundantes fueron la quercitina-3-ß-glucósido y la rutina, no detectando
kaempferol, aunque otros autores lo describen incluso como mayoritario en sus entradas de rúcola
(Jin et al., 2009; Selma et al., 2010; Bennett et al., 2006). Estas diferencias en contenido fenólico,
tanto cualitativas como cuantitativas, se pueden deber a las metodologías de análisis empleadas o
a las características y condiciones de las muestras (Brown et al., 2002). Las entradas que exhiben
mayor contenido en cuanto a los phenoles estudiados fueron HGC1 y LGC2.
La luteína fue el carotenoide más abundante, seguida de la ß-criptosantina, ß-caroteno,
zeaxantina y violaxantina, siendo de nuevo las entradas LGC2 y HGC1 las que mostraron mayor
concentración y variabilidad en carotenoides. Nuestros resultados cualitativos respecto a este
grupo de fitoquímicos concuerdan con otros trabajos previos (Ramos y Rodríguez-Amaya, 1987;
Kimura y Rodríguez-Amaya, 2003; Niizu y Rodríguez-Amaya, 2005), aunque el contenido medio
de luteína fue superior en nuestro estudio.
Teniendo en cuenta los contenidos en glucosinolatos, el rendimiento en isotiocianatos y el
de las sustancias antioxidantes fenólicas o de tipo carotenoide, podemos avanzar la idea de que
entradas similares a LGC2 son interesantes desde el punto de vista de la Mejora Genética,
fundamentalmente debido al alto contenido y variabilidad de fitoquímicos y al porcentaje de
conversión de glucosinolatos a isotiocianatos.
206
El uso de rúcola como alimento y nutracéutico puede también ser promocionado por su alto
contenido en minerales, lo que concuerda con otros autores (Bozokalfa et al., 2009). Los análisis
estadísticos mostraron diferencias significativas entre las entradas para todos los minerales
excepto para el Ca, existiendo entradas con alto contenido para uno ó varios minerales como S3,
S5, S9 o S22 entre otras (PEX-60, PEX-62, PEX-66 y PEX-83, respectivamente). Estos análisis
también mostraron diferencias significativas para todos los minerales para las entradas agrupadas
por países excepto para el Ca. Dado que las entradas se cultivaron bajo las mismas condiciones
ambientales, las diferencias en la acumulación de minerales pueden ser debidas al genotipo de
las entradas.
Las entradas de Eruca estudiadas fueron una buena fuente de minerales, particularmente
potasio y calcio. Presentaron valores medios de 496 mg/100g de tejido fresco de potasio y una
media de 395 mg/100g de tejido fresco de calcio, existiendo entradas con cantidades de calcio
cercanas a los 646 mg/100g de tejido fresco (Tabla 3, Capítulo IV). Considerando que el calcio de
la rúcola y el de la leche tiene el mismo porcentaje de absorción, dado que esta crucífera está
libre de oxalatos y fitatos que bloquean la absorción de minerales como el Fe, el Zn y el Ca
(Lucarini et al., 1999; Sandberg et al., 2002; Vilar et al., 2008) las entradas de rúcola se presentan
como una buena fuente de calcio y con una disponibilidad elevada. Por tanto, estos cultivos se
perfilan como un alimento importante en individuos con osteoporosis o con intolerancia a la
lactosa. Suponiendo que una persona consuma un plato de rúcola de unos 80 g, esto
proporcionaría un 57% de Ca, 46% de Mn, 25% de Fe, 20% K, 11.5% de Cu, 11% de Mg, 8% de
Zn y 2% de Na de los requerimientos diarios de minerales de una persona
Los resultados del contenido medio mineral de nuestro estudio fueron más altos que los
descritos en estudios previos (Kawashima y Valente-Soares, 2003; Bozokalfa et al., 2009;
Cavarianni et al., 2008).
La tecnología NIRS, es un método que permite realizar un cribado de un amplio número de
muestra de manera rápida y de bajo coste. Nuestros resultados indican que la espectroscopía en
el infrarrojo cercano puede ser utilizada como un método de muestreo rápido para la
determinación del contenido mineral de Fe, Na, K, Mg y Zn en rúcola, representando una
herramienta útil para la reducción del tiempo de análisis, de bajo coste y sin la utilización de
productos químicos tóxicos. De acuerdo con los coeficientes de determinación en la validación
cruzada, las ecuaciones NIRS desarrolladas para la predicción del contenido de Na y K fueron
características de ecuaciones que permiten una separación óptima de muestras en contenidos
altos, medios y bajos. Las ecuaciones NIRS desarrolladas para la predicción del contenido total de
minerales, Fe, Mg y Zn fueron validas para el cribado de muestras; mientras que se obtuvieron
unas pobres calibraciones para la predicción del contenido en cenizas, Cu, Mn y Ca, debido al
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estrecho rango de concentración del elemento en cuestión y/o a la baja concentración de los
componentes orgánicos asociado a dicho elemento.
La caracterización multidisciplinaria previa, tanto agro-morfológica, como sensorial y fitoquímica
de entradas de rúcola, ha constituido la base para poder llevar a cabo los objetivos últimos de
evaluación de la actividad biológica, que tienen implicaciones sobre el consumo de rúcola en la
salud humana. Se ha determinado la capacidad tumoricida y apoptótica y anti/mutagénica y
antidegenerativa en ensayos humanos y animales modelo. En el capítulo V se ha analizado la
actividad biológica de las cuatro entradas de rúcola (analizadas previamente para el perfil
fitoquímico en el capítulo III), así como del isotiocianato (ITC) sulforrafano (SF). Se estudió
mediante tres aproximaciones: 1) midiendo el efecto en la proliferación celular con las líneas HL60
(células tumorales humanas promielocíticas), PC3 (células tumorales humanas de próstata) y
PNT1A (línea celular normal de epitelio postpubertal de próstata); 2) evaluando su actividad
inductora de la apoptosis, y 3) ensayando el efecto en la expresión de la proteína p21.
La proliferación celular se ha analizado con el test de exclusión del azul tripán con la línea
celular HL60 y con el ensayo de proliferación WST-1 en las líneas celulares PC3 y PNT1A. Los
resultados del test de exclusión con el azul tripán mostraron que el SF fue altamente citotóxico,
así como las entradas con mayor contenido de isotiocianatos. Los datos de la concentración
inhibitoria 50 (IC50) fueron: 0.4, 0.42, 1 mg/ ml, y 6.5 mM para HGC2, LGC2, HGC1 y SF
respectivamente. La IC50 para la muestra con el contenido más bajo en GLs no se alcanza.
Analizando la relación entre la actividad citotóxica medida como IC50 y el contenido en SF de las
entradas de rúcola, se puede observar que la viabilidad de las células HL60 desciende conforme
aumenta el rendimiento en SF de la muestra. Los resultados del ensayo de proliferación WST-1,
sin embargo no mostraron diferencias significativas a bajas concentraciones para los diferentes
tratamientos y líneas celulares. Lo cual no concuerda con trabajos previos realizados por otros
autores (Harris y Jeffery, 2008; Traka et al., 2010). Esto puede ser debido a que el WST puede
reducirse por la presencia de fenoles en la muestra (Maioli et al., 2009; Anter et al., 2011).
Se ha detectado fragmentación nuclear como marca general de inducción de apoptosis en
los tratamientos de células tumorales HL60 a altas concentraciones de extractos de rúcola y a las
concentraciones de 8, 26 y 32 µM de SF, por lo que sugerimos que la actividad citotóxica
observada por rúcola puede estar asociada a mecanismos diferentes de la fragmentación
cromosómica. Sí hemos podido mostrar la inducción de la expresión de la proteína p21 en los
tratamientos con SF a la concentración de 15 µM coincidiendo con otros autores (Dashwood et al.,
2007; Kim et al, 2010; Melchini et al., 2009). No obstante, el extracto vegetal no fue capaz de
inducir la proteína p21, por lo que serían aconsejables futuros ensayos de expresión con
concentraciones superiores de rúcola.
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Ha sido la entrada LGC2 la que mostró el mejor comportamiento in vitro. Estos resultados
pueden relacionarse con los del capítulo III de análisis fitoquímico en el que las entradas LGC2 y
HGC1 fueron las que mostraron el mejor perfil fitoquímico. El hecho de que la entrada HGC1
tenga un bajo rendimiento en la conversión de GLs a ITC originando posiblemente otros
metabolitos sin actividad biológica promotora de salud, confirma el comportamiento único de la
línea LGC2. Podemos proponer que las diferentes actividades in vitro pueden estar relacionadas
con el contenido de ITCs, más que con el contenido de GLs, así como con la interacción de otros
compuestos fitoquímicos como fenoles y carotenoides.
Las pruebas más claras de que los vegetales y frutas están relacionados con una reducción
del riesgo de padecer cáncer vienen aportadas por los estudios epidemiológicos con crucíferas
(Gasper et al., 2007). Los ITCs provenientes de las hidrólisis de GLs más que éstos mismos,
serían los responsables de de los efectos protectores de las crucíferas. A partir de estos hechos
acerca de los efectos protectores de los ITCs nos hemos centrado en el capítulo VI en estudiar la
actividad biológica in vivo del SF y de extractos de rúcola, concretamente respecto a su papel en
la protección del ADN (ausencia de genotoxicidad y potencia antigenotóxica) y en el incremento
en la supervivencia de Drosophila. Hasta la fecha sólo existen datos de un ensayo publicado
utilizando el material vegetal de Eruca (Lamy et al., 2008), en el cual no se mostraron efectos
genotóxicos en hepatocitos en cultivo HpG2.
Ni el SF ni ninguna de las cuatro entradas de rúcola analizadas en el ensayo SMART
resultaron ser genotóxicas, a excepción de la entrada Es4 (HGC2) a la máxima concentración del
extracto ensayada (5mg/ml). Esta entrada, aun presentando el máximo contenido en
glucosinolatos y glucorrafanina, proporciona un bajo porcentaje de conversión a SF. Algunas de
las concentraciones exhibieron valores de genotoxicidad inferiores a los controles con agua. Las
alas de Drosophila melanogaster de fenotipo Serrate nos muestran el porcentaje de mutaciones
que son debidas a eventos mutacionales no recombinogénicos. Si comparamos este porcentaje
con el obtenido para mutaciones totales en alas marcadoras transheterocigóticas obtendremos el
porcentaje de recombinogénesis inducida por la entrada Es4 (HGC2) a la concentración máxima
(5 mg/ml), que es un 82%. Sabiendo que el peróxido de hidrógeno puede producir mutaciones por
recombinación en un 44% (Villatoro-Pulido et al., 2009), la entrada Es4 (HGC2) puede provocar
casi el doble de mutaciones por recombinación que la genotoxina utilizada en el ensayo. VázquezGómez et al., (2010) han descrito al sulforrafano como mutagénico en el cruce estándar del
ensayo SMART en Drosophila, aunque utilizaron concentraciones de 140 µM mientras que en
nuestro ensayo la mayor concentración fue de 12.6 µM. Esta concentración se escogió
suponiendo que el 100% del contenido el GLs de la entrada con más alto nivel (Es4) pasaría a
ITC, ya que concentraciones más altas puede que no se den en condiciones nutricionales.
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Todas las concentraciones de sulforrafano y de extractos de rúcola ensayadas fueron
antigenotóxicas en los ensayos de antigenotoxicidad, aunque no se pudo observar efecto de
dosis. Las muestras exhibieron porcentajes de inhibición del efecto mutagénico del peróxido de
hidrógeno que oscilaron entre un 0.13 para la entrada Es4 (que fue genotóxica) y un 0.93 para la
entrada Es1 a la mayor concentración ensayada (5 mg/ml). En general se puede afirmar que las
entradas con alta conversión de glucosinolatos a isotiocianatos son más seguras desde el punto
de vista de la integridad del ADN en células somáticas proliferativas de Drosophila melanogaster.
El objetivo último de estudios sobre alimentos nutracéuticos es analizar su incidencia en la
esperanza de vida. Por ello hemos llevado a cabo ensayos de supervivencia, entendiendo que
ésta es un carácter de origen multifactorial, y al mismo tiempo teniendo en cuenta que una
molécula o una sustancia compleja pueden incidir sobre diversos procesos metabólicos que
resulten finalmente en un incremento o en una disminución del life span de una especie. Con
objeto de estudiar in vivo el posible efecto de los extractos de rúcola o el sulforrafano
administrados crónicamente se realizaron ensayos de life span en Drosophila. El análisis de las
curvas de supervivencia completas mostraron que la esperanza de vida mayor correspondió al
tratamiento con la entrada Es4. A priori se pensó que la entrada Es2, puesto que no era
genotóxica y era más antigenotóxica, debería mostrar mejores resultados que la Es4. Sin
embargo, la esperanza de vida máxima del tratamiento con la entrada Es2 (LGC2) fue un 4%
mayor que el control con respecto a la entrada Es4 que mostró una esperanza de vida máxima de
7.2% mayor que el control. Esto puede ser debido a la presencia de otros compuestos con efecto
beneficioso como fenoles o carotenoides presentes en las muestras (Boyd et al., 2006; Gil et al.,
2004; Kassie et al., 2003). La esperanza de vida máxima con el tratamiento con sulforrafano
correspondió a la concentración de 1.87 µM y fue un 16.13% menor que el control.
Un estudio más detallado de las curvas de supervivencia, en las que se tienen en cuenta
los supervivientes de percentiles superiores, es decir el tiempo en el que sobrevive una gran parte
de la población (health span o calidad de vida) arroja datos más prometedores. Si se trabaja con
niveles de calidad de vida para el 75% de todos los individuos del ensayo vivos, podemos ver que
todas las concentraciones ensayadas de sulforrafano incrementaron la esperanza de vida
saludable excepto la concentración más alta (15 µM). Las concentraciones de 0.625 mg/ml de la
entrada Es2 y las concentraciones de 0.625 y 2.5 mg/ml de Es4 incrementaron también
significativamente la calidad de vida con respecto al control. Estos últimos resultados de la
entrada Es4 concuerdan con los resultados de geno/antigenotoxicidad, en los que las mismas
concentraciones de los extractos vegetales mostraron tasas de mutación más bajas. El hecho de
que no se pueda observar un efecto de dosis en los tratamientos puede ser debido a que las
crucíferas no solo producen isotiocianatos, sino también otra serie de compuestos de degradación
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de glucosinolatos como nitrilos y epitionitrilos, los cuales a altas concentraciones pueden tener
efectos nocivos degenerativos como iniciación de mutagénesis, citotoxicidad, y procesos
carcinogénicos (Martín-Dietz et al., 1991).
Las líneas de Drosophila utilizadas en este trabajo no son mutantes de vida extensible. Se
ha realizado una búsqueda in silico (FlyBase) de todo el fondo genético de las estirpes multiple
wing hair y flr3/TM3, BdS, así como de la función de estos genes y sus productos, no existiendo
ningún gen relacionado con algún efecto de vida extensible. Sin embargo, los datos de la
esperanza de vida media de los individuos estudiados son bastante superiores a los encontrados
en la literatura (Trotta et al., 2006; Mockett and Sohal, 2006; Li et al., 2008; Bahadorani and
Hilliker, 2008; Avanesian et al., 2010; Boyd et al., 2011). Esto puede ser debido a efectos de
heterosis (los individuos son transheterocigotos al igual que los usado en los ensayos de
geno/antigenotoxicidad) o a las óptimas condiciones de manejo (medio de cultivo y manipulación)
con las que se han realizado todos y cada uno de los ensayos de los tratamientos y
concentraciones.
El último capítulo de esta tesis, consiste en una aplicación práctica para demostrar la
idoneidad de los ensayos utilizados en la determinación del potencial promotor de salud de
crucíferas, tanto del sistema in vivo de Drosophila, como del modelo in vitro de citotoxicidad en
líneas celulares tumorales. Las crucíferas han demostrado una moderada-alta capacidad para
acumular metales (Pb, Cr, Cd, Ni, Zn y Cu) (Ebbs y Kochian, 1997), incluido el rábano (Raphanus
sativus L.) (Carbonell-Barrachina et al., 1999). Una contaminación en suelos por As, Pb o Cd
podría repercutir en una acumulación en los tejidos vegetales entrando a formar parte de la
cadena alimenticia humana. Estos elementos, al no poder ser eliminados del organismo, se
acumulan en los órganos vitales produciendo toxicidad progresiva (Bryce-Smith, 1997; Morales et
al., 2000; Hamers et el., 2006). En el capítulo VII se describe el estudio de la dinámica de
absorción y distribución de As, Pb y Cd en las plantas de rábano, así como el establecimiento de
las actividades genotóxicas, antigenotóxicas y citotóxicas de la parte aérea y raíz (parte
comestible) de este vegetal.
Aunque los niveles de As en el suelo fueron superiores a los límites máximos establecidos
para suelos normales (no contaminados), debido a la baja biodisponibilidad de este metaloide en
el suelo utilizado, su acumulación en las plantas fue bajo (Tabla 2, capítulo VII) estando asimismo
por debajo del límite establecido como tóxico por la legislación para el contenido de
metales(oides) en vegetales (ANZFZ, 1998; Commission Regulation 629, 2008). No obstante,
hemos encontrado una acumulación diferencial de As, Cd y Pb en raíces y parte aérea en las
plantas de rábano desarrolladas en suelos contaminados, siendo más alta en parte aérea que en
raíces. Estos resultados contrastan con los de Marchiol y colaboradores (2004) que determinaron
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una acumulación más alta de Cd y Pb en raíces que en parte aérea. En otros ensayos (Tlustos et
al., 1998; Carbonell-Barrachina, et al., 1999) se encontró que la distribución de As en la planta
depende del contenido del mismo en el suelo. Cuando el contenido de As es bajo se tiende a
acumular más cantidad en parte aérea que en raíz, y viceversa.
Los estudios de genotoxicidad y antigenotoxicidad pueden ayudar a la evaluación de la
seguridad de productos alimentarios (Bast et al., 2002). En el caso del As, es conocido que
aunque sea una genotoxina carcinogénica, estudios previos han puesto de manifiesto que en su
forma inorgánica no es genotóxico en el test SMART de Drosophila melanogaster (Rizki et al.,
2006). No obstante, hemos detectado genotoxicidad en las muestras de parte aérea desarrolladas
en suelos contaminados. El análisis del fenotipo serrate nos permitió concretar que esta
genotoxicidad es debida a actividad recombinogénica hasta en un 50%. Aunque la
carcinogenicidad de los metales(oides) no siempre está relacionada con resultados positivos de
genotoxicidad, nosotros sí hemos encontrado una clara relación entre el contenido en metales y la
genotoxicidad, que puede ser debida a la suma de las tres actividades mutagénicas del As, Cd y
Pb. Rizki y colaboradores (2006) concluyeron que el As inorgánico no fue genotóxico debido a que
Drosophila no es capaz de biotransformarlo en formas metiladas que puedan ejercer una actividad
tóxica. Nuestro estudio solventa este problema, ya que el As, al ser incorporado a una matriz
biológica como es el rábano es capaz de convertir el As inorgánico en una especie biodisponible
que resulta mutagénico en Drosophila. Estos resultados presentan una nueva manera de analizar
compuestos simples y complejos en Drosophila, alimentando las larvas no con la molécula
inactiva, sino con las moléculas bioactivadas por las plantas de la misma manera y
concentraciones en que son consumidas por los humanos.
En cuanto a la antigenotoxicidad de las muestras, se observó que las concentraciones más
altas de los extractos de muestras desarrolladas en suelos contaminados pueden inhibir el daño
causado por la genotoxina (peróxido de hidrógeno), mientras que las concentraciones más bajas
no lo hicieron. Sugerimos que un sinergismo entre el peróxido de hidrógeno y los metal(oides)
puede activar genes, que codifiquen enzimas de estrés que detoxifican los efectos tóxicos del
peróxido de hidrógeno, pero solo a las concentraciones más altas. El contenido de glucosinolatos
e isotiocianatos del rábano puede comportarse como desmutágeno por contrarrestar el efecto de
las especies reactivas de oxígeno (ROS) generadas por el peróxido de hidrógeno y los
metal(oides), pero solo cuando se alcanza una concentración umbral.
El potencial citotóxico frente a células HL60 de parte aérea y raíces conteniendo metales
pesados y raíces control aumentó conforme se incrementó la concentración. Sin embargo, no
logramos alcanzar una IC50 para las muestras de parte aérea contaminados y, en el caso de
raíces contaminadas, la IC50 fue 15 veces superior a la IC50 de los cultivos controles
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establecidos con raíces no contaminadas. El contenido en GLs e ITC de las raíces puede ser la
causa de la elevada actividad citotóxica de las raíces control (Musk et al., 1995), mientras que en
el caso de la parte aérea y raíces contaminadas, se detecta alguna actividad citotóxica, aunque no
total debido al contenido en metales pesados.
Por tanto, en base a los datos obtenidos, se concluyó que aunque el contenido de As, Cd y
Pb en las muestras de rábano no excedieron los límites establecidos como tóxicos por la
legislación, el consumo de las mismas puede causar efectos genotóxicos, así como una menor
citotoxicidad frente a células tumorales.
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CONCLUSIONES
214
Las conclusiones que se derivan de los trabajos de investigación realizados en la presente
Tesis son las que se exponen a continuación:
1. Existe una gran variabilidad agro-morfológica para la mayoría de los caracteres de
interés (longitud de hoja, alto contenido en clorofila, floración tardía y ausencia de
pubescencia).
2. Los caracteres más relevantes en la diferenciación entre entradas fueron: longitud,
forma, lobulación y grosor de la hoja y forma del ápice de la hoja. Otros caracteres
además, diferencian entre especies y subespecies de rúcola como la longitud del
peciolo, lobulación del margen de la hoja, ancho, pubescencia y rugosidad de la hoja.
3. El potencial existente para el contenido en glucorafanina entre poblaciones de rúcola
(Eruca vesicaria subesp. vesicaria) procedentes de España nos indica que es posible
utilizar este material para futuros programas de mejora genética, sugiriendo, por tanto,
una alta probabilidad de encontrar nuevos y valiosos recursos fitogenéticos en futuras
prospecciones.
4. No se encontraron correlaciones significativas entre los contenidos en glucorafanina y
el rendimiento en sulforafano en las entradas de rúcola, hecho éste que deberá ser
tenido en cuenta en futuros programas de mejora en esta especie.
5. El panel sensorial generó 26 descriptores simples clasificados en tres grupos diferentes
(7 para apariencia, 14 para sabor y 6 para textura) que permitirá el análisis sensorial de
los recursos fitogenéticos de esta especie.
6. Tanto el contenido cualitativo y cuantitativo en glucosinolatos como los atributos
sensoriales de las entradas variaron entre años, lo que fue atribuido al efecto del
ambiente. Sin embargo no fue posible correlacionar el contenido en glucosinolatos con
las características sensoriales.
7. Las entradas de Eruca fueron una buena fuente de minerales, particularmente calcio,
manganeso, hierro y potasio pudiendo representar el 57%, 46%, 25% y 20%,
respectivamente de los requerimientos diarios de una persona.
215
8. La espectroscopía en el infrarrojo cercano puede ser utilizada como un método de
muestreo rápido para la determinación del contenido mineral de Fe, Na, K, Mg y Zn en
rúcola, representando una herramienta útil para la reducción del tiempo de análisis, de
bajo coste y sin la utilización de productos químicos tóxicos.
9. El potencial tumoricida y la inducción de apoptosis de entradas de rúcola y sulforrafano
medido frente a diferentes líneas tumorales depende del perfil fitoquímico de la
entrada, especialmente de su rendimiento en isotiocianatos, aunque también del tipo
de ensayo, siendo elegible el basado en la exclusión de azul tripán.
10. El potencial protector del daño genético oxidativo de entradas con distintos contenidos
en glucosinolatos y la capacidad para incrementar los valores de “health span” se
encuentran correlacionados con su rendimiento en isotiocianatos, más que con el
contenido en glucosinolatos.
11. El test SMART es una herramienta rápida y de bajo coste analítico para evaluar la
toxicidad asociada a metal(oides) en matrices nutricionales.
12. Todas las muestras de rábano exhibieron actividad tumoricida, pero con diferentes
rangos de inhibición. La planta no tratada con metales presentó la actividad
antiproliferativa más alta. Los glucosinolatos y sus productos de hidrólisis, los
isotiocianatos de la raíz, pueden ser los principales agentes moduladores de las
actividades antigenotóxicas y citotóxicas de las plantas desarrolladas en suelos
contaminados con estos metales.
13. Sería necesario revisar los criterios para el establecimiento de los límites permitidos
por la legislación acerca de la concentración de los diferentes metales en rábano. Esta
afirmación se basa en que aunque las concentraciones de metal(oides) en rábano no
excedían los límites máximos permitidos por la legislación para su consumo (excepto la
muestra de la raíz para el caso del Cd), los ensayos de genotoxicidad desarrollados
mostraron una tasa alta de mutaciones en Drosophila.
216
217
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