Professional Documents
Culture Documents
Dirigido por:
ISBN-13: 978-84-694-5933-1
Dra. Mercedes Del Ro Celestino, Investigadora del rea de Mejora y Biotecnologa de Cultivos
del IFAPA- Centro la Mojonera, Almera,
Dr. Rafael Font Villa, Investigador del rea de Tecnologa, Postcosecha e Industria
Agroalimentaria del IFAPA- Centro la Mojonera, Almera, y
Dra. ngeles Alonso Moraga, Catedrtica del Departamento de Gentica de la Universidad de
Crdoba, tutora de esta Tesis,
INFORMAN:
Que el trabajo titulado CARACTERIZACIN NUTRICIONAL Y AGRONMICA, ANLISIS DE
LA ACTIVIDAD BIOLGICA Y SELECCIN DE CRUCFERAS PARA USO ALIMENTARIO,
realizado por Myriam Magdalena Villatoro Pulido, bajo la direccin de los doctores Mercedes Del
Ro Celestino y Rafael Font Villa, puede ser presentado para su exposicin y defensa como Tesis
Doctoral en el Departamento de Gentica de la Universidad de Crdoba.
Considerando que se encuentra concluida, dan el VB para su presentacin y lectura.
AGRADECIMIENTOS
Podra llenar pginas y pginas de agradecimientos para todas aquellas personas que han
compartido estos aos de tesis y que han colaborado este trabajo. Para empezar quisiera
agradecer a mis directores de tesis, Mercedes del Ro 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, ponenciasEstos aos no siempre han sido fciles, con
vosotros he podido ver la parte ms dura y difcil de la investigacin en Espaa, aunque tambin
la parte positiva cuando detrs hay un trabajo y un esfuerzo.
Gracias a Angelines Alonso portodo, sin ella nada de esto no hubiera sido posible.
Gracias a Andrs Muoz por toda su ayuda con el tratamiento estadstico, y por estar siempre
dispuesto con una sonrisa. A Jouad Anter por poder contar siempre con l como compaero y
amigo. Gracias a Marisol Paredes, a Fernando Calahorro y a Zahira Fernndez por los buenos
momentos y la risa durante las comidas. Gracias a
conseguido lo que cualquier persona soara para su trabajo: el confundir la llave de su casa con
la de la puerta del laboratorio, creo que esto es ms de lo dice todo.
Quisiera agradecer al IFAPA de Crdoba, a la Universidad de Crdoba, al IFAPA de
Almera, al Instituto de Investigacin Alimentaria (IFR) de Norwich y al Departamento de Qumica
Farmacetica de la Facultad de Farmacia de Gnova por la colaboracin prestada, as como a los
miembros que han colaborado. Me gustara destacar en especial al Dr. Richard Mithen, la Dra.
Mara Traka, el Dr. Michele Forina y la Dra. Carla Armanino.
Especial mencin a todos y cada uno de los colaboradores de los captulos que conforman
este trabajo, sin vosotros estos resultados no seran posibles. Adems quisiera agradecer su
apoyo al Dr. Antonio de Haro Bailn y a la Dra. M Dolores Luque de Castro.
Gracias a los miembros del tribunal por su buena disposicin y su colaboracin.
La lista de personas por nombrar sera interminable, amigos, compaeros, profesores y
tantas personas que han trabajado durante estos ltimos aos dando nimos y consejo.
Por ltimo y no menos importante quiero agradecer a mi familia su apoyo constante, su
comprensin en los momentos difciles, 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 ms difcil de este proyecto que termina, para comenzar el nuestro en
comn. Sin tu paciencia y maravillosos consejos no se cmo lo hubiera podido terminar.
Nota: el trabajo de esta tesis doctoral se presenta en parte en Ingls, ya que los captulos que la
conforman han sido publicados o enviados a diferentes revistas de investigacin y se han editado
los trabajos originales. Por esta misma razn las referencias de cada captulo aparecen al final del
mismo tal y como establecen las normas de cada una de las revistas.
10
NDICE
11
RESUMEN
INTRODUCCIN
15
20
20
21
3. La rcula
3.1. Clasificacin taxonmica y procedencia.
3.2. Referencias histricas de la rcula.
3.3. Fuentes de variabilidad gentica
3.4. Bancos de germoplasma de rcula
3.5. Situacin actual en la mejora gentica de la rcula
4. El rbano
4.1. Procedencia, referencias histricas y usos
5. Fitoqumicos presentes en Crucferas
5.1. Los glucosinolatos
5.2. Los isotiocianatos
5.3. Los compuestos fenlicos
5.4. Los carotenoides
5.5. Los carbohidratos
6. Los minerales
7. Acumulacin de metales pesados en especies de crucferas
8. Caracterizacin de los recursos genticos
8.1. Caracterizacin agro-morfolgica de Eruca
8.2. Caracterizacin organolptica de Eruca
8.3. Caracterizacin nutricional y funcional de Eruca
8.3.1. Aproximacin al papel fitoqumico de Eruca
8.3.2. La Espectroscopa por reflectancia en el infrarrojo cercano (NIRS) como
herramienta analtica en la caracterizacin del perfil de minerales en especies
vegetales.
8.3.3. Actividad biolgica de las lneas de crucferas.
22
22
23
23
24
25
26
26
27
27
29
31
33
33
34
35
35
36
37
38
38
38
OBJETIVOS DE LA TESIS
39
39
40
40
40
41
43
46
48
12
70
93
114
138
168
CAPTULO VII: Actividad biolgica del rbano, una crucfera, con contenido en
metales y metaloides
181
DISCUSIN GENERAL
202
CONCLUSIONES
213
218
REFERENCIAS
13
RESUMEN
14
nos indica que es posible utilizar este material como base para la mejora
15
16
17
18
INTRODUCCIN
19
20
fitoqumicos para el
21
crucferas (Mithen, 2001). Los glucosinolatos y sus productos de degradacin han demostrado
ser unos potentes inhibidores del crecimiento de ciertas clulas tumorales. As, se ha comprobado
que ciertos compuestos como el sulforrafano, derivado de la glucorafanina, un glucosinolato
presente en las hojas de rcula y en otras crucferas como el brcol, tienen un marcado efecto
protector contra determinadas sustancias carcinognicas (Juge et al., 2007; Singh et al., 2009;
Keum et al., 2009; Chambers et al., 2009; Traka et al., 2010).
3. La rcula
3.1. Clasificacin taxonmica y procedencia.
Bajo el trmino de rcula se incluyen varias especies de plantas pertenecientes a la
familia Cruciferae, siendo las ms comunes aquellas pertenecientes a los gneros Eruca y
Diplotaxis. Eruca es originaria de la cuenca del Mediterrneo y Asia occidental. Es una planta
anual y parcialmente algama (2n = 2x = 22), e incluye las especies Eruca stenocarpa y Eruca
vesicaria (Padulosi 1997). Esta ltima 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 (Gmez-Campo, 1993; Gmez-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 rea geogrfica ms amplia
en el mundo. Las subespecies vesicaria y pinnatifida son endmicas de Espaa y Noroeste de
frica. La subsp. longirostra (Uechtr.) Maire, aunque tambin ha sido descrita, un detallado
anlisis morfomtrico basado en las dimensiones del fruto no termina de confirmar este estatus
(Gmez-Campo, 2003).
La rcula es un cultivo conocido desde hace siglos. Su denominacin proviene del latn
uro que significa quemo, debido al sabor pungente que tiene. Es importante como cultivo de
hoja en diferentes pases circunmediterrneos entre los que se encuentran Italia, Grecia, Turqua,
Egipto y Sudn (Pimpini y Enzo, 1997). En Espaa, son varias las empresas del sector de la IV
gama que la producen desde hace algunos aos, como Verdifresh, Florette y Primaflor, entre
otras. En concreto, en Andaluca es producida y comercializada como hortaliza de hoja por la
empresa Primaflor S.L. en la provincia de Almera, 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 Amrica del Norte, sur de frica y Australia. En el resto del mundo, el rgano de consumo lo
constituyen las hojas y tallos jvenes, que se ingieren crudos en ensaladas (Stephens, 2006).
Adems se considera como planta medicinal y puede ser empleada en control biolgico de plagas
(Gmez-Campo, 1995; DAntuono et al. 2009). Como otras crucferas, contiene un amplio rango
22
23
plantas (Hawkes, 1991; Swanson, 1996). Paradjicamente, el mejorador que desarrolla nuevas y
uniformes variedades para el mercado, aumentando an ms la homogeneidad, al mismo tiempo
es absolutamente dependiente de poseer un pool gentico asequible, muy amplio y bien
conservado que le permita afrontar nuevos retos del futuro. Este pool gentico, tan necesario
para la agricultura, se halla principalmente mantenido en distintos bancos de germoplasma, tanto
nacionales como internacionales.
3.4. Bancos de germoplasma de rcula
Algunos Centros Pblicos e Internacionales poseen importantes Bancos de Germoplasma.
En Espaa, el CRF (Centro de Recursos Fitogenticos) del INIA (www.inia.es/crf), tiene la
responsabilidad de conservar duplicados un banco base de todas las colecciones espaolas de
semillas y ser centro nacional de documentacin. Actualmente, su inventario nacional recoge
informacin de 52 entradas de Eruca vesicaria (Cav) DC. De ellas 46 corresponden a Espaa (37
de Castilla-La Mancha, 5 de Comunidad de Madrid, 3 de Aragn y 1 de Castilla-Len). Respecto a
Diplotaxis tenuifolia (L.) DC. el CRF conserva 2 entradas de las cuales slo una procede de
Espaa (Castilla-La Mancha).
El inters de la rcula como hortaliza de hoja para uso alimentario (Padulosi 1995; SilvaDas, 1997) y la iniciativa del proyecto por parte del International Plant Genetic Resources Institute
(IPGRI) en Especies Mediterrneas Infrautilizadas como mejora de conservacin, deriv en el
establecimiento de la Red de Recursos Genticos de Rcula (Rocket Genetic Resources Network)
(Gmez-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
posean fuentes genticas de rcula en el mundo. Adems, la creacin de dicho proyecto en 1994
tena como objetivo promover este cultivo mediterrneo infrautilizado, supervisar las instituciones
que albergan germoplasma y salvaguardar su diversidad proporcionando as una amplia base
gentica para su explotacin (Padulosi y Pignone, 1997). Hasta entonces el conocimiento
existente sobre la variabilidad de la especie era muy escaso. La mayora de los individuos
pertenecan a la subespecie sativa y al menos un 50% del total del germoplasma proceda de
Italia estando el resto de Europa mal representada. Se cree que en las colecciones de Eruca hay
cierto grado de duplicacin y que los programas de recoleccin no han seguido estrategias bien
definidas, ni han prestado todo el apoyo econmico necesario. Adems falta informacin de la
coleccin mantenida en el USDA (United States Department of Agriculture) acerca del origen real
de las muestras y de su variabilidad gentica.
Aunque lneas nuevas son continuamente aadidas a la coleccin de germoplasma
silvestre de rcula (Pita-Vilamil et al., 2002), el gnero Eruca, comparado con otros vegetales de
la familia Cruciferae se encuentra infrautilizado desde la perspectiva de la Mejora Vegetal, ya que
24
segn el estatus de las colecciones se puede observar que la rcula ha sido objeto de escasos
programas de evaluacin. Por tanto, se hace necesario el anlisis de las caractersticas biolgicas
y agronmicas del germoplasma para optimizar su utilizacin (Chin, 1994).
El bajo nivel de
25
glucosinolatos), queda fuera de toda duda el inters potencial del cultivo y la mejora de esta
especie, y explicara el inters de empresas
Research Park en Reino Unido, Univ. Vila Real en Portugal, University of Bologna en Italia,
Saskatoon Research Centre en Canad, Universidad de Izmir en Turqua) en el desarrollo de
lneas con alto valor aadido.
26
(Nakamura et al., 2001), activacin 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), as
como induccin de enzimas de detoxificacin (Lee y Lee, 2006; Hanlon et al., 2007). En los
estudios de su actividad biolgica se han utilizado extractos o fitoqumicos especficos 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 fenlicos (Sgherri et al., 2003) y antocianinas (Liu et al.,
2008; Wang et al., 2010).
La
molcula de glucosinolato consiste en una unidad de -tioglucsido, una oxima sulfonada y una
cadena lateral variable, derivada de un aminocido (Fig. 1).
27
28
Adems de existir una gran variabilidad en nmero (hay especies que presentan un solo
glucosinolato en su composicin, mientras que otras poseen ms de 30 diferentes), existe tambin
una gran variabilidad en la concentracin de dichos compuestos dentro de una misma especie, as
como entre las distintas partes de la planta (races, tallo, hojas y semillas), o en funcin del estado
fenolgico 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 crucferas. Numerosos artculos demuestran los
efectos beneficiosos de los ITCs tales como los efectos en las enzimas de biotransformacin
implicadas en el metabolismo de carcingenos (Lampe y Peterson, 2002), prevencin del cncer,
inhibicin de enzimas de biotransformacin de fase I e induccin 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 crucferas (Mithen, 2001; Traka et al., 2010). Los ITCs ms 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
hidrlisis de la glucorrafanina, glucoerucina y glucoiberina, respectivamente. La ER adems puede
obtenerse a travs de la reduccin in vivo del sulforrafano (Melchini et al., 2009). La interconversin in vivo de estos dos glucosinolatos y su similitud estructural han sugerido una actividad
biolgica tambin semejante.
A diferencia de la mayora de los ITCs, el SF contribuye poco al sabor, y es el ITC ms
hidroflico. El SF es el ITC ms 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 tumorignesis inducida por carcingenos y se cree que los efectos
quimiopreventivos probablemente impliquen varios mecanismos, los cuales interaccionan juntos
para reducir el riesgo de carcinognesis (Juge et al., 2007). Estos mecanismos incluyen:
1) Inhibicin de enzimas citocromo P450 de fase I: los procarcingenos una vez que entran en el
organismo pueden oxidarse, as como reducirse o hidrolizarse pasando a intermediarios
altamente reactivos que pueden unirse a macromolculas como el ADN, ARN o protenas. Este
evento es llamado metabolismo de fase I (Nelson et al., 1993). Se ha demostrado adems que
29
el SF in vitro puede inhibir la formacin de los aductos de ADN inducidos por carcingenos
(Yang et al., 1994).
2) Induccin de enzimas del metabolismo de fase II: las enzimas de fase II convierten los
carcingenos en metabolitos inactivos preparados para ser excretados por el cuerpo y
previniendo su reaccin con el ADN. El SF ha recibido mucha atencin al descubrirse que es la
sustancia natural ms potente de induccin 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). Adems se
ha observado que no solo es activo en clulas carcingenas, sino tambin en clulas no
transformadas (McMahon et al., 2003).
3) Funciones antioxidantes a travs del incremento de los niveles en el tejido de glutatin: el SF
no es un antioxidante directo o prooxidante, sino que acta indirectamente para incrementar la
capacidad antioxidante de las clulas animales y su capacidad para sobrellevar el estrs
oxidativo actuando sobre el GSH, que es un tripptido importante que mantiene el equilibrio de
oxidacin-reduccin y protege a la clula 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
condensacin de la cromatina, traslocacin del la fosfatidilserina a travs de la membrana
plasmtica y fragmentacin del ADN en tratamientos con SF en clulas tumorales (GametPayrastre et al., 2000; Choi et al., 2003; Gingras, et al., 2004; Jackson et al., 2004). Adems se
ha visto una implicacin 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
tambin una implicacin de la mitocondria, la familia proteica bcl-2, de JNK/MAPK y de p53
(Juge et al., 2007).
5) Induccin del arresto del ciclo celular: hay evidencias de que el SF ejerce un mecanismo
anticarcinognico por arresto del ciclo celular a diferentes etapas de su progresin, por
regulacin de la inhibicin de CDKs, la disrupcin de microtbulos y modificacin de histonas
entre otras (Juge et al., 2007).
6) Propiedades antiinflamatorias: implicando iNOS, Cox-2 y TNF- (Juge et al., 2007).
7) Inhibicin de la angiognesis: actuando sobre la proliferacin de las clulas endoteliales (Juge
et al., 2007).
30
31
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 fenlicos de Eruca (Weckerle et al., 2001; Bennett et al., 2002; Arrabi et al., 2004).
Entre los compuestos fenlicos encontrados se pueden citar kaempferol, isohamnerin y
quercetina.
32
33
6. Los minerales
Los humanos requieren ms de 22 elementos minerales para cubrir sus necesidades
fisiolgicas alimentarias. Algunos de estos elementos son requeridos en elevadas cantidades
como son por ejemplo el Calcio (Ca) y el Fsforo (P). Sin embargo, otros como el Hierro (Fe), Zinc
(Zn) y Cobre (Cu), slo son necesarios en pequeas 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. As se estima que de 6
billones de personas, entre el 60-80% son deficientes en Fe, ms del 30 % son deficientes en Zn,
y ms del 15% son deficientes en Ca, Mg y Cu (White y Broadley, 2005).
La deficiencia en Fe es la ms extendida en el mundo, estando ligada a muchas
enfermedades como la anemia y en muchas de las muertes de mujeres embarazadas y neonatos,
adems de ser causa de abortos. Principalmente la encontramos en el sur de Asia y frica,
aunque los grupos de poblacin con ms 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, tambin est muy extendida por el mundo, siendo este un elemento
implicado en la expresin de los genes, desarrollo y replicacin celular (Hambrigde, 2000). A este
micronutriente se le atribuye el incremento de padecer diarreas, neumona y malaria, adems de
asociarse su deficiencia con un incremento en la mortalidad en nios menores de 5 aos. Las
regiones ms afectadas por esta deficiencia son el sur de Asia y frica subsahariana,
principalmente afectando a nios (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). As 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 cules indicaron su potencial uso como suplementos
nutricionales de minerales concentrados en forma de cpsulas o tabletas. En dichos trabajos las
concentraciones medias de Fe, Zn, Mn, Se, Cr, Ca, Mg y P variaron desde 2500 hasta 73000 g g-1
ps, adems dichos elementos se encontraron en una forma ms soluble y metablicamente ms
disponible que en los suplementos minerales comerciales especficos.
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), indicndose que las hojas de esta especie poseen concentraciones
significativas de los minerales principales.
35
estrategias para el diseo de variedades que incrementen la salud del consumidor de estos
vegetales.
La caracterizacin es el establecimiento de todos los caracteres posibles de un cultivo.
Entre estos enfoques podemos citar los siguientes:
- Caracterizacin agro-morfolgica: estudia cualquier rgano de la planta desde el
de los alimentos que se perciben por los sentidos de la vista, el odo, el gusto, el olfato y el
tacto, mediante personas entrenadas o instrumental analtico.
- Caracterizacin nutricional y funcional: estudia los valores nutricionales y funcionales
36
37
38
personal especializado, destruccin de la matriz analizada, y polucin del medio ambiente, debido
al uso de reactivos qumicos, entre otros.
Estos antecedentes han llevado a la bsqueda de tecnologas analticas alternativas, que
aunque perdiendo precisin en la cuantificacin del analito, permitan un muestreo rpido y a bajo
coste econmico, redundando en una importante descarga analtica para el laboratorio. Es por ello
una tcnica con posibilidades reales de ser empleada como mtodo de muestreo rpido, 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 Espectroscopa en el Infrarrojo
Cercano (NIRS), ha mostrado un alto potencial para la prediccin de minerales, tanto en matrices
orgnicas como en inorgnicas (Clark et al., 1989; Nilsson et al., 1996; Halgerson et al., 2004;
Cozzolino y Moron 2004; Petisco et al., 2005), si bien, an no se ha aplicado dicha tecnologa en
rcula.
8.3.3. Actividad biolgica de las lneas de crucferas.
Los efectos beneficiosos atribuibles a los compuestos fitoqumicos de las crucferas, as
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 evaluacin sobre modelos biolgicos que se asemejen a las
condiciones fisiolgicas humanas. Se han publicado numerosos trabajos sobre la capacidad de
diferentes especies de crucferas de inhibir procesos de tumorognesis in vitro (Verhoeven et al.,
1997; van Poppel et al., 1999; Lynn et al., 2001). Estudios enfocados al anlisis de la expresin
global gnica pueden aportar pocas pruebas del los mecanismos potenciales derivados de
experimentos in vitro e in vivo con objeto de explicar los datos epidemiolgicos a favor de que el
consumo de crucferas puede reducir el riesgo de padecer cncer (Traka et al., 2008). No
obstante, en dicho trabajo se pudo observar una perturbacin en las vas de seal implicadas en
la carcinognesis e inflamacin. Hasta ahora slo se ha realizado un estudio utilizando el material
vegetal de Eruca para analizar los efectos antigenotxicos (Lamy et al., 2008) y otro analizando el
posible efecto protector de los glucosinolatos y productos de degradacin de Eruca.
Ensayo de inhibicin del crecimiento tumoral
La utilizacin de cultivos celulares para el ensayo de las caractersticas txicas de una
sustancia qumica constituye una metodologa barata, rpida y evita el sacrificio de animales de
sangre caliente. Estas clulas se caracterizan por proliferar continuamente en suspensin, aunque
tras un periodo de cultivo, pierden su capacidad de divisin y se diferencian. Durante muchos
aos, diferentes lneas celulares han sido extensamente estudiada para esclarecer los
mecanismos de citotoxicidad y que inducen diferenciacin y apoptosis y as poder controlar su
proliferacin en los organismos vivos (Collins et al., 1978; Conte-Anazetti et al., 2003).
39
alteraciones
genticas producidas en las clulas de discos imaginales alares de la larva, que pueden
evidenciarse fenotpicamente en el tejido adulto despus de la expansin clonal y la metamorfosis,
y fue desarrollado por Graf y colaboradores en 1984. Este ensayo ha mostrado ser capaz de
detectar actividad genotxica en compuestos de estructura qumica variada, tanto mutgenos
directos como promutgenos, con diferentes mtodos de accin genotxica, como agentes
alquilantes, intercalantes o formadores de aductos, tanto slidos, como lquidos, gaseosos,
simples o mezclas complejas (Graf et al, 1984; Alonso-Moraga y Graf, 1989; Graf at al, 1994;
Osaba et al, 1999). Adems, ante esta evidencia indirecta, los estudios bioqumicos dan cuenta de
40
41
42
43
propuesto como primer objetivo en esta tesis la caracterizacin de esta coleccin para la seleccin
de lneas de Eruca con mejores caractersticas agronmicas y nutricionales, mayor capacidad
antimutagnica 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 rbano son acumuladoras de
metales pesados txicos, cualidad que puede resultar negativa para su aprovechamiento
agronmico y funcional.
44
OBJETIVOS DE LA TESIS
45
46
47
CAPTULO I
Artculo en preparacin
Phenotypic diversity of rocket (Eruca)
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 Crdoba (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 (Gmez-Campo, 1993; Gmez-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 GmezCampo 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 (Gmez-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; DAntuono 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-Das, 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 Nuz, 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 Nuz, 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.
51
52
53
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
A. Seedling stage
Fresh matter
FM
Leaf colour
LC
LBS
LML
Leaf lobation
LLo
Leaf pubescence
LP
LAS
LBT
PME
1. Narrow, 2. Enlarged
Growth rate
GR
LPL
Leaf length
LL
Leaf width
LW
LL/W
LR
B. Flowering stage
Days to first flowering
Flower Colour
DFF
FC
C. Maturity stage
Plant height
Plant growth attitude
PH
PGA
55
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 Hernndez-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 Consejera de Innovacin, Ciencia y Empresa (Junta
de Andaluca), 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. Csar GmezCampos and to the germplasm banks for providing seeds used in this work. Myriam VillatoroPulido was supported by Instituto de Investigacin y Tecnologa Agraria y Alimentaria (INIA)
contract.
60
References
-
Bennett, R. N., Mellon, F. A., Rosa, E. A. S., Perkins, L., Kroon, P.A. 2004. Profiling
Glucosinolates, Flavonoids, Alkaloids, and Other Secondary Metabolites in Tissues of
Azima tetracantha L. (Salvadoraceae). Journal of Agriculture and Food Chemistry, 2004,
52: 58565862.
Bennet, R. N., Carvalho, R., Mellon, F. A., Eagles, J., Rosa, E. A. S. 2007. Identification
and quantification of glucosinolates in sprouts derived from seeds of wild Eruca sativa L.
(salad rocket) and Diplotaxis tenuifolia L. (wild rocket) from diverse geographical locations.
Journal of Agriculture and Food Chemistry, 55: 67-74.
Bozokalfa, M. K., llbi, D.H, Asiogul, T.K. 2010. Estimates of genetic variability and
association studies in quantitative plant traits of Eruca spp. landraces. Genetika, 42: 501512.
Chandel, K. P. S., Bhandari, D. C. 1989. Collection of germplasm resources in northeastern Rajasthan. Indian Journal of Plant Genetic Resources, 2: 150-56.
DAntuono, L. P., Elementi, S., Neri, R. 2008. Glucosinolates in Diplotaxis and Eruca
leaves: Diversity, taxonomic relations and applied aspects. Phytochemistry, 69: 187-199.
Esiyok, D. 1997. Marketing and utilization of rocket in turkey. In: Padulosi S, Pignone D,
editors. Rocket: A mediterranean crop for the world. Report of a workshop 13-14 December
1996. Rome, Italy: International plant Genetic Resources Institute.
Gmez Campo, C. 1993. Eruca. In S. Castroviejo & al. (eds). Flora. Ibrica, 4: 390-392.
Jalas, J., Suominen, J., Lampinen, R. (eds.) 1996. Atlas Florae Europaeae distribution of
Vascular Plants in Europe, Vol. 11. Cruciferae (Ricotia to Raphanus). Helsinki, Finland:
Helsinki University Printing House.
IBPGR, 1990. Descriptors for Brassica and Raphanus. International Board for Plant
Genetic Resources, Rome.
IPGRI, 1999. Descriptor for Rocket (Eruca spp.). International Plant Genetic Resources
Institute. ISBN 92-9043-421-X.
Mithen, R.F., Dekker, M., Verkerk, R., Rabot, S., and Jonson, I.T., 2000. Review: The
nutritional significance, biosynthesis and bioavailability of glucosinolates in human foods. J
Sci Food Agric 80, 967-984.
Morales, M. R., Maynard, E., Janick, J., 2006. Adagio: A slowbolting Arugula.
HortScience, 41: 15061507.
Padulosi S. 1995. The Rocket Genetic Resources Network. Report of the First Meeting, 13
15 November 1994, Lisbon, Portugal. International Plant Genetic Resource Institute,
Rome, Italy.
Padulosi, S., Pignone, D. 1997. Rocket: A mediterranean crop for the world. Project on
Unterutilized Mediterranean Species. International Plant Genetic Resources Institute
(IPRGI), Roma. http://www.ipgri.cgiar.org/publications/pdf/234.pdf.
Pico, B., Nuez, F., 1999. Genetic resources of Leafy crops in the Genebank of the
Polytechic University of Valencia. In: Lebeda, E. Kfistkova (Eds.). Eucarpia Leafy
Vegetables 99: 73-74.
Pignone, D and Gmez-Campo, C., 2011. Eruca. In: Wild Crop Relatives: Genomic and
Breeding Resources, C. Kole (ed.) 149-160
Pimpini, F., Enzo, M. 1997. La coltura della rucola negli ambienti veneti. Colture protette 4:
21-32.
Podsedek, A., 2007. Natural antioxidants and antioxidant capacity of Brassica vegetables:
A review. Food Science and Technology, 40,1-11.
Silva-Das J.C. (1997). Rocket in Portugal: botany, cultivation, uses gram (Cicer arietinum)
and taramira (Eruca sativa). Indian and potential. In: Padulosi S. and Pignone D. (eds),
Rocket: a Mediterranean crop for the world. Report of a workshop, 1314 December 1996,
Legnaro (Padova), Italy. International Plant Genetic Resource Institute, Rome, Italy, pp.
8185.
62
Warwick, S. I., Gugel, R. K., Gmez-Campo, C., James, T. 2007. Genetic variation in Eruca
vesicaria (L.) Cav.. Plant Genetic Resources: Characterization and Utilization, 5: 142-153.
Yaniv, Z., 1996. Traditions, uses and research on rocket in Israel. In Rocket: A
Mediterranean Crop for the the World; Report of a workshop, 13-14 December, 1996,
Legnaro, Italy; Pignone, D., Padulosi, S., Eds.; IPGRI: Rome, pp. 76-80.
Yaniv, Z., Schafferman, D., Amar, Z., 1998. Tradition, uses and biodiversity of rocket
(Eruca sativa Brassicaceae) in Israel. Economic Botany 52: 394400.
63
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
136.5932.28de
134.931.54de
234.9436.98fg
127.318.61cd
112.2710.01cd
216.151.02fg
96.426.19bc
145.350.63df
56.314.13a
282.751.55gh
125.618.61cd
39.924.99bh
35.463.52fk
38.323.91cj
34.795.72gk
37.978.45cj
35.063.10gk
39.274.93bi
44.555.99ab
48.824.78a
36.984.11bf
34.975.24gk
1.030.15bc
1.050.20bc
1.010.10bc
0.330.03a
0.430.23a
1.170.07cd
1.220.08cd
1.200.00cd
0.810.10ab
0.640.38ab
0.900.11bc
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
1000.66b
821.28a
990.94b
1240.75c
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
122.142.08cd
127.142.08cd
145.411.10df
240.577.17fg
102.341.5bc1
92.236.39bc
287.931.55gh
93.7214.72bc
384.365.47h
234.2833.48fg
142.9711.04df
239.566.92fh
189.2044.90ef
36.566.01ek
35.23 4.08gk
38.703.08cj
40.033.66bh
43.515.74bc
37.205.23dj
31.273.45ik
36.053.94ek
35.433.81fk
35.825.63fk
38.474.79cj
36.883.25dk
34.994.51gk
nd
1.050.14bc
1.200.00cd
1.100.00cd
1.200.00cd
0.930.08bc
1.280.17de
1.330.06de
1.100.00cd
1.160.14cd
1.030.06bc
1.160.15de
1.400.00de
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
1240.60c
990.58b
PEX-63
PEX-64
PEX-65
145.828.27df
122.133.46de
112.4912.71cd
34.935.79gk
35.029.05gk
37.533.26dj
1.550.28e
1.150.21cd
1.330.06de
7 (0.0%)c
7 (0.0%)c
5 (0.0%)b
PEX-66
117.315.74cd
33.724.83ik
nd
PEX-67
140.259.12df
37.485.53dj
40.613.47bg
34.932.93cj
41.763.71be
33.86.66ik
38.918.15cj
34.64.19gk
36.94.90dk
35.185.85gk
37.313.34dj
33.224.51ik
PEX-68
89.3314.53ab
PEX-69
76.5015.35a
PEX-70
151.452.47df
PEX-71
75.580.94a
PEX-72
126.1422.46de
PEX-73
106.0113.25cd
PEX-74
150.1428.32df
PEX-75
72.1127.81a
PEX-76
174.9434.60ef
PEX-77
172.5026.73ef
Table 3. Continued.
1220.66c
991.28b
1040.89b
1010.55b
1240.86c
1090.62b
970.54b
991.35b
1010.87b
1060.57b
1060.27b
1000.47b
990.52b
1010.43b
1040.43b
1040.57b
1040.66b
1011.28b
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
P
E
E
E
P
P
P
P
E
P
I
P
E
E
E
P
I
1050.39b
99
99
105
P
I
3 (0.0%)a
990.52b
99
nd
3 (0.0%)a
1040.43b
104
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
1010.43b
1000.57b
101
100
99
82
101
82
99
82
96
82
990.89b
990.55b
990.66b
821.28a
1010.89b
820.91a
990.89b
820.91a
960.52b
820.43a
Accession
FM
LC
PH
GR
DFF
PGA
PEX-78
PEX-79
PEX-80
200.6841.25fg
148.240.32df
81.670.27ab
42.526.06bd
38.412.71cj
38.274.38cj
nd
nd
nd
5 (0.0%)b
7 (0.0%)c
7 (0.0%)c
1030.43b
920.57b
820.66a
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
124.0838.65cd
93.144.21bc
159.9118.00ef
138.774.26df
236.1186.49fg
185.920.27ef
140.8523.97df
128.7111.97de
125.618.61cd
63.9215.30a
37.345.21dj
36.625.78ek
36.216.97ek
37.496.83dj
36.305.04ek
36.864.92dk
33.193.39ik
33.606.46ik
34.354.82hk
34.414.83hk
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
821.28a
820.89a
820.91a
820.66a
821.28a
960.89b
820.43a
950.57b
1030.66b
961.28b
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
5.381.17a
5.342.24a
5.101.04b
2.881.03b
2.421.37b
5.501.16b
4.391.80b
4.571.23b
1.790.52b
2.190.82b
6.951.01b
6.300.71b
6.190.96a
3.911.28a
5.850.82a
7.111.54a
3.230.83a
6.671.41a
3.870.88a
14.051.67ef
13.302.24ef
15.585.67de
10.551.32fg
10.191.60fg
14.891.67de
13.212.54eg
12.731.50eg
10.331.84fg
8.843.34fg
14.941.45de
15.431.64de
13.141.44eg
11.891.08fg
14.011.57ef
17.363.12b
9.262.37bg
14.442.01de
11.001.04fg
2.630.52b
3.371.05a
2.930.48b
2.010.34b
1.690.28b
2.690.61b
2.700.92b
2.810.59b
2.980.62b
2.300.95b
3.200.39b
3.590.74b
2.570.34a
2.400.41a
2.740.52a
4.051.04a
2.680.63a
2.800.93a
2.770.25a
5.471.04cd
4.170.96ab
5.371.87cd
5.320.64cd
6.131.16d
5.751.29cd
5.211.39cd
4.721.21ab
3.510.49a
3.601.44a
4.690.35ab
4.420.77ab
5.150.53cd
5.091.01cd
5.291.15cd
4.390.81ab
3.591.03a
3.720.64a
3.980.36ab
LPL
LL
LW
LL/W
PEX-58
PEX-59
PEX-60
PEX-61
PEX-62
PEX-63
PEX-64
8.011.39a
4.801.47a
4.261.11a
5.010.94a
6.651.32a
7.710.59a
8.250.42a
16.611.04bc
12.651.92fg
11.212.06fg
12.921.25eg
16.721.96b
15.691.01de
17.831.21b
3.500.40a
3.020.45a
2.790.61a
2.850.41a
3.460.55a
3.160.39a
3.860.52a
4.790.53ab
4.240.78ab
4.080.65ab
4.590.54ab
4.910.76bc
5.010.53bc
4.680.67ab
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
6.051.41a
4.711.55a
4.611.19a
5.731.47a
5.482.58a
6.102.10a
5.251.32a
4.960.96a
4.651.32a
7.551.07a
6.561.24a
5.230.82a
6.831.93a
4.931.27a
5.441.55a
4.270.44a
4.851.21a
4.741.44a
4.901.41a
5.010.97a
6.201.32a
5.491.58a
5.280.64a
5.640.59a
6.070.87a
6.142.29a
15.822.04bd
23.963.55a
13.132.46eg
14.522.62de
12.744.83eg
16.702.83bc
13.523.10ef
11.951.75fg
12.532.89fg
16.701.92bc
15.033.16de
15.022.32de
16.982.22b
13.531.68ef
14.661.67de
11.181.17fg
15.527.09de
11.761.29fg
12.701.40fg
14.712.42de
14.812.15de
14.342.45de
12.942.08eg
13.502.94ef
14.371.63de
15.242.67de
3.170.43a
3.170.43a
3.020.57a
3.280.66a
2.741.28a
3.930.89a
3.080.91a
2.800.65a
3.030.67a
3.961.32a
3.280.58a
3.400.41a
3.500.88a
2.870.45a
3.300.86a
2.770.62a
3.270.54a
2.770.60a
3.180.58a
4.041.35a
3.280.57a
3.250.57a
2.650.27a
3.110.47a
3.250.43a
4.050.76a
5.050.82bc
6.890.86d
4.410.79ab
4.470.55ab
4.451.84ab
4.340.76ab
4.500.67ab
4.370.69ab
4.230.94ab
4.501.05ab
4.610.70ab
4.720.55ab
5.050.97bc
4.800.92bc
4.600.77ab
4.150.66ab
4.782.04ab
4.370.77ab
4.070.57ab
3.840.79ab
4.590.75ab
4.480.83ab
4.890.65bc
4.451.18ab
4.460.65ab
3.860.86ab
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 Duncans Multiple Range Test among groups and LL show the letters among
accession.
66
67
68
69
CAPTULO II
Artculo en preparacin
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 Ruz Prez-Cachoc, Sara Obregn-Canod, Antonio De
Haro-Bailnd, , Mercedes Del Ro-Celestinob.
a
Spain.
d
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.
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. DAntouno 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.
72
Seeds were
germinated in Petri dishes for 48 h under a minimum temperature of 25C. Pots were placed in
greenhouse under natural light, temperature of 27/18 C (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 (37 53 N; 4 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
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
Origin
Germany
Iran (Persepolis
Ruins)
Spain (Zaragoza)
Spain (Zaragoza)
Italy (Palermo)
Begium
France
United Kingdom
Spain (Navarra)
Spain
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
A. Seedling stage
Fresh matter
Number of leaves
Leaf colour
FM
NL
LC
LBS
LML
Leaf lobation
Leaf pubescence
Leaf apex shape
Leaf blade thickness
Petiole and/or
midvein enlargement
Growth rate
LLo
LP
LAS
LBT
PME
1. Narrow, 2. Enlarged
GR
LPL
Leaf length
LL-s
Leaf width
LW-s
LL/W
B. Flowering stage
Days to first flowering
DFF
PH
C. Maturity stage
Plant height
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 C, 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 L 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 m bead size, 30000 Da
exclusion limit). Desulfation was performed by addition of purified sulfatase (75 l, 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 m 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).
75
2.6.2. Assessors
Ten trained panellists (7 female, 3 male) from the University of Crdoba (Crdoba, 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 Crdoba (Crdoba, 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.
-1
(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).
76
77
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 (99106 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 (6088 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 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 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 moles of
79
80
81
glucosinolates g-1
(GRA<5 moles of
82
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 DAntuono 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
Acknowledgements
This research was supported by the Consejera de Innovacin, Ciencia y Empresa (Junta de
Andaluca), 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. Csar Gmez-Campos
and to the germplasm banks for providing vegetal material for this work. Myriam Villatoro was
supported by Instituto Nacional de Investigacin y Tecnologa Agraria y Alimentaria (INIA)
contract.
88
References
1. Padulosi S and D. Pignone, Rocket: a Mediterranean crop for the world. Report of a
workshop, 13-14 December 1996, Legnaro (Padova), Italy. International Plant Genetic
Resources Institute, Rome, Italy (1997).
2. Greuter W, Burdet HM and Long G, Med-Checklist, vol. III. Conservatoire et Jardin
Botaniques de la Ville de Genve, Genve (1986).
3. Gmez Campo C, Eruca. In S. Castroviejo & al. (eds). Flora. Ibrica, 4: 390-392 (1993).
4. Gmez-Campo C, Taxonomy. In: Gmez-Campo, C. (ed.). Biology of Brassica
coenospecies, ed. by Elsevier Science, Amsterdam, pp. 3-32 (1999).
5. Jalas J, Suominen J and Lampinen R, (eds.) Atlas Florae Europaeae distribution of
Vascular Plants in Europe, Vol. 11. Cruciferae (Ricotia to Raphanus), ed by. University
Printing House, Helsinki, (1996).
6. Al-Shehbaz IA, The genera of Brassiceae (Cruciferae, Brassicaceae) in the Southeastern. Arnold Arbor J. United. States, pp. 279-351 (1985).
7. Bennet RN, Rosa EAS, Mellon FA and Kroon PA, Ontogenic Profiling of Glucosinolates,
Flavonoids, and Other Secondary Metabolites in Eruca sativa (Salad Rocket),
Diplotaxis erucoides (Wall Rocket), Diplotaxis tenuifolia (Wild Rocket), and Bunias
orientalis (Turkish rocket). J. Agric. Food Chem, 54: 4005-4015 (2006).
8. Mithen RF, Dekker M, Verkerk R, Rabot S and Jonson IT, Review: The nutritional
significance, biosynthesis and bioavailability of glucosinolates in human foods. J Sci
Food Agric 80: 967-984 (2000).
9. Podsedek A, Natural antioxidants and antioxidant capacity of Brassica vegetables: A
review. Food Sci Technol, 40: 1-11 (2007).
10. Chamorro L and Sans F X, Life-history variation in agricultural and wild populations of
Erucastrum nasturtiifolium (Brassicaceae). Flora, 205: 2636 (2010).
11. Lefol E, Sguin-Swart G and Downey RK, Sexual hybridisation in crosses of cultivated
Brassica species with the crucifers Erucastrum gallicum and Raphanus raphanistrum:
Potential for gene introgression. Euphytica, 95: 127-139 (1997).
12. Chandra A, Gupta ML, Ahuja I, Kaur G and Banga SS, Intergeneric hybridization
between Erucastrum cardaminoides and two dipoid Brassica species. Theor Appl
Genet, 108:1620-1626 (2004).
13. Smith JSC and Smith OS, The description and assessment of distance between inbred
lines of maize: I. The use of morphological traits as descriptors. Maydica, 34: 141-150
(1989).
14. DAntuono LP, Elementi S and Neri R, Exploring new potential health-promoting
vegetables: glucosinolates and sensory attributes of rocket salads and related
Diplotaxis and Eruca species. J Sci Food Agric 89: 713-722 (2009).
89
15. IPGRI, Descriptors for rocket (Eruca spp.). ed. by IPGRI [International Plant Genetic
Resources Institute], Rome, (1999).
16. Wathelet JP, Wagstaffe PJ and Boenke A, The certification of the total glucosinolate
and sulphur contents of three rapeseed (colza), CRMs 190, 366 and 367. Comission of
the European Communities, report EUR 13339 EN, 1-75 (1991).
17. ISO norm, Rapessed-Determination of glucosinolates content- Part 1: method using
high-performance liquid chromatography. ISO 9167-1, 1-9 (1992).
18. Galn-Soldevilla H, Ruiz-Prez-Cacho MP, Serrano S, Jodral M and Bentabol A,
Development of a preliminary sensory lexicon for floral honey. Food Qual Pref, 16: 71
77 (2005).
19. Ruz Prez-Cacho MP, Galn-Soldevilla H, Len-Crespo F and Molina-Recio G,
Determination of the sensory attributes of a Spanish dry-cured sausage. Meat Sci, 71:
620-633 (2005).
20. Ruiz Prez-Cacho P, Galn-Soldevilla H, Mahattanatawee K, Elston A and Rouseff R,
Sensory lexicon for fresh squeezed and processed orange juice. Food Sci Technol Int,
14: 131-142 (2008).
21. Sensory analysis. General guidance for the design of test rooms. Organization for
Standardization, Genve. Ref. No. ISO 8589:1988.
22. Guerrero L, Gou P, Alonso P and Arnau J, Study of the physicochemical and sensory
characteristics of dry-cured hams in three pig genetic types. J. Sci. Food Agric, 70:
526530 (1996).
23. International standard 11035. Sensory analysis methodology identification and
selection of descriptors for establishing a sensory profile by a multidimensional
approach. International Organization for Standardization, Genve. Ref. No. ISO
11035:1994.
24. Sensory Analysis. Vocabulary. Organization for Standardization, Genve. Ref. No. ISO
5492:2008.
25. Egea-Gilabert C, Fernndez JA, Migliaro D, Martnez-Snchez JJ and Vicente MJ,
Genetic variability in wild vs. cultivated Eruca vesicaria populations as assessed by
morphological, agronomical and molecular analyses. Sci Hort, 121: 260266, (2009).
26. Warwick SI, Gugel RK, Gmez-Campo C and James T, Genetic variation in Eruca
vesicaria (L.) Cav. Plant Gen Res, 5: 142153 (2007).
27. Yaniv Z, Schafferman D and Amar Z, Tradition, uses and biodiversity of rocket (Eruca
sativa Brassicaceae) in Israel. Econ Bot, 52: 394400 (1998).
28. Rosa, E. A. S., Heaney, R. K., Portas, C. A. M. and Fenwick, G. R. (1996), Changes in
Glucosinolate Concentrations in Brassica Crops (Brassica oleracea and Brassica
napus) throughout Growing Seasons. Journal of the Science of Food and Agriculture,
71, (2) 237244.
90
29. Charron, CS, Arnold M, Saxton AM and Sams CE, Relationship of climate and
genotype to seasonal variation in the glucosinolate-myrosinase system. I. Glucosinolate
content in ten cultivars of Brassica oleracea grown in fall and spring seasons. J. Sci.
Food Agric, 85: 671-681 (2005).
30. Velasco P, Cartea ME, Gonzales C, Vilar M and Ords A, Factors affecting the
glucosinolate content of Kale (Brassica oleracea acephala group). J Agric Food Chem,
55: 955-962 (2007).
31. Cartea ME, Velasco P, Obregn S, Padilla G and De Haro A, Seasonal variation in
glucosinolate content in Brassica oleracea crops grown in northwestern Spain.
Phytochemistry, 69: 403-410 (2008).
32. Kim SJ and Ishii G, Glucosinolate profiles in the seeds, leaves and roots of rocket salad
(Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and purified 4methoxyglucobrassicin. Soil Sci Plant Nutr 52: 394400, (2006).
33. Daxenbichler ME, Spencer GF, Carlson DG, Rose GB Brinker AM and Powel RG,
Glucosinolate composition of seeds from 297 species of wild plants. Phytochemistry,
30: 2623-2638 (1991).
34. Agerbirk N, Warwick SI, Hansen PR and Olsen CE, Sinapis phylogeny and evolution of
glucosinolates and specific nitrile degrading enzymes. Phytochemistry, 69: 2937-2949
(2008).
35. Padilla G, Cartea ME, Velasco P, de Haro A and Ords A, Variation of glucosinolates in
vegetable crops of Brassica rapa. Phytochemistry, 68: 536-545 (2007).
36. Fenwick GR, Griffiths NM and Heaey RK, Bitterness in Brussels sprouts (Brassica
oleracea L var gemnifera): the role of glucosinolates and their breakdown products. J
Sci Food Agric, 34: 73-80 (1983).
37. Schonhof I, Krumbein A and Brckner B, Genotypic effects on glucosinolates and
sensory properties of broccoli and cauliflower. Nahrung/ Food, 48: 25-33 (2004).
38. Drewnowski A and Gomez-Carneros C, Bitter taste, phytonutrients, and the consumer:
a review. Am J Clin Nutr, 72: 1424-1435 (2000).
91
92
CAPTULO III
Artculo en preparacin:
An approach to the phytochemical profile of rocket (Eruca sativa (Mill.) Thell)
Myriam Villatoro-Pulido1, Feliciano Priego-Capote2, Beatriz lvarez-Snchez2, Shikha Saha3, Mark
Philo4, Sara Obregn-Cano5, Antonio De Haro.Bailn5, Rafael Font6, Mercedes Del Ro-Celestino6
1
IFAPA Centro-Alameda del Obispo, Department of Plant Breeding and Biotechnology, Crdoba,
Spain.
2
Phytochemicals and Health Programme, Institute of Food Research, Norwich Research Park,
Metabolomics and Mass Spectrometry, Institute of Food Research, Norwich Research Park, NR4
Almera, 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 mol/ 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 g/g of dry weight revealing a high variability. Lutein was the main
carotenoid ranging from 8.3 to 124.3 g/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 consumers 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 Rodrguez-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 (Gmez-Gonzlez 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 consumers 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,
Almera (south Spain). Seeds of Eruca sativa LGC1 (commercial variety Sky), LGC2, HGC1 and
HGC2 were obtained from Tozer Seeds Lyd (Cobham, Surrey, U.K.), Facult 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 C. Pots were placed in greenhouse under natural light at 27/18 C
(day/night) and a relative humidity of 50/70% (day/night). When the plants reached 812 cm, they
were transferred to a field in Crdoba, 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 C 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 75C for 15 min in 2.5 mL of 70:30
methanolwater and 200 L 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 m bead size, 30000 Da exclusion limit). Desulfation was carried out by addition of 75
L 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
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 (LCMS)
Freeze-dried leaves (40 mg) were hydrolyzed in 1 mL phosphate saline buffer (PBS),
incubated at 37C for 2 h, and then centrifuged (at 13,000 g, 30 min at 4C) 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 m 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.
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 C, 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
FolinCiocalteu 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 FolinCiocalteu
reagent and 0.3 mL of Na2CO3 (20% w/v) were mixed and heated at 50 C for 5 min. After 30 min,
the absorbance was measured at 765 nm against a blank similarly prepared, but containing 70:30
ethanolwater mixture (pH 3.2) instead of extract. Sodium carbonate (Panreac), FolinCiocalteu
reagent (FCR) and gallic acid (both from SigmaAldrich) were used to determine the total phenol
fraction. The absorbance was measured with a ThermoSpectronic UVvisible 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 ethanolwater
mixture at pH 3.2 fixed with formic acid (Heimler et al., 2007). Prior to LCMS analysis, 100 L of
extract was evaporated to dryness and reconstituted in 100 L of initial mobile phase for injection
of 10 L 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 m 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) acetonitrilemethanol, respectively. The
flow rate and the column oven temperature were set at 1 mL/min and 35 C, 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 ESIQqQ, were: flow rate and
temperature of drying gas 10 mL/min and 325 C, 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--D glucoside and myricetin) and the flavone glycoside rutin
hydrate, which were purchased from SigmaAldrich (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 C,
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 L of an acetonitrile
methanoldichloromethane (45:20:35 v/v) solution, filtered through a 0.22 m PTFE syringe filter
(Millipore) directly to sample vials, and 10 L 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 m) and a dual
absorbance detector (model 2487), controlled by Breeze workstation.
The initial mobile phase consisted of acetonitrilemethanol (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 30 C. A 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 (-carotene, -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
ethanolwater mixture at pH 3.2 fixed with formic acid as in the determination of the phenolic
fraction (Heimler et al., 2007). A 150 L aliquot of this extract was evaporated to dryness and
reconstituted in 150 L of derivatization solution, which consisted of 50-L pyridine from Merck
(Darmstadt, Germany), 98-L N,OBis(trimethylsilyl)trifluoroacetamide (BSTFA) and 2 L
trimethylchlorosilane (TMCS) from SigmaAldrich. The reaction mixture was vortexed at room
temperature for 1 h, before the GCMS 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 m) from Varian (Palo
Alto, USA). Thus, after derivatization, 1 L 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 A; transfer line, ion trap and manifold temperatures
were kept at 280, 200 and 50 C, respectively. The MSMS process was carried out by collisioninduced dissociation (CID) in non-resonant excitation mode. Table 2 shows the optimal MSMS
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).
101
102
HGC1 and HGC2 accessions (14.90 and 12.64 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 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 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 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 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 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 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 mols/g dw for glucoerucin and glucobrassicin respectively. Nevertheless
the level of glucoraphanin (1.25 mols/g dw) and total GL content (11 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 mols/g dw) and glucosativin (ranging from 15.34 to 30.67 mols/g
dw) in contrast to our values (ranging from 0.14 to 4.03 mols/g dw, and from 8.10 to 11.40
mols/g dw for glucoerucin and glucosativin respectively). Bennet and collaborators (2007) reported
higher values of total GLs (55.4 mols/g dw), glucoerucin (32.0 mols/g dw), glucosativin (31.3
mols/g dw), but lower values of glucoraphanin (6.1 mols/g dw) and glucobrassicanapin (0.2
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). Pearsons correlation of glucoraphanin and
sulforaphane was not significant in leaves of rocket (-0.38, P >0.05), which suggested differences
103
including
methodology
(all
reports
used
different
approaches
for
extraction,
104
105
106
content > 300 g/g dw in food can be considered high. Therefore, the flavonol content was low in
accessions of rocket (<20 g/g dw).
Apart from flavonoids, ferulic acid, a phenolic acid (ranging from 30.60 to 48.00 g/g dw)
was detected in the rocket accessions. The mean content of total phenolic ranged from 4474.5 to
32700 g/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 g/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.
Compound
Neoxantin
Violaxanthin
Xantophylls
Lutein
Zeaxanthin
-Cryptoxanthin
Carotene
-Carotene
Total Content of Carotenoids
a
LGC1
a
nd
0.200.01
8.300.10
1.910.01
8.010.04
0.900.03
19.510.41
LGC2
2.200.00
8.200.10
115.200.40
24.100.20
93.700.10
20.710.20
263.910.30
b
HGC1
0.800.01
13.000.20
124.300.40
25.810.03
81.710.21
14.620.10
260.310.50
HGC2
0.300.01
2.500.02
55.600.20
12.810.01
1.700.10
14.900.10
87.910.20
nd: non-detected.
107
Concentrations, expressed as g/g dw, are meansstandard error of at least three independent
extractions and analyses of ten plants.
In previous works (Ramos and Rodrguez- Amaya, 1987; Kimura and Rodrguez-Amaya,
2003; Niizu and Rodrguez-Amaya 2005) lutein and -carotene were reported as the most
abundant carotenoids with mean contents of 50 g/g for lutein and 30 g/g for -carotene. The
contents of lutein in our work are higher than those found in previous works, but lower than the
values found for -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 carotene, -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 (meanstandard deviation) isolated from rocket leaves.
Classification
Elemental
composition
C5H10O5
Analyte
Monosaccharide
C6H12O6
Arabinose
Mannose
Fructose
Glucose
Galactose
Disaccharide
C12H22O11
Sucrose
LGC1
HGC1
HGC2
nd
0.150.11
0.010.03
nd
0.190.02 1.940.31
1.240.02
0.360.11
nd
5.480.22
nd
nd
31.190.10 38.550.65 35.871.01 38.460.04
1.440.13 2.140.04
1.630.21
1.860.10
2.060.05
108
LGC2
5.970.18
2.960.21
1.750.01
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.
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 consumers health.
Acknowledgments
This work was supported by the project P06-AGR-02230 (CICE, Junta de Andaluca) and
FEDER funds. Myriam Villatoro was supported by Instituto Nacional de Investigacin y Tecnologa
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 Crdoba, Spain) and Dr. M Dolores Luque de Castro,
(Department of Analytical Chemistry, University of Crdoba, Spain), for insightful advice in all
aspects of the work.
(Belgium), Dipartimento di Scienze Botaniche of Palermo, Italy and Botanischer Garten der
Universitat of Karlsruhe, Germany, for the supply of vegetal material.
109
References
-
Arrabi, P., Genovese, M. I., Lajolo, F. M. (2004). Flavonoids in vegetable foods commonly
consumed in Brazil and estimated ingestion by the Brazilian population. Journal of
Agricultural and Food Chemistry, 52, 1124-1131.
Ashraf, M. (1994). Organic substances responsible for salt tolerance in Eruca sativa.
Biology Plantarum. 36, 255259.
Bennett, R., Mellon, F., Botting, N., Eagles, J., Rosa, E., Williamson, G. (2002).
Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca
sativa L. (salad rocket). Phytochemistry, 61, 25-30.
Bennett, R. N., Rosa, E. A. S., Mellon, F. A., Kroon, P. A. (2006). Ontogenic Profiling of
Glucosinolates, Flavonoids, and Other Secondary Metabolites in Eruca sativa (Salad
Rocket), Diplotaxis erucoides (Wall Rocket), Diplotaxis tenuifolia (Wild Rocket), and Bunias
orientalis (Turkish Rocket). Journal of Agricultural and Food Chemistry, 54, 40054015.
Bennett, R. N., Carvalho, R., Mellon, F. A., Eagles, J., Rosa, E. A. S. (2007). Identification
and Quantification of Glucosinolates in Sprouts Derived from Seeds of Wild Eruca sativa L.
(Salad Rocket) and Diplotaxis tenuifolia L. (Wild Rocket) from Diverse Geographical
Locations. Journal of Agricultural and Food Chemistry, 55, 6774.
Blaevic, I., Mastelic, J. (2008). Free and bound volatiles of rocket (Eruca sativa Mill.).
Flavour and Fragrance Journal, 23, 278285
Bones, A. M., Rossiter, J. T. (2006). The enzymic and chemically induced decomposition of
glucosinolates. Phytochemistry, 67, 1053-1067.
Brown, A. F., Yousef, G. G., Jeffery, E. H., Klein, B. P., Wallig, M. A., Kushad, M. M., Juvik,
J. A. (2002). Glucosinolate profiles in broccoli: Variation in levels and implications in
breeding for cancer chemoprotection. Journal of the American Society for Horticultural
Science, 127, 807-813.
Font, R., Del Ro-Celestino, M., Cartea, M. E., De Haro-Bailon, A. (2005). Quantification of
glucosinolates in leaves of leaf rape (Brassica napus ssp. pabularia) by near-infrared
spectroscopy. Phytochemistry, 66, 175185.
mass
spectrometry
(GC-MS/MS)
after
ultrasound-assisted
110
Heimler, D., Isolani, L., Vignolini, P., Tombelli, S., Romani, A. (2007). Polyphenol content
and antioxidative activity in some species of freshly consumed salads. Journal of
Agricultural and Food Chemistry, 55, 1724-1729.
Hertog, M. G. L., Kromhout, D., Aravanis, C et al. (1995). Flavonoid intake and long-term
risk of coronary heart disease and cancer in the Seven Countries Study. Archives of
Internal Medicine, 155, 381386.
Jin, J., Koroleva, O. A., Gibson, T., Swanston, J., Magan, J., Zhang, Y., Rowland, I. R.,
Wagstaff, C. (2009). Analysis of Phytochemical Composition and Chemoprotective
Capacity of Rocket (Eruca sativa and Diplotaxis tenuifolia). Leafy Salad Following
Cultivation in Different Environments. Journal of Agricultural and Food Chemistry, 57,
52275234.
Kim, S. J., Ishii, G. (2006). Glucosinolate profiles in the seeds, leaves and roots of rocket
salad (Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and purified 4methoxyglucobrassicin. Soil Science and Plant Nutrition, 52, 394400.
Matusheski, N. V., Swarup, R., Juvik, J. A., Mithen, R., Bennet, M., Jeffery, E. H. (2006).
Epithiospecifier protein from Brocoli (Brassica oleracea L. Ssp. italica) inhibits formation of
the anticancer agent sulforaphane. Journal of Agricultural and Food Chemistry, 54, 2069207.
Melchini, A., Costa, C., Traka, M., Miceli, N., Mithen, R., DePasquale, R., Trovato, A.
(2009). Erucin, a new promising cancer chemopreventive agent from rocket salads, shows
anti-proliferative activity on human lung carcinoma A549 cells. Food and Chemical
Toxicology, 47, 14301436.
111
Pignatelli, P., Ghiselli, A., Buchetti, B., Carnevale, R., Natella, F., German, G., Fimognari,
F., Di Santo, S., Lenti, L., Violi, F. (2006). Polyphenols synergistically inhibit oxidative
stress in subjects given red and white wine. Atherosclerosis, 188, 7783.
Seddon, J. M., Ajani, U. A., Sperduto, R. D., Hiller, R., Blair, N., Burton, T. C., Farber, M.
D., Gragoudas, E. S., Haller, J., Miller, D. T., Yannuzzi, L. A., Willett, W. C. (1994). Dietary
carotenoids, vitamins A, C and E and advanced macular degeneration. Journal of the
American Medical Association, 272, 1413-1420.
Selma, M. V., Martnez-Snchez, A., Allende, A., Ros, M., Hernndez, M. T., Gil, M. (2010).
Impact of Organic Soil Amendments on Phytochemicals and Microbial Quality of Rocket
Leaves (Eruca sativa). Journal of Agricultural and Food Chemistry, 58, 83318337.
Singleton, V. L., Rossi, J. A. (1965). Colorimetry of Total Phenolics with PhosphomolybdicPhosphotungstic Acid Reagents. American Journal of Enology and Viticulture, 16, 144-158.
Tadmor, Y., Larkov, O., Meir, A., Minkoff, M., Lastochkin, E., Edelstein, M., (2000).
Reversed-phase high performance liquid chromatographic determination of vitamin E
components in maize kernels. Phytochemical Analysis, 11, 370374.
Van den Berg, H., Faulks, R., Fernando-Granado, H., Hirschberg, J., Olmedilla, B.,
Sandmann, G., Southon, S., Stahl, W. (2000). The potential for the improvement of
carotenoid levels in foods and the likely systemic effects. Journal of the Science of Food
and Agriculture, 80, 880-912.
Wathelet, J. P., Wagstaffe, P., Boenke, A. (1991). The certification of the total glucosinolate
and sulphur contents of three rapeseed (colza). CRMs, 190, 366-367.
Weckerle, B., Michel, K., Balazs, B., Schreier, P., Toth, G. (2001). Three new quercetin
3,3',4'-tri-O--D-glucopyranosides from the leaves of Eruca sativa (500 g). Phytochemistry,
57, 547-55.
112
113
CAPTULO IV
Caracterizacin y prediccin por espectroscopa por reflectancia en el
infrarrojo cercano (NIRS) de la composicin mineral de rcola (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, Andrs Muoz-Serranoc, Vanessa Cardeosaa,
Manuel ngel Amaro Lpezb, Rafael Fontd, Mercedes Del Ro-Celestinod.
a
Spain.
c
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 (meanse) were: 23.550.15
mg ashes/100 g, 27.330.42 mg Fe/100 g, 1.810.04 mg Cu/100 g, 283.384.69 mg Na/100 g,
7158101.71 mg K/100 g, 6461121.97 mg Ca/100 g, 68911.40 mg Mg/100 g, 10.160.12 mg
Mn/100 g, and 6.710.04 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.
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
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
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
16-20
in order to assess the degree of reliability. The sensitivity was defined as being the
119
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
Fig. 1. Near infrared mean spectrum (a) and standard deviation (b) of rocket samples.
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
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
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
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
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
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
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
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): 918 Fe, 1.1 Cu, 3100 K, 79.5 Zn, 1300-1500 Na, 300350 Mg,
1.8-2.3 Mn, 9001000 Ca
33
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
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 (400700 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
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.); Birds-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
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
(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 Consejera de Innovacin, Ciencia y
Empresa (Junta de Andaluca), 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 Investigacin y Tecnologa Agraria y
Alimentaria (INIA) contract.
131
Ash
Fig. 4. NIRS predicted data vs. chemical reference data for mineral in rocket (dry weight).
Fe
Cu
Na
132
Fig. 4. Continued.
Ca
Mg
Mn
Zn
133
References
1.
Human Vitamin and Mineral Requirements, Report of a joint FAO/WHO expert consultation.
Mithen RF, Dekker M, Verkerk R, Rabot S and Jonson IT, Review: The nutritional
significance, biosynthesis and bioavailability of glucosinolates in human foods. J Sci Food Agric,
80: 967-984 (2000).
3.
Spectroscopy: Methodology and Potential for Predicting Trace Elements in Plants. Methods
Biotechnol, 23: 205-217 (2007).
4.
Orser CS, Salt DE, Pickering IJ, Prince RC, Epstein A and Ensley BD, Brassica Plants to
and their natural occurrence within the Mediterranean region (pp. 20-21), in The Rocket Genetic
Resources Network, ed. by Padulosi B, Report of the First Meeting in Lisbon, Portugal. Rome.
International Plant Genetic Resource Institute, Rome (1994).
7.
Bozokalfa MK, Yagmur B, Ilbi H, Esiyok D and Kavak S, Genetic variability for mineral
concentration of Eruca sativa L. and Diplotaxis tenuifolia L. accessions. Crop Breed Appl
Biotechnol, 9: 372-381 (2009).
8.
Munter RC, Quality assurance for plant tissue analysis by ICP-AES. Commun Soil Sci Plant
Shenk JS and Westerhaus MO, The application of near infrared reflectance spectroscopy
(NIRS) to forage analysis, in Forage Ouality, Evaluation and Utilization, ed. by Fahey GC,
Collins M, Mertens DR and Moser LE, American Society of Agronomy, Crop Science Society of
America, Soil Science Society of America, Madison, WI, USA. pp. 406450 (1994).
10. Clark DH, Mayland HF and Lamb RC, Mineral analysis of forages with near infrared
reflectance spectroscopy. Agron J, 79: 485490 (1987).
11. Halgerson JL, Sheaffer CC, Martin NP, Peterson PR and Weston SJ, Near-infrared
reflectance spectroscopy prediction of leaf and mineral concentrations in alfalfa. Agron J, 96:
344351 (2004).
12. Cozzolino D and Moron A, Exploring the use of near infrared reflectance spectroscopy to
predict trace minerals in legumes. Anim Feed Sci Technol, 11: 161-173 (2004).
13. Font R, Del Ro-Celestino M, Vlez D, Montoro R and De Haro-Bailn A, Use of nearinfrared spectroscopy for determining the total arsenic content in prostrate amaranth. Sci Total
Environ, 327: 93-104 (2004).
134
14. Stoltz MA, Provisional assessment of quality components in lucerne (Medicago sativa) and
white clover (Trifolium repens) using a near-infrared reflectance spectrophotometer. S Afri J
Plant Soil, 7: 105-112 (1990).
15. Saiga S, Sasaki T, Nonaka K, Takahashi K, Watanabe M and Watanabe K, Prediction of
mineral concentrations of orchard grass (Dactylis glomerata L.) with near infrared reflectance
spectroscopy. J Japan Soc Grass Sci, 35: 228233 (1989).
16. Moreno-Rojas R, Sanchez-Segarra PJ, Garca-Martnez M, Gordillo-Otero MJ and Amaro
Lopez MA, Mineral composition of skimmed milk fruit-added yoghurts, nutritional assessment.
Milchwissenschaft, 55: 510512 (2000).
17. Analytical Methods Committee, Recommendations for definition, estimation and use of
detection limit. Analyst, 112: 199204 (1987).
18. AOAC (Association of Official Analytical Chemist), Official methods of analysis, 15th ed;
2nd. supplement, 991.25: 101102 (1991).
19. Horwitz W, Albert R, Deutsch MJ and Thompson JN, Precision parameters of methods of
analysis required for nutrition labelling. J Assoc Offic Anal Chem Int, 73: 661-680 (1990).
20. Long OL and Winefordner SD, Limit of detection: a closer look at the IUPAC definition. Anal
Chem, 55: 712A724A (1983).
21. Barnes RJ, Dhanoa MS and Lister SJ, Standard normal variate transformation and detrending of near-infrared diffuse reflectance spectra. Appl Spectrosc, 43: 772-777 (1989).
22. Shenk JS, Workman J and Westerhaus M, Application of NIR spectroscopy to agricultural
products, in Handbook of Near Infrared Analysis, 2nd Edition, ed. by Burns DA and Ciurczac
EW. Marcel Dekker, Nueva York, USA, pp. 419-474 (2001).
23. SAS, General lineal model (GLM) procedures. SAS/STAT Users Guide, 4 th edn (pp.45
52). Cary, NC: SAS Institute Inc (1989).
24. Osborne BG, Fearn T and Hindle PH, Practical NIR spectroscopy with applications in food
and beverage analysis, ed. by Longman Scientific & Technical, Essex, England, p. 227 (1993).
25. Tkachuk R and Kuzina FD, Chlorophyll analysis of whole rapeseed kernels by near infrared
reflectance. Can J Plant Sci, 62: 875 884 (1982).
26. Murray I, The NIR spectra of homologous series of organic compounds, in Proceedings of
the International Near Infrared Diffuse Reflectance/Transmittance Spectroscopy Conference, ed.
by Kaftka KJ, Akademic Kiado, Budapest, Hungary, pp. 13-28 (1989).
27. Garnsworthy PC, Wiseman J and Fegeros K, Prediction of chemical, nutritive and
agronomic characteristics of wheat by near infrared spectroscopy. J Agric Sci, 135: 409417
(2000).
28. Kawashima LM and Valente-Soares LM, Mineral profile of raw and cooked leafy vegetables
consumed in Southern Brazil. J Food Comp Anal, 16: 605-611 (2003).
135
29. Cavarianni RL, Filho ABC, Cazetta JO, May A and Corradi MM, Nutrient contents and
production of rocket as affected by nitrogen concentrations in the nutritive solution. Scientia
Agricola 65: 652-658 (2008).
30. Sandberg AS, Bioavailability of minerals in legumes. Br J Nutr 88: S281S285 (2002).
31. Lucarini M, Canali R, Cappelloni M, Di Lullo G and Lombarda-Boccia G, In vitro calcium
availability from brassica vegetables (Brassica oleracea L.) and as consumed in composite
dishes. Food Chem, 64: 519-529 (1999).
32. Kalac P and Svoboda L, A review of trace element concentrations in edible mushrooms.
Food Chem, 69: 273-281 (2000).
33. Cuervo M, Abete I, Baladia E, Corbaln M, Manera M, Basulto J and Martnez A, Ingestas
dietticas de referencia (IDR) para la poblacin espaola. Federacin Espaola de Sociedades
de Nutricin, Alimentacin y Diettica (FESNAD), Ediciones Universidad de Navarra, (2010).
34. Gencelep H, Uzun Y, Tuntrk Y and Demirel K, Determination of mineral contents of
wild-grown edible mushrooms. Food Chem, 113: 1033-1036 (2009).
35. Clark DH, Cary EE and Mayland HF, Analysis of trace elements in forages by near infrared
reflectance spectroscopy. Agron J, 81: 9195 (1989).
36. Vazquez de Aldana BR, Garcia-Criado B, Garcia-Ciudad A and Perez-Corona ME,
Estimation of mineral content in natural grasslands by near infrared reflectance spectroscopy.
Comm Soil Sci Plant Anal, 26: 1383-1396 (1995).
37. Williams PC and Sobering DC, Comparison of commercial near infrared transmittance and
reflectance instruments for analysis of whole grains and seeds, J Near Inf Spec, 1: 2532
(1993).
38. Castro P, Baez D and Roca AI, Analysis of macronutrients in mixed swards samples by
NIR spectroscopy. In: Pastos : fuente natural de energa : 4 Reunin Ibrica de Pastos y
Forrajes, Zamora. Miranda do Douro, Alfredo Calleja Surez. Len: Universidad de Len, rea
de Publicaciones. Espaa: Sociedad Espaola para el Estudio de los Pastos. pp. 279-284
(2010).
39. Poji M, Mastilovi J, Pali D and Pestori M, The development of near-infrared
spectroscopy (NIRS) calibration for prediction of ash content in legumes on the basis of two
different reference methods. Food Chem, 123: 800-805 (2010).
40. Font R, Del Ro-Celestino M and De Haro A, Use near-infrared reflectance spectroscopy
(NIRS) to evaluate heavy metal content in Brassica juncea cultivated on the polluted soils of the
Guadiamar river area. Fresen environ bull, 11: 777-781 (2002).
136
137
CAPTULO V
Artculo en preparacin
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 Fernndez-Bdmar4, Rafael Font3,
Andrs Muoz-Serrano4, Richard Mithen2, ngeles Alonso-Moraga4, Mercedes Del Ro- Celestino3
1
2
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,
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 (Calabr 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
Rossiter, 2001). Carotenoids have also shown antioxidant activity depending on the localisation
and concentration (Van den Berg et al., 2000). The carotenoid -carotene and the xantophyll lutein
act both as antioxidants. -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
consumers 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.
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 -80 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 L, 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 m bead size, 30000 Da exclusion limit). Purified
sulfatase (75 L) 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 m 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- 3m (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
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 37C. 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.
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
0.140.00c(1)
3.640.00c
9.150.30a
14.020.30b
2.700.03b
6.060.03b
8.100.80a
19.401.04b
4.030.03a
12.640.20a
8.100.80a
27.650.30a
0.630.08c
14.901.46a
11.400.08a
28.241.61a
0.150.02b
0.750.09a
0.020.00c
nd(2)
5.900.33a
0.970.01a
1.550.12a
0.010.00
1.330.07b
0.340.01a
1.370.03a
nd
0.810.01b
0.150.00a
0.570.01b
nd
Polyphenols
Isoquercitrin
Rutin
Quercetin
Myricetin
Ferulic acid
Total content
102312.01b
nd
nd
nd
33.001.50a
32248.5a
774.012.01b
18.001.50b
nd
3.003.02a
30.603.02a
18750.1b
1621.59.01a
12.010.03b
nd
nd
nd
32700.2a
168015.00a
27.001.50a
13.5015a
3.000.01a
48.004.50a
4474.5a
Carotenoids
-Carotene
Lutein
Total content
0.900.03
8.300.10c
19.510.41c
14.900.10b
55.600.20b
87.910.20b
14.620.10b
124.300.40a
260.310.50a
Glucosinolates
Glucoerucin
Glucoraphanin
Glucosativin
Total content
Isothiocyanates
Sulforaphane
Sulforaphane- nitrile
Iberin
Erucin
(1)
20.710.20a
115.200.40a
263.910.30a
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)
weight (dw). Content of polyphenols and carotenoids expressed as g/g dw. Data expressed as
meanstandard deviation.
HGC1 and HGC2 accessions showed the highest glucoraphanin content and total content
of glucosinolates with approximately 14 and 28 mol /g dry weight, respectively. Accessions
showed differences in the hydrolysis of glucoraphanin and formation of SF ranging from 4.12%
145
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 (meanSD values from three
independent experiments).
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 M. 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 M; lane 3, 8.7
M; lane 4, 17.5 M; lane 5, 25 M; lane 6, 50 M; lane 7, 100 M.
(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 M 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
M) 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 (<30mol/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 consumers
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 M. 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 (1525 M) 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 M
(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 (12.5M) 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 mol /g dw) and the highest SF
and total carotenoid content among the accessions analysed, with 5.90 mol /g dw and 264 g /g
dw, respectively. On the other hand, the HGC2 accession had high glucosinolate content (27.6
mol /g dw), low SF content (1.3 mol /g dw) and the highest total polyphenol content with 32700.2
g /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 -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 Consejera de Innovacin, Ciencia y Empresa (Junta de
Andaluca, Spain) for funding the Project [P06-AGR-02230] and to Gloria Fernndez, (IAS-CSIC,
152
Cordoba) for technical assistance in the analysis of plants. We also acknowledge to the Facult
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
Investigacin y Tecnologa Agraria y Alimentaria (INIA) contract.
153
References
- Androutsopoulos VP, Papakyriakou A, Vourloumis D, Tsatsakis AM, Spandidos DA. Dietary
flavonoids in cancer therapy and prevention: Substrates and inhibitors of cytochrome P450 CYP1
enzymes. Pharm Ther 2010;126:9-20.
- Anter J, Romero-Jimnez M, Fernndez-Bedmar Z, Villatoro-Pulido M, Analla M, Alonso-Moraga
cyclodextrin interactions in fully aqueous solution: Spectroscopic studies and biological assays. J
Pharmaceut Biomed 2005;36:10191027.
- Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. Terminal differentiation of human
promyelocytic leukaemia cells induced by dimethyl sulfoxide and other polar compounds. Proc
Medical Sciences Natl Acad Sci USA 1978;75:2458-2462.
- Conte-Anazetti M, Silva-Melo P, Duran N, Haun M. Comparative cytotoxicity of dimethylamide-
crotonin in the promyelocytic leukemia cell line (hl60) and human peripheral blood mononuclear
cells. Toxicology 2003;188:261274.
- Cussenot O, Berthon P, Berger R, Mowszowics I, Faille A. Hojman F, et al. Immortalization of
human adult normal prostate epithelial cells by liposomes containing large T-SV40 gene. J Urol
1991;146:881886.
- Dashwood RH, Ho E. Dietary histone deacetylase inhibitors: from cells to mice to man, Semin.
2007;635:90-104.
- Gasper AV, Traka M, Bacon JR, Smith JA, Tailor MA, Hawkey CJ, Barret DA, Mithen R.
Consuming broccoli does not induce genes associated with xenobiotic metabolism and cell cycle
control in human gastric mucosa. J Nutr 2007;137:1718-1724.
- Harris KE, Jeffery HE. Sulforaphane and erucin increase MRP1 and MRP2 in human carcinoma
154
- Heijnen CG, Haenen GR, Van Acker FA, Van der Vijgh WJ, Bast A. Flavonoids as peroxynitrite
of a human prostate carcinoma cell line (PC-3). Invest Urol 1979,17:16-23, 1979.
- Kassie F, Knasmller S. Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl
DH. Sulforaphane Increases Cyclin-Dependent Kinase Inhibitor, p21 Protein in Human Oral
Carcinoma Cells and Nude Mouse Animal Model to Induce G2/M Cell Cycle Arrest. J Clin
Biochem Nutr 2010;46:6067.
- Kong AN, Owuor E, Yu R. Induction of xenobiotic enzymes by the MAP kinase pathway and the
antioxidant or electrophile response element (ARE/EpRE). Drug Metab Rev 2001;33: 255-27.
- Lamy E, Schrder J, Paulus S, Brenk P, Stahl T, Mersch-Sundermann V. Antigenotoxic
properties of Eruca sativa (rocket plant), erucin and erysolin in human hepatoma (HepG2) cells
towards benzo(a)pyrene and their mode of action. Food Chem Toxicol 2008;46:24152421.
- Maioli E, Torricelli C, Fortino V, Carlucci F, Tommassini V, Pacini A. Critical Appraisal of the MTT
Assay in the Presence of Rottlerin and Uncouplers. Biol Proced Online 2009;11: 227-240.
- Makris DP, Rossiter JT. Comparison of quercetin and a non-orthohydroxy
flavonol as
promising cancer chemopreventive agent from rocket salads, shows anti-proliferative activity on
human lung carcinoma A549 cells. Food Chem Toxicol 2009;47(7):1430-1436.
- Mithen RF, Dekker M, Verkerk R, Rabot S. The nutritional significance, biosynthesis and
155
- Prasain JK, Carlson SH, Wyss JM. Flavonoids and age-related disease: Risk, benefits and critical
based lead discovery for cancer chemoprevention: the case of resveratrol. J Med Chem
2009;52:1963-74.
- Ramos S. Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention.
TRAIL in prostate cancer orthotopic model through regulation of apoptosis, metastasis, and
angiogenesis. Clin Cancer Res 2008;14:68556866.
- Seddon JM, Ajani UA, Sperduto RD, Hiller R, Blair N, Burton TC, Farber MD, Gragoudas ES,
Haller J, Miller DT, Yannuzzi LA, Willet W. Dietary carotenoids, vitamins A, C and E and
advanced age-related macular degeneration. J Am Med Assoc 1994;272:14131420.
- Shimmyo Y, Kihara T, Akaike A, Niidome T, Sugimoto H. Three distinct neuroprotective functions
of myricetin against glutamate-induced neuronal cell death: involvement of direct inhibition of
caspase-3. J Neurosci Res 2008;86:18361845.
- Surh, YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003; 3:768780.
- Tang L, Zhang Y. Dietary isothiocyanates inhibit the growth of human bladder carcinoma cells. J
Nutr 2004;134:2004-2010.
- Traka MH, Spinks CA, Doleman JF, Melchini A, Ball RY, Mills RD, Mithen RF. The dietary
isothiocyanate sulforaphane modulates gene expression and alternative gene splicing in a PTEN
null preclinicla murine model of prostate cancer. Mol Cancer 2010;9:189-212.
- Van den Berg H, Faulks R, Fernando-Granado H, Hirschberg J, Olmedilla B, Sandmann G,
Southon S, Stahl W. The potential for the improvement of carotenoid levels in foods and the likely
systemic effects. J Sci Food Agr 2000;80:880-912.
- Villatoro-Pulido M, Font R, De Haro-Bravo MI, Romero-Jimenez M, Anter J, De Haro Bailon A,
Alonso-Moraga A, Del Rio-Celestino M. Modulation of genotoxicity and cytotoxicity by radish
grown in metal-contaminated soils, Mutagenesis 2009;24:5157.
- Wittstock, U, Kliebenstein, D, Lambrix, VM, Reichelt, M, Gershenzon, J. Glucosinolate hydrolysis
and its impact on generalists and spacialists insect herbivores. In: Romeo, J.T. (ed.) Molecular
ecology recent advances in phytochemistry, pp 101-126. 2003, Elsevier, Amsterdam.
- Wu, X, Zhou, Q, Xu, K. Are isothiocyanates potential anti-cancer drugs?. Acta Pharmacol Sin
2009;30: 501512.
- Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of
cyclin kinases. Nature 1993;366:701704.
156
- Yang, J, Guo, J., & Yuan, J. In vitro antioxidant properties of rutin. Food Sci Technol
2008;41:1060-1066.
- Yao, H, Wang, H, Zhang, Z, Jiang, BH, Luo, J, Shi, X. Sulforaphane inhibited expression of
hypoxia-inducible factor-1alpha in human tongue squamous cancer cells and prostate cancer
cells. Int J Cancer 2008;123:12551261.
- Yeh, C, Yen, G. Effect of sulforaphane on metallothionein expression and induction of apoptosis
in human hepatoma HepG2 cells. Carcinogenesis 2005;26:21382148.
157
158
CAPTULO VI
Actividad in vivo de extractos de rcola (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, Obregn-Cano4, S., J. Anter5, Zahira Fernndez-Bdmar5,
A. De Haro Bailn4, A. Alonso-Moraga5, M. Del Ro- Celestino2
1
Phytochemicals and Health Programme, Institute of Food Research, Norwich Research Park,
Council for Scientific Research (CSIC), Alameda del Obispo s/n, 14080 Crdoba, Spain
5
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 M) 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.
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-Jimnez 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
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 m bead size, 30000 Da exclusion limit). Purified
sulfatase (75 L) 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 m 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- 3m (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 C and 65% humidity.
163
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-Jimnez
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 Faures 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 Wrgler, 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 KaplanMeier 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) mol /g of dry weight (dw). Glucoraphanin content ranged from 3.24 (Es1) to
14.90 (Es4) mol /g of vegetal tissue, whereas SF content ranged from 0.14 (Es1) to 5.90 (Es2)
mol /g dw. Pearsons 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 mols/g dw) and total GL
content (11 mols/g dw), which were lower than those we found (minimum mean values of
glucoraphanin of 3.24 mol /g and 14.79 mol /g of total glucosinolate content). Bennet and
collaborators (2006) reported values of glucoraphanin (6.1 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 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
14.791.20
20.101.12
27.20.98
30.520.90
Glucoraphanin
3.240.15
6.300.15
12.880.28
16.961.11
0.140.07
6.250.15
0.820.09
1.240.13
Glucosinolates
Isothiocyanate
Sulforaphane
Content of GLs and ITCs expressed as mol /g of dw. Data expressed as meanstandard
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
Twin spots
Total spots
of wings
(1-2 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 (M)
(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 Wrgler (Frei and Wrgler, 1988): + (positive), -
167
(2)
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
Twin spots
Total spots
Inhibition
of wings
(1-2 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)+
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 (M)
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 Wrgler (Frei and Wrgler, 1988): +
(positive), - (negative) and i (inconclusive). Significance levels = = 0.05.
accuracy of the genotoxicity and antigenotoxicity assays was ensured (Romero-Jimnez 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 mol/g) (Fig. 2) and with different
concentrations of Es4 accession extracts (low SF: 1.24 mol/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 M) to 104 days (for the concentration of 3.75 M). 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 M), 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 (P0.05).
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 (<30mol/L) and higher concentrations can
make disappear this protective effect (Tang and Zhang, 2004).
Recently, Vzquez-Gmez 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 M of SF, while in our work
the highest concentration was 12.6 M 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 activitycalculated 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 mol /g) and total content of GLs (30.52 mol/g), although the yielding SF
content was low (1.24 mol /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- Jimnez 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 mol/g and high SF: 6.25 mol/g content) should result healthier
than Es4 (with high GL: 30.52 mol/g and low SF: 1.24 mol/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 Consejera de Innovacin, Ciencia y Empresa (Junta de
Andaluca, Spain) for funding the Project P06-AGR-02230 and to Gloria Fernndez, (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. Andrs
Muoz-Serrano for advice in statistical analysis. Myriam Villatoro was supported by Instituto
Nacional de Investigacin y Tecnologa Agraria y Alimentaria (INIA) contract.
175
References
- Abraham, S.K., 1994. Antigenotoxicity of coffee in the Drosophila assay for somatic mutation
28,463-499.
- Avanesian, A., Khodayari, B., Felgner, J.S., Jafari, M., 2010. Lamotrigine extends lifespan but
Glucosinolates, Flavonoids, and Other Secondary Metabolites in Eruca sativa (Salad Rocket),
Diplotaxis erucoides (Wall Rocket), Diplotaxis tenuifolia (Wild Rocket), and Bunias orientalis
(Turkish Rocket). J. Agric. Food Chem. 54, 40054015.
- Boyd, O., Weng, P., Sun, X., Alberico, T., Laslo, M., Obenland, D.M., Kern, B., Zou, S., 2011.
Nectarine promotes longevity in Drosophila melanogaster. Free Rad Biol Med, (in press).
- Chavous, D.A., Jackson, F.R., O'Connor, C.M., 2001. Extension of the Drosophila lifespan by
Res. 635,90-104.
- Fleming, J.E., Reveillaud, I., Niedzwiecki, A., 1992. Role of oxidative stress in Drosophila
from Drosophila assays indicate a positive, negative, or inconclusive result. Mutat. Res.
203,297-308.
- Gasper, A.V., Traka, M., Bacon, J.R., Smith, J.A., Tailor, M.A., Hawkey, C.J., Barret, D.A.,
Mithen, R., 2007. Consuming broccoli does not induce genes associated with xenobiotic
metabolism and cell cycle control in human gastric mucosa. J. Nutr. 137,1718-1724.
- Gill, C. I.R., Haldar, S., Porter, S., Matthews, S., Sullivan, S., Coulter, J., McGlynn, H.,
Rowlnad, I. 2004. The effect of cruciferous and leguminous sprouts on genotoxicity, in vitro
and in vivo. Cancer Epidemiol. Biomarkers Prev. 13, 1199-1205.
- Graf, U., Wrgler, F.E., Katz, A.J., Frei, H., Juon, H., Hall, C.B. Kale, P.G., 1984. Somatic
176
- Graf, U., Abraham, S.K., Guzmn-Rincn, J. Wrgler, F.E., 1998. Antigenotoxicity studies in
11,298-300.
- Jin, J., Koroleva, O.A., Gibson, T., Swanston, J., Magan, J., Zhang, Y., Rowland, I.R.,
(Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and purified 4methoxyglucobrassicin. Soil Sci. Plant Nutr. 52,394400.
- Lamy, E., Schrder, J., Paulus, S., Brenk, P., Stahl, T., Mersch-Sundermann, V., 2008.
Antigenotoxic properties of Eruca sativa (rocket plant), erucin and erysolin in human hepatoma
(HepG2) cells towards benzo(a)pyrene and their mode of action. Food Chem. Toxicol. 46,
24152421.
- Li, Y.M., Chan, H.Y.E, Yao, X.Q., Huang, Y., Chen, Z.Y., 2008. Green tea catechins and
Radical Theory of Aging, in: Masoro, E.J., Austad, S.N. (Eds.), Handbook of the biology of
aging (7th Edition). Academic Press, San Diego, pp.177-202.
- Martin Dietz, H., Panigrahi, S., Harris, R.V. 1991. Toxicity of hydrolysis Products from 3-
Epithiospecifier protein from Brocoli (Brassica oleracea L. ssp. italica) inhibits formation of the
anticancer agent sulforaphane. J. Agric. Food Chem. 54,2069-2076.
- Melchini, A., Costa, C., Traka, M., Miceli, N., Mithen, R., DePasquale, R., Trovato, A., 2009.
Erucin, a new promising cancer chemopreventive agent from rocket salads, shows anti-
177
proliferative activity on human lung carcinoma A549 cells. Food Chem. Toxicol. 47,1430
1436.
- Mithen, R., 2001. Glucosinolates- biochemistry, genetics and biological activity. Plant growth
regul. 34,91-103.
- Mockett, R.J., Sohal, R.S., 2006. Temperature-dependent trade-offs between longevity and
In vitro antiproliferative activity of isothiocyanates and nitriles generated by myrosinasemediated hydrolysis of glucosinolates from seeds of cruciferous vegetables. J. Agric. Food
Chem. 48,35723575.
- Ren, N., Charlton, J., Adler, P.N., 2007. The flare gene, which encodes the AIP1 protein of
A., 2005. Genotoxicity and antigenotoxicity of some traditional medicinal herbs. Mutat. Res.
585,147-155.
- Schumacker, P.T., 2006. Reactive oxygen species in cancer cells: Live by the sword, die by
J. Nutr.
135,18651872.
- Trotta, V., Calboli, F.C., Ziosi, M., Guerra, D., Pezzoli, M.C., David, J.R., Cavicchi, S., 2006.
Bailon, A., Alonso-Moraga, A., Del Rio-Celestino, M., 2009. Modulation of genotoxicity and
cytotoxicity by radish grown in metal-contaminated soils, Mutagenesis 24,5157.
178
- Wathelet, J.P., Wagstaffe, P., Boenke, A., 1991. The certification of the total glucosinolate and
179
180
CAPTULO VII
Actividad biolgica del rbano, una crucfera, 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-Jimnez,
Jaouad Anter, Antonio De Haro Bailn, ngeles Alonso-Moraga, and Mercedes Del Ro-Celestino
Mutagenesis vol. 24 no. 1 pp. 5157, 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 (47). 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 120 mg/kg of As in the soil (10), 2300
mg/kg of Pb in the soil and 0.012 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 Aznalcllar, 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
vegetables roots and shoots.
184
The contaminated soil was obtained from the experimental area El Vicario (37 2621N,
61300W) within the Green Corridor, close to the pyrite mine of Aznalcllar (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 1C. 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 /mwh flr3) were removed and mounted on slides using Faures 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,
antibioticantimycotic 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 37C (Shel Lab, Cornelius, OR, USA) (31). Cultures were passed every 23
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.
Pb (mg/ kg)
Cd (mg/ kg)
As (mg/ kg)
DPTA extractable
Control
Contaminated
Control
Contaminated
41.80.1
0.20.0
9.10.1
83.20.1*
0.40.0*
74.80.1**
4.20.1
0.010.0
0.100.0
14.10.2*
0.080.0*
3.80.1*
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
Cd (mg/ kg)
As (mg/ kg)
Shoot 0.80.4
1.60.7*
0.50.1
0.90.2*
0.70.0
3.90.8*
Root 0.60.5
0.20.1ns
0.20.0
0.40.0ns
0.20.2
1.20.2*
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-Jimnez 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
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 (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 (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)
44
Raphanus sativus
L(mg/ml)
NMTR(3)
(1)
22
0.275 (11) +
0.25 (10) +
0.125 (5)
0.25 (10) +
0.125 (5)
16
50
50
Statistical diagnoses according to Frei and Wrgler (32): + (positive), - (negative) and i
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
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
40
40
40
0.025 (1) i
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 Wrgler (32): + (positive), - (negative) and i
Non metal treated root, NMTR; metal treated root, MTR; and metal treated shoot, MTS.
191
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 Aznalcllar (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 g/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 g/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
Consejera de Agricultura y Pesca (Junta de Andaluca, Spain) (C03-070).
Acknowledgements
The authors thank Gloria Fernndez (IAS-CSIC, Crdoba) for technical assistance in the analysis
of plants.
Conflict of interest statement: None declared.
196
References
1. Guttormsen, G., Singh, B. R. and Jeng, A. S. (1995) Cadmium concentrations in vegetable
crops grown in a sandy soil as affected by Cd levels in fertiliser and soil pH. Fert. Res., 41, 2732.
2. Singh, B. R. and Steinnes, E. (1994) Soil and water contamination by heavy metals. In Lal, R.
and Stewart, B. A. (eds), Soil Processes and Water Quality. Boca Raton, FL, Lewis Publishers,
CRC Press, pp. 233270.
3. Salim, R., Al-Subu, M. M., Douleh, A., Chenavier, L. and Hagemeyer, J. (1992) Effects of root
and foliar treatments on carrot plants with lead and cadmium on the growth, uptake and the
distribution of metals in treated plants. J. Environ. Sci. Health Part A, 27, 17391758.
4. Chen, C. J., Chiou, H. Y., Chiang, M. H., Lin, T. M. and Tai, T. Y. (1996) Dose-response
relationship between ischemic heart disease mortality and long-term arsenic exposure.
Arterioscler. Tromb. Vasc. Biol., 16, 504510.
5. Morales, K. H., Ryan, L., Kuo, T. L., Wu, M. M. and Chen, C. J. (2000) Risk of internal cancers
from arsenic in drinking water. Environ. Health Perspect., 108, 655661.
6. Rahman, M. (2002) Arsenic and contamination of drinking water in Bangladesh: a public health
perspective. J. Health Popul. Nutr., 20, 193197.
7. Srivastava, M., Ahmad, N., Gupta, S. and Mukhtar, H. (2001) Involvement of Bcl-2 and Bax in
photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes Rif 1 cells to
photodynamic therapy apoptosis. J. Biol. Chem., 276, 1548115488.
8. Bryce-Smith, D. (1997) Heavy metals as contaminants of human environ. (eds) Peter G. Publ
Edu. Tech. Subgroup, The Chemical Society London. pp. 2123.
9. Hamers, T., Van den Berg, J. H. J., Van Gestel, C. A. M., Van Schooten, F. J., amd Murk, A. J.
(2006) Risk assessment of metals and organic pollutants for herbivorous and carnivorous small
mammal food chains in a polluted floodplain (Biesbosch, The Netherlands). Environ. Pollut., 144,
581595.
10. Wauchope, R. D. (1983) Uptake, translocation and phytotoxicity of arsenic in plants. In
Lederer, W. H. and Fensterheim, R. J. (eds), Arsenic: Industrial, Biomedical, Environmental
Perspectives. Van Nostrand Reinhold, New York, pp. 348375.
11. Bowen, H. J. M. (1979) Environmental Chemistry of the Elements. Academic Press, London, p.
333.
12. Larsen, E. H., Moseholm, L. and Nielsen, M. M. (1992) Atmospheric deposition of trace
elements around point sources and human risk assessment. II. Uptake of arsenic and chromium
by vegetables grown near a wood preservation factory. Sci. Total Environ., 126, 263275.
13. Ebbs, S. D. and Kochian, L. V. (1997) Toxicity of zinc and copper to Brassica species:
implications for phytoremediation. J. Environ. Qual., 26, 776781.
14. Carbonell-Barrachina, A. A., Burlo, F., Lo pez, E. and Mart nez-Sa nchez, F. (1999) Arsenic
toxicity and accumulation in radish as affected by arsenic chemical speciation. Environ. Sci.
Health, 34, 661679.
197
15. Simn, M., Ortiz, I., Garcia, I., Fernndez, E., Fernndez, J., Dorronsoro, C. and Aguilar, J.
(1999) Pollution of soils by the toxic spill of a pyrite mine (Aznalco llar, Spain). Sci. Total Environ.,
242, 105115.
16. Del Ro, M., Font, R., Almela, C., Velez, D., Montoro, R. and De Haro, A. (2002) Heavy metals
and arsenic uptake by wild vegetation in the Guadiamar river area after the toxic spill of the
Aznalcllar mine. J. Biotechnol., 98, 125137.
17. Bast, A., Chandler, R. F., Choy, P. C. et al. (2002) Botanical health products, positioning and
requirements for effective and safe use. Environ. Toxicol. Pharmacol., 12, 195211.
18. Graf, U., Wu rgler, F. E., Katz, A. J., Frei, H., Juon, H., Hall, C. B. and Kale, P. G. (1984)
Somatic mutation and recombination test in Drosophila melanogaster. Environ. Mutagen., 6, 153
188.
19. Graf, U., Abraham, S. K., Guzmn-Rincn, J. and Wrgler, F. E. (1998) Antigenotoxicity
studies in Drosophila melanogaster. Mutat. Res., 402, 203209.
20. Zimmering, S., Olvera, O., Hernndez, M. E., Cruces, M. P., Arceo, C. and Pimental, E. (1990)
Evidence for a radioprotective effect of chlorophyllin in Drosophila. Mutat. Res., 245, 4749.
21. Graf, U., Alonso-Moraga, A., Castro, R. and Diaz, E. (1994) Genotoxicity testing of different
types of beverages in the wing somatic mutation and recombination test. Food Chem. Toxicol., 32,
423430.
22. Romero-Jime nez, M., Campos-Snchez, J., Analla, M., Muoz-Serrano, A. and AlonsoMoraga, A. (2005) Genotoxicity and antigenotoxicity of some traditional medicinal herbs. Mutat.
Res., 585, 147155.
23. Rizki, M., Kossatz, E., Velzquez, A., Creus, A., Farina, M., Fortaner, S., Sabbioni, E. and
Marcos, R. (2006) Metabolism of arsenic in Drosophila melanogaster and the genotoxicity of
dimethylarsinic acid in the Drosophila wing spot test. Environ. Mol. Mutagen., 47, 162168.
24. Collins, S. J., Ruscetti, F. W., Gallagher, R. E. and Gallo, R. C. (1978) Terminal differentiation
of human promyelocytic leukaemia cells induced by dimethyl sulfoxide and other polar compounds.
Proc. Natl Acad. Sci. USA, 75, 24582462.
25. Conte-Anazetti, M., Silva-Melo, P., Duran, N. and Haun, M. (2003) Comparative cytotoxicity of
dimethylamide-crotonin in the promyelocytic leukemia cell line (hl60) and human peripheral blood
mononuclear cells. Toxicology, 188, 261274.
26. Santos, A., Alonso, E., Callejo n, M. and Jimnez, J. C. (2002) Heavy metal content and
speciation in groundwater of the Guadiamar river basin. Chemosphere, 48, 279285.
27. Muoz, O., Devesa, V., Suer, M. A., Vlez, D., Montoro, R., Urieta, I., Macho, M. L. and Jalo
n, M. (2000) Total and inorganic arsenic in fresh and processed fish products. J. Agric. Food
Chem., 48, 43694376.
28. Guzman, G., Alcantara, E., Barron, V. and Torrent, J. (1994) Phytoavailability of phosphate
adsorbed on ferrihydrite, hematite, and goethite. Plant Soil, 159, 219225.
198
29. Griepink, B. and Muntau, H. (1988) The Certification of the Contents (Mass Fractions) of As,
Cd, Cu, Pb, Se and Zn in a Sea Lettuce (Ulva lactuca). CRM 279. Report no EUR 11185 EN,
Luxembourg: Commission of the European Communities.
30. Lindsley, D. L. and Zimm, G. G. (1992) The Genome of Drosophila melanogaster. Academic
Press Inc., San Diego, CA.
31. Gallagher, R., Collins, S., Trujillo, J. et al. (1979) Characterization of the continuous,
differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukaemia. Blood,
54, 713733.
32. SPSS (2000). SPSS for Windows, Version 10.0. (19891999) SPSS Inc., Chicago.
33. Frei, H. and Wrgler, F. E. (1988) Statistical methods to decide whether mutagenicity test data
from Drosophila assays indicate a positive, negative, or inconclusive result. Mutat. Res., 203, 297
308.
34. Abraham, S. K. (1994) Antigenotoxicity of coffee in the Drosophila assay for somatic mutation
and recombination. Mutagenesis, 9, 383386.
35. Allen, R. G. and Tresini, M. (2000) Oxidative stress and gene regulation. Free Radic. Biol.
Med., 28, 463499.
36. Lozano-Baena, M. D., Tasset, I., De-Haro, A., Glvez, C., Campos-Snchez, J., MuozSerrano, A. and Alonso-Moraga, A. (2005) Tumoricide and antigenotoxic effects of Olive Oil, Seed
Oils and Fresh Plant of Borago officinalis and Brassica carinata. International Conference on
Industrial Crops and Rural Development. AAIC Annual Meeting, Murcia, Spain.
37. De Andaluca, J. (2001) Corredor Verde del Guadiamar. In Corredor Verde del Guadiamar
(ed.), Consejer a de Medio Ambiente de la Junta de Andaluca. Junta de Andaluca, Sevilla,
Spain, pp. 170.
38. Marchiol, L., Assolari, S., Sacco, P. and Zerbi, G. (2004) Phytoextraction of heavy metals by
canola (Brassica napus) and radish (Raphanus sativus) grown on multicontaminated soil. Environ.
Pollut., 132, 2127.
39. Tlustos, P., Balik, J., Szakova, J. and Pavlikova, D. (1998) The accumulation of arsenic in
radish biomass when different forms of As were applied in the soil (Czech). Rostlinna Vyroba, 44,
713.
40. Commission Regulation (EC) No 629/2008 of 2 July 2008 Amending Regulation (EC) No
1881/2006 Setting Maximum Levels for Certain Contaminants in Foodstuffs (1). Official Journal of
the European Union, (2008).
41. ANZFZ (1998) Australian Food Standard Code. (Issue 41) Camberra: Australia NewZealand
Food Authority.
42. Donma, O. and Donma, M. (2005) Cadmium, lead and phytochemicals. Med. Hypotheses, 65,
699702.
43. Girardot, F., Monnier, V. and Tricoire, H. (2004) Genome wide analysis of common and
specific stress response in adult Drosophila melanogaster. BMC Genomics, 5, 7489.
199
44. Hei, T. K. and Filipic, M. (2004) Role of oxidative damage in the genotoxicity of arsenic. Free
Radic. Biol. Med., 37, 574581.
45. Musk, S. R. R., Smith, T. K. and Johnson, I. T. (1995) On the cytotoxicity and genotoxicity of
allyl and phenethyl isothiocyanates and their parent glucosinolates sinigrin and gluconasturtiin.
Mutat. Res., 348, 1923.
46. Wang, J. P., Qi, L., Moore, M. R. and Ng, J. C. (2002) A review of animal models for the study
of arsenic carcinogenesis. Toxicol. Lett., 133, 1731.
47. Waisberg, M., Joseph, P., Hale, B. and Beyersmann, D. (2003) Molecular and cellular
mechanisms of cadmium carcinogenesis. Toxicology, 192, 95117.
48. Exposure of Children to Chemical Hazards in Food. Copenhagen, WHO Regional Office for
Europe. (2007) (ENHIS fact sheet 4.4)
http://www.euro.who.int/Document/EHI/ENHIS_Factsheet_4_4.pdf (accessed 30 May 2006).
49. Model of Spanish diet for the determination of the exposition of the consumer to chemical
substances. Ministry of Health and Consumption, Spanish Agency of Food Safety, Madrid, Spain
http://www.aesan.msc.es/ AESAN/docs/docs/notas_prensa modelo_dieta_espanola.pdf (accessed
30 May 2006).
200
201
DISCUSIN GENERAL
202
203
PEX-61 (sativa) presentaron similar peso fresco que la variedad comercial PEX-55, y fueron
significativamente ms altas que las variedades comerciales PEX-16 y PEX-56 (Table 3). As
mismo, entradas (PEX-7, PEX-52 PEX-93) pertenecientes a la subp. vesicaria, presentaron una
floracin ms tarda cuando se compararon con las tres variedades comerciales.
Respecto a otros caracteres morfolgicos fue posible seleccionar las entradas PEX-14,
PEX-58, PEX-61 (sativa) y PEX-7, PEX-9, PEX-52 y PEX-53 (vesicaria), las cules mostraron
caractersticas interesantes (hoja de pequeo tamao, alto contenido en clorofila, ausencia de
pubescencia en sativa y alta lobulacin de las hojas en vesicaria) desde el punto del consumidor.
Para obtener entradas de Eruca con valor aplicable en Mejora Vegetal que puedan competir
adaptndose a la lucha de los mercados nos hemos planteado, no slo la seleccin de lneas con
un deseable comportamiento agronmico robusto, sino que posean un valor alimenticio que
atraiga al consumidor. La rcola es preferida en consumo crudo frente a otros vegetales por
diversas caractersticas sensoriales: su aspecto (color, vellosidad y forma de las hojas), por su
textura, aromas en boca y por sus caractersticas sensaciones trigeminales. Estas ltimas
caractersticas sensoriales vienen dadas en ltima instancia por la hidrlisis de los glucosinolatos
(mediado por la enzima mirosinasa) a isotiocianatos y otros metabolitos. En el captulo II se han
mostrado datos preliminares acerca del contenido en glucosinolatos y los atributos sensoriales,
as como el desarrollo de un lxico especfico para el anlisis sensorial de un total de 10 entradas
de rcola y Erucastrum.
Ciertas caractersticas sensoriales como las de fase visual arrojaron diferencias entre las
entradas (color prpura en venacin, tamao 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 ltimas
debidas posiblemente al muestreo necesariamente reducido en cata. Respecto al resto de fases
sensoriales, se ha encontrado una variedad de descriptores como csped, rbano, piel de limn y
almendra, que arrojan diferencias entre entradas. Sin embargo, hemos detectado un patrn de
diferencias para ciertas notas (coles, rbano, trbol, tomate y alcachofa ente otros) que arrojan
resultados estadsticamente 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 caracterstica diferencial de las crucferas que estudiamos, se cuantific
el contenido de glucosinolatos. Entre los glucosinolatos alifticos, los mayoritarios fueron la
glucoerucina y glucorrafanina, este ltimo con valores de hasta 17.9 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 moles of glucosinolates/ g. Es importante resaltar la presencia de este
204
glucosinolato por sus propiedades anticancergenas (Farham et al., 2004; Sun-Ju y Gensho,
2006). El glucosinolato indlico mayoritario fue la glucosativina que probablemente es derivado de
una desmetilacin de la glucoerucina (Bennet et al., 2006), lo que explicara los bajos niveles de
glucoerucina que muestran las hojas; y el nico glucosinolato aromtico 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
no
se
ha
podido
relacionar
un
determinado
contenido
de
27.6 y 28.2
205
moles / g (peso seco) de tejido liofilizado para las muestras denominadas LGC1, LGC2, HGC1 y
HGC2 respectivamente. Se ha analizado adems el contenido de 13 de los glucosinolatos
contenidos en las muestras de rcola.
En cuanto al rendimiento en isotiocianatos, el sulforrafano fue el mayoritario, seguido de la
erucina e iberina, siendo la entrada LGC2 la que present ms variabilidad y el valor ms alto en
isotiocianatos. El contenido de sulforrafano nitrilo tambin fue determinado. Las entradas
mostraron diferencias significativas en la hidrlisis de glucorrafanina y formacin de sulforrafano
con un porcentaje de conversin 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
detoxificacin a diferencia del sulforrafano sera conveniente estudiar la seleccin de entradas con
bajos niveles o ausencia de la protena epitioespecfica (cofactor responsable de la generacin de
epitionitrilos y tiocianatos orgnicos) y altos niveles de mirosinasa (Matusheski et al., 2006) y
comprobar que estas lneas exhiben alto rendimiento de sulforrafano, con lo que se incrementara
el valor aadido 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 ms abundantes fueron la quercitina-3--glucsido y la rutina, no detectando
kaempferol, aunque otros autores lo describen incluso como mayoritario en sus entradas de rcola
(Jin et al., 2009; Selma et al., 2010; Bennett et al., 2006). Estas diferencias en contenido fenlico,
tanto cualitativas como cuantitativas, se pueden deber a las metodologas de anlisis empleadas o
a las caractersticas 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 lutena fue el carotenoide ms abundante, seguida de la -criptosantina, -caroteno,
zeaxantina y violaxantina, siendo de nuevo las entradas LGC2 y HGC1 las que mostraron mayor
concentracin y variabilidad en carotenoides. Nuestros resultados cualitativos respecto a este
grupo de fitoqumicos concuerdan con otros trabajos previos (Ramos y Rodrguez-Amaya, 1987;
Kimura y Rodrguez-Amaya, 2003; Niizu y Rodrguez-Amaya, 2005), aunque el contenido medio
de lutena fue superior en nuestro estudio.
Teniendo en cuenta los contenidos en glucosinolatos, el rendimiento en isotiocianatos y el
de las sustancias antioxidantes fenlicas o de tipo carotenoide, podemos avanzar la idea de que
entradas similares a LGC2 son interesantes desde el punto de vista de la Mejora Gentica,
fundamentalmente debido al alto contenido y variabilidad de fitoqumicos y al porcentaje de
conversin de glucosinolatos a isotiocianatos.
206
El uso de rcola como alimento y nutracutico puede tambin ser promocionado por su alto
contenido en minerales, lo que concuerda con otros autores (Bozokalfa et al., 2009). Los anlisis
estadsticos mostraron diferencias significativas entre las entradas para todos los minerales
excepto para el Ca, existiendo entradas con alto contenido para uno varios minerales como S3,
S5, S9 o S22 entre otras (PEX-60, PEX-62, PEX-66 y PEX-83, respectivamente). Estos anlisis
tambin mostraron diferencias significativas para todos los minerales para las entradas agrupadas
por pases excepto para el Ca. Dado que las entradas se cultivaron bajo las mismas condiciones
ambientales, las diferencias en la acumulacin 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, Captulo IV). Considerando que el calcio de
la rcola y el de la leche tiene el mismo porcentaje de absorcin, dado que esta crucfera est
libre de oxalatos y fitatos que bloquean la absorcin 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 rcola 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 rcola de unos 80 g, esto
proporcionara 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 ms altos que los
descritos en estudios previos (Kawashima y Valente-Soares, 2003; Bozokalfa et al., 2009;
Cavarianni et al., 2008).
La tecnologa NIRS, es un mtodo que permite realizar un cribado de un amplio nmero de
muestra de manera rpida y de bajo coste. Nuestros resultados indican que la espectroscopa en
el infrarrojo cercano puede ser utilizada como un mtodo de muestreo rpido para la
determinacin del contenido mineral de Fe, Na, K, Mg y Zn en rcola, representando una
herramienta til para la reduccin del tiempo de anlisis, de bajo coste y sin la utilizacin de
productos qumicos txicos. De acuerdo con los coeficientes de determinacin en la validacin
cruzada, las ecuaciones NIRS desarrolladas para la prediccin del contenido de Na y K fueron
caractersticas de ecuaciones que permiten una separacin ptima de muestras en contenidos
altos, medios y bajos. Las ecuaciones NIRS desarrolladas para la prediccin 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 prediccin del contenido en cenizas, Cu, Mn y Ca, debido al
207
estrecho rango de concentracin del elemento en cuestin y/o a la baja concentracin de los
componentes orgnicos asociado a dicho elemento.
La caracterizacin multidisciplinaria previa, tanto agro-morfolgica, como sensorial y fitoqumica
de entradas de rcola, ha constituido la base para poder llevar a cabo los objetivos ltimos de
evaluacin de la actividad biolgica, que tienen implicaciones sobre el consumo de rcola en la
salud humana. Se ha determinado la capacidad tumoricida y apopttica y anti/mutagnica y
antidegenerativa en ensayos humanos y animales modelo. En el captulo V se ha analizado la
actividad biolgica de las cuatro entradas de rcola (analizadas previamente para el perfil
fitoqumico en el captulo III), as como del isotiocianato (ITC) sulforrafano (SF). Se estudi
mediante tres aproximaciones: 1) midiendo el efecto en la proliferacin celular con las lneas HL60
(clulas tumorales humanas promielocticas), PC3 (clulas tumorales humanas de prstata) y
PNT1A (lnea celular normal de epitelio postpubertal de prstata); 2) evaluando su actividad
inductora de la apoptosis, y 3) ensayando el efecto en la expresin de la protena p21.
La proliferacin celular se ha analizado con el test de exclusin del azul tripn con la lnea
celular HL60 y con el ensayo de proliferacin WST-1 en las lneas celulares PC3 y PNT1A. Los
resultados del test de exclusin con el azul tripn mostraron que el SF fue altamente citotxico,
as como las entradas con mayor contenido de isotiocianatos. Los datos de la concentracin
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 ms bajo en GLs no se alcanza.
Analizando la relacin entre la actividad citotxica medida como IC50 y el contenido en SF de las
entradas de rcola, se puede observar que la viabilidad de las clulas HL60 desciende conforme
aumenta el rendimiento en SF de la muestra. Los resultados del ensayo de proliferacin WST-1,
sin embargo no mostraron diferencias significativas a bajas concentraciones para los diferentes
tratamientos y lneas 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 fragmentacin nuclear como marca general de induccin de apoptosis en
los tratamientos de clulas tumorales HL60 a altas concentraciones de extractos de rcola y a las
concentraciones de 8, 26 y 32 M de SF, por lo que sugerimos que la actividad citotxica
observada por rcola puede estar asociada a mecanismos diferentes de la fragmentacin
cromosmica. S hemos podido mostrar la induccin de la expresin de la protena p21 en los
tratamientos con SF a la concentracin de 15 M 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 protena p21, por lo que seran aconsejables futuros ensayos de expresin con
concentraciones superiores de rcola.
208
Ha sido la entrada LGC2 la que mostr el mejor comportamiento in vitro. Estos resultados
pueden relacionarse con los del captulo III de anlisis fitoqumico en el que las entradas LGC2 y
HGC1 fueron las que mostraron el mejor perfil fitoqumico. El hecho de que la entrada HGC1
tenga un bajo rendimiento en la conversin de GLs a ITC originando posiblemente otros
metabolitos sin actividad biolgica promotora de salud, confirma el comportamiento nico de la
lnea LGC2. Podemos proponer que las diferentes actividades in vitro pueden estar relacionadas
con el contenido de ITCs, ms que con el contenido de GLs, as como con la interaccin de otros
compuestos fitoqumicos como fenoles y carotenoides.
Las pruebas ms claras de que los vegetales y frutas estn relacionados con una reduccin
del riesgo de padecer cncer vienen aportadas por los estudios epidemiolgicos con crucferas
(Gasper et al., 2007). Los ITCs provenientes de las hidrlisis de GLs ms que stos mismos,
seran los responsables de de los efectos protectores de las crucferas. A partir de estos hechos
acerca de los efectos protectores de los ITCs nos hemos centrado en el captulo VI en estudiar la
actividad biolgica in vivo del SF y de extractos de rcola, concretamente respecto a su papel en
la proteccin del ADN (ausencia de genotoxicidad y potencia antigenotxica) y en el incremento
en la supervivencia de Drosophila. Hasta la fecha slo existen datos de un ensayo publicado
utilizando el material vegetal de Eruca (Lamy et al., 2008), en el cual no se mostraron efectos
genotxicos en hepatocitos en cultivo HpG2.
Ni el SF ni ninguna de las cuatro entradas de rcola analizadas en el ensayo SMART
resultaron ser genotxicas, a excepcin de la entrada Es4 (HGC2) a la mxima concentracin del
extracto ensayada (5mg/ml). Esta entrada, aun presentando el mximo contenido en
glucosinolatos y glucorrafanina, proporciona un bajo porcentaje de conversin 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 recombinognicos. Si comparamos este porcentaje
con el obtenido para mutaciones totales en alas marcadoras transheterocigticas obtendremos el
porcentaje de recombinognesis inducida por la entrada Es4 (HGC2) a la concentracin mxima
(5 mg/ml), que es un 82%. Sabiendo que el perxido de hidrgeno puede producir mutaciones por
recombinacin en un 44% (Villatoro-Pulido et al., 2009), la entrada Es4 (HGC2) puede provocar
casi el doble de mutaciones por recombinacin que la genotoxina utilizada en el ensayo. VzquezGmez et al., (2010) han descrito al sulforrafano como mutagnico en el cruce estndar del
ensayo SMART en Drosophila, aunque utilizaron concentraciones de 140 M mientras que en
nuestro ensayo la mayor concentracin fue de 12.6 M. Esta concentracin se escogi
suponiendo que el 100% del contenido el GLs de la entrada con ms alto nivel (Es4) pasara a
ITC, ya que concentraciones ms altas puede que no se den en condiciones nutricionales.
209
210
de glucosinolatos como nitrilos y epitionitrilos, los cuales a altas concentraciones pueden tener
efectos nocivos degenerativos como iniciacin de mutagnesis, citotoxicidad, y procesos
carcinognicos (Martn-Dietz et al., 1991).
Las lneas de Drosophila utilizadas en este trabajo no son mutantes de vida extensible. Se
ha realizado una bsqueda in silico (FlyBase) de todo el fondo gentico de las estirpes multiple
wing hair y flr3/TM3, BdS, as como de la funcin de estos genes y sus productos, no existiendo
ningn gen relacionado con algn 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 ptimas condiciones de manejo (medio de cultivo y manipulacin)
con las que se han realizado todos y cada uno de los ensayos de los tratamientos y
concentraciones.
El ltimo captulo de esta tesis, consiste en una aplicacin prctica para demostrar la
idoneidad de los ensayos utilizados en la determinacin del potencial promotor de salud de
crucferas, tanto del sistema in vivo de Drosophila, como del modelo in vitro de citotoxicidad en
lneas celulares tumorales. Las crucferas han demostrado una moderada-alta capacidad para
acumular metales (Pb, Cr, Cd, Ni, Zn y Cu) (Ebbs y Kochian, 1997), incluido el rbano (Raphanus
sativus L.) (Carbonell-Barrachina et al., 1999). Una contaminacin en suelos por As, Pb o Cd
podra repercutir en una acumulacin 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 rganos vitales produciendo toxicidad progresiva (Bryce-Smith, 1997; Morales et
al., 2000; Hamers et el., 2006). En el captulo VII se describe el estudio de la dinmica de
absorcin y distribucin de As, Pb y Cd en las plantas de rbano, as como el establecimiento de
las actividades genotxicas, antigenotxicas y citotxicas de la parte area y raz (parte
comestible) de este vegetal.
Aunque los niveles de As en el suelo fueron superiores a los lmites mximos establecidos
para suelos normales (no contaminados), debido a la baja biodisponibilidad de este metaloide en
el suelo utilizado, su acumulacin en las plantas fue bajo (Tabla 2, captulo VII) estando asimismo
por debajo del lmite establecido como txico por la legislacin para el contenido de
metales(oides) en vegetales (ANZFZ, 1998; Commission Regulation 629, 2008). No obstante,
hemos encontrado una acumulacin diferencial de As, Cd y Pb en races y parte area en las
plantas de rbano desarrolladas en suelos contaminados, siendo ms alta en parte area que en
races. Estos resultados contrastan con los de Marchiol y colaboradores (2004) que determinaron
211
una acumulacin ms alta de Cd y Pb en races que en parte area. En otros ensayos (Tlustos et
al., 1998; Carbonell-Barrachina, et al., 1999) se encontr que la distribucin de As en la planta
depende del contenido del mismo en el suelo. Cuando el contenido de As es bajo se tiende a
acumular ms cantidad en parte area que en raz, y viceversa.
Los estudios de genotoxicidad y antigenotoxicidad pueden ayudar a la evaluacin de la
seguridad de productos alimentarios (Bast et al., 2002). En el caso del As, es conocido que
aunque sea una genotoxina carcinognica, estudios previos han puesto de manifiesto que en su
forma inorgnica no es genotxico en el test SMART de Drosophila melanogaster (Rizki et al.,
2006). No obstante, hemos detectado genotoxicidad en las muestras de parte area desarrolladas
en suelos contaminados. El anlisis del fenotipo serrate nos permiti concretar que esta
genotoxicidad es debida a actividad recombinognica hasta en un 50%. Aunque la
carcinogenicidad de los metales(oides) no siempre est relacionada con resultados positivos de
genotoxicidad, nosotros s hemos encontrado una clara relacin entre el contenido en metales y la
genotoxicidad, que puede ser debida a la suma de las tres actividades mutagnicas del As, Cd y
Pb. Rizki y colaboradores (2006) concluyeron que el As inorgnico no fue genotxico debido a que
Drosophila no es capaz de biotransformarlo en formas metiladas que puedan ejercer una actividad
txica. Nuestro estudio solventa este problema, ya que el As, al ser incorporado a una matriz
biolgica como es el rbano es capaz de convertir el As inorgnico en una especie biodisponible
que resulta mutagnico en Drosophila. Estos resultados presentan una nueva manera de analizar
compuestos simples y complejos en Drosophila, alimentando las larvas no con la molcula
inactiva, sino con las molculas 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 observ que las concentraciones ms
altas de los extractos de muestras desarrolladas en suelos contaminados pueden inhibir el dao
causado por la genotoxina (perxido de hidrgeno), mientras que las concentraciones ms bajas
no lo hicieron. Sugerimos que un sinergismo entre el perxido de hidrgeno y los metal(oides)
puede activar genes, que codifiquen enzimas de estrs que detoxifican los efectos txicos del
perxido de hidrgeno, pero solo a las concentraciones ms altas. El contenido de glucosinolatos
e isotiocianatos del rbano puede comportarse como desmutgeno por contrarrestar el efecto de
las especies reactivas de oxgeno (ROS) generadas por el perxido de hidrgeno y los
metal(oides), pero solo cuando se alcanza una concentracin umbral.
El potencial citotxico frente a clulas HL60 de parte area y races conteniendo metales
pesados y races control aument conforme se increment la concentracin. Sin embargo, no
logramos alcanzar una IC50 para las muestras de parte area contaminados y, en el caso de
races contaminadas, la IC50 fue 15 veces superior a la IC50 de los cultivos controles
212
establecidos con races no contaminadas. El contenido en GLs e ITC de las races puede ser la
causa de la elevada actividad citotxica de las races control (Musk et al., 1995), mientras que en
el caso de la parte area y races contaminadas, se detecta alguna actividad citotxica, aunque no
total debido al contenido en metales pesados.
Por tanto, en base a los datos obtenidos, se concluy que aunque el contenido de As, Cd y
Pb en las muestras de rbano no excedieron los lmites establecidos como txicos por la
legislacin, el consumo de las mismas puede causar efectos genotxicos, as como una menor
citotoxicidad frente a clulas tumorales.
213
CONCLUSIONES
214
215
216
217
REFERENCIAS
218
Anter, J., Romero-Jimnez, M., Fernndez-Bedmar, Z., Villatoro-Pulido, M., Analla, M., AlonsoMoraga, A., Muoz-Serrano, A. (2011). Antigenotoxicity, Cytotoxicity, and Apoptosis Induction
by Apigenin, Bisabolol, and Protocatechuic Acid. Journal of Medicinal Food 14: 276-283.
ANZFZ (1998) Australian Food Standard Code. (Issue 41) Camberra: Australia NewZealand
Food Authority.
Arrabi, P., Genovese, M. I., Lajolo, F. M. (2004). Flavonoids in vegetable foods commonly
consumed in Brazil and estimated ingestion by the Brazilian population. Journal of
Agricultural and Food Chemistry 52: 1124-1131.
Avanesian, A., Khodayari, B., Felgner, J.S., Jafari, M. (2010). Lamotrigine extends lifespan but
compromises health span in Drosophila melanogaster. Biogerontology 11: 4552.
Bahadorani, S., Hilliker, A.J. (2008). Cocoa confers life span extension in Drosophila
melanogaster. Nutrition Research 28: 377382.
Bagli, E., Stefaniotou, M., Morbidelli, L., Ziche, M., Psillas, K., Murphy, C., Fotsis, T. (2004).
Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of
endothelial cell survival and proliferation by targeting phosphatidylinositol 30 -kinase activity.
Cancer Research 64: 7936 7946.
Baker, A. J. M., Brooks, R. R. (1989). Terrestrial higher plants which hyperaccumulate metallic
elements - A review of their distribution, ecology and phytochemistry. Biorecovery 1: 81-126.
Banuelos, G. S., Cardon, G., Mackey, B., Ben-Asher, J., Wu, L., Beuselinck, P., Akohoue, S.,
Zambrzuski, S. (1993). Plant and Environment Interactions. Boron and selenium removal in
boronladen soils by four sprinkler irrigated plant species. Journal of Environmental Quality 22:
786-792.
Barley, S. (2010). 2000-year-old pills found in Greek shipwreck. The New Scientist 207: 14.
Bast, A., Chandler, R. F., Choy, P. C. et al. (2002) Botanical health products, positioning and
requirements for effective and safe use. Environmental Toxicology and Pharmacology 12:
219
195211.
-
Beevi, S. S., Mangamoori, L. N., Subathra, M., Edula, J. R. (2010). Hexane extract of
Raphanus sativus L. roots inhibits cell proliferation and induces apoptosis in human cancer
cells by modulating genes related to apoptotic pathway. Plant Foods for Human Nutrition 65:
200209.
Bellostas, N., Srensen, J. C., Srensen, H. (2007). Profiling glucosinolates in vegetative and
reproductive tissues of four Brassica species of the U-Triangle for their biofumigation
potential. Journal of Science of Food Agriculture 87: 1586-1594.
Ben Salah-Abbs, J., Abbs, S., Ouanes, Z., Abdel-Wahhab, M. A., Bacha, H., Oueslati, R.,
(2009). Isothiocyanate from the Tunisian radish (Raphanus sativus) prevents genotoxicity of
Zearalenone in vivo and in vitro. Mutation Research 677: 5965.
Bendich, A. (1989). Carotenoids and the immune response. Journal of Nutrition 119: 112-115.
Bennett, R., Mellon, F., Botting, N., Eagles, J., Rosa, E., Williamson, G. (2002). Identification of
the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad
rocket). Phytochemistry 61: 25-30.
Bennett, R. N., Rosa, E. A. S., Mellon, F. A., Kroon, P. A. (2006). Ontogenic Profiling of
Glucosinolates, Flavonoids, and Other Secondary Metabolites in Eruca sativa (Salad Rocket),
Diplotaxis erucoides (Wall Rocket), Diplotaxis tenuifolia (Wild Rocket), and Bunias orientalis
(Turkish Rocket). Journal of Agricultural and Food Chemistry 54: 40054015.
Bennett, R. N., Carvalho, R., Mellon, F. A., Eagles, J., Rosa, E. A. S. (2007). Identification and
Quantification of Glucosinolates in Sprouts Derived from Seeds of Wild Eruca sativa L. (Salad
Rocket) and Diplotaxis tenuifolia L. (Wild Rocket) from Diverse Geographical Locations.
Journal of Agricultural and Food Chemistry 55: 6774.
Bernal, M. P., McGrath, S. P. (1994). Effects of pH and heavy metal concentrations in solution
culture on the proton release, growth and elemental composition of Alyssum murale and
Raphanus sativus L. Plant and Soil 166: 83-92.
Blaevic, I., Mastelic, J. (2008). Free and bound volatiles of rocket (Eruca sativa Mill.). Flavour
and fragrance journal 23: 278-285.
Boyd, O., Weng, P., Sun, X., Alberico, T., Laslo, M., Obenland, D. M., Kern, B., Zou, S. (2011).
Nectarine promotes longevity in Drosophila melanogaster. Free Radical Biology and Medicine
50: 1669-1678.
Bones, A. M., Rossiter, J. T. (2006). The enzymic and chemically induced decomposition of
glucosinolates. Phytochemistry 67: 1053-1067.
Bozokalfa, M.K., Yagmur, B., Ilbi, H., Esiyok, D., Kavak, S. (2009). Genetic variability for
mineral concentration of Eruca sativa L. and Diplotaxis tenuifolia L. accessions. Crop
Breeding and Applied Biotechnology 9: 372-381.
220
Brown, S. L., Chaney, R. L., Angle, J. S., Baker, A. J. M. (1995). Zinc and cadmium uptake by
hyperaccumulator Thlaspi caerulescens grown in nutrient solution. Soil Science Society of
America Journal 59: 125-133.
Brown, A. F., Yousef, G. G., Jeffery, E. H., Klein, B. P., Wallig, M. A., Kushad, M. M., Juvik, J.
A. (2002). Glucosinolate profiles in broccoli: Variation in levels and implications in breeding
for cancer chemoprotection. Journal of the American Society for Horticultural Science, 127:
807-813.
Bryce-Smith, D. (1997). Heavy metals as contaminants of human environ. (eds) Peter G. Publ
Edu. Tech. Subgroup, The Chemical Society London. pp. 2123.
Buchanan, B. B., Cruissem, W., Jones R. L. (2000). Chemistry and Molecular Biology of
Plants. Rockville, Maryland: John Wiley and Sons Inc (Chapter 24).
isothiocyanates
is
followed
by
transporter-mediated
export
as
Carbonell-Barrachina, A. A., Burlo, F., Lpez, E., Martnez-Snchez, F. (1999). Arsenic toxicity
and accumulation in radish as affected by arsenic chemical speciation. Environmental
Science Health 34: 661679.
Cardone, M., Mazzoncini, M., Menini, S., Rocco, V., Senatore, A., Seggiani, M., Vitolo, S.
(2003). Brassica carinata as an alternative oil crop for the production of biodiesel in Italy:
agronomic evaluation, fuel production by transesterification and characterization. Biomass
Bioenergy 25: 623-636.
Cartea, M. E., Velasco, P., Obregn, S., Padilla, G., De Haro, A. (2008). Seasonal variation in
glucosinolate
content
in
Brassica
oleracea
crops
grown
in
northwestern
Spain.
Caulfield, L. E., Back, R. E. (2004). Zinc deficiency. In Ezzati, M., Lopez, A.D., Rodgers, A.,
Murray, C. J. L. (Eds.). Comparative quantification of health risks: Global and regions burden
of diseases attribution to selected major risk factors, Vol I.
Causse, M., Lecompte, L., Baffert, N., Duffe, P., Hospital, F. (2001). Market-assisted selection
for the transfer of QTLs controlling fruit quality traits into tomato elite lines. In Dore, C.,
Dosba, F., Baril, C. Acta Horticulturae 546.
Cavarianni, R. L., Filho, A. B. C, Cazetta, J. O., May, A., Corradi, M. M. (2008). Nutrient
contents and production of rocket as affected by nitrogen concentrations in the nutritive
solution. Scientia Agricola 65: 652-658.
Chambers, K. F., Bacon, J. R., Kemsley, E. K., Mills, R. D., Ball, R. Y., Mithen, R. F., Traka, M.
H. (2009). Gene expression profile of primary prostate epithelial and stromal cells in response
to sulforaphane or iberin exposure. Prostate 69: 1411-1421.
221
Charron, C. S., Arnold, M., Saxton, A. M., Sams, C. E. (2005). Relationship of climate and
genotype to seasonal variation in the glucosinolate-myrosinase system. I. Glucosinolate
content in ten cultivars of Brassica oleracea grown in fall and spring seasons. Journal of
Science of Food and Agriculture 85: 671-681.
Chen, K. C., Calzone, L., Csikasz-Nagy, A., Cross, F. R., Novak, B., Tyson J. J. (2004).
Integrative Analysis of Cell Cycle Control in Budding Yeast. Molecular Biology of the Cell 15:
38413862.
Chevalier, F., Chobert, J. M., Genot, C., Haertl, T. (2001). Scavenging of free radicals,
antimicrobial, and cytotoxic activities of the Maillard reaction products of -lactoglobulin
glycated with several sugars. Journal of Agriculture and Food Chemistry 49: 5031-5038.
Chin H. F. (1994). Seed banks: conserving the past for the future. Seed Science and
Technology 22: 385 400.
Clark, D. H., Cary, E. E., Mayland, H. F. (1989). Analysis of trace elements in forages by near
infrared reflectance spectroscopy. Agronomy Journal 81: 9195.
Clifford, M. N., Brown, J. E. (2006). Flavonoids and Health. In Andersen, O. M., Markham, K.
R. (Eds.). Flavonoids: Chemistry, biochemistry and applications (pp.319-370). Taylor and
Francis Group Inc. New York.
Collins, S. J., Ruscetti, F. W., Gallagher, R. E. and Gallo, R. C. (1978). Terminal differentiation
of human promyelocytic leukaemia cells induced by dimethyl sulfoxide and other polar
compounds. Proceedings of the National Academy of Sciences USA 75: 24582462.
Conte-Anazetti, M., Silva-Melo, P., Duran, N., Haun, M. (2003). Comparative cytotoxicity of
dimethylamide-crotonin in the promyelocytic leukemia cell line (hl60) and human peripheral
blood mononuclear cells. Toxicology 88: 261274.
Cozzolino, D., Moron, A. (2004). Exploring the use of near infrared reflectance spectroscopy to
predict trace minerals in legumes. Animal Feed Science and Technology 11: 161-173.
DAntuono, L. P., Elementi, S., Neri, R. (2009). Exploring new potential health-promoting
222
vegetables: glucosinolates and sensory attributes of rocket salads and related Diplotaxis and
Eruca species. Journal of the Science of Food and Agriculture 89: 713-722.
-
Dashwood, R. H., Ho, E. (2007). Dietary histone deacetylase inhibitors: from cells to mice to
man. Seminars in Cancer Biology 17: 363369.
Del Ro, M., Font, R., Fernndez-Martnez, J. M., Domnguez, J., De Haro, A. (2000) Fields
trials of Brassica carinata and B. juncea in polluted soils of the Guadiamar river area.
Fresenius Environmental Bulletin 9: 328- 332.
Del Ro, M., Font, R., De Haro, A. (2005). Differential accumulation of Pb, Zn and Cu by
Brassica species grown in the polluted soils of Aznalcllar (Souther Spain). In: Del Valls, T.
A., Blasco, J. (Eds). Integrated assessment and management of the ecosystems affected by
the Aznalcllar mining spill (SW, SPAIN), pp 55-60. Unesco, Pars.
Ebbs, S. D., Kochian, L. V. (1997). Toxicity of zinc and copper to Brassica species: implications
for phytoremediation. Journal of Environmental Quality 26: 776781.
Edge, R., McGarvey, D. J., Truscott, T. G. (1997). The carotenoids as antioxidants: a review.
Journal of Photochemistry and Photobiology 41: 189-200.
Elless, M. P., Blaylock, M. J., Huang, J. W., Gussman, C. D. (2000). Plants as a natural source
of concentrated mmineral nutritional supplements. Food Chemistry 71: 181-188.
Farnham, M. W., Wilson, P. E., Stephenson, K. K., Fahey, J. V. (2004). Genetic and
environmental effects on glucosinolate content and chemoprotective potency of broccoli.
Plant Breeding 123: 6065.
Felix, H. (1997). Field trials for in situ decontamination of heavy metal polluted soils using crops
of metal-accumulating plants. Z. Pflanzenernhr. Bodenk. 160: 525-529.
Fenwick, G. R., Griffiths, N. M., Heaey, R. K. (1983). Bitterness in Brussels sprouts (Brassica
oleracea L var gemnifera): the role of glucosinolates and their breakdown products. Journal of
the Science of Food and Agriculture 34: 73-80 (1983).
Font, R., Del Ro, M., Fernndez-Martnez, J. M., De Haro-Bailn, A. (2004). Use of near infrared spectroscopy for screening the individual and total glucosinolate content in indian mustard
seed (Brassica juncea L. Czern. & coss). Journal of Agricultural and Food Chemistry 52:
3563-3569.
Font, R., Del Ro-Celestino, M., Cartea, M. E., De Haro-Bailn, A. (2005). Quantification of
glucosinolates in leaves of leaf rape Brassica napus var. pabularia) by near-infrared
223
Font, R., Del Ro-Celestino, M., De Haro-Bailn, A. (2006). Near Infra-Red Spectroscopy:
Methodology and potential for predicting trace elements in plants. Phytorremediation
Methods and Reviews, pp. 205-217. Ed. Willey, Neil, Humana Press Inc.
Fraga, C. G. (2007). Plant polyphenols: How to translate their in vitro antioxidant actions to in
vivo conditions. IUBMB Life 59: 308-315.
Gamet-Payrastre, L. , Li, P., Lumeau, S., Cassar, G., Dupont, M. A., Chevolleau, S., Gasc, N.,
Tulliez, J., Terce, F. (2000). Sulforaphane, a naturally occurring isothiocyanate, induces cell
cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Research 60: 1426
1433.
Gasper, A. V., Traka, M., Bacon, J. R., Smith, J. A., Tailor, M. A., Hawkey, C. J., Barret, D. A.,
Mithen, R. (2007). Consuming broccoli does not induce genes associated with xenobiotic
metabolism and cell cycle control in human gastric mucosa. Journal of Nutrition 137: 17181724.
Gill, C. I. R., Haldar, S., Porter, S., Matthews, S., Sullivan, S., Coulter, J., McGlynn, H.,
Rowlnad, I. (2004). The effect of cruciferous and leguminous sprouts on genotoxicity, in vitro
and in vivo. Cancer Epidemiology Biomarkers and Prevention 13, 1199-1205.
Gingras, D., Gendron, M., Boivin, D., Moghrabi, A., Theoret, Y., Beliveau, R. (2004). Induction
of medulloblastoma cell apoptosis by sulforaphane, a dietary anticarcinogen from Brassica
vegetables. Cancer Letters 203: 35 43.
Glew, R.H. (2005). The nutrient content of three edible plants of the Republic of Niger. Journal
of Food Composition and Analysis, 18: 1527.
Gmez, J. A. (2002). Solventando los problemas habituales de la rcula. Horticultura 164: 88.
Gmez-Campo, C., (1993). Eruca. In Castroviejo, S. al. (Eds). Flora. Ibrica, 4: 390- 392.
Brassica
Graf, U., Wrgler, F. E., Katz, A. J., Frei, H., Juon, H., Hall, C. B. and Kale, P. G. (1984).
224
Graf, U., Alonso-Moraga, A., Castro, R. and Diaz, E. (1994). Genotoxicity testing of different
types of beverages in the wing somatic mutation and recombination test. Food and Chemical
Toxicology 32: 423430.
Gutirrez, R. M., Perez, R. L. (2004). Raphanus sativus (radish): their chemistry and biology.
Scientific World Journal 4: 811837.
Halgerson, J. L., Sheaffer, C. C., Martin, N. P., Peterson, P. R., Weston, S. J. (2004). Nearinfrared reflectance spectroscopy prediction of leaf and mineral concentrations in alfalfa.
Agronomy Journal 96: 344351.
Halliwell, B. (2008). Are polyphenols antioxidants or pro-oxidants? What do we learn from cell
culture and in vivo studies?. Archives of Biochemistry and Biophysics 476: 107-112.
Hamers, T., Van den Berg, J. H. J., Van Gestel, C. A. M., Van Schooten, F. J., amd Murk, A. J.
(2006). Risk assessment of metals and organic pollutants for herbivorous and carnivorous
small mammal food chains in a polluted floodplain (Biesbosch, The Netherlands).
Environmental Pollution 144: 581595.
Hampson, C. R., Quamme, H. A., Hall, J. W., MacDonald, R. A., King, M. C., Cliff, M. A.
(2000). Sensory evaluation as a selection tool in apple breeding. Euphytica 111: 79-90.
Hanlon, P. R., Webber, D. M., Barnes, D. M. (2007). Aqueous extract from spanish black
radish (Raphanus sativus L. var. niger) induces detoxification enzymes in the HepG2 human
hepatoma cell line. Journal of Agriculture and Food Chemistry 55: 643946.
Harker, F. R., Gunson, F. A., Jaeger, F. R. (2003). The case of fruit quality: and interpretative
review of consumer attitudes and preferences for apples. Postharvest Biology and
Technology 28: 333-347.
Harris, K. E., Jeffery, H. E. (2008). Sulforaphane and erucin increase MRP1 and MRP2 in
human carcinoma cell lines. Journal of Nutritional Biochemistry 19: 246254.
Heijnen, C. G., Haenen, G. R., Van Acker, F. A., Van der Vijgh, W. J., Bast, A. (2001).
Flavonoids as peroxynitrite scavengers: the role of the hydroxyl groups. Toxicology In Vitro
15: 3-6.
225
House, W. A. (1999). Element bioavailability as exemplified by iron and zinc. Field Crops
Research 60: 115-141.
IPGRI (2002). Descrittori per la rucola (Eruca spp.). Instituto Internazionale per le Risorse
Fitogenetiche, Roma, Italia.
ILSI. (2004) Conceptos sobre los alimentos funcionales. ILSI Europe Concise Monograph
Series, USA.
Jadhav, U., Vaughn, S. F., Berhow, M. A., Sanjeeva, M. (2007). Iberin induces cell cycle arrest
and apoptosis in human neuroblastoma cells. International Journal of Molecular Medicine 19:
353-361.
Jin, J., Koroleva, O. A., Gibson, T., Swanston, J., Magan, J., Zhang, Y., Rowland, I. R.,
Wagstaff, C. (2009). Analysis of Phytochemical Composition and Chemoprotective Capacity
of Rocket (Eruca sativa and Diplotaxis tenuifolia). Leafy Salad Following Cultivation in
Different Environments. Journal of Agricultural and Food Chemistry 57: 52275234.
Jirovetz, L., Smith, D., Buchbauer, G. (2002). Aroma compound analysis of Eruca sativa
(Brassicaceae) SPME headspace leaf samples using GC, GC-MS, and olfactometry. Journal
of Agricultural and Food Chemistry 50: 4643-4646.
Jones, M. A., Grotewiel, M., (2011). Drosophila as a model for age-related impairment in
locomotor and other behaviors. Experimental Gerontology 46: 320-325.
Kassie, F., Knasmller, S. (2000). Genotoxic effects of allyl isothiocyanate (AITC) and
phenethyl isothiocyanate (PEITC). Chemical-Biological International 127: 163-180.
Kawashima, L. M., Valente-Soares, L. M. (2003). Mineral profile of raw and cooked leafy
vegetables consumed in Southern Brazil. Journal of Food Composition and Analasys 16: 605611.
Kerr, J. F. R., Wyllie, A. H., Currie, A. R. (1972). Apoptosis: A Basic Biological Phenomenon
with Wide-ranging Implications in Tissue Kinetics. British Journal of Cancer 26: 239257.
Keum, Y. S., Khor, T. O., Lin, W., Shen, G., Kwon, K. H., Barve, A., Li, W., Kong, A. N. (2009).
Pharmacokinetics and pharmacodynamics of broccoli sprouts on the suppression of prostate
cancer in transgenic adenocarcinoma of mouse prostate (TRAMP) mice: implication of
induction of Nrf2, HO-1 and apoptosis and the suppression of Akt-dependent kinase pathway.
Pharmaceutical Research, 26: 2324-2331.
226
Kim, J. H., Han Kwon, K., Jung, J. Y., Han, H. S., Hyun Shim, J., Oh, S., Choi, K. H., Choi, E.
S., Shin, J. A., Leem, D. H. (2010). Sulforaphane Increases Cyclin-Dependent Kinase
Inhibitor, p21 Protein in Human Oral Carcinoma Cells and Nude Mouse Animal Model to
Induce G /M Cell Cycle Arrest. Journal of Clinical Biochemistry and Nutrition 46: 6067.
2
Kim, S. J., Ishii, G. (2006). Glucosinolate profiles in the seeds, leaves and roots of rocket salad
(Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and purified 4methoxyglucobrassicin. Soil Science and Plant Nutrition 52: 394400.
Kong, A. N., Owuor, E., Yu, R. (2001). Induction of xenobiotic enzymes by the MAP kinase
pathway and the antioxidant or electrophile response element (ARE/EpRE). Drug Metabolism
Reviews 33: 255-27.
Koukounaras, A., Siomos, A., Sfakiotakis, E. (2007). Postharvest CO2 and ethylene production
and quality of rocket (Eruca sativa Mill.) leaves as affected by leaf age and storage
temperature. Postharvest Biology and Technology 46: 167-173.
Lampe, J. W., Peterson, S., (2002). Brassica biotransformation and cancer risk: genetic
polymorphisms alter the preventive effects of cruciferous vegetables. Journal of Nutrition 132:
29912994.
Lamy, E., Schrder, J., Paulus, S., Brenk, P., Stahl, T., Mersch-Sundermann, V. (2008).
Antigenotoxic properties of Eruca sativa (rocket plant), erucin and erysolin in human
hepatoma (HepG2) cells towards benzo(a)pyrene and their mode of action. Food and
Chemical Toxicology 46: 24152421.
Lee, S. O., Lee, I. S. (2006). Induction of quinone reductase, the phase 2 anticarcinogenic
marker enzyme, in Hepa1c1c7 cells by radish sprouts, Raphanus sativus L.. Journal of Food
Science 71: 144148.
41. Li, Y. M., Chan, H. Y. E, Yao, X. Q., Huang, Y., Chen, Z. Y. (2008). Green tea catechins and
broccoli reduce fat-induced mortality in Drosophila melanogaster. Journal of Nutrition and
Biochemistry 19: 376383.
42. Lucarini, M., Canali, R., Cappelloni, M., Di Lullo, G., Lombarda-Boccia, G. (1999). In vitro
calcium availability from brassica vegetables (Brassica oleracea L.) and as consumed in
composite dishes. Food Chemistry 64: 519-529.
43. Lugasi, A., Blazovics, A., Hagymasi, K., Kocsis, I., Kery, A. (2005). Antioxidant effect of
squeezed juice from black radish (Raphanus sativus L. var niger) in alimentary
hyperlipidaemia in rats. Phytotherapy Research 19: 58791.
227
44. Liu, L., Shelp, B. J., Spiers, G. A. (1992). Boron distribution and mobility in field grown broccoli
(Brassica oleracea var. italica). Canadian Journal of Plant Science 73: 587-600.
45. Liu, Y., Murakami, N., Wang, L., Zhang, S. (2008). Preparative high-performance liquid
chromatography for the purification of natural acylated anthocyanins from red radish
(Raphanus sativus L.). Journal of Chromatographic Science 46: 7436.
46. Lynn, S., Van Remmen, H., Epstein, C. J., Huang, T. T. (2001). Investigation of mitochondrial
DNA deletions in post-mitotic tissues of the heterozygous superoxide dismutase 2 knockout
mouse: effect of ageing and genotype on the tissue-specific accumulation. Free Radical
Biology & Medicine 31: S58.
47. MacKendrick, P. L., Howe, H. M., Classics in Translation, Volume I: Greek Literature. 1952.
University of Wisconsin Press.
48. Maioli, E., Torricelli, C., Fortino, V., Carlucci, F., Tommassini, V., Pacini, A. (2009). Critical
Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers. Biological
Procedures Online 11: 227-240.
49. Mandiki, S. N. M., Derycke, G., Bister, J. L., Paquay, A., Mabon, N., Wathelet, P., Marlier, M.
(2000). Les potentialits du tourteau de colza pour l' engrissement de jeunes rumiants.
Presses Universitaires de Namur. Belgique.
50. Marchiol, L., Assolari, S., Sacco, P. and Zerbi, G. (2004). Phytoextraction of heavy metals by
canola (Brassica napus) and radish (Raphanus sativus) grown on multicontaminated soil.
Environmental Pollution 132: 2127.
51. Martin Dietz, H., Panigrahi, S., Harris, R. V. (1991). Toxicity of hydrolysis products from 3butenyl glucosinolate in rats. Journal of Agriculture and Food Chemistry 39: 311315.
52. Martnez-Snchez, J. J., Conesa, E. Vicente, M. J., Jimnez, A. Franco, J. A. (2006).
Germination responses of Juncus acutus (Juncaceae) and Schoenus nigricans (Cyperaceae)
to light and temperature. Journal of Arid Environments 66: 187191.
53. Mth-Gaspar, G. y Anton, A. (2002). Heavy metal uptake by two radish varieties. Acta
Biologica Szegediensis 46: 113114.
54. Mathews-Roth, M. M. (1990). Plasma concentration of carotenoids after large doses of betacarotene. American Journal of Clinical Nutrition 52: 500-501.
55. Matusheski, N. V., Swarup, R., Juvik, J. A., Mithen, R., Bennet, M., Jeffery, E. H. (2006).
Epithiospecifier protein from Brocoli (Brassica oleracea L. Ssp. italica) inhibits formation of the
anticancer agent sulforaphane. Journal of Agricultural and Food Chemistry 54: 2069-2070.
56. McMahon, M., Itoh, K., Yamamoto, M., Hayes, J. D. (2003). Keap1-dependent proteasomal
degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant
response element-driven gene expression. Journal of Biological Chemistry 278: 2159221600.
57. Melchini, A., Costa, C., Traka, M., Miceli, N., Mithen, R., De Pascuale, R., Trovato, A. (2009).
Erucin, a new promising cancer chemopreventive agent from rocket salads, shows antiproliferative activity on human lung carcinoma A549 cells. Food and Chemical Toxicology 47:
228
1430-1436.
58. Miller, J. 1987. Bioavailable iron in raw and cooked spinach and broccoli. Nutrition Reports
International 36: 435-440.
59. Mithen, R. F., Dekker, M., Verkerk, R., Rabot, S., Jonson, I. T. (2000). Review: The nutritional
significance, biosynthesis and bioavailability of glucosinolates in human foods. Journal of
Science of Food and Agriculture 80: 967-984.
60. Mithen, R. (2001). Glucosinolates-biochemistry, genetics and biological activity. Plant Growth
Regulation, 34: 91-103.
61. Miyazawa, M., Maehara, T., Kurose, K. (2002). Composition of the essential oil from the
leaves of Eruca sativa. Flavour and Fragrance Journal, 17: 187190.
62. Mockett, R. J., Sohal, R. S., (2006). Temperature-dependent trade-offs between longevity and
fertility in the Drosophila mutant, Methuselah. Experimental Gerontology 41: 6566-6573.
63. Morales, K. H., Ryan, L., Kuo, T. L., Wu, M. M. and Chen, C. J. (2000). Risk of internal
cancers from arsenic in drinking water. Environmental Health Perspectives 108: 655661.
64. Morales, M. R., Janick, J. (2002). Arugula: A promising specialty leaf vegetable. In: Janick, J.,
Whipkey, A. (Eds.). Trends in new crops and new uses, pp. 418423.. ASHS Press, Alexandria, Va.
65. Morales, M. R., Maynard, E., Janick, J., (2006). Adagio: A slowbolting Arugula. Horticultural
Science 41: 15061507.
66. Moskowitz, H. R. (1993). Sensory analysis procedures and viewpoints: Intellectual history,
current debates, future outlooks. Journal of Sensory Studies 8: 241-256.
67. Muller, H. J. (1927). Artificial transmutation of the gene. Science 66: 84-87.
68. Munday, R, Munday, C. M. (2004). Induction of phase II detoxification enzymes in rats by
plant-derived isothiocyanates: comparison of allyl isothiocyanate with sulforaphane and
related compounds. Journal of Agriculture and Food Chemistry 52: 18671871.
69. Musk, S. R. R., Smith, T. K. and Johnson, I. T. (1995). On the cytotoxicity and genotoxicity of
allyl and phenethyl isothiocyanates and their parent glucosinolates sinigrin and gluconasturtiin.
Mutation Research 348, 1923.
70. Nakamura, Y., Iwahashi, T., Tanaka, A., Koutani, J., Matsuo, T., Okamoto, S., Sato, K.,
Ohtsuki, K. (2001). 4-(methylthio)-3-butenyl isothiocyanate, a principal antimutagen in daikon
(Raphanus sativus; Japanese white radish). Journal of Agriculture and Food Chemistry 49:
57555760.
71. Nanda-Kumar, P. B. A., Dushenkov, V., Ensley, B. D. (1995). The use of crop Brassica
phytoextraction: a subject of phytoremediation to remove toxic metals from soils. In
Proceedings of the 9th International Rapeseed Conference, pp. 753-756. Cambrigde, Reino
Unido, 4-7 julio 1995.
72. Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P., Estabrook, R. W., Feyereisen,
R., Gonzalez, F. J., Coon, M. J., Gunsalus, I. C., Gotoh, O. (1993). The P450 superfamily :
229
update on new sequences, gene mapping, accession numbers, early trivial names of
enzymes, and nomenclature. DNA Cell Biology 12: 1 51.
73. Nielsen, T., Bergstrm, B., Borch, E. (2008). The origin of off-odours in packaged rucola
(Eruca sativa). Food Chemistry 110: 96105
74. Niizu, P. Y., Rodrguez-Amaya, D. B. (2005). New data on the carotenoid composition of raw
salad vegetables. Journal of Food Composition and Analysis, 18: 739749.
75. Nishino, H. (1998). Cancer prevention by carotenoids. Mutation Research 402: 159-163.
76. Nilsson, M.B., Dabakk, E., Korsman, T. Renberg, I. Environ. Sci. Technol., 30, 2586-2590,
1996.
77. Nuez, F., Hernndez-Bermejo, J. E. (2009). Hortcolas marginadas. Al otro lado del Atlntico :
Espaa. http://www.rlc.fao.org/es/agricultura/produ/cdrom/contenido/libro09/Cap5-4.htm
78. Nuez, F., Ruiz, J. J. (1999a). Conservacin y Utilizacin de Recursos Fitogenticos. Servicio
de publicaciones de la Universidad Politcnica de Valencia, Valencia, Spain (ISBN: 84-7721758-0).
79. Nuez, F., Ruiz, J. J. (1999b). La Biodiversidad Agrcola Valenciana: estrategias para su
conservacin y utilizacin. (Premio Bancaja Estudios sobre el Agroentorno 1998, modalidad
investigacin). Servicio de publicaciones de la Universidad Politcnica de Valencia, Valencia,
Spain (ISBN 84-7721-742-4).
80. O'Leary, K. A., de Pascual-Tereasa, S., Needs, P. W., Bao, Y. P., O'Brien, N. M., Williamson,
G. (2004). Effect of flavonoids and vitamin E on cyclooxygenase-2 (COX-2) transcription.
Mutation Research 551: 245-254.
81. Onwukaeme, D. N., Ikuegbvweha, T. B., Asonye, C. C. (2007). Evaluation of phytochemical
constituents, antibacterial activities and effect of exudates of Pycanthus Angolensis weld warb
(Myristicaceae) on corneal ulcers in rabbits. Tropical Journal of Pharmaceutical Research 6:
725-730.
82. Oraguzie, N. C., Whitworth, C., Fraser, J., Alspach, P. A., Morgan, C. G. T. (2003). First
generation of recurrent selection in apple: estimation of genetic parameters. In Janick, J. (ed.),
Acta Horticulturae 622: 213-220.
83. Ortiz-Monasterio, J. I., Palacios-Rojas, N., Meng, E., Pixley, K., Trethowan, R., Pea, R. J.
(2007). Enhancing the mineral and vitamin content of wheat and maize through plant
breeding. Journal of Ceral Science 46: 293-307.
84. Osaba, L., Aguirre, A., Alonso, A., Graf, U. (1999). Genotoxicity testing of six insecticides in
two crosses of the Drosophila wing spot test. Mutation Research 439: 49-61.
85. Padilla, G., Cartea, M. E., Velasco, P., de Haro, A., Ords, A. (2007). Variation of
glucosinolates in vegetable crops of Brassica rapa. Phytochemistry 68: 536-545.
86. Padulosi, S., Pignone, D. (1997). Rocket: A Mediterranean Crop for the World; International
Plant Genetic Resources Institute: Rome, Italy.
87. Papi, A., Orlandi, M., Bartolini, G., Barillari, J., Iori, R., Paolini, M., Ferroni, F., Grazia-Fumo,
230
M., Pedulli, G. F., Valgimigli, L. (2008). Cytotoxic and antioxidant activity of 4-methylthio-3butenyl isothiocyanate from Raphanus sativus L. (Kaiware Daikon) sprouts. Journal of
Agriculture and Food Chemistry 56: 87583.
88. Petisco, C., Garcia-Criado, B., de Aldana, B. R. V., Zabalgogeazcoa, I., Mediavilla, S., GarciaCiudad, A. (2005). Use of near-infrared reflectance spectroscopy in predicting nitrogen,
phosphorus and calcium contents in heterogeneous woody plant species. Analytical and
Bioanalytical Chemistry 382: 458465.
89. Pietta, P. G. (2000). Flavonoids as antioxidants. Journal of Natural Products 63: 1035-1042.
90. Pic, B, Ruiz-Quian, J. J. (2000). In Nuez, F., Carrillo, J. M. (Eds.). Clonacin Posicional y
Mapeo Comparativo, Los Marcadores Genticos en la Mejora Vegetal, p 441-512. Editorial
U.P.V., Valencia, Espaa.
91. Pimpini, F., Enzo, M., (1997). La coltura della rucola negli ambienti veneti. Colture protette 4:
21-32.
92. Pita-Villamil, J. M. P., Perez-Garcia, F., Martinez-Laborde, J. B. (2002). Time of seed
collection and germination in rocket, Eruca vesicaria (L.) Cav. (Brassicaceae). Genetic
Resources and Crop Evolution 45: 47-51.
93. Pitrat, M. (2002). Gene list for melon. Cucurbit Genetic Coop Reports 25: 76-93.
94. Prasain, J. K., Carlson, S. H., Wyss, J. M. (2010). Flavonoids and age-related disease: Risk,
benefits and critical windows. Maturitas 66: 163-171.
95. Podsedek, A., (2007). Natural antioxidants and antioxidant capacity of Brassica vegetables: A
review. Food Science and Technology 40: 1-11.
96. Prochaska, H. J., Santamaria, A. B., Talalay, P. (1992). Rapid detection of inducers of
enzymes that protect against carcinogens. Proceedings of the National Academy of Sciences
USA 89: 2394 2398.
97. Ramos, D. M. R., Rodriguez-Amaya, D. B., (1987). Determination of the vitamin A value of
common Brazilian leafy vegetables. Journal of Micronutrient Analysis 3: 147155.
98. Ramos S. (2007). Effects of dietary flavonoids on apoptotic pathways related to cancer
chemoprevention. Journal of Nutrition and Biochemistry 18: 427-442.
99. Raskin, I., Kumar, P. B. A. N., Dushenkov, S., Salt, D. E. (1994). Bioconcentration of heavy
metals by plants. Current Opinion in Biotechnology 5: 285-290.
100.
Rizki, M., Kossatz, E., Velzquez, A., Creus, A., Farina, M., Fortaner, S., Sabbioni, E.,
Marcos, R. (2006). Metabolism of arsenic in Drosophila melanogaster and the genotoxicity of
dimethylarsinic acid in the Drosophila wing spot test. Environmental Molecular Mutagenesis
47: 162-168.
101.
102.
231
Salt, D. E., Blaylock, M., Nanda Kumar, P. B. A., Dushenkov, V., Ensley, B. D. Chet, L.,
Raskin, I. (1995). Phytoremediation: A novel strategy for the removal of toxic metals from the
environment using plants. Biotechnology 13: 468-474.
104.
105.
Santamaria, P., Elia, A., Conversa, G. (1994). Broccoli growth and yield in a long term
vegetable crop sequence. N and herbicides effect. [Accrescimento e produzione di cavolo
broccolo (Brassica oleracea L. var. italica Plenck) in una successione orticola. Effetti dell'azoto
e dei diserbanti]. Rivista di Agronomia 28: 141-147.
106.
Scalbert, A., Johnson, I. T., Slatmarsh, M. (2005). Polyphenols: antioxidants and beyond.
American Journal of Clinical Nutrition 81: 215-217.
107.
Schubert, B. A., Jahren, A. H., (2011). Fertilization trajectory of the root crop Raphanus
sativus across atmospheric pCO estimates of the next 300 years. Agriculture, Ecosystem &
2
Selma, M. V., Martnez-Snchez, A., Allende, A., Ros, M., Hernndez, M. T., Gil, M. (2010).
Impact of Organic Soil Amendments on Phytochemicals and Microbial Quality of Rocket
Leaves (Eruca sativa). Journal of Agricultural and Food Chemistry, 58: 83318337.
109.
Sgherri, C., Cosi, E., Navari-Izzo, F. (2003). Phenols and antioxidative status of Raphanus
sativus grown in copper excess. Plant Physiology 118: 21-28.
110.
Shukla, S., Chatterji, S., Mehta, S., Rai, P. K., Singh, R. K., Yadav, D. K., Watal, G. (2011).
Antidiabetic effect of Raphanus sativus root juice. Pharmaceutical Biology 49: 32-37.
111.
Sikdar, S. R., Chatterjee, G., Das, S. (1987). Regeneration of plants from mesophyll
protoplasts of the wild crucifer Eruca sativa Lam. Plant Cell Reports 6: 486-489.
112.
113.
Singh, S. V., Warin, R., Xiao, D., Powolny, A. A., Stan, S. D., Arlotti, J. A., Zeng, Y., Hahm,
E. R., Marynowski, S. W., Bommareddy, A., Desai, D., Amin, S., Parise, R. A., Beumer, J. H.,
Chambers, W. H. (2009). Sulforaphane inhibits prostate carcinogenesis and pulmonary
metastasis in TRAMP mice in association with increased cytotoxicity of natural killer cells.
Cancer Research 69: 2117-2125.
114.
115.
Spitz, M. R., Duphorne, C. M., Detry, M. A., Pillow, P. C., Amos, C. I., Lei, L., de Andrade,
M., Gu, X., Hong, W. K., Wu, X. (2000). Dietary intake of isothiocyanates: evidence of a joint
232
effect with glutathione S-transferase polymorphisms in lung cancer risk. Cancer Epidemiology
Biomarkers and Prevention 9: 1017-1020.
116.
Staack, R., Kingston, S., Wallig, M. A., Jeffery, E. H. (1998). A comparison of the individual
and collective effects of four glucosinolate breakdown products from brussels sprouts on
induction of detoxification enzymes. Toxicology and Applied Pharmacology 149: 1723.
117.
Stephens, J. (2006). Arrugula - Eruca sativa Mill. Fact Sheet HS-543. Horticultural Sciences
119.
Sun-Ju, K., Gensho, I. (2006). Glucosinolate profiles in the seeds, leaves and roots of
rocket salad (Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and purified
4-methoxyglucobrassicin. Soil Science and Plant Nutrition 52: 394400.
120.
121.
Tlustos, P., Balik, J., Szakova, J. and Pavlikova, D. (1998). The accumulation of arsenic in
radish biomass when different forms of As were applied in the soil (Czech). Rostlinna Vyroba
44: 713.
122.
Traka, M. H., Spinks, C. A., Doleman, J. F., Melchini, A., Ball, R. Y., Mills, R. D., Mithen, R.
F. (2010). The dietary isothiocyanate sulforaphane modulates gene expression and alternative
gene splicing in a PTEN null preclinical murine model of prostate cancer. Molecular Cancer 9:
189.
123.
Trotta, V., Calboli, F. C., Ziosi, M., Guerra, D., Pezzoli, M. C., David, J. R., Cavicchi, S.
(2006). Thermal plasticity in Drosophila melanogaster: A comparison of geographic
populations. BMC Evolutionary Biology 6: 67.
124.
Van den Berg, H., Faulks, R., Fernando-Granado, H,. Hirschberg, J., Olmedilla, B.,
Sandmann, G., Southon, S., Stahl, W. (2000). The potential for the improvement of carotenoid
levels in foods and the likely systemic effects. Journal of the Science of Food and Agriculture
80: 880-912.
125.
Van Poppel, G., Verhoeven, D. T., Verhagen, H., Goldbohm, R. A. (1999). Brassica
vegetables and cancer prevention. Epidemiology and mechanisms. Advances in Experimental
Medicine and Biology 472: 159-68.
126.
Vavilov, N. I. (1935). The phytogeographical basis for plant breeding. Theoretical. Basis
Plant Breeding, 1: 17-75.
127.
233
128.
Verhoeven, D. T. H., Verhagen, H., Goldbohm, R. A., van den Brandt, P. A., van Poppel, G.
(1997). A review of mechanisms underlying anti carcinogenicity by brassica vegetables.
Chemico- Biological Interactions 103: 79129.
129.
Velasco, P., Cartea, M. E., Gonzlez, C., Vilar, M., Ords, A. (2007). Factors affecting the
glucosinolate content of kale (Brassica oleracea acephala group) Journal of Agriculture and
Food Chemistry 55: 955962.
130.
Vilar, M., Cartea, M. E., Padilla, G., Soengas, P., Velasco, P. (2008). The potential of kales
as a promising vegetable crop. Euphytica 159: 153-165.
131.
Wang, J. P., Qi, L., Moore, M. R. Ng, J. C. (2002). A review of animal models for the study
of arsenic carcinogenesis. Toxicology Letters 133: 1731.
132.
Wang, L. S., Sun, X. D., Cao, Y., Wang, L., Li, F. J., Wang, Y. F. (2010). Antioxidant and
pro-oxidant properties of acylated pelargonidin derivatives extracted from red radish
(Raphanus sativus var. niger, Brassicaceae). Food Chemical Toxicology 48: 27122718.
133.
Warwick, S. I., Gugel, R. K., Gmez-Campo, C., James, T. (2007). Genetic variation in
Eruca vesicaria (L.) Cav. Plant Genetic Resources: Characterization and Utilization 5: 142
153.
134.
Weckerle, B., Michel, K., Balzs, B., Schreier, P., Tth, G. (2001). Quercetin 3, 3, 4-tri-O-D-glucopyranosides from leaves of Eruca sativa (Mill.). Phytochemistry 57: 547551.
135.
Welch, R. M., Graham, R. D. (2004). Breeding for micronutrients in staple food crops from a
human nutrition perspective. Journal of Experimental Botany 55: 353-364.
136.
White P. J., and Broadley M. R. (2005). Biofortifying crops with essential mineral elements,
Trends Plant Science 10: 586-593.
137.
Wismer, W. V., Harker, F. R., Gunson, F. A., Rossiter, K. L., Lau, K., Seal, A. G., Lowe, R.
G., Beatson, R. (2005). Identifying flavour targets for fruit breeding: A kiwifruit example.
Euphytica 141: 93-104.
138.
Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R., Beach, D. (1993). p21 is a
universal inhibitor of cyclin kinases. Nature 366: 701704.
139.
Yamasaki, M., Omi, Y., Fujii, N., Ozaki, A., Nakama, A., Sakakibara, Y., Suiko, M.,
Nishiyama, K. (2009). Mustard oil in Shibor i aikon a var iety of Japanese radish, selectively
inhibits the proliferation of H-ras-transfor med 3Y1 cells. Bioscience, Biotechnology and
Biochemistry 73: 221721.
140.
Yang, C. S., Smith, T. J., Hong, J. Y. (1994). Cytochrome P- 450 enzymes as targets for
chemoprevention against chemical carcinogenesis and toxicity : opportunities and limitations.
Cancer Research 54: 1982s-1986s.
141.
Yang, X. E., Chen, W. R., Feng, Y. (2007). Improving human micronutrient utrition through
biofortification in the soil-plant system: China as a case study. Environmental Geochemistry
Health 29: 413-428.
142.
234
Yaniv, Z., Scha.erman, D., Amar, Z., 1998. Tradition, uses and biodiversity of rocket (Eruca
144.
145.
Zhang, W., Fu, Q., Dai, X., Bao, M. (2008). The culture of isolated microspores of
ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo
regeneration. Science Horticulture 117: 69-72.
235