universidad de granada facultad de ciencias · caracterizaciÓn y estudios metabolÓmicos de...
TRANSCRIPT
UNIVERSIDAD DE GRANADA
FACULTAD DE CIENCIAS
Departamento de Química Analítica
Centro de Investigación y Desarrollo del Alimento Funcional
CARACTERIZACIÓN Y ESTUDIOS METABOLÓMICOS DE COMPUESTOS
FENÓLICOS BIOACTIVOS MEDIANTE TÉCNICAS SEPARATIVAS
ACOPLADAS A ESPECTROMETRÍA DE MASAS
presentada por:
Rosa Mª Quirantes Piné
para optar al grado de:
Doctor Internacional por la Universidad de Granada
Tesis doctoral dirigida por:
D. Alberto Fernández Gutiérrez
D. Antonio Segura Carretero
Granada, 2012
Editor: Editorial de la Universidad de GranadaAutor: Rosa María Quirantes PinéD.L.: GR 188-2013ISBN: 978-84-9028-292-2
Esta tesis doctoral ha sido realizada gracias a una beca predoctoral de
formación de profesorado universitario concedida por el Ministerio de
Educación y Ciencia (AP2007 03246) y a la financiación con cargo a fondos del
Centro de Investigación y Desarrollo del Alimento Funcional (CIDAF)
procedentes de diferentes proyectos, contratos y subvenciones de las
Administraciones central y autonómica, plan propio de investigación de la
UGR, así como de empresas interesadas en los resultados de la investigación.
CARACTERIZACIÓN Y ESTUDIOS METABOLÓMICOS DE COMPUESTOS
FENÓLICOS BIOACTIVOS MEDIANTE TÉCNICAS SEPARATIVAS
ACOPLADAS A ESPECTROMETRÍA DE MASAS
por
Rosa Mª Quirantes Piné
Visado en Granada a 15 de Junio de 2012
Fdo: Prof. Dr. D. Alberto Fernández Gutiérrez
Catedrático del Departamento de Química Analítica
Facultad de Ciencias. Universidad de Granada
Fdo: Prof. Dr. D. Antonio Segura Carretero
Catedrático del Departamento de Química Analítica
Facultad de Ciencias. Universidad de Granada
Memoria para optar al grado de Doctor Internacional por la UGR:
Fdo: Rosa Mª Quirantes Piné
El Prof. Dr. D. ALBERTO FERNÁNDEZ GUTIÉRREZ, Catedrático de Universidad en
el Departamento de Química Analítica “Profesor Fermín Capitán” y en el
Instituto de Nutrición y Tecnología de los Alimentos “José Mataix” y Director del
Centro de Investigación y Desarrollo del Alimento Funcional (CIDAF) de la
Universidad de Granada,
CERTIFICA
Que el trabajo que se presenta en esta tesis doctoral con el título:
“CARACTERIZACIÓN Y ESTUDIOS METABOLÓMICOS DE COMPUESTOS
FENÓLICOS BIOACTIVOS MEDIANTE TÉCNICAS SEPARATIVAS ACOPLADAS A
ESPECTROMETRÍA DE MASAS”, que ha sido realizado bajo mi dirección y la del
Prof. Dr. D. Antonio Segura Carretero en los laboratorios del Centro de
Investigación y Desarrollo del Alimento Funcional (Parque Tecnológico de
Ciencias de la Salud) y también parcialmente, en las instalaciones que la
compañía Bruker Daltonik GMBH tiene en Bremen (Alemania), reúne todos los
requisitos legales, académicos y científicos para hacer que el doctorando Dña.
Rosa Mª Quirantes Piné pueda optar al grado de Doctor Internacional por la
Universidad de Granada.
Y para que así conste, expido y firmo el presente certificado en Granada a
15 de Junio de 2012:
ÍNDICE
Índice
11
ÍNDICE GENERAL
ABREVIATURAS ................................................................................................... 17
RESUMEN ........................................................................................................... 23
SUMMARY .......................................................................................................... 29
OBJETIVOS.......................................................................................................... 35
INTRODUCCIÓN.................................................................................................. 39
1. Alimentos funcionales y nutracéuticos................................................................. 41
2. Fuentes vegetales para el desarrollo de nutracéuticos ........................................ 44
2.1. Lippia citriodora ............................................................................................. 45
2.2. Hoja de olivo .................................................................................................. 46
3. Compuestos fenólicos como compuestos bioactivos de origen vegetal.............. 48
3.1. Estructura y clasificación de los compuestos fenólicos ................................. 49
3.2. Efectos saludables de los compuestos fenólicos ........................................... 51
3.2.1. Actividad antioxidante de los compuestos fenólicos........................... 53
3.2.2. Actividad antiinflamatoria de los compuestos fenólicos ..................... 55
3.2.3. Actividad anticancerígena de los compuestos fenólicos...................... 56
3.3. Absorción y metabolismo de los compuestos fenólicos................................ 58
4. El papel de la metabolómica en el estudio de los compuestos fenólicos ............ 61
4.1. Concepto de metabolómica........................................................................... 62
4.2. Estrategias en metabolómica......................................................................... 63
4.3. Aproximaciones metabolómicas al estudio de compuestos fenólicos.......... 65
5. Tratamiento de muestra para la determinación de compuestos fenólicos ......... 67
Índice
12
5.1. Muestras de origen vegetal ........................................................................... 67
5.2. Muestras de origen biológico ........................................................................ 70
6. Técnicas separativas para la determinación de compuestos fenólicos................ 72
6.1. Electroforesis capilar (CE) .............................................................................. 73
6.1.1. Fundamentos .................................................................................. 73
6.1.2. Separación de compuestos fenólicos mediante CE ............................. 76
6.2. Cromatografía líquida (LC) ............................................................................. 78
6.2.1. Fundamentos .................................................................................. 78
6.2.2. Separación de compuestos fenólicos mediante HPLC y UPLC/RRLC ..... 82
6.2.3. Separación de compuestos fenólicos mediante nanoLC...................... 83
7. Sistemas de detección para el acoplamiento con técnicas separativas ............... 85
7.1. Espectroscopía de absorción UV Vis.............................................................. 86
7.2. Espectrometría de masas (MS) ...................................................................... 88
7.2.1. Sistema de ionización: Ionización por electrospray (ESI) ..................... 91
7.2.1.1. Interfase ESI para el acoplamiento CE MS................................... 93
7.2.1.2. Interfase ESI para el acoplamiento HPLC MS ............................... 95
7.2.1.3. Interfase ESI para el acoplamiento nanoLC MS............................ 96
7.2.2. Analizadores de masas..................................................................... 97
7.2.2.1. Trampa de iones (IT) ................................................................. 98
7.2.2.2. Tiempo de vuelo (TOF) ............................................................ 100
7.2.2.3. Cuadrupolo tiempo de vuelo (QTOF) ........................................ 102
Bibliografía ............................................................................................................... 105
Índice
13
PARTE EXPERIMENTAL ..................................................................................... 127
Bloque I. Caracterización de fuentes de compuestos fenólicos bioactivos .. 129
Capítulo 1. Identificación de los compuestos fenólicos de un extracto de Lippia
citriodoramediante HPLC DAD ESI TOF/IT MS................................................... 131
Capítulo 2. Caracterización de los compuestos fenólicos y otros compuestos
polares de un extracto de Lippia citriodoramediante CE ESI MS.......................141
Capítulo 3. HPLC ESI QTOF MS como una potente herramienta analítica para
caracterizar los compuestos fenólicos de extractos de hoja de olivo.................153
Bloque II. Estudios metabolómicos de compuestos fenólicos bioactivos..... 179
Capítulo 4. Evaluación de diferentes estrategias de extracción para la
determinación de compuestos fenólicos y sus metabolitos en plasma mediante
nanoLC ESI TOF MS ............................................................................................. 181
Capítulo 5. Los fenilpropanoides y sus metabolitos son los principales
compuestos responsables de la protección de las células sanguíneas contra el
estrés oxidativo tras la administración de un extracto de Lippia citriodora .......205
Capítulo 6. Evaluación de la absorción y metabolismo de compuestos fenólicos de
hojas de olivo en células SKBR3 mediante perfilado metabólico con HPLC ESI
QTOF MS.............................................................................................................. 229
CONCLUSIONES ................................................................................................ 251
FINAL CONCLUSIONS ........................................................................................ 257
Índice
14
ÍNDICE DE TABLAS
Tabla 1.Modos de separación en electroforesis capilar .................................... 76
Tabla 2. Principales diferencias entre los distintos tipos de LC .......................... 80
Tabla 3.Modos de LC en función de la fase estacionaria empleada. ................. 81
Tabla 4. Bandas de absorción características de diversas familias de compuestos
fenólicos ............................................................................................................. 88
Tabla 5. Sistemas de ionización y analizadores de masas más utilizados en el
acoplamiento HPLC MS y CE MS. ....................................................................... 90
Índice
15
ÍNDICE DE FIGURAS
Figura 1. Tallo de Lippia citriodora con detalle de las flores y las hojas. ............ 45
Figura 2. Olivo con detalle de las ramas y las hojas............................................ 46
Figura 3. Clasificación de las familias de compuestos bioactivos más comunes 48
Figura 4. Clasificación de las principales familias de compuestos fenólicos....... 50
Figura 5. Clasificación de los flavonoides ........................................................... 51
Figura 6. Bioactividad de los compuestos fenólicos ........................................... 52
Figura 7. Esquema de la absorción de polifenoles glicosados (PF–azúcar) en los
enterocitos y sus transformaciones metabólicas................................................ 60
Figura 8. Diagrama conceptual de las ciencias “ómicas”.................................... 63
Figura 9. Diagrama de los principales sistemas de extracción............................ 69
Figura 10. Proteínas precipitadas en una muestra de plasma............................ 70
Figura 11. Cartuchos de extracción en fase sólida y placas de pocillos. ............. 72
Figura 12. Esquema de un instrumento de electroforesis capilar ..................... 74
Figura 13. Esquema simplificado de un equipo de LC junto a uno comercial..... 79
Figura 14. Equipo de nanoLC comercial.............................................................. 84
Figura 15. Esquema de las partes de un espectrómetro de masas..................... 89
Índice
16
Figura 16. Plataformas analíticas empleadas en la presente memoria .............. 91
Figura 17. Diagrama de formación del electrospray........................................... 92
Figura 18. Esquema de una interfase ESI con flujo adicional.............................. 95
Figura 19. Esquema de un espectrómetro de masas IT. ..................................... 98
Figura 20. Diagrama del modo de trabajo MS/MS en la trampa de iones........ 100
Figura 21. Esquema detallado de las partes de las que consta un espectrómetro
de masas TOF.................................................................................................... 101
Figura 22. Esquema detallado de las partes de las que consta un espectrómetro
de masas QTOF. ................................................................................................ 103
ABREVIATURAS
Abreviaturas
19
APCI: ionización química a presión atmosférica
CAT: catalasa
CBG: ß glucosidasa citosólica
CE: electroforesis capilar
CF FAB: ionización por flujo continuo y bombardeo con átomos rápidos
CGE: electroforesis capilar en gel
CIEF: isoelectroenfoque capilar
CITP: isotacoforesis capilar
COMT: catecol O metiltransferasas
CZE: electroforesis capilar en zona
DAD: detector de bateria de diodos
DNA: ácido desoxirribonucleico
EOF: flujo electroosmótico
ESI: ionización por electrospray
FT ICR: analizador de masas de transformada de Fourier resonancia ciclotrónica
FWHM: anchura de pico a mitad de su altura máxima
GC: cromatografía de gases
GPx: glutatión peroxidasa
GRed: glutatión reductasa
HPLC: cromatografía líquida de alta resolución
Abreviaturas
20
ICP: plasma acoplado inductivamente
IL 1ß: interleuquina 1ß
IL 6: interleuquina 6
IT: analizador de masas de trampa de iones
LC: cromatografía líquida
LLE: extracción líquido líquido
LPH: lactasa floricin hidrolasa
MAE: extracción asistida por microondas
MALDI: desorción/ionización asistida por una matriz
MEKC: cromatografía capilar electrocinética micelar
MS: espectrometría de masas
MSPE:microextracción en fase sólida
nanoLC: nanocromatografía líquida
PLE: extracción con fluidos presurizados
Q: analizador de masas cuadrupolo
QqQ: analizador de masas triple cuadrupolo
Q TOF: analizador de masas cuadrupolo tiempo de vuelo
RF: radiofrecuencia
RRLC: cromatografía líquida de resolución rápida
SDS: dodecilsulfato sódico
SFC: cromatografía de fluidos supercríticos
Abreviaturas
21
SFE: extracción con fluidos supercríticos
SGLT1: transportador de glucosa dependiente de sodio
SLE: extracción sólido líquido
SOD: superóxido dismutasa
SPE: extracción en fase sólida
SULT: sulfotransferasas
TLC: cromatografía en capa fina
TNF : factor de necrosis tumoral
TOF: analizador de masas de tiempo de vuelo
TS: ionización por termonebulización
UGTs: uridin 5 O difosfato glucuronosiltransferasas
UPLC: cromatografía líquida de ultra alta resolución
UV Vis: ultravioleta visible
RESUMEN
Resumen
25
En esta memoria se reúnen los resultados obtenidos durante la realización de la
tesis doctoral titulada “Caracterización y estudios metabolómicos de
compuestos fenólicos bioactivos mediante técnicas separativas acopladas a
espectrometría de masas”, donde se evalúa del potencial de Lippia citriodora y
las hojas de Olea europaea como fuentes de compuestos fenólicos bioactivos
para el desarrollo de nutracéuticos. La memoria se ha dividido en dos secciones:
introducción y parte experimental.
En la introducción se describen, en primer lugar, las principales características
de los nutracéuticos y las diferencias que existen entre éstos y los alimentos
funcionales. A continuación, se detallan las principales características de las dos
fuentes vegetales bajo estudio, Lippia citriodora y Olea europaea, las
propiedades de los compuestos fenólicos (estructura, clasificación, efectos
saludables, absorción y metabolismo) y el papel que juega la metabolómica en
el estudio de estos compuestos. Por último, se describen las diferentes etapas
del procedimiento analítico para la determinación de compuestos fenólicos que
se han empleado en la parte experimental: tratamiento de muestra (tanto para
muestras vegetales como biológicas), separación (CE, HPLC y nanoLC) y
detección (absorción UV Vis, IT MS, TOF MS y QTOF MS).
La parte experimental se divide en dos bloques que a su vez constan de tres
capítulos cada uno: el primero centrado en la caracterización de las fuentes
vegetales de compuestos fenólicos bajo estudio, es decir, Lippia citriodora y
hoja de olivo, y el segundo aborda estudios de absorción y metabolismo de los
compuestos fenólicos de dichas fuentes in vitro e in vivo mediante enfoques
metabolómicos.
Resumen
26
El bloque I se ha centrado en la caracterización exhaustiva de los extractos de
las dos fuentes de compuestos fenólicos bioactivos estudiadas, Lippia citriodora
y las hojas de Olea europaea. En el capítulo 1, se emplea HPLC DAD ESI TOF/IT
MS para estudiar la composición del extracto de Lippia citriodora. El uso de una
columna de pequeño tamaño de partícula proporciona gran resolución,
permitiendo la separación de varios isómeros. La información complementaria
proporcionada por los distintos detectores, familia del compuesto indicada por
los máximos de absorción, masas exactas y distribución isotópica proporcionada
por el analizador TOF MS y el patrón de fragmentación obtenido con el
analizador IT MS/MS, permiten la identificación tentativa de compuestos
fenólicos en Lippia citriodora de los que no se dispone de patrones comerciales.
Como continuación de este estudio, en el capítulo 2 se optimiza un método de
CE ESI IT/TOF MS para completar la caracterización del extracto de Lippia
citriodora. Los principales parámetros electroforéticos así como los de la
transferencia al analizador de masas se estudian de forma pormenorizada hasta
conseguir la máxima resolución y sensibilidad. De esta forma, el método
optimizado permite la identificación de algunos compuestos que no se habían
caracterizado mediante el método cromatográfico descrito en el capítulo 1,
demostrando la complementariedad de ambas técnicas separativas. Para
finalizar el bloque, el capítulo 3, realizado en colaboración con el Instituto de
Investigación en Ciencias de la Alimentación del Consejo Superior de
Investigaciones Científicas de Madrid, se centra en el estudio de la composición
mediante HPLC ESI QTOF MS de dos extractos de hoja de olivo obtenidos
mediante PLE utilizando etanol y agua como disolventes de extracción. La
elevada exactitud de masas y distribución isotópica tanto de los espectros de
masas como de masas/masas proporcionada por el analizador QTOF MS
permite la identificación de un gran número de compuestos fenólicos en ambos
Resumen
27
extractos, incluyendo secoiridoides, fenoles simples, flavonoides, derivados de
ácidos cinámicos y ácidos benzoicos.
En el bloque II, llevado a cabo en colaboración con el Instituto de Biología
Molecular y Celular (IBMC) de la Universidad Miguel Hernández, se estudia la
absorción y metabolismo in vitro o in vivo de los compuestos fenólicos de
ambos extractos mediante enfoques metabolómicos. En el capítulo 4 se evalúan
diversos procedimientos de tratamiento de muestra y extracción para el análisis
de compuestos fenólicos en plasma mediante nanoLC ESI TOF MS, incluyendo
precipitación de proteínas en diferentes condiciones, digestión enzimática y SPE
utilizando diferentes fases sólidas y valores de pH. El procedimiento
desarrollado se aplica al análisis de muestras de plasma de rata tras la
administración del extracto de Lippia citriodora. En el capítulo 5, se estudia el
efecto del extracto de Lippia citriodora sobre la actividad de las enzimas
antioxidantes CAT, GPx y GRed en linfocitos, eritrocitos y neutrófilos, así como
la actividad de MPO en neutrófilos (marcador del daño vascular inflamatorio) en
ratas wistar tras la ingesta aguda del extracto. El efecto del extracto sobre estas
actividades enzimáticas se correlaciona con los metabolitos identificados en
plasma mediante HPLC ESI TOF MS para intentar establecer qué compuesto o
compuestos son los responsables de esta actividad y por tanto, los que podrían
resultar de interés para el desarrollo de nutracéuticos. Por último, en el capítulo
6, se estudia la absorción de los compuestos fenólicos de hoja de olivo por parte
de la línea celular de cáncer de mama SKBR3 para intentar establecer qué
compuestos fenólicos son los responsables de la actividad citotóxica
previamente mostrada en esta línea celular. Para ello, en primer lugar, el
extracto de hoja de olivo se caracteriza cuantitativamente mediante HPLC ESI
QTOF MS, y a continuación se analiza mediante el mismo método el perfil
metabólico del citoplasma de células SKBR3 incubadas con el extracto de hoja
Resumen
28
de olivo durante diferentes tiempos (15 min, 1, 2, 24 y 48 h). El trabajo
experimental incluido en este capítulo se desarrolló durante una estancia de 4
meses en la sede de la empresa Bruker Daltonik en Bremen, Alemania.
SUMMARY
Summary
31
This work is a summary of all the results obtained for the PhD thesis:
“Caracterización y estudios metabolómicos de compuestos fenólicos
bioactivos mediante técnicas separativas acopladas a espectrometría de
masas (Characterization and metabolomic studies of bioactive phenolic
compounds by separative techniques coupled to mass spectrometry)”, where
the potential of Lippia citriodora and Olea europaea leaves as bioactive phenolic
compounds source for the development of nutraceuticals is assessed. The
current work can be divided in two sections: introduction and the experimental
ones.
The introduction includes outstanding information about the main features of
nutraceuticals and the differences between these ones and functional foods.
Then, both plant sources under study, Lippia citriodora and Olea europaea are
described, as well as the properties of phenolic compounds (structure,
classification, healthy effects, absorption and metabolism) and the role that
metabolomics play in the study on these compounds. Finally, the different
stages of an analytical procedure for the determination of phenolic compounds
used in the experimental section are described: sample treatment (for plant and
biological samples), separation (CE, HPLC and nanoLC) and detection (UV Vis
spectroscopy, IT MS, TOF MS and QTOF MS).
The experimental section is divided in two sections with three chapters each
one: the first one is focused on the characterization of the plant sources of
phenolic compounds under study, Lippia citriodora and olive leaves, and the
second one studies the in vitro or in vivo absorption and metabolism of phenolic
compounds by metabolomic approaches.
Summary
32
The section I is focused in the comprehensive characterization of the bioactive
phenolic compounds from both studied plant sources, Lippia citriodora and Olea
europaea leaves. In the chapter 1, HPLC DAD ESI TOF/IT MS is used to study the
composition of the Lippia citriodora extract. The use of a small particle size
column provides a great resolution, making possible the separation of several
isomers. The complementary information supplied by the different detectors,
class of phenolic compound delimited by UV–visible spectroscopy, accurate
mass measurements and true isotopic pattern provided by TOF MS, and
fragmentation pattern obtained by IT MS/MS, enabled the tentative
identification of phenolic compounds from Lippia citriodora when commercial
standards are not available. As a continuation of this work, in the chapter 2, a
CE ESI IT/TOF MS method is optimized to complete the characterization of the
Lippia citriodora extract. The main electrophoretic conditions as well as the ion
transfer parameters of the mass analyzer are carefully studied to achieve the
maximum resolution and sensitivity. In this way, the optimized method enables
the identification of some compounds which had not been characterized by the
chromatographic method described in chapter 1, proving the complementarity
of both separation techniques. To finish this section, chapter 3 is focused on the
study by HPLC ESI QTOF MS of the composition of two olive leaf extracts
obtained by PLE using ethanol and water as extraction solvents. The high mass
accuracy and true isotopic pattern in both MS and MS/MS spectra provided by
QTOF MS analyzer enable the identification of a wide range of phenolic
compounds in both extracts, including secoiridoids, simple phenols, flavonoids,
cinnamic acid derivatives, and benzoic acids. This work was carried out in
collaboration with the Institute of Food Science Research from the Superior
Council of Scientific Research (CIAL CSIC).
Summary
33
The setion II has been carried out in collaboration with the Institute of
Molecular and Cellular Biology (IBMC) of the Miguel Hernández University, and
it is focused on the study of in vitro or in vivo absorption and metabolism of the
phenolic compounds from both extracts by metabolomic approaches. Chapter 4
assesses different sample treatments and extraction procedures to the analysis
of phenolic compounds in plasma by nanoLC ESI TOF MS. The tested
procedures include protein precipitation at different conditions, enzymatic
digestion and SPE using several solid phases and pH values. The developed
procedure is applied to the analysis of rat plasma samples after administration
of the Lippia citriodora extract. Chapter 5 studies the effect of Lippia citriodora
extract intake on antioxidant enzymes CAT, GPx and GRed activities in
lymphocytes, erythrocytes and neutrophils, as well as MPO activity in
neutrophils (marker for inflammatory vascular damage) in wistar rats after
acute intake of the extract. The effect of the extract on these enzymatic
activities is correlated with the phenolic metabolites identified in plasma by
HPLC ESI TOF MS to establish which compound or compounds are responsible
for this activity and therefore, may be interesting in nutraceuticals
development. Finally, chapter 6 evaluates the in vitro uptake of phenolic
compounds from olive leaf by breast cancer cell line SKBR3 to determine which
phenolic compounds are responsible for the citotoxic activity showed in this cell
line. For this aim, firstly, the olive leaf extract is quantitatively characterized by
HPLC ESI QTOF MS, and then a metabolite profiling approach based on the
same HPLC ESI QTOF MS method is used to identify the intracellular phenolic
compounds at different incubation times (15 min, 1, 2, 24 and 48 h). This work
has been developed during a stay of four months in the Applications
Department of the company Bruker Daltonik in Bremen (Germany).
OBJETIVOS
Objetivos
37
En los últimos años se ha despertado un creciente interés por el estudio de los
compuestos fenólicos debido, fundamentalmente, a las numerosas evidencias
de sus efectos beneficiosos para la salud. Es por esta demostrada bioactividad
que gran parte de la investigación en el ámbito del desarrollo de nutracéuticos
se ha centrado en estos compuestos, prestando especial atención a la búsqueda
de fuentes vegetales ricas en compuestos fenólicos que presenten un
determinado efecto biológico. Por todo ello, el objetivo global de la presente
tesis doctoral es la evaluación del potencial de Lippia citriodora y las hojas de
Olea europaea como fuente de compuestos fenólicos bioactivos para el
desarrollo de nutracéuticos. Este ambicioso objetivo se puede concretar en
otros dos objetivos más específicos correspondientes a los dos bloques en los
que se ha dividido la parte experimental de esta tesis:
En el primer bloque se pretende caracterizar en profundidad los
extractos de las dos fuentes de compuestos fenólicos bioactivos bajo
estudio, Lippia citriodora y las hojas de Olea europaea. Para llevar a cabo
esta caracterización se emplearán diversas combinaciones de potentes
plataformas analíticas. En el caso del extracto de Lippia citriodora, se
estudiará su composición mediante cromatografía líquida de alta
resolución (HPLC) acoplada a diversos sistemas de detección que
proporcionan información complementaria, como es el caso de la
espectroscopía UV/visible con detector de batería de diodos (DAD) y la
espectrometría de masas con analizadores de tiempo de vuelo (TOF MS)
y trampa de iones (IT MS), así como mediante electroforesis capilar (CE)
acoplada a ambos analizadores de masas. En el caso del extracto de hoja
de olivo, se caracterizará mediante HPLC acoplada a espectrometría de
masas con analizador cuadrupolo tiempo de vuelo (HPLC ESI QTOF MS).
Objetivos
38
En el segundo bloque de la tesis se pretende estudiar la absorción y
metabolismo in vitro o in vivo de los compuestos fenólicos de ambos
extractos mediante enfoques metabolómicos. Para ello, en primer lugar,
se desarrollará un procedimiento de extracción para el análisis de
compuestos fenólicos en plasma mediante nanoLC ESI TOF MS y el
procedimiento desarrollado se aplicará al análisis de muestras de plasma
de rata tras la administración del extracto de Lippia citriodora. A
continuación, se estudiará el efecto del extracto de Lippia citriodora
sobre las enzimas antioxidantes en ratas wistar tras la ingesta aguda del
extracto y se correlacionará con los metabolitos identificados en plasma
mediante HPLC ESI TOF MS para intentar establecer qué compuestos son
los responsables de esta actividad y por tanto, los que podrían resultar de
interés para el desarrollo de nutracéuticos. Para finalizar, se estudiará la
absorción y metabolismo de los compuestos fenólicos de hoja de olivo
por parte de la línea celular de cáncer de mama SKBR3 mediante HPLC
ESI QTOF MS para intentar establecer qué compuestos fenólicos son los
responsables de la actividad citotóxica previamente mostrada en esta
línea celular.
INTRODUCCIÓN
Introducción
41
1. ALIMENTOS FUNCIONALES Y NUTRACÉUTICOS
Desde los orígenes de las primeras civilizaciones se ha sospechado la relación
existente entre los hábitos alimenticios y la salud. Ya en el siglo V a.C.,
Hipócrates afirmaba: “deja que el alimento sea tu medicina y que la medicina
sea tu alimento”, sin ser consciente de como su principio seguiría teniendo
vigencia 2.500 años después. No obstante, las conexiones concretas no han sido
fundamentadas hasta el siglo XIX. Durante el siglo XIX y mitad del siglo XX, la
observación médica y la experimentación animal y bioquímica han ido
demostrando el importante papel que juega la alimentación en diversos
procesos bioquímicos. Por un lado, la dieta suministra los nutrientes necesarios
para satisfacer los requerimientos metabólicos de un individuo. Pero más allá de
los beneficios nutricionales aceptados, la alimentación puede producir una serie
de efectos fisiológicos beneficiosos al modular funciones específicas, por lo que
puede no sólo ayudar a alcanzar una salud óptima, sino desempeñar además
una función importante reduciendo los riesgos de padecer determinadas
enfermedades. De hecho, en la última década se han acumulado numerosas
evidencias científicas que demuestran el papel de ciertos componentes de la
dieta en la prevención de enfermedades cardiovasculares, osteoporosis,
obesidad, condiciones inflamatorias y algunos tipos de cáncer. De igual modo,
se ha dedicado especial atención al papel de la dieta en la modulación del
sistema inmune, el retraso en el proceso de envejecimiento así como su
influencia en el rendimiento físico e intelectual [1].
Ciertas tendencias de la sociedad actual han favorecido el desarrollo de la
investigación centrada en el efecto fisiológico de una alimentación equilibrada,
como son:
Introducción
42
El creciente costo sanitario.
El aumento paulatino de la esperanza de vida con el consiguiente
aumento de la población mayor de 65 años.
El deseo de una mejor calidad de vida.
Un mayor conocimiento de la relación entre la dieta y la salud.
Como consecuencia de estos cambios en la Ciencia de la Nutrición ha surgido
toda una nueva generación de productos así como de conceptos muy similares
entre sí como son los alimentos funcionales, nutracéuticos, complementos
alimenticios, alimentos enriquecidos, medicinales y saludables, entre otros, de
forma que la terminología relativa a este tipo de alimentos no se ha
consensuado hasta hace poco tiempo. Los factores que diferencian unos
términos de otros son: la propia naturaleza del alimento, el efecto esperado
sobre la salud, la forma de presentación, a quién va dirigido y su procesado.
En Europa, el primer documento consensuado sobre conceptos científicos en
relación con los alimentos funcionales fue elaborado en 1999 por un grupo de
expertos coordinados por el ILSI (Internacional Life Sciences Institute) según el
cual se define un alimento funcional como “aquel que contiene un componente,
nutriente o no nutriente, con efecto selectivo sobre una o varias funciones del
organismo, con un efecto añadido por encima de su valor nutricional y cuyos
efectos positivos justifican que pueda reivindicarse su carácter funcional o
incluso saludable. Debe resaltarse que los alimentos funcionales son
considerados como alimentos, demostrando su efecto en cantidades que se
encuentren de forma normal en la dieta y se consumen como parte de unos
hábitos alimenticios comunes” [2].
Introducción
43
En 1989 Stephen DeFelice, promotor de la Fundación para la Innovación en
Medicina (FIM), acuñó el término nutracéutico y lo definió como “un alimento o
parte de un alimento que proporciona beneficios médicos o saludables
incluyendo la prevención y/o tratamiento de enfermedades. Estos productos
pueden ir desde nutrientes aislados, complementos alimenticios, alimentos
diseñados genéticamente o alimentos funcionales, hasta productos herbales y
alimentos procesados” [3]. De la definición anterior puede deducirse que
cualquier alimento o parte de un alimento puede ser un nutracéutico si
presenta beneficios para la salud. Sin embargo, la tendencia actual es
diferenciar el concepto de nutracéutico del de alimento funcional, por lo que
suele considerarse como aquel suplemento dietético que proporciona una
forma concentrada de un agente presumiblemente bioactivo proveniente de un
alimento, presentado en una matriz no alimenticia y utilizado para incrementar
la salud en dosis que exceden a aquellas que pudieran ser obtenidas del
alimento normal [4]. Según esta definición, englobaría a los complementos
alimenticios pero no a los alimentos funcionales.
Por tanto, aunque no existe consenso respecto a las diferencias entre estos dos
términos, el ámbito de los nutracéuticos puede considerarse significativamente
distinto al de los alimentos funcionales por varias razones. En primer lugar,
porque mientras que los nutracéuticos participan en la prevención y el
tratamiento de enfermedades, los alimentos funcionales son relevantes sólo en
la reducción de enfermedades. Por otro lado, mientras que los nutracéuticos se
presentan en formatos similares a los fármacos, como comprimidos o jarabes,
los alimentos funcionales se presentan en forma de alimento ordinario.
A pesar de las diferencias existentes entre ambos, el primer paso en el
desarrollo tanto de un alimento funcional como de un nutracéutico, es el
Introducción
44
mismo, identificar una interacción entre uno o varios componentes de un
alimento y una función en el organismo potencialmente beneficiosa para la
salud [5]. Para ello es necesario, por un lado, conocer en profundidad los
componentes de dicho alimento, y por otro, demostrar en modelos relevantes
(in vitro e in vivo) la interacción que se produce.
2. FUENTES VEGETALES PARA EL DESARROLLO DE NUTRACÉUTICOS
A lo largo de la historia, un gran número de plantas se han empleado en la
medicina popular para la prevención y tratamiento de diversas enfermedades
tanto en humanos como en animales. La capacidad de algunos alimentos de
origen vegetal para reducir el riesgo de padecer ciertas enfermedades se ha
asociado, al menos en parte, a los metabolitos secundarios o fitoquímicos
presentes en las plantas, los cuales han demostrado ejercer una amplia gama de
actividades biológicas. Es por ello que en los últimos años una gran parte de la
investigación en el ámbito de los nutracéuticos se ha centrado en estos
fitoquímicos, estudiando en detalle tanto la composición como la bioactividad
de diversas especies vegetales tradicionalmente empleadas como plantas
medicinales.
En la presente memoria se ha estudiado el potencial de las hojas de Lippia
citriodora y Olea europaea como fuentes de compuestos bioactivos para el
desarrollo de nutracéuticos, por lo que a continuación se describen las
principales características de estas dos especies.
Introducción
45
2.1. Lippia citriodora
El género Lippia, perteneciente a la familia Verbenaceae que a su vez forma
parte del orden de las lamiales, incluye aproximadamente 200 especies de
hierbas, arbustos y pequeños árboles. En general, las distintas especies que
forman parte de este género vegetal presentan una composición química
similar, así como actividades farmacológicas y usos tradicionales característicos
[6].
La Lipia citriodora, comúnmente
conocida como hierbaluisa, es un
arbusto caducifolio, de hasta 2 m
de altura (Figura 1). Es originaria
de Sudamérica, donde crece de
forma silvestre, y se introdujo en
Europa a finales del siglo XVII de
forma que hoy día se cultiva en la
región mediterránea. Despide una
fuerte fragancia a limón y florece
en verano, formando pequeñas
flores de color pálido o lila.
La Lippia citriodora tradicionalmente se ha empleado como especia y planta
medicinal para el tratamiento de asma, fiebre, desórdenes gastrointestinales y
dolencias cutáneas [7]. Su fracción de aceite esencial se utiliza ampliamente en
la industria cosmética y en perfumería, y sus hojas se usan como ambientadores
debido a su fuerte fragancia a limón. Asimismo, las hojas y la parte superior de
Figura 1. Tallo de Lippia citriodora con detalle de lasflores y las hojas.
Introducción
46
las flores de esta planta se usan para preparar tés y otro tipo de bebidas, y
como ingrediente en algunos postres.
Las hojas y tallos de la Lippia citriodora son ricos en un aceite esencial cuyo
componente principal es el citral (neral y geranial), responsable de su aroma, y
que contiene además limoneno, cineol, geraniol, cariofileno, espatulenol y
citronelol [8]. En menor concentración, también puede encontrarse en este
aceite esencial y pineno, terpineno, linalol, camfor, terpineol, geraniol,
geranilacetato, farneseno y curcumeno [9]. Las hojas contienen además un
elevado número de compuestos polares tales como derivados de ácidos
hidroxicinámicos, flavonoides e iridoides glicosados, siendo el verbascósido o
acteósido el más abundante [8, 10, 11]. Es a estos compuestos polares, en su
mayoría fenólicos, y en especial al verbascósido, a los que se han asociado las
propiedades medicinales de esta planta.
2.2. Hoja de olivo
El olivo (Olea europaea) es un árbol perennifolio de tamaño medio
perteneciente a la familia Oleaceae que a su vez forma parte del orden de las
lamiales (Figura 2). Sus hojas, fruto y aceite tienen una extensa historia de usos
nutricionales, medicinales
y ceremoniales. Es uno de
los cultivos más
importantes en la Cuenca
Mediterránea, que
produce el 98% del total
(aproximadamente 11 Figura 2. Olivo con detalle de las ramas y las hojas.
Introducción
47
millones de toneladas), siendo uno de los pilares fundamentales de la
agricultura de esta región. Los principales países productores son España, Italia,
Francia, Grecia, Marruecos, Túnez y Turquía.
El olivo se cultiva fundamentalmente para la producción de aceite, una actividad
agroindustrial de vital importancia económica en los países de la Cuenca del
Mediterráneo. Durante la producción del aceite de oliva se producen grandes
cantidades de subproductos. Se ha estimado, que tan sólo en la poda, se
producen 25 kg de ramas y hojas por año y árbol, y que las hojas suponen un 5%
del peso de las aceitunas recolectadas para la producción del aceite de oliva.
Químicamente, las hojas de olivo se caracterizan por la presencia de
secoiridoides como la oleuropeína y el ligustrósido; flavonas tanto glucosadas
como la luteolina 7 O glucósido, la apigenina 7 O glucósido y la diosmetina 7 O
glucósido, como agliconas tales como la luteolina y la diosmetina; derivados de
ácidos hidroxicinámicos como el verbascósido; flavonoles como la rutina; flavan
3 oles como la catequina, y fenoles sustituidos como el tirosol, el hidroxitirosol,
la vanillina, el ácido vanílico y el ácido cafeico. El compuesto más abundante es
la oleuropeína, seguida del hidroxitirosol, la luteolina 7 O glucósido, la
apigenina 7 O glucósido y el verbascósido [12 15]. Históricamente, la hoja de
olivo se ha usado como remedio en la medicina tradicional para combatir los
síntomas febriles de la malaria. Estas propiedades medicinales se han atribuido
a los compuestos fenólicos presentes en las hojas de esta planta,
principalmente a la oleuropeína y al hidroxitirosol.
Introducción
48
3. COMPUESTOS FENÓLICOS COMO COMPUESTOS BIOACTIVOS DE
ORIGEN VEGETAL
Los compuestos bioactivos son aquellos que inducen efectos metabólicos
derivados de su actividad biológica que se asocian a efectos beneficiosos sobre
la salud humana, como una mejora de ciertas funciones o una reducción del
riesgo de alguna enfermedad. Existe una gran variedad de compuestos
bioactivos con distintas estructuras químicas y actividades biológicas, algunos
de los cuales se recogen en la Figura 3.
Los compuestos fenólicos son una de las familias de compuestos bioactivos más
estudiadas por su amplia distribución en la naturaleza y por su elevada
diversidad tanto en lo referente a estructura química como en cuanto a
actividad biológica.
Figura 3. Clasificación de las familias de compuestos bioactivos más comunes
Introducción
49
3.1. Estructura y clasificación de los compuestos fenólicos
Los compuestos fenólicos constituyen uno de los grupos de sustancias más
numeroso y ampliamente distribuido en el reino vegetal, con más de 8.000
estructuras conocidas actualmente. Presentan una gran diversidad tanto en su
estructura como en sus funciones, pero generalmente constan de un anillo
aromático con uno o más sustituyentes hidroxilo. Surgen biogenéticamente de
dos principales rutas sintéticas: la ruta del ácido shikímico y la ruta del acetato.
En la Figura 4 se indican las principales familias de compuestos fenólicos junto
con su estructura básica.
Como puede verse en esta figura, pueden encontrarse desde moléculas sencillas
como los ácidos fenólicos, a compuestos altamente polimerizados como los
taninos. Normalmente se encuentran en forma conjugada, con uno o más
residuos de azúcar unidos a los grupos hidroxilo, aunque también pueden darse
enlaces directos de la unidad de azúcar a un carbón aromático. Los azúcares
asociados pueden presentarse como monosacáridos, disacáridos e incluso
oligosacáridos. Aunque la glucosa es el residuo de azúcar al que se unen más
comúnmente, también pueden conjugarse con galactosa, ramnosa, xilosa y
arabinosa, así como con ácidos glucurónico y galacturónico, entre otros.
También son comunes las conjugaciones con otros compuestos como ácidos
carboxílicos y orgánicos, aminas, lípidos e incluso con otros compuestos
fenólicos [16].
Introducción
50
Figura 4. Clasificación de las principales familias de compuestos fenólicos
Introducción
51
Los flavonoides constituyen la familia más importante y ampliamente
distribuida en la naturaleza y a su vez se puede subdividir en trece clases que se
recogen en la Figura 5.
Figura 5. Clasificación de los flavonoides
3.2. Efectos saludables de los compuestos fenólicos
El creciente interés que despiertan los compuestos fenólicos se debe,
fundamentalmente, a las numerosas evidencias de sus efectos beneficiosos
para la salud. Este interés aumentó de forma exponencial tras la publicación de
diversos estudios epidemiológicos que indicaron una relación inversa entre la
ingesta de alimentos ricos en compuestos fenólicos y la incidencia de
Introducción
52
enfermedades cardiovasculares [17 21], neurodegenerativas [22 27], diabetes
[28] y cáncer [29, 30].
Estas evidencias epidemiológicas han fomentado el estudio del papel protector
de los compuestos fenólicos sobre estas enfermedades, por lo que se han
llevado a cabo numerosos estudios tanto in vitro, como en animales e incluso en
humanos para establecer los diferentes mecanismos de acción que expliquen
dichos efectos. En la Figura 6 se muestran los principales efectos saludables
descritos para los compuestos fenólicos.
Figura 6. Bioactividad de los compuestos fenólicos (Adaptado de [31])
Los compuestos fenólicos de las dos fuentes vegetales que se han estudiado en
la presente memoria, Lippia citriodora y Olea europea han mostrado una
potente actividad antioxidante y antiinflamatoria en el caso de la hierbaluisa, y
Introducción
53
anticancerígena en el caso de las hojas de olivo. Es por ello, que a continuación
se detallan las actividades antioxidantes, antiinflamatorias y anticancerígenas
que se han descrito para compuestos fenólicos.
3.2.1. Actividad antioxidante de los compuestos fenólicos
El daño oxidativo a los componentes de la célula, al DNA, a las proteínas y a los
lípidos, se acumula con la edad y contribuye a la degeneración de las células
somáticas y a la patogénesis de diversas enfermedades. Los compuestos
fenólicos presentes en los alimentos pueden ayudar a limitar este daño
actuando directamente en las especies de oxígeno reactivas o estimulando los
sistemas de defensa endógenos. [32].
La capacidad antioxidante de los compuestos fenólicos se ha evaluado in vitro
midiendo su habilidad para captar radicales libres y reducir otros productos
químicos. Esta capacidad se compara con la de una sustancia de referencia,
generalmente Trolox (un derivado soluble en agua de la vitamina E), ácido gálico
o catequina. En todos los casos, la reacción estudiada es la reducción de un
oxidante por acción de los compuestos fenólicos
La ingesta de compuestos fenólicos produce un aumento de la capacidad
antioxidante del plasma, tal como demuestran numerosos estudios, donde se
este aumento de la capacidad antioxidante se observó de forma sistemática
durante las horas posteriores a la ingesta de bebidas y alimentos ricos en
compuestos fenólicos como té, cerveza, vino, fresas y espinacas [33 35]. Este
aumento en la capacidad antioxidante del plasma puede explicarse tanto por la
presencia compuestos fenólicos y sus metabolitos, que actuarán como
Introducción
54
reductores, como por su efecto sobre la absorción de componentes de los
alimentos pro oxidantes, tales como hierro.
Se han llevado a cabo numerosos estudios para determinar la eficacia de estos
compuestos para mejorar la protección de los componentes celulares contra el
estrés oxidativo. En algunos estudios, se observó una reducción de la
concentración plasmática de los productos de oxidación de lípidos en humanos
tras la ingesta de alimentos ricos en compuestos fenólicos como té, chocolate y
zumos de grosella negra y manzana [36 38]. Respecto a la protección del ADN,
los estudios in vitro han puesto de manifiesto que los compuestos fenólicos
pueden tener efectos tanto perjudiciales como protectores. En presencia de
metales como Cu (II) y Fe (III), pueden inducir la rotura del DNA ya que reducen
estos metales que en su forma reducida catalizan la reacción de Fenton que da
lugar a la formación del radical hidroxilo (OH ) [39]. Esta rotura del DNA puede
considerarse beneficiosa (por proporcionar efectos citotóxicos y apoptóticos en
células tumorales) o tóxica (por causar efectos mutagénicos en células
normales).
Sin embargo, los compuestos fenólicos también pueden proteger al DNA de la
degradación inducida por agentes citotóxicos. Estudios in vitro han demostrado
que pueden inhibir la formación de aductos entre hidrocarburos policíclicos
activados y el DNA [40]. Diversos estudios también han demostrado el efecto
protector del consumo de polifenoles contra el daño en el DNA tanto en
animales [41, 42] como en humanos [43].
Por otro lado, también se ha demostrado que los compuestos fenólicos pueden
estimular los sistemas de defensa endógenos mediante la activación de enzimas
antioxidantes como superóxido dismutasa (SOD), catalasa (CAT), glutatión
Introducción
55
peroxidasa (GPx) y glutatión reductasa (GRed). Estudios in vitro e in vivo han
demostrado que diversos flavonoides como la rutina, la quercitrina, la
miricetina y el kaempferol, así como los polifenoles del vino, las antocianinas,
los ácidos fenólicos, el hidroxitirosol y el verbascósido pueden aumentar las
actividades de estas enzimas [44 50].
3.2.2. Actividad antiinflamatoria de los compuestos fenólicos
La inflamación es una respuesta del organismo ante la exposición a agentes
infecciosos, estímulos antigénicos o lesiones físicas, que involucra a los sistemas
nervioso, vascular e inmunológico. Inicialmente tiene una función homeostática
de protección o defensa, no obstante si el proceso es ineficiente y se hace
crónico, se transforma en un proceso patológico.
Diversos estudios han demostrado que algunos compuestos fenólicos presentan
importantes propiedades antiinflamatorias. Algunos flavonoides han mostrado
un efecto inhibidor de la producción de citoquinas inflamatorias (TNF , IL 1 ,
IL 6), que participan activamente en el desarrollo de enfermedades relacionadas
con la inflamación crónica, así como de enzimas generadoras de eicosanoides,
inhibición que reduce en consecuencia la concentración de prostanoides y
leucotrienos, que también participan en los procesos de inflamación crónica
[51].
Varios estudios tanto in vitro como in vivo han mostrado la actividad
antiinflamatoria del verbascósido. Los estudios in vitro han mostrado un efecto
similar al de los flavonoides, inhibiendo citoquinas y leucotrienos [52, 53].
También se ha mostrado esta actividad antiinflamatoria in vivo usando el
modelo de edema de pata inducido por carragenina [54, 55], basado en la
Introducción
56
medida de la disminución de la inflamación al administrar el extracto rico en
verbascósido después de la inducción de un edema de pata.
3.2.3. Actividad anticancerígena de los compuestos fenólicos
El cáncer es un conjunto de enfermedades asociadas a una perturbación en el
control del crecimiento y metabolismo celular. De hecho, el desequilibrio en el
control de la proliferación celular es una característica primaria de las células
cancerosas y, por tanto, cualquier molécula capaz de inhibir la proliferación de
las células cancerosas puede ser útil como potencial agente quimio preventivo.
Hay muchos tipos diferentes de cáncer, aunque el de mama, de pulmón,
colorrectal y de próstata representan más de la mitad de los casos nuevos.
En los últimos 20 años, diversos estudios epidemiológicos han indicado una
correlación inversa entre el consumo regular de frutas y vegetales, y el
desarrollo de varios tipos de cáncer [29, 30]. Más recientemente, los datos de
los estudios de cohortes de gran tamaño han confirmado de alguna forma estas
asociaciones epidemiológicas [56, 57]. También se ha evaluado la relación
existente entre el consumo de bebidas ricas en compuestos fenólicos como
café, té y vino, y el riesgo de padecer cáncer. El consumo de café se ha asociado
con un menor riesgo de cáncer de colon, pero no con otros tipos de cáncer [58].
Diversas evidencias experimentales sugieren un papel protector del té contra el
cáncer aunque los estudios epidemiológicos no han sido concluyentes. Si bien se
ha observado una relación inversa entre el riesgo de padecer cáncer de colon o
de estómago y el consumo de té [59, 60], otros estudios concluyen que no tiene
un efecto claro sobre el cáncer [61, 62].
Introducción
57
Sin embargo, a nivel celular se han encontrado grandes evidencias de que los
compuestos fenólicos presentes en el té, el vino tinto, el cacao, los zumos de
frutas y el aceite de oliva tienen influencia en la carcinogénesis y desarrollo de
tumores [63]. Estos compuestos pueden interaccionar con intermediarios
reactivos [64] o carcinógenos y mutágenos activados [65], pueden modular la
actividad de proteínas clave que intervienen en la progresión del control del
ciclo celular [66] e influir en la expresión de muchos genes asociados al cáncer
[67].
Los efectos anticancerígenos de los compuestos fenólicos también están bien
documentados en animales. La administración de compuestos fenólicos a ratas
o ratones antes y/o después del tratamiento con un agente cancerígeno o de la
implantación de una línea celular humana de cáncer, mostraron en su mayoría
un efecto protector, induciendo una reducción del número de tumores o de su
crecimiento. Estos efectos se han observado en varios tipos de cáncer,
incluyendo de boca, estómago, duodeno, colon, hígado, pulmón, mama, o piel
[68]. Se han probado diversos compuestos fenólicos como quercetina,
catequinas, isoflavonas, lignanos, flavanonas, ácido elágico, polifenoles del vino
tinto, resveratrol o curcumina y todos ellos han mostrado efectos protectores
en algún modelo [32].
Especialmente notables son las propiedades anticancerígenas de los flavonoides
del té verde que se han demostrado en modelos animales [69], en líneas
celulares humanas [70], así como en estudios de intervención en humanos [71].
En estos estudios se ha puesto de manifiesto que el consumo de té verde
reduce significativamente el riesgo de cáncer del tracto biliar [72], de vejiga
[73], de mama [74] y de colon [75]. Muchas de las propiedades anticancerígenas
asociadas con el té verde se atribuyen al flavanol epigalocatequina galato, que
Introducción
58
induce apoptosis e inhibe el crecimiento de células cancerosas mediante la
alteración de la expresión de proteínas reguladoras del ciclo celular y la
actividad de las proteínas de señalización implicadas en la proliferación celular,
transformación y metástasis [76].
También resulta prometedora la actividad anticancerígena mostrada in vitro por
los compuestos fenólicos del aceite de oliva, principalmente alcoholes fenólicos,
lignanos y secoiridoides, en diversos tipos de cáncer, entre ellos el de mama
[77] y colon [78]. Las hojas de olivo también han mostrado una actividad
antitumoral similar a la del aceite de oliva, inhibiendo la proliferación celular en
líneas celulares de cáncer de mama como MCF 7, SKBR3 y JIMT 1 [14, 79 81].
3.3. Absorción y metabolismo de los compuestos fenólicos
Los estudios comentados en el apartado anterior demuestran el papel de los
compuestos fenólicos en la prevención de diversas enfermedades. Sin embargo,
para poder establecer una relación directa entre el consumo de compuestos
fenólicos y un determinado efecto saludable, es necesario que estos
compuestos sean absorbidos y alcancen los tejidos diana donde actuarán. Es por
ello que la absorción, metabolismo y biodisponibilidad de los compuestos
fenólicos es un campo de estudio de gran interés.
El metabolismo y absorción de los compuestos fenólicos se produce a través de
un mecanismo común. Las agliconas pueden absorberse en el intestino delgado,
sin embargo, la mayoría de los compuestos fenólicos se encuentran en sus
fuentes de origen en forma de ésteres, glicósidos, o polímeros que no pueden
ser absorbidos en su forma nativa. Es por ello que la absorción en el intestino
Introducción
59
delgado normalmente se asocia a la hidrólisis previa que da lugar a la
correspondiente aglicona mediante la acción de la lactasa floricin hidrolasa
(LPH) en el borde en cepillo de las células epiteliares del intestino delgado. La
LPH muestra una elevada especificidad por los flavonoides O D glucósidos
como sustratos y la aglicona resultante puede entrar en las células epiteliares
mediante difusión pasiva como resultado del incremento en su lipofilicidad. Una
forma de hidrólisis alternativa es aquella producida por la glucosidasa
citosólica (CBG) en el interior de los enterocitos. Para que esta hidrólisis
catalizada por la CBG tenga lugar, los compuestos glucosados deben ser
transportados al interior de las células epiteliares, posiblemente con
participación del transportador de glucosa dependiente de sodio (SGLT1). Por
tanto, se ha aceptado que hay dos posibles rutas por las cuales los compuestos
fenólicos conjugados con glucosa se hidrolizan para dar lugar a las
correspondientes agliconas en las células epiteliares, a las que se denomina
“LPH/difusión” y “transporte/CBG”, respectivamente [82].
Antes de pasar al torrente sanguíneo, las agliconas sufren una serie de
transformaciones previas conocidas como metabolismo de fase I y de fase II.
Las reacciones que se producen en la fase I del metabolismo suelen ser
oxidaciones y reducciones llevadas a cabo por el sistema enzimático del
citocromo P450. El metabolismo de fase II consiste en reacciones de
conjugación que dan lugar a metabolitos sulfatados, glucuronidados y/o
metilados por acción de las sulfotransferasas (SULT), las uridin 5 O difosfato
glucuronosiltransferasas (UGTs) y las catecol O metiltransferasas (COMT),
respectivamente (Figura 7).
Introducción
60
Este es un proceso de desintoxicación metabólica común a muchos xenobióticos
que restringe sus efectos tóxicos potenciales y facilita su eliminación biliar y
urinaria mediante el aumento de su hidrofilicidad. Los mecanismos de
conjugación son altamente eficientes, y las agliconas están por lo general
ausentes en la sangre o presentes en bajas concentraciones después del
consumo de dosis nutricionales, si bien pueden encontrarse en concentración
elevada tras el consumo de dosis farmacológicas, debido a una posible
saturación de los mecanismos de conjugación [83]. Los metabolitos formados en
los enterocitos pueden pasar de vuelta al lumen del intestino delgado por
acción de los transportadores ABC (cassette de unión a ATP), pero lo más común
es que pasen al torrente sanguíneo, mediante el cual llegan al hígado
rápidamente, donde pueden sufrir posteriores tranformaciones metabólicas
tanto de fase I como de fase II.
Figura 7. Esquema de la absorción de polifenoles glicosados (PF–azúcar) en los enterocitos ysus transformaciones metabólicas.
Introducción
61
Sólo las agliconas y algunos glucósidos pueden ser absorbidos en el intestino
delgado. Por tanto, cantidades sustanciales de los compuestos fenólicos
ingeridos pasan del intestino delgado al grueso donde la microbiota del colon
hidroliza los conjugados dando lugar a las agliconas. Cuando la microflora está
implicada, la eficiencia de absorción se reduce puesto que también degrada las
agliconas que libera y produce diversos ácidos aromáticos simples en el proceso.
Las moléculas generadas por la acción de la microbiota pueden ser absorbidas
aunque con menos facilidad debido a que la zona de intercambio en el colon es
más pequeña y hay una menor densidad de sistemas de transporte. Un vez
absorbidas, posteriormente sufren transformaciones metabólicas en el hígado
antes de ser excretadas en la orina.
4. EL PAPEL DE LA METABOLÓMICA EN EL ESTUDIO DE LOS
COMPUESTOS FENÓLICOS
La metabolómica, entendida como el conjunto de ciencias integradas dirigidas al
estudio de los metabolitos de un sistema biológico, se ha convertido en una
herramienta importante en muchas áreas de investigación, entre ellas el análisis
de plantas, el desarrollo de fármacos, o la nutrición, entre otras. En el ámbito de
los compuestos fenólicos, se ha empleado para el análisis exhaustivo de las
fuentes vegetales de las que provienen, así como para caracterizar los efectos
que producen tanto en cultivos celulares como en animales de experimentación
y en humanos.
Introducción
62
4.1. Concepto de metabolómica
El término metaboloma fue usado por primera vez por Oliver y colaboradores
en 1998 para describir el conjunto de metabolitos sintetizados por un
organismo de forma análoga al genoma y proteoma [84]. El metaboloma refleja
el estado metabólico de un sistema vivo y se ve influido tanto por factores
internos como externos, ya que los metabolitos son contextuales y reflejan el
entorno de éstos. El metaboloma puede, de esta forma, imaginarse como una
lente a través de la cual se muestra información referente al estado fisiológico y
patológico así como al desarrollo de un sistema biológico.
En 2001 Fiehn definió el término metabolómica como el análisis exhaustivo en
el que todos los metabolitos de un organismo son identificados y cuantificados
[85]. Los estudios inmediatamente posteriores pusieron de manifiesto que la
metabolómica, en el sentido estricto propuesto por Fiehn, es una tarea
imposible debido a la complejidad del metaboloma, tanto en lo referente a la
diversidad química como al amplio rango dinámico. Es por ello, que otros
investigadores como Villas Bôas y colaboradores consideran la metabolómica
como una nueva área de la ciencia en lugar de una aproximación analítica [86].
Según esta corriente podría definirse la metabolómica como el conjunto de
ciencias integradas que van dirigidas a conocer (identificar y cuantificar) el
conjunto de metabolitos (como intermediarios o productos finales de las rutas
metabólicas) tanto intracelulares como extracelulares de un sistema biológico,
siendo todos ellos parte del metaboloma completo. Por tanto, la Química
Analítica y en concreto, la Bioanalítica, definida como la aplicación de las
técnicas instrumentales utilizadas en la Química Analítica para el estudio de las
sustancias presentes en los organismos vivos y de las reacciones químicas en las
Introducción
63
que se basan los procesos vitales, puede
considerarse como una de las ciencias que
integran la metabolómica [87].
La metabolómica puede considerarse la
etapa final en el estudio de las ciencias
“ómicas”, tal como se muestra en la Figura
8, donde se encuentra en una primera
aproximación la genómica (estudio del
funcionamiento, contenido y evolución del
genoma), seguida de la transcriptómica
(estudio de la expresión de los genes) y de
la proteómica (estudio de la estructura y
función de las proteínas).
4.2. Estrategias en metabolómica
Existen diferentes estrategias analíticas para afrontar un estudio metabolómico,
cada una diseñada para contestar cuestiones específicas. Fiehn [88] clasificó las
distintas estrategias analíticas de la siguiente forma:
Análisis diana o dirigido (target analysis), centrado exclusivamente en un
metabolito concreto que resulte de interés.
Perfil metabólico (metabolite profiling), en el que el procedimiento
analítico se restringe a la identificación y cuantificación de un número
predefinido de metabolitos que pueden pertenecer a una clase de
compuestos específica, como compuestos fenólicos, o estar limitados a
miembros de una ruta concreta. Esta estrategia se emplea con frecuencia
Figura 8. Diagrama conceptual de lasciencias “ómicas”.
Introducción
64
en el contexto de la investigación de fármacos para estudiar la
degradación catabólica de dicho fármaco.
Metabolómica propiamente dicha (metabolomics), en la que se realiza
un análisis exhaustivo en el que todos los metabolitos de un sistema
biológico son identificados y cuantificados, revelando de esta forma el
metaboloma del sistema biológico bajo estudio.
Huella dactilar metabólica (metabolic fingerprinting), que se emplea
para clasificar muestras de forma rápida según su origen o relevancia
biológica, sin necesidad de determinar el nivel individual de cada
metabolito.
Otra clasificación de las estrategias en metabolómica propuesta por Villas Bôas
y colaboradores [89] considera perfil metabólico (metabolite profiling) como
una aproximación enfocada a identificar de forma cualitativa un número
elevado de metabolitos en un sistema biológico (superior a 100), incluyendo la
huella dactilar metabólica (metabolic fingerprinting) como el análisis de los
metabolitos intracelulares (endometaboloma) y la huella metabólica (metabolic
footprinting) como análisis de los metabolitos extracelulares
(extrametaboloma), y el análisis dirigido (target analysis) dedicado a la
identificación y cuantificación de metabolitos determinados o un conjunto de
ellos.
En cualquiera de los casos, la metabolómica pretende proporcionar información
de los metabolitos de cualquier sistema biológico, su identificación y
cuantificación sin ambigüedades utilizando para ello técnicas analíticas
robustas, reproducibles, resolutivas y sensibles aplicando los factores y
parámetros necesarios para incorporar de forma sencilla los procedimientos y
Introducción
65
resultados en redes bioquímicas modelo para su aplicación en la identificación
de biomarcadores, prevención, diagnóstico o seguimiento de enfermedades,
prever efectos tóxicos de nuevos fármacos y tener un mejor conocimiento de
las rutas metabólicas que tienen lugar en un sistema biológico.
4.3. Aproximaciones metabolómicas al estudio de compuestos
fenólicos
Desde sus comienzos, la metabolómica se ha empleado como herramienta para
intentar esclarecer las complejas relaciones entre la ingesta de determinados
fitoquímicos y sus efectos sobre la salud [90]. De esta forma, se han descrito
numerosas aplicaciones de estrategias metabolómicas al estudio de compuestos
fenólicos.
Por un lado, se han desarrollado perfiles metabólicos de alimentos de origen
vegetal para esclarecer la ingesta diaria de compuestos fenólicos. Éste es el caso
de estudios donde se han determinado un elevado número de metabolitos
secundarios en diferentes variedades de tomate [91].
Por otro lado, diversos estudios metabolómicos han permitido establecer
biomarcadores de la ingesta de determinados compuestos y alimentos. Se ha
utilizado una estrategia metabolómica mediante el uso de HPLC MS para
caracterizar el metaboloma urinario de ratas a las que se les había administrado
diferentes compuestos fenólicos, ácido ferúlico, ácido sinápico o ligninos [92].
Esta estrategia permitió obtener huellas dactilares características para cada
dieta, identificando un gran número de metabolitos fenólicos que
proporcionaron nueva información del metabolismo de estos compuestos.
También se han descrito aplicaciones de análisis de perfil metabólico de
Introducción
66
compuestos fenólicos en orina y plasma tras la ingesta de cacao ha permitido
identificar diversos ácidos fenilacéticos como biomarcadores de la ingesta de
alimentos ricos en flavanoles [93]. Otros estudios metabolómicos basados en
GC MS también permitieron identificar diversos ácidos fenólicos en orina y
heces humanas, formados por la degradación microbiana de flavonoides tras de
la ingesta de té, vino o zumo de uva [94, 95].
La metabolómica además permite caracterizar los cambios metabólicos globales
que resultan de una intervención nutricional, sin necesidad de restringir el
estudio a una vía metabólica seleccionada a priori. Esta estrategia abierta puede
conducir a la generación de nuevas hipótesis sobre los mecanismos de acción de
los compuestos fenólicos y al descubrimiento de nuevos biomarcadores de los
efectos inducidos por estos compuestos. El análisis de los perfiles urinarios de
voluntarios sanos tras el consumo de té ha puesto de manifiesto un aumento
significativo en los niveles de varios compuestos intermediarios del ciclo del
ácido cítrico, como citrato, piruvato y oxalacetato, sugiriendo un efecto de los
flavanoles del té en el metabolismo energético oxidativo [94]. El consumo de
infusiones de manzanilla ricas en compuestos fenólicos también demostró
disminuir la excreción urinaria de creatinina y aumentar la de hipurato y glicina,
lo que indica un posible efecto de la manzanilla en el metabolismo de la
microflora intestinal [96]. Éstos son sólo algunos ejemplos del uso de la
metabolómica para el estudio de los efectos y mecanismos de acción de los
compuestos fenólicos.
Introducción
67
5. TRATAMIENTO DE MUESTRA PARA LA DETERMINACIÓN DE
COMPUESTOS FENÓLICOS
La etapa de tratamiento de muestra puede considerarse una de las más
determinantes del proceso analítico y de ella va a depender en gran medida la
calidad y fiabilidad de los resultados obtenidos. Se puede llevar a cabo con
distintas finalidades: con el objetivo de extraer los analitos, en este caso los
compuestos fenólicos, de la matriz en la que se encuentren, ya sea vegetal
(hojas, tallos, etc.) o biológica (plasma, orina, citoplasma, etc.) o con el objetivo
de convertirlo en un extracto compatible con la técnica analítica que se vaya a
emplear eliminando a su vez los componentes de la matriz que pudieran
interferir en el análisis. Por otro lado, en el caso de compuestos que se
encuentren en baja concentración, como suele ocurrir en estudios
metabolómicos, el proceso de extracción puede incluir un paso de
preconcentración para alcanzar los límites de detección de la técnica analítica
aplicada.
A continuación se resumen los principales procedimientos de tratamiento de
muestra utilizados para el análisis de compuestos fenólicos y sus metabolitos,
distinguiendo entre matrices de origen vegetal y matrices de origen biológico.
5.1. Muestras de origen vegetal
El tratamiento de muestras vegetales para el análisis de compuestos fenólicos
es una tarea compleja debido a múltiples factores como la gran diversidad
estructural de estos compuestos, que les confiere distintas propiedades
químicas y la complejidad de la matriz donde se encuentren. Además, la
distribución de los compuestos fenólicos en las plantas a nivel tisular, celular y
Introducción
68
subcelular no es uniforme. Por un lado, las capas externas de las plantas
contienen niveles más altos de compuestos fenólicos que las internas. Por otro
lado, en el interior de la célula, estos compuestos se localizan principalmente en
las vacuolas aunque los polifenoles insolubles se acumulan en las paredes
celulares [97]. Esta distribución desigual complica aún más el proceso de
extracción, que requiere que se rompan las células vegetales para poder
acceder a los compuestos fenólicos.
Por lo general, las muestras vegetales se homogenizan en primer lugar y
posteriormente se secan, se liofilizan o se congelan con nitrógeno líquido. El
siguiente paso es la extracción de los compuestos fenólicos, para lo cual se
pueden emplear diversos sistemas de extracción, como extracción sólido líquido
(SLE), extracción y microextracción en fase sólida (SPE), extracción con fluidos
supercríticos (SFE), con fluidos presurizados (PLE), asistida por microondas
(MAE) y métodos de filtración, que se recogen la Figura 9.
La estrategia más empleada consiste en una extracción con disolventes (SLE),
que en algunos casos puede ir seguida de una SPE, generalmente con fases
sólidas C 18 u otros materiales de fase reversa como C 8. La elección del
disolvente para la extracción sólido líquido no es una tarea sencilla puesto que
la solubilidad de los compuestos fenólicos viene determinada por su estructura
y además pueden establecerse interacciones entre estos compuestos y otros
componentes de las plantas como carbohidratos y proteínas. La solubilidad
también se ve afectada por la polaridad del disolvente utilizado, así que no
resulta fácil desarrollar un procedimiento de extracción adecuado para
recuperar todos los compuestos fenólicos. Los disolventes más empleados son
metanol, etanol, propanol, dimetilformamida, acetona, acetato de etilo, y sus
combinaciones, a menudo con diferentes proporciones de agua. La
Introducción
69
recuperación de los compuestos fenólicos en estas extracciones sólido líquido
depende de diversos factores como tiempo de extracción, el número de etapas
de extracción, la relación de volumen de disolvente por cantidad de muestra,
tamaño de partícula de la muestra, temperatura, etc. [98]
Figura 9. Diagrama de los principales sistemas de extracción
Intro
70
5.2.
Ante
orige
citop
podr
líqui
colu
el ca
suer
de p
com
esta
La p
med
o ac
desn
med
proc
estu
al an
meta
dest
meta
may
meta
de
desp
oducción
Muestr
es de lleva
en biológic
plasma, es
rían interfe
da como t
mna. Este
aso de las
ro o plasma
proteínas,
puestos fe
blecen ent
precipitació
diante el us
etonitrilo,
naturalizan
diante dist
cedimiento
dios en los
nálisis me
anol es la
acar que
abolómico
or número
abólicos de
estos ana
proteinizac
ras de orig
r a cabo e
co con con
necesaria
erir en el a
técnica se
paso prev
s muestras
a que cont
y resulta
enólicos de
tre estos c
ón de las
so de disolv
disminuye
ndo las pro
tintas com
os (Figura
s que se co
diante LC
más efec
estos e
, es decir,
o de metab
e compues
alitos que
ción, que s
gen biológ
el análisis
ntenido pr
una elimin
análisis, e
parativa, q
vio de desp
s derivada
ienen una
determina
ebido a las
ompuesto
proteínas
ventes org
endo el pH
oteínas por
mbinacione
10). Se
omparan d
MS, en lo
tiva y rep
estudios s
, sin busc
bolitos pos
stos fenóli
se obtie
se pueden
gico
de compu
roteico, co
nación efic
incluso en
quedar ret
proteinizac
as de la sa
mayor con
ante en el
s interaccio
s y las pro
s puede c
gánicos com
H con distin
r calentam
es de los
han real
iversos mé
os que se
roducible
se llevaro
ar analitos
sible. Sin em
cos, es ne
enen con
n ver afect
estos fenó
omo es el
ciente de la
n el caso d
tenidas de
ción es esp
angre com
ncentració
análisis d
ones que s
oteínas [99
conseguirs
mo metano
ntos ácido
miento o bi
s anterior
izado var
étodos de d
concluye
[100, 101
on a cab
s concreto
mbargo, en
cesario est
los distin
tadas por
ólicos en u
caso de p
as proteína
e emplear
forma irr
pecialment
mo
n
e
se
].
se
ol
s,
ien
res
ios
desprotein
que la pre
]. Sin emb
bo desde
os, sino la
n caso de r
tudiar las
ntos proce
el grado
Figurprecimues
una muestr
lasma, sue
as ya que é
r cromatog
reversible e
te relevant
nización pre
ecipitación
bargo, hay
un enf
detección
realizar pe
recuperaci
edimiento
de interac
ra 10. Proteínipitadas en ustra de plasm
ra de
ero o
éstas
grafía
en la
te en
evios
n con
y que
oque
n del
rfiles
iones
s de
cción
nasunama.
Introducción
71
entre las proteínas y estos compuestos así como por su solubilidad en el
disolvente seleccionado.
Debido a la gran complejidad de las muestras biológicas, la desproteinización no
es suficiente para evitar que se produzca efecto matriz en su análisis ya que se
pueden producir co eluciones de los componentes de la matriz que afecten a la
ionización de los analitos cuando se emplea espectrometría de masas. Es por
ello que en muchos casos es necesario una etapa de limpieza o extracción de los
compuestos de interés posterior a la precipitación de proteínas, o en algunos
casos en sustitución de ésta, que se puede llevar a cabo mediante los diversos
sistemas de extracción citados en la sección anterior, como LLE, SPE, SFE, PLE,
MAE o métodos de filtración como la diálisis o la ultracentrifugación [102].
La LLE se ha empleado con frecuencia como sistema de extracción de
compuestos fenólicos en fluidos biológicos, empleando acetato de etilo,
especialmente en orina [103 106], aunque también se ha aplicado en algunos
estudios a muestras de plasma [107 109].
Sin embargo, la técnica de extracción más empleada para el análisis de
compuestos fenólicos y sus metabolitos en fluidos biológicos es la SPE.
Actualmente se pueden encontrar una gran variedad de materiales de relleno
para SPE disponibles comercialmente como es el caso de sílice, sílice alquilada
(C 18, C 8, etc.), fases basadas en carbono, materiales de cambio iónico,
materiales poliméricos o fases de exclusión por tamaños para eliminar las
macromoléculas. Aunque las fases de sílice modificada C 18 son las más
ampliamente usadas, sobre todo en el análisis de orina [110 112], los
materiales poliméricos han despertado un creciente interés en los últimos años,
especialmente las resinas poliméricas funcionalizadas que contienen grupos
Introducción
72
funcionales polares sobre un esqueleto polimérico no polar, ya sea en formato
convencional con cartuchos [113, 114], o empleando placas de microextracción
[115] (Figura 11). Otro tipo de fases más novedosas son los materiales de modo
mixto que se caracterizan porque la retención de los analitos se produce
mediante múltiples mecanismos gracias a la incorporación de diferentes
ligandos en un absorbente.
Estos mecanismos múltiples de
retención permiten que puedan
extraerse una gran variedad de
metabolitos, por lo que se
emplean normalmente para
análisis de huella dactilar
metabólica, no para el estudio
de perfiles metabólicos de
compuestos fenólicos.
6. TÉCNICAS SEPARATIVAS PARA LA DETERMINACIÓN DE
COMPUESTOS FENÓLICOS
Para separar los componentes de mezclas complejas, como los extractos de
plantas o de plasma, la Química Analítica dispone de numerosas técnicas que se
basan en las diferencias existentes en las propiedades físico químicas de los
distintos componentes de la muestra. Generalmente se utilizan técnicas
continuas, es decir, en las que los analitos se detectan de manera continua (on
line) tras la separación. Dentro de estas técnicas se pueden diferenciar dos
grandes grupos, las cromatográficas, como la cromatografía en capa fina (TLC),
la cromatografía líquida (LC), de gases (GC) o de fluidos supercríticos (SFC), y
las no cromatográficas, como la electroforesis capilar (CE).
Figura 11. Cartuchos de extracción en fase sólida yplacas de pocillos.
Introducción
73
A continuación se describen brevemente los fundamentos y principales
aplicaciones al análisis de compuestos fenólicos de las técnicas separativas
empleadas en esta memoria, CE y LC.
6.1. Electroforesis capilar (CE)
6.1.1. Fundamentos
La electroforesis constituye una técnica separativa basada en la diferencia en la
velocidad de migración de distintos solutos al ser sometidos a la acción de un
campo eléctrico. En electroforesis capilar, los componentes de una mezcla se
transportan a través de un tubo capilar, normalmente de sílice fundida, por
efecto de un elevado potencial de corriente continua que se aplica a lo largo de
la longitud del tubo. Los extremos del capilar se colocan en dos viales rellenos
de disolución de separación que contienen cada uno de ellos un electrodo,
ambos conectados a una fuente de alto voltaje (Figura 12). La muestra se
inyecta dentro del capilar sustituyendo temporalmente el vial inicial con
disolución de separación (normalmente el del ánodo) por un vial que contiene la
muestra a separar, aplicando un potencial eléctrico (inyección electrocinética) o
una presión externa (inyección hidrodinámica) durante unos segundos. Después
se vuelve a reemplazar el vial de muestra por el inicial conteniendo la disolución
de separación y se aplica un potencial eléctrico a lo largo del capilar que
produce la separación. Los analitos pueden ser detectados directamente con
detección óptica a través de la ventana en el capilar o al final del capilar
mediante el uso de otras técnicas de detección como la espectrometría de
masas.
Introducción
74
Figura 12. Esquema de un instrumento de electroforesis capilar
La fuerza motora de la separación en CE es el voltaje aplicado. Los iones
disueltos o suspendidos en un electrolito y sujetos a un voltaje son obligados a
migrar al electrodo con carga opuesta. Las separaciones ocurren cuando los
cationes se mueven en una dirección y los aniones en otra, o cuando las
velocidades de migración de iones del mismo signo son diferentes. Por tanto, la
separación ocurre gracias a las diferencias en las velocidades de los iones, y esta
velocidad de migración (vef) depende tanto del campo eléctrico aplicado como
de la movilidad electroforética de cada ión. La movilidad electroforética ( e)
viene dada por la siguiente expresión:
r6q
e
Introducción
75
donde q es la carga del ión, r su radio, y es la viscosidad de la disolución. Esto
significa que los iones con carga del mismo signo en un sistema electroforético
concreto se separarán entre sí según su relación q/r.
En las separaciones electroforéticas hay que tener en cuenta además otro
fenómeno que se produce denominado electroósmosis. El uso de capilares de
sílice provoca un flujo electroosmótico (EOF) que hace que los iones y moléculas
presentes en el medio electroforético se muevan hacia el cátodo. A pH superior
a 6, en la superficie interna de los capilares de sílice, que contiene grupos
hidroxilo, se forma una capa de protones cerca de la pared cargada
negativamente. Estas cargas positivas incluyendo las moléculas de solvatación
de agua son atraídas hacia el cátodo provocando este flujo electroosmótico, que
mueve igualmente a las especies neutras que se encuentran en el medio
electroforético. Su componente de velocidad es mayor, en general, que las
componentes de migración electroforéticas de los solutos a separar de forma
que esta doble capa mueve todos los iones en conjunto, tanto cationes como
aniones, hacia el cátodo produciendo un movimiento electroosmótico. Los
cationes se mueven a lo largo del capilar con una velocidad mayor que la del
flujo electroosmótico, puesto que su movimiento se ve acelerado por la
atracción electroforética al electrodo negativo. Los aniones, por el contrario, se
mueven más lentamente que el EOF debido a que son repelidos por el cátodo,
de hecho, en algunos casos las especies negativas pueden moverse en la
dirección opuesta al flujo de disolvente. Los solutos neutros se mueven a través
del capilar con el flujo electroosmótico y no se separarán entre sí, salvo que se
usen agentes micelares como aditivos del medio electroforético dando lugar a
un modo de CE conocido como cromatografía capilar electrocinética micelar
[116].
Introducción
76
La electroforesis capilar es una técnica muy versátil debido en parte, a los
distintos modos de separación disponibles. Los modos de CE más comunes se
resumen en la Tabla 1 junto con su principio de separación. Los diferentes
mecanismos de separación empleados hacen que estos modos sean
complementarios entre sí, e incluso en algunos casos, una separación puede
llevarse a cabo de forma adecuada por más de un modo electroforético.
Además, una de las grandes ventajas de esta técnica es que para cambiar de
modo puede bastar simplemente con variar la composición de la disolución
reguladora empleada.
Tabla 1.Modos de separación en electroforesis capilar
Modo de CE Principio de Separación
Electroforesis capilar en zona (CZE) Relación carga/masa
Cromatografía capilar electrocinética micelar(MEKC)
Interacción hidrofóbica/iónica con micelasdel surfactante
Isoelectroenfoque capilar (CIEF) Punto isoeléctrico
Isotacoforesis capilar (CITP)Capacidad de migración entre tampones de
distinta naturaleza
Electroforesis capilar en gel (CGE) Tamaño molecular
6.1.2. Separación de compuestos fenólicos mediante CE
Aunque las técnicas cromatográficas son las más empleadas en la separación de
compuestos fenólicos, la CE está ganando popularidad y representa un método
alternativo para el análisis de estos compuestos tanto en matrices vegetales
como alimentarias, fundamentalmente gracias a su velocidad, resolución y
relativo bajo coste por análisis. De esta forma, la CE se ha aplicado al análisis de
Introducción
77
compuestos fenólicos en aceite de oliva [117], extractos de plantas [118], vino
[119], té [120] y zumos [121], entre muchas otras matrices.
Los modos de CE más utilizados para la separación de compuestos fenólicos son
la electroforesis capilar en zona (CZE) y la cromatografía capilar electrocinética
micelar (MEKC). Para ionizar los compuestos fenólicos y así poder separarlos
mediante CZE, puesto que son ácidos relativamente débiles, normalmente se
emplean tampones de borato o acetato amónico con un pH entre 9 y 10. De
esta forma se pueden separar los compuestos fenólicos cargados en función de
su relación carga/masa. En la MEKC se suele emplear como tensioactivo
dodecilsulfato sódico (SDS), y en estos casos la separación se basa en la
hidrofobicidad de los compuestos que determina el reparto de los analitos entre
la fase acuosa (que se mueve con el EOF) y las micelas (que al estar cargadas
migran con una velocidad diferente). Los compuestos fenólicos menos polares,
como los flavonoides, interactúan fuertemente con las micelas y, por tanto, es
posible desarrollar métodos bastante selectivos modificando la fase micelar.
Wang y colaboradores compararon el comportamiento electroforético de trece
flavonoides en estos dos modos [122]. La MEKC mostró una mayor selectividad
ya que el comportamiento electroforético en CZE se ve afectado por diversos
factores como el grado de saturación y la estereoquímica del anillo C, el número
y posición de grupos hidroxilo, la metilación o glicosilación de los grupos
hidroxilo, así como la complejación de los flavonoides con el tampón borato.
Para superar estos problemas de selectividad, algunos estudios ha empleado CE
no acuosa para separar flavonoides, usando metanol a pH elevado [123].
A pesar de los numerosos métodos electroforéticos que se han desarrollado
para el análisis de compuestos fenólicos en alimentos y plantas, no se ha
Introducción
78
descrito ninguno para la separación de estos compuestos en matrices
biológicas.
6.2. Cromatografía líquida (LC)
6.2.1. Fundamentos
La cromatografía líquida (LC) es una técnica de separación donde los
componentes de la muestra se distribuyen entre una fase móvil (el disolvente) y
una fase estacionaria (el relleno de la columna). La separación se produce en
función de las distintas afinidades de los constituyentes de la muestra hacia
cada fase. La fase estacionaria puede ser un sólido poroso, generalmente en
forma particulada, o bien una fina capa de sustancia ligada a un soporte sólido,
contenido en el interior de un tubo habitualmente metálico que da lugar a la
columna cromatográfica. La fase móvil es un disolvente o mezcla de ellos a los
que en algunas ocasiones se les modifica el pH mediante adición de ácidos,
bases o disoluciones reguladoras.
En una separación por LC, la fase móvil impulsada por la bomba transporta una
banda de muestra a través de la columna cromatográfica. Al entrar la muestra
en la columna, sus componentes interaccionan en extensión variable con la fase
estacionaria y se reparten de manera distinta entre ambas fases de tal forma
que aquellos que sean más afines con la fase móvil serán menos retenidos por
la fase estacionaria y eluirán antes. Por el contrario, aquellos que tengan más
afinidad por la fase estacionaria avanzarán más lentamente a través de la
columna y eluirán más tarde. Cuando cada analito eluye del final de la columna,
entra en el detector y produce de alguna forma una señal medible. La
intensidad y duración de la señal estará relacionada con la cantidad o naturaleza
Introducción
79
del analito. Generalmente, la señal es amplificada y registrada por un integrador
electrónico, un ordenador o por otros medios mediante los cuales se obtiene el
cromatograma que permite identificar y cuantificar el analito.
Figura 13. Esquema simplificado de un equipo de LC junto a uno comercial
Un equipo de LC está formado por una serie de componentes básicos que son
comunes a todos los modelos: bomba, inyector, columna cromatográfica, horno
termostatizado, detector y sistema de adquisición de datos. En la Figura 13 se
representa el esquema básico de un equipo de LC y se muestra el aspecto de
uno comercial. Sin duda, el componente más determinante en la separación es
la columna, cuyas características van a definir el tipo y modo de LC.
Por un lado, en función del diámetro interno de la columna y de los caudales
utilizados, las técnicas de LC se pueden clasificar en diversas categorías que se
muestran en la Tabla 2. Generalmente se considera HPLC cuando la separación
cromatográfica se lleva a cabo en columnas de diámetro interno de entre 1,5 y
Introducción
80
4,5 mm, para columnas con diámetros alrededor de 800 μm se define como
microLC, cuando se usan columnas de diámetro entre 100 y 500 μm se conoce
como LC capilar y en caso de columnas de entre 10 y 100 μm se denomina
nanoLC.
Tabla 2. Principales diferencias entre los distintos tipos de LC
También se puede distinguir otra categoría que se diferencia en el tamaño de
partícula, de forma que se denomina comúnmente cromatografía líquida de
resolución rápida (RRLC) o cromatografía líquida de ultra alta resolución (UPLC)
cuando se emplean columnas con tamaños de partícula inferiores a 2 m. En
estos casos, normalmente la longitud de la columna es menor y se usan flujos
relativamente elevados. Este tipo de LC permite realizar análisis más rápidos
manteniendo la eficacia constante, o bien conseguir eficacias mayores
manteniendo el mismo tiempo de análisis. La combinación del uso de pequeños
diámetros de partícula y presiones de hasta 1.250 bares (la presión más alta que
soporta la instrumentación comercial actualmente disponible) proporciona una
eficacia de hasta 100.000 platos teóricos [124] frente a los 15.000–25.000 platos
teóricos de la HPLC convencional.
UPLC/RRLC HPLC Micro LC LC capilar Nano LC
Diámetro interno dela columna (mm)
1,5 4,5 1,5 4,5 0,8 0,18 0,32 0,075 0,1
Longitud de lacolumna (cm)
3 15 3 30 5 25 5 25 5 15
Tamaño de partícula( m)
< 2 3 40 3 5 3 5 3 5
Flujo de fase móvil0,2 5ml/min
0,2 2,5ml/min
10 100l/min
1 10 l/min0,1 1l/min
Introducción
81
Por otro lado, se pueden diferenciar distintos modos de cromatografía líquida
según la naturaleza de la fase estacionaria, tal y como se recoge en la Tabla 3.
Tabla 3.Modos de LC en función de la fase estacionaria empleada.
Modo de LC Fase estacionaria
Cromatografía de partición Líquido retenido por un sólido soporte
Cromatografía de adsorción Sólido con propiedades superficiales
Cromatografía de cambio iónicoSólido con propiedades cambiadoras de
iones
Cromatografía de exclusión por tamaños Sólido con porosidad controlada
Cromatografía de afinidadSólido con propiedades de retención
bioespecíficas
Cromatografía quiralReactivo quiral unido a fase móvil o al
soporte sólido
La cromatografía de partición es la más ampliamente utilizada y en la práctica,
pueden diferenciarse dos modalidades, dependiendo de la polaridad de las dos
fases:
Cromatografía en fase normal, donde la fase estacionaria es de
naturaleza polar y la fase móvil es apolar. En este caso, las muestras
polares son retenidas más fuertemente por la columna permitiendo, por
tanto, la elución de componentes apolares en primer lugar.
Cromatografía en fase inversa, donde la fase estacionaria es de
naturaleza apolar y el disolvente de elución o fase móvil es polar. En este
Introducción
82
caso ocurre lo contrario que en fase normal, los compuestos apolares
serán retenidos durante más tiempo en la columna.
6.2.2. Separación de compuestos fenólicos mediante HPLC y UPLC/RRLC
Los compuestos fenólicos se separan, fundamentalmente, mediante
cromatografía de partición en fase inversa, ya que son ácidos débiles que en su
forma neutra presentan relativa hidrofobicidad, aunque la cromatografía en
fase normal se ha usado para la separación de proantocianidinas [125]. La
mayoría de las aplicaciones emplean elución en gradiente, aunque también se
han desarrollado métodos cromatográficos isocráticos para la separación de
compuestos fenólicos pertenecientes a una misma familia [126, 127].
En general, la elución se lleva a cabo con una fase móvil de polaridad elevada
como es el caso de disoluciones acuosas de ácidos débiles que suelen emplearse
en un sistema binario con disolventes orgánicos polares como acetonitrilo o
metanol. Los gradientes de elución que emplean como fase orgánica metanol,
normalmente comienzan con un porcentaje de 5 10% (v/v) y terminan con 40
100%, mientras que los que emplean acetonitrilo suelen comenzar con 0 10% y
terminar con 30 90% [128]. La separación de los compuestos fenólicos más
polares, como los ácidos fenólicos, depende mucho del pH de la fase móvil
[129]. La presencia del ácido débil en la fase móvil evita que estos compuestos
se ionicen, mejorando de esta forma la separación en la columna de fase
inversa. Otros compuestos menos polares, como los flavonoides, presentan una
menor tendencia a ionizarse y por tanto, se pueden separar en fases móviles
neutras, sin la presencia de ácido. Sin embargo, el tipo de ácido y concentración
depende del sistema de detección que se emplee. Cuando se usan detectores
de absorción UV Vis, pueden utilizarse ácidos más fuertes, como el fosfórico, y
Introducción
83
ajustarse valores de pH menores, mientras que si se emplea espectrometría de
masas como sistema de detección, la concentración de ácido debe ser mucho
menor. Cuyckens y colaboradores concluyeron que cuando se emplea
acetonitrilo como fase orgánica, el ácido fórmico es preferible al acético o al
trifluoroacético, con una concentración óptima de 0,1% para el modo de
ionización negativo y 0,5% para el positivo. Sin embargo, cuando se usa
metanol, una concentración de ácido acético de 1% ofrecía mayor sensibilidad
[130].
Las columnas más empleadas para la separación de compuestos fenólicos son
las rellenas de sílice modificada químicamente con cadenas de hidrocarburos
como C 8 (n octilo) o más comúnmente, C 18 (n octadecilo). Existe una gran
diversidad de columnas disponibles comercialmente, que se diferencian,
fundamentalmente, en sus dimensiones, es decir, longitud y diámetro, así como
en el tamaño de partícula del relleno. Tradicionalmente, para el análisis de
compuestos fenólicos se han empleado columnas convencionales de HPLC, es
decir, con un tamaño de partícula de entre 3 y 5 m, pero la tendencia actual se
centra en la disminución del tamaño de partícula. Por tanto, el uso de la RRLC o
UPLC se ha extendido ampliamente en la última década para la separación de
estos compuestos, tanto en matrices vegatales como biológicas [131 134].
6.2.3. Separación de compuestos fenólicos mediante nanoLC
La nano cromatografía líquida (nanoLC) fue desarrollada en 1988 por Karlsson y
Novotny [135] y desde entonces ha ido emergiendo como una herramienta
analítica potente, sobre todo en determinados campos como el de la
proteómica, debido principalmente a la reducción en la cantidad de muestra
requerida. Además, desde un punto de vista teórico, se ha demostrado que una
Introducción
84
reducción en el diámetro interno de la columna aumenta la sensibilidad debido
a que se produce una menor dilución del analito [136] y se consigue una mayor
eficacia [137].
Las columnas capilares de nano LC suelen ser de sílice fundida, empleando las
mismas fases estacionarias utilizadas normalmente en HPLC. En cuanto al
tamaño de partícula de estas fases estacionarias
normalmente suelen estar comprendidos entre 3 y 5
m aunque hoy día ya se están utilizando fases
estacionarias con tamaños de partícula de 1.5 1.8 m
similares a las utilizadas en RRLC o UPLC. Las fases
móviles empleadas también son las comúnmente
utilizadas en HPLC [138].
Las principales características que presenta la técnica nanoLC y que la
diferencian de la HPLC convencional se pueden resumir en los siguientes
puntos:
Uso de columnas capilares, con un diámetro interno muy pequeño.
Bajo consumo de fases móviles, ya que utiliza flujos comprendidos entre
100 y 500 nl/min, lo que supone una reducción de costes además de
beneficios medioambientales.
Posibilidad de disminuir los volúmenes de inyección de muestra.
Aumento teórico de sensibilidad, sobre todo cuando se acopla a
espectrometría de masas.
Estas características hacen que el empleo de esta técnica pueda resultar de
especial interés en aplicaciones donde se requiera de elevada sensibilidad y se
Figura 14. Equipo denanoLC comercial
Introducción
85
disponga de una cantidad de muestra limitada, como es el caso de ciertos
estudios metabolómicos.
Como ya se ha comentado, la nanoLC se ha aplicado mayoritariamente en
proteómica [139], aunque poco a poco se ha ido ampliando su campo de
aplicación, de forma que en los últimos años se ha empleado en diversas áreas
de investigación como la farmacología [140], la alimentación [141, 142], el
análisis medioambiental [143] y de enantiómeros [144, 145], aunque en menor
extensión. Sólo se han descrito algunas aplicaciones para el análisis de
compuestos fenólicos en matrices alimenticias [142, 146, 147], vegetales [148] y
biológicas [78], pero los resultados obtenidos hacen de la nanoLC una técnica
prometedora en el ámbito de los compuestos fenólicos.
7. SISTEMAS DE DETECCIÓN PARA EL ACOPLAMIENTO CON
TÉCNICAS SEPARATIVAS
Toda técnica separativa requiere un detector que produzca de alguna forma una
señal medible cuando un analito eluya del capilar o de la columna
cromatográfica. Se pueden emplear detectores muy diversos, en su mayoría
comunes para CE y LC, pero idealmente todos deberían cumplir los siguientes
requisitos:
Aumento teórico de sensibilidad, sobre todo cuando se acopla a
espectrometría de masas.
Elevada sensibilidad.
Límites de detección bajos, para lo cual se requiere la combinación de
una elevada sensibilidad con una baja fluctuación de la señal de fondo.
Respuesta rápida ante un cambio en la concentración de analito.
Introducción
86
Su presencia no debe perjudicar a la eficacia de la separación.
Proporcionar señales fiables, es decir, reproducibles y estables en el
tiempo.
Amplio intervalo lineal.
Idealmente, la señal debe ser nula en ausencia de analito. Para que la
señal de fondo no perturbe la señal correspondiente a los analitos
conviene que sea lo más pequeña y constante posible.
Para algunas aplicaciones, es necesario que el detector presente elevada
selectividad.
Los detectores que se emplean en CE y LC, como ya se ha comentado, son de
naturaleza muy diversa, pero sin duda, los más empleados para el análisis de
compuestos fenólicos son la detección espectrofotométrica UV Vis y la
espectrometría de masas (MS).
7.1. Espectroscopía de absorción UV Vis
Los sistemas de detección basados en la absorción de radiación UV/visible son
los más comunes en los equipos de CE y HPLC comerciales. La detección de los
analitos se fundamenta en la interacción entre la radiación UV/visible y la
materia que da lugar a un fenómeno de absorción de determinadas longitudes
de onda de esa radiación por parte de los compuestos. Los detectores de
absorción UV Vis pueden ser de longitud de onda fija, de longitud de onda
variable o de batería de diodos o como son más conocidos “diodo array” (DAD).
Estos últimos son los que se emplean en los equipos de CE y HPLC actuales, y
Introducción
87
adquieren los espectros completos de absorción UV Vis de muestras que pasan
rápidamente por una celda de medida.
Los múltiples enlaces conjugados presentes en los compuestos fenólicos los
convierten en cromóforos que presentan bandas de absorción en la región UV e
incluso en el visible como es el caso de las antocianidinas y algunos flavonoles.
Son muchas las moléculas que pueden absorber radiación de esta zona del
espectro electromagnético por lo que este detector se puede considerar
universal. Este comportamiento presenta la ventaja de que puede emplearse
para resolver una gran cantidad de problemas analíticos pero también puede
resultar un inconveniente en aquellos casos en los que se requiere selectividad.
La detección UV/visible es muy robusta, por lo que es uno de los mejores
detectores para llevar a cabo cuantificaciones, si bien presenta el inconveniente
de no proporcionar información estructural y por tanto, no permite identificar
de forma inequívoca compuestos si no se dispone de patrones comerciales. Sin
embargo, sí puede resultar útil en la determinación de compuestos fenólicos
para acotar la familia a la que pertenecen los analitos ya que cada familia posee
unas bandas de absorción características, tal como se muestra en la Tabla 4.
.
Introducción
88
Tabla 4. Bandas de absorción características de diversas familias de compuestos fenólicos
Compuestos Bandas UV (nm)
Ácidos benzoicos 270 280
Ácidos cinámicos 305 325
Cumarinas 220 230 310 350
Chalconas 220 270 340 390
Dihidrochalconas ~ 220 ~ 280
Auronas 240 270 340 370
Flavonas 250 270 330 350
Flavonoles 250 270 350 380
Flavanonas 270 295
Flavanoles 270 280
Antocianidinas 240 280 450 560
Isoflavonas 245 270 300 340
Proantocianidinas ~ 280
7.2. Espectrometría de masas (MS)
En los últimos años han aumentado exponencialmente las aplicaciones de la
espectrometría de masas (MS) como sistema de detección acoplado a diversas
técnicas separativas. Este auge de la técnica se debe principalmente a su
selectividad y al hecho de que es uno de los pocos sistemas de detección que
proporciona información estructural, evitando la inherente falta de sensibilidad
de la resonancia magnética nuclear. Este acoplamiento también presenta la
Introducción
89
ventaja de que proporciona una segunda dimensión de separación ya que tras
separar los compuestos según su tiempo de migración o retención, se produce
en el espectrómetro de masas una separación en función de la relación m/z.
La espectrometría de masas se basa en la separación a vacío de iones en fase
gaseosa de acuerdo con su relación masa/carga (m/z). Existen diversos tipos de
espectrómetros de masas, pero todos incluyen los siguientes elementos: un
sistema de introducción de muestra, un sistema de ionización, un acelerador de
iones mediante un campo eléctrico, un analizador que disperse de iones de
acuerdo a su relación masa/carga y un detector de los iones junto con un
procesador adecuado de la señal (Figura 15). El acelerador de iones, el
analizador y el detector deben estar a una presión inferior a 10 4 10 8 Torr para
evitar colisiones entre los iones de interés y otras sustancias como radicales o
moléculas neutras [149].
Figura 15. Esquema de las partes de un espectrómetro de masas
Introducción
90
Existen diversos tipos de sistemas de ionización y analizadores de masas. Los
más empleados en los acoplamientos HPLC MS y CE MS se recogen en la Tabla
5.
Tabla 5. Sistemas de ionización y analizadores de masas más utilizados en el acoplamientoHPLC MS y CE MS.
SISTEMAS DE IONIZACIÓN ANALIZADORES DE MASAS
Ionización química a presión atmosférica
(APCI)Cuadrupolo (Q)
Ionización por electrospray (ESI) Triple cuadrupolo (QqQ)
Desorción/ionización asistida por una matriz
(MALDI)Trampa de iones (IT)
Flujo continuo y bombardeo con átomos
rápidos (CF FAB)Tiempo de vuelo (TOF)
Plasma acoplado inductivamente (ICP) Cuadrupolo tiempo de vuelo (Q TOF)
Ionización por termonebulización (TS)Transformada de Fourier resonancia
ciclotrónica (FT ICR)
En la presente memoria el sistema de ionización que se ha empleado para todos
los acoplamientos CE/HPLC/nanoLC MS ha sido la ionización por electrospray y
los analizadores de masas empleados han sido trampa de iones, tiempo de
vuelo y cuadrupolo tiempo de vuelo. Los acoplamientos empleados se muestran
en la Figura 16.
Introducción
91
Figura 16. Plataformas analíticas empleadas en la presente memoria
7.2.1. Sistema de ionización: Ionización por electrospray (ESI)
Para llevar a cabo el acoplamiento de técnicas que trabajan en fase líquida,
como son la electroforesis capilar y la cromatografía líquida, con un
espectrómetro de masas, en el que las sustancias para ser analizadas deben
entrar en fase gaseosa, es necesaria una interfase adecuada. Se han
desarrollado diferentes interfases que se recogen en la Tabla 5, pero la más
empleada para el análisis de compuestos fenólicos es la de ionización por
electrospray (ESI) ya que es eficaz en el análisis de compuestos polares, lábiles
y/o con alto peso molecular.
Introducción
92
En el proceso de formación del electrospray, que se lleva a cabo a presión
atmosférica, intervienen diversos mecanismos al mismo tiempo, tal como se
muestra en la Figura 17. La muestra, procedente del capilar o de la columna,
pasa través de un fino capilar de acero inoxidable cuyo extremo se encuentra a
un potencial eléctrico elevado (3 6 kV). Este elevado potencial unido al pequeño
radio de curvatura al final del capilar crean un fuerte campo eléctrico, que
produce reacciones de oxidación reducción y hace que el líquido emerja como
finas gotas cargadas eléctricamente. Esta nebulización normalmente es asistida
por un flujo de nitrógeno gaseoso (el gas nebulizador) que fluye a través de un
tubo coaxial al capilar principal. En la formación de las gotas cargadas resultan
críticas la velocidad del flujo, la tensión superficial y la conductividad de la
solución. La conductividad debe ser baja lo que implica concentraciones de
electrolitos menores que 10 4 M.
Figura 17. Diagrama de formación del electrospray
Las gotas cargadas atraviesan una serie de cámaras con vacío creciente. A
medida en que las gotas pasan por las cámaras, se evaporan y se hacen cada vez
más pequeñas debido a la desolvatación. Al mismo tiempo, dado que el área
superficial de las gotas se reduce, aumenta la densidad de carga eléctrica sobre
la superficie. En cierto momento, la repulsión de los iones se hace mayor que la
Introducción
93
tensión superficial de las gotas, alcanzándose el denominado límite de Rayleigh,
en ese punto las gotas se vuelven inestables y estallan en las conocidas como
explosiones de Coulomb. Como resultado, se forman una serie de gotas más
pequeñas cargadas que seguirán sufriendo procesos de evaporación y explosión
sucesivos hasta que finalmente se forman iones que pasan a fase gaseosa y son
atraídos hacia la entrada del espectrómetro de masas como consecuencia del
voltaje aplicado [150, 151].
En el proceso de ionización se pueden formar iones mono o multicargados, lo
que permite detectar compuestos con pesos moleculares muy altos empleando
analizadores de masas que trabajan con un intervalo limitado de valores m/z.
El diseño específico de la interfase ESI es distinto según la técnica separativa a la
que se acople. A continuación se describen las interfases ESI utilizadas en la
presente tesis doctoral para el acoplamiento CE MS, HPLC MS y nanoLC MS.
7.2.1.1. Interfase ESI para el acoplamiento CE MS
En el acoplamiento CE ESI MS, el primer problema que se plantea es la
incompatibilidad del flujo procedente del capilar de separación (de hasta 100
nl/min), con el flujo necesario para la formación de un electrospray estable (1
200 l/min). Para solucionar este problema se han propuesto dos estrategias, la
primera de ellas es el uso de un flujo adicional, y la segunda, el uso de las
llamadas interfases micro o nano ESI, con las cuales se va a poder trabajar con
flujos extremadamente pequeños.
Introducción
94
El segundo problema es mantener el circuito eléctrico que se requiere para
llevar a cabo la separación en CE y que se forma entre los extremos del capilar al
aplicar la diferencia de potencial. Otro problema que aparece en el
acoplamiento CE ESI MS es la compatibilidad de los diferentes modos de
electroforesis capilar con el espectrómetro de masas, ya que dependiendo del
modo de CE, se va a requerir o no el empleo de sustancias poco compatibles con
ESI (como iones borato, fosfato, detergentes para la formación de micelas,
ciclodextrinas, etc.), las cuales por su escasa volatilidad contribuyen de forma
significativa al incremento del ruido de fondo en la detección por MS,
disminuyendo la sensibilidad, y llegando en algunos casos a obstruir y/o
contaminar el sistema de detección.
Todas las dificultades indicadas del acoplamiento CE MS han hecho que se
hayan desarrollado diversos tipos de interfases para CE ESI MS teniendo como
objetivo la formación de un electrospray estable y el mantenimiento de la
corriente eléctrica en el interior del capilar [152, 153]. Con esta idea, se han
desarrollado fundamentalmente tres tipos de interfases ESI: sin flujo adicional,
con flujo adicional y con unión líquida. La interfase ESI con flujo adicional
actualmente es la más utilizada en el acoplamiento CE MS, siendo hasta la fecha
la única disponible comercialmente y es la que se ha empleado en la presente
tesis doctoral.
La interfase ESI con flujo adicional está formada por tres tubos concéntricos,
como se muestra en la Figura 18. El primero de ellos es el propio capilar de
separación que se encuentra rodeado de un tubo de acero inoxidable por el que
se hace fluir el líquido adicional, y por un tercer tubo por el cual se introduce el
gas nebulizador que favorece la formación del electrospray .
Introducción
95
Figura 18. Esquema de una interfase ESI con flujo adicional. (Adaptado de [154])
El flujo que eluye del capilar se aumenta mediante el empleo del líquido
adicional, facilitando el acoplamiento CE MS y dando lugar a la formación de un
electrospray estable. Además, el flujo adicional facilita el cierre del circuito
eléctrico con el interior del capilar, ya que el líquido añadido permite el
contacto entre el electrolito de separación que fluye del interior del capilar y el
tubo metálico (conectado a tierra) que rodea el capilar de separación.
Se trata de una interfase robusta y de fácil manejo, aunque presenta algunas
limitaciones, como por ejemplo la disminución de la sensibilidad resultante de la
dilución que el líquido adicional produce sobre las bandas de los analitos que
salen del capilar. Por otro lado, es necesario considerar otros parámetros como
son la composición y el flujo de la disolución adicional, presión del gas
nebulizador, situación del capilar con respecto al tubo concéntrico que lo rodea,
naturaleza del electrolito de separación, etc., ya que van a influir tanto sobre la
intensidad de la señal MS como sobre la resolución de la separación [154].
7.2.1.2. Interfase ESI para el acoplamiento HPLC MS
En el acoplamiento HPLC ESI MS, al contrario de lo que ocurre en CE, el
principal problema es el excesivo flujo de fase móvil que eluye de la columna, en
Introducción
96
muchos casos comprendido entre 0,5 y 3 ml/min. Las interfases ESI comerciales
empleadas en el acoplamiento HPLC MS admiten flujos comprendidos entre 1
μl/min y 1 ml/min, aunque realmente el flujo recomendado a la entrada del
espectrómetro de masas está entre 0,2 0,5 ml/min, dependiendo del
analizador. Para solucionar el problema de los flujos elevados el diseño de la
interfase ESI no se modifica, sino que se suelen emplear divisores de flujo entre
la salida de la columna cromatográfica y la entrada de la interfase, aunque esto
supone una disminución de la sensibilidad producida por la eliminación de parte
de la muestra. Otra posible solución es utilizar columnas cromatográficas más
estrechas con flujos de 0,2 ml/min, que sí se pueden introducir directamente en
el espectrómetro de masas.
7.2.1.3. Interfase ESI para el acoplamiento nanoLC MS
En el acoplamiento nanoLC ESI MS al igual que ocurre con CE el flujo que
proviene de la columna capilar es demasiado pequeño (del orden de los
nanolitros) para conseguir un electrospray estable. Para solucionar este
problema se han utilizado interfases ESI con flujo adicional similares a las
empleadas en el acoplamiento CE ESI MS aunque existen pocas referencias
bibliográficas, ya que pronto se desarrollaron las interfases nanoESI sin flujo
adicional que permiten trabajar con flujos muy pequeños (50 500 nl/min)
obteniendo alta sensibilidad. Son fuentes con un diseño diferente al utilizado en
otro tipo de acoplamientos (HPLC ESI MS o CE ESI MS), ya que están
especialmente diseñadas para flujos bajos. En vez de un nebulizador utilizan una
aguja o capilar interno para transportar la muestra desde la columna capilar
hasta la cámara de nebulización. La diferencia de potencial que se genera entre
la punta del capilar y la entrada del espectrómetro de masas produce la
Introducción
97
ionización y en muchos casos no se requiere de un gas nebulizador adicional
debido a los pequeños volúmenes de disolvente utilizados. Los capilares
internos pueden ser de sílice, con algún tipo de unión conductora que permita
establecer el potencial necesario para la formación del electrospray, o pueden
estar fabricados de acero inoxidable. Muchas aplicaciones encontradas en
bibliografía utilizan los capilares de sílice, que además presentan la ventaja de
que la columna cromatográfica se puede empaquetar directamente en ellos y
de esta forma eliminar los volúmenes muertos post columna. Los capilares de
sílice tienen un diámetro en la punta de 1 2 μm y producen sprays estables en el
rango de 20 300 nl/min. En algunos trabajos se utilizan fuentes nanoESI
disponibles comercialmente pero insertando capilares preparados en el propio
laboratorio, para solucionar problemas concretos.
En general en las fuentes nanoESI la eficacia de la ionización es mayor ya que los
iones se producen con más eficacia a medida que el tamaño medio de las
microgotas del spray disminuye. Además el pequeño tamaño del spray formado
y la proximidad de la punta del capilar a la entrada del espectrómetro de masas
permiten la introducción de una fracción mayor de los iones hacia el interior.
7.2.2. Analizadores de masas
Una vez que en la interfase se ha llevado a cabo la transferencia de los iones
procedentes del capilar desde la fase líquida a la fase gaseosa, los iones son
dirigidos hacia el analizador de masas. Los analizadores de masas permiten la
separación, detección y cuantificación de los analitos en estudio con un grado
de sensibilidad y selectividad muy elevado, proporcionando información sobre
su masa molecular.
Introducción
98
Existen diversos tipos de analizadores: cuadrupolos, trampas de iones, sectores
magnéticos, analizadores de tiempo de vuelo, etc. En el desarrollo experimental
de esta memoria, se han utilizados los analizadores trampa de iones (IT), tiempo
de vuelo (TOF) y cuadrupolo tiempo de vuelo (QTOF).
7.2.2.1. Trampa de iones (IT)
En la Figura 19 se puede ver el esquema de un espectrómetro de masas IT con
sus distintas partes.
Figura 19. Esquema de un espectrómetro de masas IT.
En el esquema se pueden diferenciar las siguientes partes: la cámara de
nebulización, la denominada zona de transporte y focalización de iones,
formada por skimmers, octopolos y lentes; el analizador (IT); y el detector.
Introducción
99
La primera zona es la cámara de nebulización (la interfase), donde se nebuliza e
ioniza la solución de la muestra tal y como se ha detallado en la sección
anterior. La zona de transporte y focalización de iones se divide en cuatro zonas
de alto vacío. Los iones pasan a través del capilar de vidrio y el skimmer elimina
el gas de secado y las especies neutras, entonces los iones pasan al octopolo
que los transporta, focaliza y guía desde el skimmer hasta la trampa de iones
atravesando una serie de lentes. Los iones formados en la fuente entran en el
analizador que consta de un electrodo hiperbólico en forma de anillo y otros
dos que actúan como tapas a los que se aplican diferencias de potencial de
signo contrario al electrodo anular con componentes de corriente directa y
alterna. De esta forma se genera un campo cuadrupolar tridimensional en la
cavidad de la trampa donde los iones con trayectorias estables son capturados,
oscilando a frecuencias que dependen de su masa y carga. Durante la detección,
los potenciales de los electrodos se alteran sometiéndolos a una rampa lineal de
radiofrecuencia (RF) para provocar inestabilidad en las trayectorias de los iones
y expulsarlos en la dirección axial en función de su relación m/z dando lugar a
un espectro de masas. Después del analizador, los iones pasan al detector, que
tiene también una serie de lentes y un sínodo que dirigen los iones hasta del
propio detector.
Una vez que los iones se encuentran atrapados dentro de este analizador se
puede llevar a cabo bien el análisis de sus masas, obteniéndose el espectro de
MS o bien el aislamiento de uno o varios iones precursores y su posterior
fragmentación, dando lugar a los espectros de MS/MS. En la Figura 20 se
muestra un diagrama del proceso de aislamiento de iones precursores y
fragmentación de éstos.
Introducción
100
Figura 20. Diagrama del modo de trabajo MS/MS en la trampa de iones
Las principales especificaciones de la trampa de iones son que puede analizar un
rango de masas de 50 2.200 m/z y que permite realizar análisis MS/MS y MSn.
7.2.2.2. Tiempo de vuelo (TOF)
El analizador de tiempo de vuelo (TOF) separa los iones según la distinta
velocidad que adquieren en su interior en función de su relación m/z. En primer
lugar, los iones son extraídos de la cámara de ionización y focalizados hacia el
tubo de vuelo donde se aceleran mediante un campo electrostático que les
aporta una elevada energía cinética. Los iones de mayor m/z “volarán” a menor
velocidad que los de menor m/z. La resolución entre los iones de diferente m/z
será mayor cuanto mayor sea longitud del tubo (habrá una mayor separación de
los iones en el tiempo) y cuanto menor sea la dispersión en las energías
cinéticas de los iones.
Introducción
101
La muestra disuelta entra en la cámara de nebulización donde tiene lugar la
formación del spray. Los iones formados atraviesan la unidad de desolvatación,
que separa las zonas a presión atmosférica de la primera zona de alto vacío, y
que consta de un calentador del gas de secado y un capilar de cristal. Se llega a
través de ella a la zona de transmisión o transferencia óptica que consta de tres
módulos que están a alto vacío, separados entre sí por dos skimmers. Los dos
hexapolos son los que transfieren los iones hasta la zona de alto vacío, mientras
que las lentes enfocan o dirigen dichos iones.
Figura 21. Esquema detallado de las partes de las que consta un espectrómetro de masas TOF
Introducción
102
La siguiente unidad es el analizador TOF propiamente dicho, que a su vez consta
de tres zonas. La primera de ellas es la zona de aceleración ortogonal donde se
aceleran los iones hacia el tubo de vuelo aplicando un campo eléctrico
intermitente. Los iones volarán a lo largo del tubo de vuelo hasta alcanzar la
segunda zona, el reflector, que permite corregir la dispersión en la energía
cinética de los iones, aumentando de esta forma la resolución. El último
componente del analizador TOF es el detector de impacto electrónico que
consiste en una serie de placas a alto voltaje que convierten el impacto de los
iones en señales eléctricas. En el detector hay millones de pequeños poros
internamente recubiertos con una capa semiconductora, de forma que cada
uno de ellos trabaja como un multiplicador de electrones independiente.
El analizador TOF es rápido y sensible, y su principal característica es que gracias
a su elevada resolución permite obtener valores de masa muy exactos. La
combinación de los datos de masas exactas con la determinación de la
distribución isotópica, permite poder determinar la fórmula molecular del
compuesto.
Las principales especificaciones del TOF son que puede analizar un rango de
masas de 50 3.000 m/z, presenta una resolución de entre 10.000 y 15.000
FWHM, y una exactitud de 3 ppm con calibración interna y 5 ppm con
calibración externa.
7.2.2.3. Cuadrupolo tiempo de vuelo (QTOF)
El analizador cuadrupolo tiempo de vuelo (QTOF) está formado básicamente
por las mismas partes que un analizador TOF con la diferencia de que se
Introducción
103
introduce un cuadrupolo donde se pueden seleccionar determinados iones para
posteriormente fragmentarlos en una celda de colisión con ayuda de un gas de
colisión, normalmente N2. Los iones fragmentados se separan en el analizador
TOF función de su relación m/z de la misma forma que se ha descrito en el
apartado anterior. Además se sustituyen los skimmers de la zona de trasmisión
de iones por funnels (anillos concéntricos apilados en forma de embudo) que
evitan la pérdida de iones durante la transmisión iónica, aumentando de esta
forma la sensibilidad.
Figura 22. Esquema detallado de las partes de las que consta un espectrómetro de masasQTOF.
El analizador QTOF, con la posibilidad de hacer MS/MS, añade un tercer nivel de
información, que se une a la exactitud de masas y a la distribución isotópica,
para llevar a cabo la identificación de compuestos.
Introducción
104
Las principales especificaciones de este analizador son un rango de masas de
50 20.000 m/z, una resolución de entre 17.500 y 20.000 FWHM, la posibilidad
de llevar acabo análisis MS/MS, y una exactitud de 3 ppm con calibración
interna y 5 ppm con calibración externa tanto de iones precursores como de
fragmentos.
BIBLIOGRAFÍA
Bibliografía
107
[1] I.S. Arvanitoyannis, M. Van Houwelingen Koukaliaroglou; Functional foods: Asurvey of health claims, pros and cons, and current legislation, Crit. Rev. FoodSci. Nutr. 45 (2005) 385 404.
[2] A. Diplock, P. Aggett, M. Ashwell, F. Bornet, E. Fern, M. Roberfroid; Scientific
concepts of functional foods in Europe consensus document, Br. J. Nutr. 81
(1999) S1 S27.
[3] S. DeFelice; The nutraceutical revolution: its Impact on food industry R&D,
Trends Food Sci. Technol. 6 (1995) 59 61.
[4] S. Barnes, J. Prasain; Current progress in the use of traditional medicines and
nutraceuticals, Curr. Opin. Plant Biol. 8 (2005) 324 328.
[5] M.B. Roberfroid; What is beneficial for health? The concept of functional
food, Food Chem. Toxicol. 37 (1999) 1039 1041.
[6] M.E. Pascual, K. Slowing, E. Carretero, D. Sánchez Mata, A. Villar; Lippia:
traditional uses, chemistry and pharmacology: a review, J. Ethnopharmacol. 76
(2001) 201 214.
[7] C.A. Newall, L.A. Anderson, J.D. Phillipson; Herbal medicines. A guide for
health care professionals, The Pharmaceutical Press, London (1996).
[8] A. Carnat, A.P. Carnat, D. Fraisse, J.L. Lamaison; The aromatic and
polyphenolic composition of lemon verbena tea, Fitoterapia 70 (1999) 44 49.
[9] N. Kim, D. Lee; Headspace solid phase microextraction for characterization
of fragrances of lemon verbena (Aloysia triphylla) by gas chromatography mass
spectrometry, J. Sep. Sci. 27 (2004) 96 100.
[10] A.R. Bilia, M. Giomi, M. Innocenti, S. Gallori, F.F. Vincieri; HPLC DAD ESI MS
analysis of the constituents of aqueous preparations of verbena and lemon
verbena and evaluation of the antioxidant activity, J. Pharm. Biomed. Anal. 46
(2008) 463 470.
Bibliografía
108
[11] P. Valentão, P.B. Andrade, F. Areias, F. Ferreres, R.M. Seabra; Analysis of
vervain flavonoids by HPLC/diode array detector method. Its application to
quality control, J. Agric. Food Chem. 47 (1999) 4579 4582.
[12] O. Benavente García, J. Castillo, J. Lorente, A. Ortuño, J. Del Río;
Antioxidant activity of phenolics extracted from Olea europaea L. leaves, Food
Chem. 68 (2000) 457 462.
[13] M. Laguerre, L.J.L. Giraldo, G. Piombo, M.C. Figueroa Espinoza, M. Pina, M.
Benaissa, A. Combe, A.R. Castera, J. Lecomte, P. Villeneuve; Characterization of
olive leaf phenolics by ESI MS and evaluation of their antioxidant capacities by
the CAT assay, J. Am. Oil Chem. Soc. 86 (2009) 1215 1225.
[14] S. Fu, D. Arráez Román, A. Segura Carretero, J.A. Menéndez, M.P.
Menéndez Gutiérrez, V. Micol, A. Fernández Gutiérrez; Qualitative screening of
phenolic compounds in olive leaf extracts by hyphenated liquid chromatography
and preliminary evaluation of cytotoxic activity against human breast cancer
cells, Anal. Bioanal. Chem. 397 (2010) 643 654.
[15] J. Meirinhos, B.M. Silva, P. Valentão, R.M. Seabra, J.A. Pereira, A. Dias, P.B.
Andrade, F. Ferreres; Analysis and quantification of flavonoidic compounds from
Portuguese olive (Olea europaea L.) leaf cultivars, Nat. Prod. Res. 19 (2005) 189
195.
[16] L. Bravo; Polyphenols: chemistry, dietary sources, metabolism, and
nutritional significance, Nutr. Rev. 56 (1998) 317 333.
[17] U. Peters, C. Poole, L. Arab; Does tea affect cardiovascular disease? A meta
analysis, Am. J. Epidemiol. 154 (2001) 495 503.
[18] G. De Gaetano, H.S. Rotondo; The relationship between wine consumption
and vascular risk: from epidemiological evidence to biological plausibility, Arch.
Hellenic Med. 17 (2000) A57 A63.
Bibliografía
109
[19] M. Hertog, D. Kromhout, C. Aravanis, H. Blackburn, R. Buzina, F. Fidanza, S.
Giampaoli, A. Jansen, A. Menotti, S. Nedeljkovic, M. Pekkarinen, B. Simic, H.
Toshima, E. Feskens, P. Hollman, M. Katan; Flavonoid intake and long term risk
of coronary heart disease and cancer in the 7 Countries Study, Arch. Intern.
Med. 155 (1995) 381 386.
[20] P. Knekt, R. Jarvinen, A. Reunanen, J. Maatela; Flavonoid intake and
coronary mortality in Finland: a cohort study, Br. Med. J. 312 (1996) 478 481.
[21] I. Arts, P. Hollman, E. Feskens, H. de Mesquita, D. Kromhout; Catechin
intake might explain the inverse relation between tea consumption and
ischemic heart disease: the Zutphen Elderly Study, Am. J. Clin. Nutr. 74 (2001)
227 232.
[22] J. Orgogozo, J. Dartigues, S. Lafont, L. Letenneur, D. Commenges, R.
Salamon, S. Renaud, M. Breteler; Wine consumption and dementia in the
elderly: a prospective community study in the Bordeaux area, Rev. Neurol. 153
(1997) 185 192.
[23] J. Lindsay, D. Laurin, R. Verreault, R. Hebert, B. Helliwell, G. Hill, I.
McDowell; Risk factors for Alzheimer's disease: a prospective analysis from the
Canadian Study of Health and Aging, Am. J. Epidemiol. 156 (2002) 445 453.
[24] Q. Dai, A.R. Borenstein, Y. Wu, J.C. Jackson, E.B. Larson; Fruit and vegetable
juices and Alzheimer's disease: the Kame Project, Am. J. Med. 119 (2006) 751
759.
[25] D. Commenges, V. Scotet, S. Renaud, H. Jacqmin Gadda, P. Barberger
Gateau, J. Dartigues; Intake of flavonoids and risk of dementia, Eur. J. Epidemiol.
16 (2000) 357 363.
Bibliografía
110
[26] H. Checkoway, K. Powers, T. Smith Weller, G. Franklin, W. Longstreth, P.
Swanson; Parkinson's disease risks associated with cigarette smoking, alcohol
consumption, and caffeine intake, Am. J. Epidemiol. 155 (2002) 732 738.
[27] L. Letenneur, C. Proust Lima, A. Le Gouge, J.F. Dartigues, P. Barberger
Gateau; Flavonoid intake and cognitive decline over a 10 year period, Am. J.
Epidemiol. 165 (2007) 1364 1371.
[28] R. van Dam, E. Feskens; Coffee consumption and risk of type 2 diabetes
mellitus, Lancet 360 (2002) 1477 1478.
[29] S. Franceschi, M. Parpinel, C. La Vecchia, A. Favero, R. Talamini, E. Negri;
Role of different types of vegetables and fruit in the prevention of cancer of the
colon, rectum, and breast, Epidemiology 9 (1998) 338 341.
[30] C. La Vecchia, L. Chatenoud, S. Franceschi, M. Soler, F. Parazzini, E. Negri;
Vegetables and fruit and human cancer: update of an Italian study, Int. J. Cancer
82 (1999) 151 152.
[31] X. Han, T. Shen, H. Lou; Dietary polyphenols and their biological
significance, Int. J. Mol. Sci. 8 (2007) 950 988.
[32] A. Scalbert, C. Manach, C. Morand, C. Rémésy, L. Jiménez; Dietary
polyphenols and the prevention of diseases, Crit. Rev. Food Sci. Nutr. 45 (2005)
287 306.
[33] R. Leenen, A. Roodenburg, L. Tijburg, S. Wiseman; A single dose of tea with
or without milk increases plasma antioxidant activity in humans, Eur. J. Clin.
Nutr. 54 (2000) 87 92.
[34] G. Cao, R. Russell, N. Lischner, R. Prior; Serum antioxidant capacity is
increased by consumption of strawberries, spinach, red wine or vitamin C in
elderly women, J. Nutr. 128 (1998) 2383 2390.
Bibliografía
111
[35] A. Ghiselli, F. Natella, A. Guidi, L. Montanari, P. Fantozzi, C. Scaccini; Beer
increases plasma antioxidant capacity in humans, J. Nutr. Biochem. 11 (2000)
76 80.
[36] K. Nakagawa, M. Ninomiya, T. Okubo, N. Aoi, L. Juneja, M. Kim, K.
Yamanaka, T. Miyazawa; Tea catechin supplementation increases antioxidant
capacity and prevents phospholipid hydroperoxidation in plasma of humans, J.
Agric. Food Chem. 47 (1999) 3967 3973.
[37] J. Young, S. Nielsen, J. Haraldsdottir, B. Daneshvar, S. Lauridsen, P.
Knuthsen, A. Crozier, B. Sandstrom, L. Dragsted; Effect of fruit juice intake on
urinary quercetin excretion and biomarkers of antioxidative status, Am. J. Clin.
Nutr. 69 (1999) 87 94.
[38] D. Rein, S. Lotito, R. Holt, C. Keen, H. Schmitz, C. Fraga; Epicatechin in
human plasma: in vivo determination and effect of chocolate consumption on
plasma oxidation status, J. Nutr. 130 (2000) 2109S 2114S.
[39] S. Shirahata, H. Murakami, K. Nishiyama, K. Yamada, G. Nonaka, I. Nishioka,
H. Omura; Dna breakage by flavan 3 ols and procyanidins in the presence of
cupric ion, J. Agric. Food Chem. 37 (1989) 299 303.
[40] R. Vance, R. Teel; Effect of tannic acid on rat liver S9 mediated mutagenesis,
metabolism and DNA binding of benzo[a]pyrene, Cancer Lett. 47 (1989) 37 44.
[41] C. Casalini, M. Lodovici, C. Briani, G. Paganelli, S. Remy, V. Cheynier, P.
Dolara; Effect of complex polyphenols and tannins from red wine (WCPT) on
chemically induced oxidative DNA damage in the rat, Eur. J. Nutr. 38 (1999) 190
195.
[42] M. Lodovici, C. Casalini, C. De Filippo, E. Copeland, X. Xu, M. Clifford, P.
Dolara; Inhibition of 1,2 dimethylhydrazine induced oxidative DNA damage in
Bibliografía
112
rat colon mucosa by black tea complex polyphenols, Food Chem. Toxicol. 38
(2000) 1085 1088.
[43] S. Boyle, V. Dobson, S. Duthie, J. Kyle, A. Collins, Absorption and DNA
protective effects of flavonoid glycosides from an onion meal, Eur. J. Nutr. 39
(2000) 213 223.
[44] L. Funes, L. Carrera Quintanar, M. Cerdán Calero, M.D. Ferrer, F. Drobnic,
A. Pons, E. Roche, V. Micol; Effect of lemon verbena supplementation on
muscular damage markers, proinflammatory cytokines release and neutrophils'
oxidative stress in chronic exercise, Eur. J. Appl. Physiol. 111 (2011) 695 705.
[45] N. Doronicheva, H. Yasui, H. Sakurai; Chemical structure dependent
differential effects of flavonoids on the catalase activity as evaluated by a
chemiluminescent method, Biol. Pharm. Bull. 30 (2007) 213 217.
[46] L. Carrera Quintanar, L. Funes, E. Viudes, J. Tur, V. Micol, E. Roche, A. Pons;
Effect of lemon verbena supplementation on muscular damage markers,
proinflammatory cytokines release and neutrophils’ oxidative stress in chronic
exercise, Scand J Med Sci Sports. IN PRESS (2010) doi: 10.1111/j.1600
0838.2010.01244.x.
[47] M.S. Fernández Pachón, G. Berna, E. Otaolaurruchi, A.M. Troncoso, F.
Martín, M.C. García Parrilla; Changes in antioxidant endogenous enzymes
(activity and gene expression levels) after repeated red wine intake, J. Agric.
Food Chem. 57 (2009) 6578 6583.
[48] N.M. Aymoto Hassimotto, M.D.S. Pinto, F.M. Lajolo; Antioxidant status in
humans after consumption of blackberry (Rubus fruticosus L.) juices with and
without defatted milk, J. Agric. Food Chem. 56 (2008) 11727 11733.
[49] H. Jemai, I. Fki, M. Bouaziz, Z. Bouallagui, A. El Feki, H. Isoda, S. Sayadi;
Lipid lowering and antioxidant effects of hydroxytyrosol and its triacetylated
Bibliografía
113
derivative recovered from olive tree leaves in cholesterol fed rats, J. Agric. Food
Chem. 56 (2008) 2630 2636.
[50] C. Yeh, G. Yen; Induction of hepatic antioxidant enzymes by phenolic acids
in rats is accompanied by increased levels of multidrug resistance associated
protein 3 mRNA expression, J. Nutr. 136 (2006) 11 15.
[51] H. Kim, K. Son, H. Chang, S. Kang; Anti inflammatory plant flavonoids and
cellular action mechanisms, J. Pharmacol. Sci. 96 (2004) 229 245.
[52] A.M. Díaz, M.J. Abad, L. Fernández, A.M. Silván, J. De Santos, P. Bermejo;
Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro anti
inflammatory activity, Life Sci. 74 (2004) 2515 2526.
[53] J. Lee, E. Woo, K. Kang; Inhibition of lipopolysaccharide inducible nitric
oxide synthase expression by acteoside through blocking of AP 1 activation, J.
Ethnopharmacol. 97 (2005) 561 566.
[54] Y. Akcos, N. Ezer, I. Calis, R. Demirdamar, B. Tel; Polyphenolic compounds of
Sideritis lycia and their anti inflammatory activity, Pharm. Biol. 37 (1999) 118
122.
[55] M. Deepak, S.S. Handa; Antiinflammatory activity and chemical composition
of extracts of Verbena officinalis, Phytother. Res. 14 (2000) 463 465.
[56] D. Feskanich, R. Ziegler, D. Michaud, E. Giovannucci, F. Speizer, W. Willett,
G. Colditz; Prospective study of fruit and vegetable consumption and risk of lung
cancer among men and women, J. Natl. Cancer Inst. 92 (2000).
[57] V. Benetou, P. Orfanos, P. Lagiou, D. Trichopoulos, P. Boffetta, A.
Trichopoulou; Vegetables and fruits in relation to cancer risk: evidence from the
Greek EPIC cohort study, Cancer Epidemiol. Biomarkers Prev. 17 (2008) 387 392.
Bibliografía
114
[58] A. Tavani, C. La Vecchia; Coffee, decaffeinated coffee, tea and cancer of the
colon and rectum: a review of epidemiological studies, 1990 2003, Cancer
Cause. Control 15 (2004) 743 757.
[59] L. Su, L. Arab; Tea consumption and the reduced risk of colon cancer
results from a national prospective cohort study, Public Health Nutr. 5 (2002)
419 425.
[60] C. Sun, J. Yuan, M. Lee, C. Yang, Y. Gao, R. Ross, M. Yu; Urinary tea
polyphenols in relation to gastric and esophageal cancers: a prospective study
of men in Shanghai, China, Carcinogenesis 23 (2002) 1497 1503.
[61] W. Blot, J. McLaughlin, W. Chow; Cancer rates among drinkers of black tea,
Crit. Rev. Food Sci. Nutr. 37 (1997) 739 760.
[62] C. Yang, P. Maliakal, X. Meng; Inhibition of carcinogenesis by tea, Annu.
Rev. Pharmacol. Toxicol. 42 (2002) 25 54.
[63] E. Middleton, C. Kandaswami, T. Theoharides; The effects of plant
flavonoids on mammalian cells: implications for inflammation, heart disease,
and cancer, Pharmacol. Rev. 52 (2000) 673 751.
[64] S. Duthie, V. Dobson; Dietary flavonoids protect human colonocyte DNA
from oxidative attack in vitro, Eur. J. Nutr. 38 (1999) 28 34.
[65] M. Calomme, L. Pieters, A. Vlietinck, D. Vanden Berghe; Inhibition of
bacterial mutagenesis by Citrus flavonoids, Planta Med. 62 (1996) 222 226.
[66] B. Plaumann, M. Fritsche, H. Rimpler, G. Brandner, R. Hess; Flavonoids
activate wild type p53, Oncogene 13 (1996) 1605 1614.
[67] M. van Erk, P. Roepman, T. van der Lende, R. Stierum, J. Aarts, P. van
Bladeren, B. van Ommen; Integrated assessment by multiple gene expression
analysis of quercetin bioactivity on anticancer related mechanisms in colon
cancer cells in vitro, Eur. J. Nutr. 44 (2005) 143 156.
Bibliografía
115
[68] C. Yang, J. Landau, M. Huang, H. Newmark; Inhibition of carcinogenesis by
dietary polyphenolic compounds, Annu. Rev. Nutr. 21 (2001) 381 406.
[69] W. Khan, Z. Wang, M. Athar, D. Bickers, H. Mukhtar; Inhibition of the skin
tumorigenicity of (+/ ) 7 ,8 dihydroxy 9 ,10 epoxy 7,8,9,10
tetrahydrobenzo[a]pyrene by tannic acid, green tea polyphenols and quercetin
in Sencar mice, Cancer Lett. 42 (1988) 7 12.
[70] M. Takada, Y. Nakamura, T. Koizumi, H. Toyama, T. Kamigaki, Y. Suzuki, Y.
Takeyama, Y. Kuroda; Suppression of human pancreatic carcinoma cell growth
and invasion by epigallocatechin 3 gallate, Pancreas 25 (2002) 45 48.
[71] M. Inoue, K. Tajima, M. Mizutani, H. Iwata, T. Iwase, S. Miura, K. Hirose, N.
Hamajima, S. Tominaga; Regular consumption of green tea and the risk of breast
cancer recurrence: follow up study from the Hospital based Epidemiologic
Research Program at Aichi Cancer Center (HERPACC), Japan, Cancer Lett. 167
(2001) 175 182.
[72] M. Takada, Y. Ku, K. Habara, T. Ajiki, Y. Suzuki, Y. Kuroda; Inhibitory effect of
epigallocatechin 3 gallate on growth and invasion in human biliary tract
carcinoma cells, World J. Surg. 26 (2002) 683 686.
[73] K.M. Rieger Christ, R. Harley, C. Loclowsky, T. Bernier, P. Vemulapalli, M.
Roth, J. Kim, A.S. Yee, S.M. Le, P.J. Marie, J.A. Libertino, I.C. Surnmerhayes; The
green tea compound, ( ) epigallocatechin 3 gallate downregulates N cadherin
and suppresses migration of bladder carcinoma cells, J. Cell. Biochem. 102
(2007) 377 388.
[74] H. Leong, P.S. Mathur, G.L. Greene; Inhibition of mammary tumorigenesis
in the C3(1)/SV40 mouse model by green tea, Breast Cancer Res. Treat. 107
(2008) 359 369.
Bibliografía
116
[75] C.A. Larsen, R.H. Dashwood; Suppression of Met activation in human colon
cancer cells treated with ( ) epigallocatechin 3 gallate: minor role of hydrogen
peroxide, Biochem. Biophys. Res. Commun. 389 (2009) 527 530.
[76] N. Khan, F. Afaq, M. Saleem, N. Ahmad, H. Mukhtar; Targeting multiple
signaling pathways by green tea polyphenol ( ) epigallocatechin 3 gallate,
Cancer Res. 66 (2006) 2500 2505.
[77] C. Oliveras Ferraros, S. Fernández Arroyo, A. Vázquez Martín, J. Lozano
Sánchez, S. Cufi, J. Joven, V. Micol, A. Fernández Gutiérrez, A. Segura Carretero,
J.A. Menéndez; Crude phenolic extracts from extra virgin olive oil circumvent de
novo breast cancer resistance to HER1/HER2 targeting drugs by inducing
GADD45 sensed cellular stress, G2/M arrest and hyperacetylation of Histone H3,
Int. J. Oncol. 38 (2011) 1533 1547.
[78] S. Fernández Arroyo, A. Gómez Martínez, L. Rocamora Reverte, R.
Quirantes Piné, A. Segura Carretero, A. Fernández Gutiérrez, J.A. Ferragut;
Application of nanoLC ESI TOF MS for the metabolomic analysis of phenolic
compounds from extra virgin olive oil in treated colon cancer cells; J. Pharm.
Biomed. Anal. 63 (2012) 128 134.
[79] J. Han, T.P.N. Talorete, P. Yamada, H. Isoda; Anti proliferative and apoptotic
effects of oleuropein and hydroxytyrosol on human breast cancer MCF 7 cells,
Cytotechnology 59 (2009) 45 53.
[80] V. Goulas, V. Exarchou, A.N. Troganis, E. Psomiadou, T. Fotsis, E. Briasoulis,
I.P. Gerothanassis; Phytochemicals in olive leaf extracts and their
antiproliferative activity against cancer and endothelial cells, Mol. Nutr. Food
Res. 53 (2009) 600 608.
[81] Z. Bouallagui, J. Han, H. Isoda, S. Sayadi; Hydroxytyrosol rich extract from
olive leaves modulates cell cycle progression in MCF 7 human breast cancer
cells, Food Chem. Toxicol. 49 (2011) 179 184.
Bibliografía
117
[82] A. Crozier, D. Del Río, M.N. Clifford; Bioavailability of dietary flavonoids and
phenolic compounds, Mol. Aspects Med. 31 (2010) 446 467.
[83] A. Scalbert, C. Morand, C. Manach, C. Rémésy; Absorption and metabolism
of polyphenols in the gut and impact on health, Biomed. Pharmacother. 56
(2002) 276 282.
[84] S. Oliver, M. Winson, D. Kell, F. Baganz; Systematic functional analysis of
the yeast genome, Trends Biotechnol. 16 (1998) 373 378.
[85] O. Fiehn; Combining genomics, metabolome analysis, and biochemical
modelling to understand metabolic networks, Comp. Funct. Genomics 2 (2001)
155 168.
[86] S. Villas Bôas, S. Rasmussen, G. Lane; Metabolomics or metabolite
profiles?, Trends Biotechnol. 23 (2005) 385 386.
[87] A. Fernández Gutiérrez; De Monardes y Tswett a la bioanalítica, Discurso de
ingreso en la Academia de Ciencias Matemáticas, Físico Químicas y Naturales de
Granada (2011).
[88] O. Fiehn; Metabolomics the link between genotypes and phenotypes,
Plant Mol. Biol. 48 (2002) 155 171.
[89] S. Villas Bôas, S. Mas, M. Akesson, J. Smedsgaard, J. Nielsen; Mass
spectrometry in metabolome analysis, Mass Spectrom. Rev. 24 (2005) 613 646.
[90] C. Manach, J. Hubert, R. Llorach, A. Scalbert; The complex links between
dietary phytochemicals and human health deciphered by metabolomics, Mol.
Nutr. Food Res. 53 (2009) 1303 1315.
[91] S. Moco, R.J. Bino, O. Vorst, H.A. Verhoeven, J. De Groot, T.A. Van Beek, J.
Vervoort, C.H. Ric De Vos; A liquid chromatography mass spectrometry based
metabolome database for tomato, Plant Physiol. 141 (2006) 1205 1218.
Bibliografía
118
[92] A. Fardet, R. Llorach, A. Orsoni, C. Martin, E. Pujos Guillot, C. Lapierre, A.
Scalbert; Metabolomics provide new insight on the metabolism of dietary
phytochemicals in rats, J. Nutr. 138 (2008) 1282 1287.
[93] M. Urpi Sarda, M. Monagas, N. Khan, R. Llorach, R.M. Lamuela Raventós, O.
Jáuregui, R. Estruch, M. Izquierdo Pulido, C. Andrés Lacueva; Targeted
metabolic profiling of phenolics in urine and plasma after regular consumption
of cocoa by liquid chromatography tandem mass spectrometry, J. Chromatogr.
A 1216 (2009) 7258 7267.
[94] F.A. Van Dorsten, C.A. Daykin, T.P.J. Mulder, J.P.M. Van Duynhoven;
Metabonomics approach to determine metabolic differences between green
tea and black tea consumption, J. Agric. Food Chem. 54 (2006) 6929 6938.
[95] C.H. Grun, F.A. van Dorsten, D.M. Jacobs, M. Le Belleguic, E.J.J. van Velzen,
M.O. Bingham, H. Janssen, J.P.M. van Duynhoven; GC MS methods for
metabolic profiling of microbial fermentation products of dietary polyphenols in
human and in vitro intervention studies, J. Chromatogr. B 871 (2008) 212 219.
[96] Y. Wang, H. Tang, J. Nicholson, P. Hylands, J. Sampson, E. Holmes; A
metabonomic strategy for the detection of the metabolic effects of chamomile
(Matricaria recutita L.) ingestion, J. Agric. Food Chem. 53 (2005) 191 196.
[97] M. Naczk, F. Shahidi; Phenolics in cereals, fruits and vegetables: occurrence,
extraction and analysis, J. Pharm. Biomed. Anal. 41 (2006) 1523 1542.
[98] K. Robards; Strategies for the determination of bioactive phenols in plants,
fruit and vegetables, J. Chromatogr. A 1000 (2003) 657 691.
[99] A. Papadopoulou, R. Frazier; Characterization of protein polyphenol
interactions, Trends Food Sci. Technol. 15 (2004) 186 190.
[100] E. Want, G. O'Maille, C. Smith, T. Brandon, W. Uritboonthai, C. Qin, S.
Trauger, G. Siuzdak; Solvent dependent metabolite distribution, clustering, and
Bibliografía
119
protein extraction for serum profiling with mass spectrometry, Anal. Chem. 78
(2006) 743 752.
[101] S.J. Bruce, P. Jonsson, H. Antti, O. Cloarec, J. Trygg, S.L. Marklund, T.
Moritz; Evaluation of a protocol for metabolic profiling studies on human blood
plasma by combined ultra performance liquid chromatography/mass
spectrometry: from extraction to data analysis, Anal. Biochem. 372 (2008) 237
249.
[102] K. Dettmer, P.A. Aronov, B.D. Hammock; Mass spectrometry based
metabolomics, Mass Spectrom. Rev. 26 (2007) 51 78.
[103] M. Gonthier, J. Donovan, O. Texier, C. Felgines, C. Rémésy, A. Scalbert;
Metabolism of dietary procyanidins in rats, Free Radic. Biol. Med. 35 (2003) 837
844.
[104] L.I. Mennen, D. Sapinho, H. Ito, S. Bertrais, P. Galan, S. Hercberg, A.
Scalbert; Urinary flavonoids and phenolic acids as biomarkers of intake for
polyphenol rich foods, Br. J. Nutr. 96 (2006) 191 198.
[105] M.T. Díez, P. García del Moral, J.A. Resines, M.J. Arín; Determination of
phenolic compounds derived from hydrolysable tannins in biological matrices by
RP HPLC, J. Sep. Sci. 31 (2008) 2797 2803.
[106] R. García Villalba, A. Carrasco Pancorbo, E. Nevedomskaya, O.A.
Mayboroda, A.M. Deelder, A. Segura Carretero, A. Fernández Gutiérrez;
Exploratory analysis of human urine by LC ESI TOF MS after high intake of olive
oil: understanding the metabolism of polyphenols, Anal. Bioanal. Chem. 398
(2010) 463 475.
[107] E. Azzini, P. Vitaglione, F. Intorre, A. Napolitano, A. Durazzo, M.S. Foddai,
A. Fumagalli, G. Catasta, L. Rossi, E. Venneria, A. Raguzzini, L. Palomba, V.
Bibliografía
120
Fogliano, G. Maiani; Bioavailability of strawberry antioxidants in human
subjects, Br. J. Nutr. 104 (2010) 1165 1173.
[108] S. Shahrzad, I. Bitsch; Determination of gallic acid and its metabolites in
human plasma and urine by high performance liquid chromatography, J.
Chromatogr. B 705 (1998) 87 95.
[109] Y. Liu, X. Li, Y. Li, L. Wang, M. Xue; Simultaneous determination of
danshensu, rosmarinic acid, cryptotanshinone, tanshinone IIA, tanshinone I and
dihydrotanshinone I by liquid chromatographic mass spectrometry and the
application to pharmacokinetics in rats, J. Pharm. Biomed. Anal. 53 (2010) 698
704.
[110] L. Ríos, M. Gonthier, C. Rémésy, L. Mila, C. Lapierre, S. Lazarus, G.
Williamson, A. Scalbert; Chocolate intake increases urinary excretion of
polyphenol derived phenolic acids in healthy human subjects, Am. J. Clin. Nutr.
77 (2003) 912 918.
[111] R.L. Prior, T.R. Rogers, R.C. Khanal, S.E. Wilkes, X. Wu, L.R. Howard;
Urinary excretion of phenolic acids in rats fed cranberry, J. Agric. Food Chem. 58
(2010) 3940 3949.
[112] A. Tsarbopoulos, E. Gikas, N. Papadopoulos, N. Aligiannis, A. Kafatos;
Simultaneous determination of oleuropein and its metabolites in plasma by
high performance liquid chromatography, J. Chromatogr. B 785 (2003) 157 164.
[113] V. Ruiz Gutiérrez, M. Juan, A. Cert, J. Planas; Determination of
hydroxytyrosol in plasma by HPLC, Anal. Chem. 72 (2000) 4458 4461.
[114] K. Hosoda, T. Furuta, A. Yokokawa, K. Ogura, A. Hiratsuka, K. Ishii; Plasma
profiling of intact isoflavone metabolites by high performance liquid
chromatography and mass spectrometric identification of flavone glycosides
Bibliografía
121
daidzin and genistin in human plasma after administration of kinako, Drug
Metab. Disposition 36 (2008) 1485 1495.
[115] M. Suárez, M. Romero, A. Maciá, R.M. Valls, S. Fernández, R. Soia, M.
Motilva; Improved method for identifying and quantifying olive oil phenolic
compounds and their metabolites in human plasma by microelution solid phase
extraction plate and liquid chromatography tandem mass spectrometry, J.
Chromatogr. B 877 (2009) 4097 4106.
[116] A. Fernández Gu érrez, A. Segura Carretero, A. Carrasco Pancorbo;
Fundamentos teóricos y modos de separación, en: A. Segura Carretero y A.
Fernández Gu érrez (Eds.), Electroforesis Capilar: aproximación según la técnica
de detección, 1ª ed., Ed. Universidad de Granada, España, (2005) 11 54.
[117] A. Carrasco Pancorbo, D. Arráez Román, A. Segura Carretero, A.
Fernández Gutiérrez; Capillary electrophoresis electrospray ionization mass
spectrometry method to determine the phenolic fraction of extra virgin olive
oil, Electrophoresis 27 (2006) 2182 2196.
[118] J. Chen, S. Li, P. Li, Y. Song, X. Chai, D. Ma; Qualitative and quantitative
analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis
coupled with solid phase extraction, J. Sep. Sci. 28 (2005) 365 372.
[119] L. Arce, M. Tena, A. Ríos, M. Valcárcel; Determination of trans resveratrol
and other polyphenols in wines by a continuous flow sample clean up system
followed by capillary electrophoresis separation, Anal. Chim. Acta 359 (1998)
27 38.
[120] L. Arce, A. Ríos, M. Valcárcel; Determination of anti carcinogenic
polyphenols present in green tea using capillary electrophoresis coupled to a
flow injection system, J. Chromatogr. A 827 (1998) 113 120.
Bibliografía
122
[121] Y. Peng, F. Liu, Y. Peng, J. Ye; Determination of polyphenols in apple juice
and cider by capillary electrophoresis with electrochemical detection, Food
Chem. 92 (2005) 169 175.
[122] S. Wang, J. Zhang, X. Chen, Z. Hu; Study of the electrophoretic behaviour
of flavonoids, Chromatographia 59 (2004) 507 511.
[123] Z. Demianova, H. Siren, R. Kuldvee, M. Riekkola; Nonaqueous capillary
electrophoretic separation of polyphenolic compounds in wine using coated
capillaries at high pH in methanol, Electrophoresis 24 (2003) 4264 4271.
[124] G. Desmet, D. Cabooter, P. Gzil, H. Verelst, D. Mangelings, Y. Vander
Heyden, D. Clicq; Future of high pressure liquid chromatography: Do we need
porosity or do we need pressure?, J. Chromatogr. A 1130 (2006) 158 166.
[125] R. Prior, L. Gu; Occurrence and biological significance of
proanthocyanidins in the American diet, Phytochemistry 66 (2005) 2264 2280.
[126] N. Vanbeneden, F. Delvaux, F.R. Delvaux; Determination of
hydroxycinnamic acids and volatile phenols in wort and beer by isocratic high
performance liquid chromatography using electrochemical detection, J.
Chromatogr. A 1136 (2006) 237 242.
[127] M. Iranshahi, Y. Amanzadeh; Rapid isocratic HPLC analysis of caffeic acid
derivatives from Echinacea purpurea cultivated in Iran, Chem. Nat. Comp. 44
(2008) 190 193.
[128] J.M. Harnly, S. Bhagwat, L. Lin; Profiling methods for the determination of
phenolic compounds in foods and dietary supplements, Anal. Bioanal. Chem.
389 (2007) 47 61.
[129] L. Rehova, V. Skerikova, P. Jandera; Optimisation of gradient HPLC analysis
of phenolic compounds and flavonoids in beer using a CoulArray detector, J.
Sep. Sci. 27 (2004) 1345 1359.
Bibliografía
123
[130] F. Cuyckens, M. Claeys; Optimization of a liquid chromatography method
based on simultaneous electrospray ionization mass spectrometric and
ultraviolet photodiode array detection for analysis of flavonoid glycosides, Rapid
Commun. Mass Spectrom. 16 (2002) 2341 2348.
[131] C. Cavaliere, P. Foglia, R. Gubbio , P. Sacche , R. Samperi, A. Laganà,
Rapid resolution liquid chromatography/mass spectrometry for determination
and quantitation of polyphenols in grape berries., Rapid Commun. Mass
Spectrom. 22 (2008) 3089 3099.
[132] J. Castro Pérez, R. Plumb, J.H. Granger, I. Beattie, K. Joncour, A. Wright;
Increasing throughput and information content for in vitro drug metabolism
experiments using ultra performance liquid chromatography coupled to a
quadrupole time of flight mass spectrometer, Rapid Commun. Mass Spectrom.
19 (2005) 843 848.
[133] A. Serra, A. Maciá, M. Romero, M. Salvado, M. Bustos, J. Fernández
Larrea, M. Motilva; Determination of procyanidins and their metabolites in
plasma samples by improved liquid chromatography tandem mass
spectrometry, J. Chromatogr. B 877 (2009) 1169 1176.
[134] M. Gómez Romero, A. Segura Carretero, A. Fernández Gutiérrez;
Metabolite profiling and quantification of phenolic compounds in methanol
extracts of tomato fruit, Phytochemistry 71 (2010) 1848 1864.
[135] K. Karlsson, M. Novotny; Separation efficiency of slurry packed liquid
chromatography microcolumns with very small inner diameters, Anal. Chem. 60
(1988) 1662 1665.
[136] J. Vissers; Recent developments in microcolumn liquid chromatography, J.
Chromatogr. A 856 (1999) 117 143.
Bibliografía
124
[137] S. Hsieh, J. Jorgenson; Preparation and evaluation of slurry packed liquid
chromatography microcolumns with inner diameters from 12 to 33 mu m, Anal.
Chem. 68 (1996) 1212 1217.
[138] J. Hernández Borges, Z. Aturki, A. Rocco, S. Fanali; Recent applications in
nanoliquid chromatography, J. Sep. Sci. 30 (2007) 1589 1610.
[139] Y. Ishihama; Proteomic LC MS systems using nanoscale liquid
chromatography with tandem mass spectrometry, J. Chromatogr. A 1067 (2005)
73 83.
[140] S. Fanali, Z. Aturki, G. D'Orazio, A. Rocco; Separation of basic compounds
of pharmaceutical interest by using nano liquid chromatography coupled with
mass spectrometry, J. Chromatogr. A 1150 (2007) 252 258.
[141] A. Rocco, S. Fanali; Analysis of phytosterols in extra virgin olive oil by
nano liquid chromatography, J. Chromatogr. A 1216 (2009) 7173 7178
[142] R. García Villalba, A. Carrasco Pancorbo, G. Zurek, M. Behrens, C.
Baessmann, A. Segura Carretero, A. Fernández Gutiérrez; Nano and rapid
resolution liquid chromatography electrospray ionization time of flight mass
spectrometry to identify and quantify phenolic compounds in olive oil, J. Sep.
Sci. 33 (2010) 2069 2078.
[143] K. Buonasera, G. D'Orazio, S. Fanali, P. Dugo, L. Mondello; Separation of
organophosphorus pesticides by using nano liquid chromatography, J.
Chromatogr. A 1216 (2009) 3970 3976.
[144] G. D'Orazio, Z. Aturki, M. Cristalli, M. Quaglia, S. Fanali; Use of vancomycin
chiral stationary phase for the enantiomeric resolution of basic and acidic
compounds nano liquid chromatography, J. Chromatogr. A 1081 (2005) 105
113.
Bibliografía
125
[145] G. D'Orazio, A. Cifuentes, S. Fanali; Chiral nano liquid chromatography
mass to amino acids analysis for orange spectrometry applied juice profiling,
Food Chem. 108 (2008) 1114 1121.
[146] C. Fanali, L. Dugo, G. D'Orazio, M. Lirangi, M. Dacha, P. Dugo, L. Mondello;
Analysis of anthocyanins in commercial fruit juices by using nano liquid
chromatography electrospray mass spectrometry and high performance liquid
chromatography with UV vis detector, J. Sep. Sci. 34 (2011) 150 159.
[147] C. Fanali, A. Rocco, Z. Aturki, L. Mondello, S. Fanali; Analysis of
polyphenols and methylxantines in tea samples by means of nano liquid
chromatography utilizing capillary columns packed with core shell particles, J.
Chromatogr. A 1234 (2012) 38 44.
[148] S. Fanali, Z. Aturki, G. D'Orazio, A. Rocco, A. Ferranti, L. Mercolini, M.A.
Raggi; Analysis of aloe based phytotherapeutic products by using nano LC MS, J.
Sep. Sci. 33 (2010) 2663 2670.
[149] J.T. Watson, O.D. Sparkman; Introduction to Mass Spectrometry, John
Wiley & Sons, Inc., New York (2007).
[150] C. Simó, A. Cifuentes, Electroforesis Capilar: Detección mediante
espectrometría de masas, en: A. Segura Carretero y A. Fernández Gu érrez
(Eds.), Electroforesis Capilar: Aproximación Según La Técnica De Detección, 1ª
ed., Ed. Universidad de Granada, España (2005) 409 438.
[151] R. Martin Smith; Instrumentation, in: Busch, K.L. (Ed.), Understanding
Mass Spectra. A Basic Approach, 1ª ed., John Wiley & Sons, USA, (1999).
[152] P. Schmitt Kopplin, M. Frommberger; Capillary electrophoresis mass
spectrometry: 15 years of developments and applications, Electrophoresis 24
(2003) 3837 3867.
Bibliografía
126
[153] E. Gelpi; Interfaces for coupled liquid phase separation/mass
spectrometry techniques. An update on recent developments, J. Mass
Spectrom. 37 (2002) 241 253.
[154] C. Simó, C. Barbas, A. Cifuentes; Capillary electrophoresis mass
spectrometry in food analysis, Electrophoresis 26 (2005) 1306 1318.
PARTE EXPERIMENTAL
Bloque I. Caracterización de
fuentes de compuestos fenólicos
bioactivos
High performance liquid chromatography with diode array
detection coupled to electrospray time of flight and ion trap
tandem mass spectrometry to identify phenolic compounds
from a lemon verbena extract
Identificación de los compuestos fenólicos de un extracto de
Lippia citriodora mediante HPLC DAD ESI TOF/IT MS
Capítulo 1
Journal of Chromatography A, 1216 (2009) 5391–5397
Contents lists available at ScienceDirect
Journal of Chromatography A
journa l homepage: www.e lsev ier .com/ locate /chroma
High-performance liquid chromatography with diode array detection coupled to
electrospray time-of-flight and ion-trap tandem mass spectrometry to identify
phenolic compounds from a lemon verbena extract
R. Quirantes-Pinéa, L. Funesb, V. Micolb, A. Segura-Carreteroa,∗, A. Fernández-Gutiérreza
a Department of Analytical Chemistry, University of Granada, c/Fuentenueva s/n, 18003 Granada, Spainb Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avenida de la Universidad s/n, E-03202 Elche, Alicante, Spain
a r t i c l e i n f o
Article history:
Received 18 February 2009
Received in revised form 13 May 2009
Accepted 14 May 2009
Available online 21 May 2009
Keywords:
Lemon verbena
Phenolic compounds
High-performance liquid chromatography
Mass spectrometry
a b s t r a c t
High-performance liquid chromatography with diode array and electrospray ionization mass spectro-
metric detection was used to carry out the comprehensive characterization of a lemon verbena extract
with demonstrated antioxidant and antiinflammatory activity. Two different MS techniques have been
coupled to HPLC: on one hand, time-of-flight mass spectrometry, and on the other hand, tandem mass
spectrometry on an ion-trap. The use of a small particle size C18 column (1.8 �m) provided a great res-
olution and made possible the separation of several isomers. The UV–visible spectrophotometry was
used to delimit the class of phenolic compound and the accurate mass measurements on time-of-
flight spectrometer enabled to identify the compounds present in the extract. Finally, the fragmentation
pattern obtained in MS–MS experiments confirmed the proposed structures. This procedure was able
to determine many well-known phenolic compounds present in lemon verbena such as verbascoside
and its derivatives, diglucuronide derivatives of apigenin and luteolin, and eukovoside. Also gardoside,
verbasoside, cistanoside F, theveside, campneoside I, chrysoeriol-7-diglucuronide, forsythoside A and
acacetin-7-diglucuronide were found for the first time in lemon verbena.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Lippia citriodora, also called lemon verbena, is a deciduous
shrub originated in South America. It was introduced into Europe
at the end of the 17th century so it is nowadays cultivated in
the Mediterranean area [1]. The leaves of lemon verbena con-
tain phenolic compounds, mainly flavonoids, phenolic acids and
phenylpropanoids [2–4]. Some pharmacological properties have
been attributed to these compounds. In fact, lemon verbena has tra-
ditionally been used in infusions for the treatment of fever, stomach
ache, indigestion and other gastrointestinal disorders, besides it can
act as diuretic [5], antiinflammatory [6] and analgesic [7]. Moreover,
lemon verbena products and their compounds can be considered
into the food category.
As a result of these properties, a bioactive plant extract from the
aerial part of Lippia citriodora has been developed (Monteloeder,
Elche). Its main component is the phenylethanoid glycoside ver-
bascoside, also known as acteoside, which has previously been
reported to have biological activity [6–11]. Several in vitro and in
vivo assays have shown that this lemon verbena extract has antiox-
∗ Corresponding author. Tel.: +34 958 243296; fax: +34 958 249510.
E-mail address: [email protected] (A. Segura-Carretero).
idant and antiinflammatory effects [12]. This antiinflammatory
activity might enable the use of this plant extract for the preven-
tion and treatment of diseases related with inflammation such as
osteoarthritis and rheumatoid arthritis among others.
In spite of this activity is attributed to verbascoside, other polar
compounds from the leaves can be present in the extract, mainly
phenolic compounds. In this way, we cannot assume that the whole
biological activity of lemon verbena extract are due to just one sin-
gle compound since several researches have shown interactions
between phenolic compounds, mainly synergistic and antagonis-
tic effects [13–16]. As a result, we have carried out the qualitative
characterization of this commercial foodstuff.
Phenolic compounds identification in plant matrix can be a com-
plex task as there is a wide variety of structures. They can also
get bonded to five different sugar moieties or get conjugated to
form dimers and trimers. Besides, a lot of polyphenol standards
are not commercially available. Several separative techniques have
been used to determine phenolic compounds in vegetable matrix
such as gas chromatography (GC), capillary electrophoresis (CE)
and mainly high-performance liquid chromatography (HPLC), all
of them coupled to different detection system, mostly mass spec-
trometry [2,17–19]. Recently, an improvement in chromatographic
performance has been achieved by the use of columns packed
with small particles (sub-2 �m) [20] which provide a higher peak
0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2009.05.038
______Capítulo 1_______
5392 R. Quirantes-Piné et al. / J. Chromatogr. A 1216 (2009) 5391–5397
capacity, greater resolution, increased sensitivity and high speed of
analysis [21–25].
We have developed a methodology for qualitative charac-
terization of complex plant matrix consisting of the coupling
of reversed-phase high-performance liquid chromatography (RP-
HPLC) eqquiped with a small particle size column, with two
different detection systems: photodiode array (DAD) and mass
spectrometry with time-of-flight (TOF) and ion-trap (IT) analyz-
ers. UV–visible spectrophotometry is a valuable tool for identifying
the class of phenolic compounds, whereas MS data are useful for
their structural characterization. The sensitivity together with mass
accuracy and true isotopic pattern of TOF–MS analyzer provided the
most probable molecular formula. On the other hand, tandem mass
spectrometry carried out with IT–MS analyzer was used to deter-
mine or to corroborate structures based on fragmentation patterns.
In this work we have applied the described methodology to carry
out the comprehensive characterization of a lemon verbena extract.
2. Experimental
2.1. Chemicals
All chemicals were of analytical reagent grade and used as
received. Formic acid and acetonitrile for HPLC were purchased
from Fluka, Sigma–Aldrich (Steinheim, Germany) and Lab-Scan
(Gliwice, Sowinskiego, Poland) respectively. Solvents were filtered
using a Solvent Filtration Apparatus 58061 (Supelco, Bellefonte,
PA, USA). Water was purified by a Milli-Q system from Millipore
(Bedford, MA, USA).
2.2. Instrumentation
Analyses were carried out using an Agilent 1200 Series Rapid
Resolution LC system (Agilent Technologies, Palo Alto, CA, USA),
including a standard autosampler and a diode array detector.
The HPLC column used was a Zorbax Eclipse Plus C18 (1.8 �m,
150 mm × 4.6 mm). The HPLC system was coupled to a microTOF
mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped
with an ESI interface. MS–MS analysis was performed using a Bruker
Daltonics Esquire 2000 IT mass spectrometer (Bruker Daltonics,
Bremen, Germany) also equipped with an ESI interface.
2.3. Sample
Lemon verbena commercial extract was kindly provided by
Monteloeder. Briefly, the manufacture procedure consisted of
lemon verbena aerial part drying and subsequent extraction in
an overheated water extractor through maceration/percolation by
exhaustive recirculation of water (≤80 ◦C) to solvent saturation.
Then, aqueous solution was concentrated by high vacuum at low
temperature up to 20◦ Brix and filtered through a silica–cellulose
press filter (1 �m) to eliminate insoluble materials. Concentration
process was continued by vacuum up to 70◦ Brix and final syrup
was dried by using industrial hot plate vacuum oven system. Final
ratio of extract to raw material was approximately 1:7.
The commercial extract manufactured according to the above
described procedure was dissolved in water to obtain a final con-
centration of 1000 �g ml−1. This solution was filtered and injected
directly into the HPLC system.
2.4. Chromatographic procedure
The separation of the compounds from lemon verbena extract
was carried out at room temperature with a gradient elution pro-
gram at a flow rate of 0.5 ml min−1. The mobile phases consisted in
water:acetonitrile (90:10, v/v) with 1% of formic acid (A) and ace-
tonitrile (B). The following multi-step linear gradient was applied:
0 min, 5% B; 25 min, 20% B; 30 min, 40% B; 35 min, 5% B. The ini-
tial conditions were held for 10 min. The injection volume in the
HPLC system was 20 �l. The UV–vis detection was performed in
the 190–450 nm range.
2.5. ESI–TOF–MS detection
As the flow rate at chromatographic conditions was set at
0.5 ml min−1, to split the flow was required when ESI interface was
used. In order to achieve reproducible results, ionization condi-
tions need to be constant and that is not possible at regular HPLC
conditions since the flow is too high. In the current paper the efflu-
ent from the HPLC column was reduced using a “T” type splitter
before being introduced into the mass spectrometer (split ratio
1:3). Thus the flow which arrived to the ESI–TOF–MS detector was
125 �l min−1.
The HPLC system was coupled to a TOF mass spectrometer
equipped with an ESI interface operating in negative ion mode
using a capillary voltage of +4 kV. The other optimum values of the
ESI–TOF parameters were drying gas temperature, 190 ◦C; drying
gas flow, 7 l min−1, and nebulizing gas pressure, 1.5 bar. The detec-
tion was carried out considering a mass range of 50–1000 m/z.
The accurate mass data of the molecular ions were processed
through the software DataAnalysis 4.0 (Bruker Daltonics), which
provided a list of possible elemental formulas by using Generate-
MolecularFormula Editor. The GenerateMolecularFormula Editor
uses a CHNO algorithm, which provides standard functionalities
such as minimum/maximum elemental range, electron config-
uration and ring-plus double bonds equivalents, as well as a
sophisticated comparison of the theoretical with the measured
isotope pattern (Sigma Value) for increased confidence in the
suggested molecular formula [26]. The widely accepted accuracy
threshold for confirmation of elemental compositions has been
established at 5 ppm [27]. We also have to say that even with very
high mass accuracy (<1 ppm) many chemically possible formulae
are obtained depending on the mass regions considered. So, high
mass accuracy alone is not enough to exclude enough candidates
with complex elemental compositions. The use of isotopic abun-
dance patterns as a single further constraint removes >95% of false
candidates. This orthogonal filter can condense several thousand
candidates down to only a small number of molecular formulas.
During the development of the HPLC method, external instru-
ment calibration was performed using a 74900-00-05 Cole Palmer
syringe pump (Vernon Hills, Illinois, USA) directly connected to
the interface, with a sodium formate cluster solution passing
through containing 5 mM sodium hydroxide and 0.2% formic acid in
water:isopropanol (1:1, v/v). The calibration solution was injected
at the beginning of each run and all the spectra were calibrated prior
to the compound identification. By using this method, an exact cal-
ibration curve based on numerous cluster masses each differing by
68 Da (NaCHO2) was obtained. Due to the compensation of tem-
perature drift in the microTOF, this external calibration provided
accurate mass values (better than 5 ppm) for a complete run with-
out the need for a dual sprayer setup for internal mass calibration.
2.6. ESI–IT–MS–MS detection
The mass spectrometer was run in the negative ion mode and the
capillary voltage was set at 3000 V. The IT scanned at 50–1000 m/z
range. The other parameters were dry temperature, 300 ◦C; drying
gas flow, 7 l min−1; nebulizing gas pressure, 1.5 bar. The instrument
was controlled by a personal computer running Esquire NTsoftware
from Bruker Daltonics.
Bloque I_______
R. Quirantes-Piné et al. / J. Chromatogr. A 1216 (2009) 5391–5397 5393
Fig. 1. UV chromatogram obtained at 280 nm (a) and BPC (50–1000 m/z) (b) for aqueous solution of lemon verbena extract.
3. Results and discussion
Fig. 1 shows the UV chromatogram registered at 280 nm since
most of the phenolic compounds absorb at this wavelength. The
figure also shows the ESI–TOF base peak chromatogram of aque-
ous solution of the lemon verbena extract where the peaks are
identified with numbers (1–19) considering the elution order. All
the compounds were identified by the interpretation of their mass
spectra obtained by the TOF–MS and the MS–MS spectra acquired
with the IT–MS and also taking into account the data provided by
the literature and their absorption spectra in UV–visible region.
Table 1 summarizes UV–visible bands and MS data including exper-
imental and calculated m/z for the provided molecular formulas,
error, sigma value and the main fragments obtained by MS–MS, as
well as the proposed compound for each peak. Fig. 2 shows the
structures of the proposed compounds.
Compound 1 was identified as gardoside taking into account
the molecular formula provided for its accurate mass. This iri-
doid glycoside has been reported previously in other species from
genus Lippia, for instance Lippia alba [28]. MS–MS spectrum of
this compound showed fragments at m/z 211, 193, 167 and 149
corresponding to [M−glucose]− (211) and the successive losses of
water (193) and CO2 (167), respectively, from the main fragment,
as well as the simultaneous elimination of water and CO2 (149)
(see Fig. 3a). Another fragment was found at m/z 123, correspond-
ing to [M−glucose-88]−, which was obtained by the loss of the
Table 1MS and MS–MS data and UV–visible bands for each compound and their proposed structures.
Peak tR (min) m/z Experimental m/z Calculated Error (ppm) Molecular formula MS–MS fragments �max (nm) Proposed compound
1 3.97 373.1147 373.1140 1.9 C16H21O10 211, 193, 167, 149, 123 234 Gardoside
2 4.33 461.1670 461.1664 1.3 C20H29O12 315, 297, 161, 135 238 Verbasoside
3 4.84 487.1433 487.1457 4.9 C21H27O13 179 240, 328 Cistanoside F
4 6.36 389.1096 389.1089 −1.6 C16H21O11 371, 345, 209, 179, 121 234 Theveside
5 9.05 387.1656 387.1661 1.1 C18H27O9 369, 225, 207, 163 239 Unknown 1
6 9.25 433.2065 433.2079 3.4 C20H33O10 387, 225 240 Unknown 2
7 10.71 639.1878 639.1931 8.2 C29H35O16 621, 459 242, 330 �-hydroxy-verbascoside/�-
hydroxy-isoverbascoside
8 11.09 639.1936 639.1931 −0.9 C29H35O16 621, 459 242, 330
9 11.46 637.1013 637.1046 5.2 C27H25O18 351, 285 253, 266 sh, 347 Luteolin-7-diglucuronide
10 15.07 621.1118 621.1097 3.3 C27H25O17 351 244 sh, 266, 334 Apigenin-7-diglucuronide
11 15.66 653.2139 653.2087 7.9 C30H37O16 635, 621, 459 246, 330 Campneoside I or isomer
12 15.81 653.2097 653.2087 1.6 C30H37O16 621 246, 330
13 16.94 651.1228 651.1203 −3.8 C28H27O18 395, 351 252, 266 sh, 345 Chrysoeriol-7-diglucuronide
14 17.28 623.2038 623.1981 −9.1 C29H35O15 461 237, 296 sh, 330 Verbascoside
15 19.45 623.1979 623.1981 0.5 C29H35O15 461, 315 244, 290 sh, 326 Isoverbascoside
16 20.10 623.1969 623.1981 2.0 C29H35O15 461 246, 290 sh, 330 Forsythoside A
17 22.04 637.2174 637.2138 −5.6 C30H37O15 491, 475, 461, 315 245, 290 sh, 330 Eukovoside
18 25.62 635.1278 635.1254 −3.8 C28H27O17 501, 351 245 sh, 267, 330 Acacetin-7-diglucuronide
19 28.28 651.2280 651.2294 2.1 C31H39O15 505, 475, 457 246, 329 Martinoside
Note: sh, shoulder.
______Capítulo 1_______
5394 R. Quirantes-Piné et al. / J. Chromatogr. A 1216 (2009) 5391–5397
Fig. 2. Chemical structures of the proposed compounds.
3-oxopropanic acid molecule according to the fragmentation path-
way shown in Fig. 4. These fragments were also similar to previous
data reported for other iridoid glycosides [29]. Also theveside was
identified in the sample (compound 4), another iridoid glycoside.
This compound presented fragments at m/z 371, 345 and 209 which
were consistent with the loss of water, CO2 and glucose moiety
respectively (see Fig. 3d). The fragment at m/z 121 corresponded
to the elimination of the 3-oxopropanic acid molecule from the
main fragment [M-glucose]− according to the fragmentation path-
way previously described for gardoside. This was also corroborated
since theveside is present in other plants from genus Lippia [30].
Both compounds showed an UV absorbance band at 234 nm which
is characteristic of these iridoid glycosides [31].
Compound 2 corresponded to verbasoside also known as decaf-
feoylverbascoside. Its MS–MS spectrum showed fragments at m/z
315 and 297 corresponding to the loss of rhamnose moiety fol-
lowed by water elimination, respectively. Other fragments were
detected at m/z 161 and 135. The ion at m/z 161 was consistent
with deoxyhexose group and the fragment at m/z 135 represented
the hydroxytyrosol moiety after the loss of water (see Fig. 3b). This
compound has been detected previously in the genus Lippia [32,33].
Peak 3 MS–MS spectrum gave a fragment at m/z 179 which cor-
responded to caffeic acid (see Fig. 3c). Cistanoside F was proposed as
structure taking into account the molecular formula provided, the
presence of caffeic acid in the structure and the naturally occur-
rence of this compound in Lippia alba [34]. This compound has
Bloque I_______
R. Quirantes-Piné et al. / J. Chromatogr. A 1216 (2009) 5391–5397 5395
Fig. 3. Most representative MS–MS spectra of compounds. The spectra correspond to: (a) gardoside, (b) verbasoside, (c) cistanoside F, (d) theveside, (e) �-hydroxyverbascoside
or �-hydroxyisoverbascoside, (f) luteolin-7-diglucuronide, (g) campneoside I or isomer, (h) verbascoside, (i) eukovoside, (j) martinoside.
showed previously vasorelaxant activity [35] as well as radical scav-
enging capacity against DPPH [30]. Therefore, it could contribute to
the antioxidant activity of the extract. However, the fact that it is a
minor component in the extract must be taken into account.
It was not possible to identify compounds 5 and 6 since the
molecular formulas and their possible structures did not match any
previous evidence in nature. Moreover, the obtained fragments did
not provide any useful information to propose a tentative identifi-
cation.
Several verbascoside derivatives were present in the extract such
as �-hydroxyverbascoside and �-hydroxyisoverbascoside (com-
pounds 7 and 8). They both showed the same fragmentation pattern
with fragments at m/z 621 and 459 (see Fig. 3e), so it was not pos-
sible to distinguish between them. The ion found at m/z 621 was
consistent with [M−H2O]−, whereas the fragment at m/z 459 cor-
responded to the loss of the caffeic acid moiety. The presence of
these verbascoside derivatives in lemon verbena extracts has been
previously reported in the literature [33,36].
MS–MS fragmentation of compounds 9, 10, 13 and 18 pre-
sented the same ion at m/z 351 which is characteristic of
diglucuronide group after water elimination. As well, all these com-
pounds showed similar UV–visible spectra typical of flavonoids
although hypsochromic shifts were observed by O-glycosylation
[37]. According to their molecular formulas, they were identified as
luteolin-7-diglucuronide, apigenin-7-diglucuronide, chrysoeriol-
7-diglucuronide and acacetin-7-diglucuronide, respectively. In the
______Capítulo 1_______
5396 R. Quirantes-Piné et al. / J. Chromatogr. A 1216 (2009) 5391–5397
Fig. 4. Proposed fragmentation pathway for gardoside.
case of luteolin-7-diglucuronide, the fragment at m/z 285 cor-
responding to luteolin aglycone was also present (see Fig. 3f).
Luteolin-7-diglucuronide and apigenin-7-diglucuronide have been
previously reported in lemon verbena [33,36].
The software provided the same molecular formula for peaks
11 and 12 and they showed similar absorption spectra. They were
tentatively identified as campneoside I and isomer although it was
not possible to assign what peak corresponded to campneoside I.
They both gave a fragment at m/z 621 which represented the loss
of methoxyl group. Compound 11 also showed an ion at m/z 635
(see Fig. 3g), consistent with water elimination, and another one at
m/z 459 corresponding to the loss of the caffeoyl moiety from the
main fragment (m/z 621). Campneoside I has showed antibacterial
activity in previous works [38].
As expected, the main peak (compound 14) was identified as
verbascoside, according to MS data and by comparison with reten-
tion time and MS–MS spectrum of a standard (see Fig. 3h). Besides,
two verbascoside isomers were found in the lemon verbena extract.
Compound 15 corresponded to isoverbascoside. It was consistent
with its fragmentation pattern, very similar to verbascoside. The
fragments found at m/z 461 and 315 corresponded to the loss of
the caffeoyl moiety and the successive loss of rhamnose, respec-
tively. Peak 16 corresponded to another verbascoside isomer which
showed the same MS–MS profile. It was tentatively identified as
forsythoside A since it is usually present in natural products [39–41]
and it showed UV bands according to the data provided by the
literature [42].
Compound 17 corresponded to eukovoside. Its assignment was
consistent with the presence of fragments found at m/z 491, 461
and 315 which represented the loss of rhamnose moiety, feruloyl
group and their successive elimination (see Fig. 3i). Martynoside
was the proposed structure for compound number 19. In its MS–MS
spectrum, a fragment due to the loss of rhamnose moiety at m/z 505
was observed (see Fig. 3j). Other fragments were detected at m/z
475 and 457 corresponding to the feruloyl unit elimination and the
further loss of water, respectively. These phenylethanoid glycosides
were also corroborated by the data reported in the literature [33,35].
4. Conclusions
A powerful analytical method has been used to carry out the
comprehensive characterization of a lemon verbena extract. The
combined use of HPLC separation with a small particle size col-
umn assisted by UV–vis and mass spectrometric detections with
different mass analyzers, such as TOF or IT, has proved to be an use-
ful tool in the identification of secondary metabolites produced by
plants. The utilized method simultaneously separated a wide range
of iridoid glycosides, flavonoid glycosides and phenylethanoid gly-
cosides and the successfully identification of the major compounds
of this extract was done in less than 30 min.
It is also important to highlight that, to our knowledge,
the compounds gardoside, verbasoside, cistanoside F, theveside,
campneoside I, chrysoeriol-7-diglucuronide, forsythoside A and
acacetin-7-diglucuronide were described for the first time in lemon
verbena.
Acknowledgements
The authors are grateful to the Spanish Ministry of Education
and Science for two projects (AGL2007-62806 and AGL2008-05108-
C03-03) and a grant (FPU, AP2007-03246) and to Andalusian
Regional Government Council of Innovation and Science for the
project P07-AGR-02619. This investigation has been partially sup-
ported too by private funds from Monteloeder, S.L. The authors also
thank Monteloeder, S.L. for providing the lemon verbena extract.
References
[1] C. Argyropoulou, D. Daferera, P.A. Tarantilis, C. Fasseas, M. Polissiou, Biochem.Syst. Ecol. 35 (2007) 831.
Bloque I_______
R. Quirantes-Piné et al. / J. Chromatogr. A 1216 (2009) 5391–5397 5397
[2] P.B. Andrade, R.M. Seabra, P. Valentão, F. Areias, J. Liq. Chromatogr. Rel. Technol.21 (1998) 2813.
[3] A. Carnat, A.P. Carnat, D. Fraisse, J.L. Lamaison, Fitoterapia 70 (1999) 44.[4] P. Valentão, P.B. Andrade, F. Areias, F. Ferreres, R.M. Seabra, J. Agric. Food Chem.
47 (1999) 4579.[5] M.E. Pascual, K. Slowing, E. Carretero, D. Sánchez Mata, A. Villar, J. Ethnophar-
macol. 76 (2001) 201.[6] M. Deepak, S.S. Handa, Phytother. Res. 14 (2000) 463.[7] T. Nakamura, E. Okuyama, A. Tsukada, M. Yamazaki, M. Satake, S. Nishibe, T.
Deyama, A. Moriya, M. Maruno, H. Nishimura, Chem. Pharm. Bull. 45 (1997)499.
[8] A.M. Díaz, M.J. Abad, L. Fernández, A.M. Silván, J. De Santos, P. Bermejo, Life Sci.74 (2004) 2515.
[9] T. Ohno, M. Inoue, Y. Ogihara, I. Saracoglu, Biol. Pharm. Bull. 25 (2002) 666.[10] P. Akbay, I. Calis, U. Ündeger, N. Basaran, A. Ahmet Basaran, Phytother. Res. 16
(2002) 593.[11] P. Wang, J. Kang, R. Zheng, Z. Yang, J. Lu, J. Gao, Z. Jia, Biochem. Pharmacol. 51
(1996) 687.[12] O. Laporta, L. Pérez-Fons, K. Balan, D. Paper, V. Cartagena, V. Micol, Agro Food
Ind. Hi-Tech 15 (2004) 30.[13] A.S. Meyer, M. Heinonen, E.N. Frankel, Food Chem. 61 (1998) 71.[14] S.I. Kayano, H. Kikuzaki, N. Fukutsuka, T. Mitani, N. Nakatani, J. Agric. Food Chem.
50 (2002) 3708.[15] M.N. Peyrat-Maillard, M.E. Cuvelier, C. Berset, J. Am. Oil Chem. Soc. 80 (2003)
1007.[16] E.M. Becker, G. Ntouma, L.H. Skibsted, Food Chem. 103 (2007) 1288.[17] C. Proestos, D. Sereli, M. Komaitis, Food Chem. 95 (2006) 44.[18] R. Tsao, Z. Deng, J. Chromatogr. B 812 (2004) 85.[19] G. Cimpan, S. Gocan, J. Liq. Chromatogr. Rel. Technol. 25 (2002) 2225.[20] T. Yoshida, R.E. Majors, J. Sep. Sci. 29 (2006) 2421.[21] L.W. Qi, X.D. Wen, J. Cao, C.Y. Li, P. Li, L. Yi, Y.X. Wang, X.L. Cheng, X.X. Ge, Rapid
Commun. Mass Spectrom. 22 (2008) 2493.[22] L. Zhao, Z.Y. Lou, Z.Y. Zhu, H. Zhang, G.Q. Zhang, Y.F. Chai, Biomed. Chromatogr.
22 (2008) 64.[23] P. de la Iglesia, G. Giménez, J. Diogène, J. Chromatogr. A 1215 (2008) 116.
[24] C. Cavaliere, P. Foglia, R. Gubbiotti, P. Sacchetti, R. Samperi, A. Laganà, RapidCommun. Mass Spectrom. 22 (2008) 3089.
[25] I. Ferrer, E.M. Thurman, J.A. Zweigenbaum, Rapid Commun. Mass Spectrom. 21(2007) 3869.
[26] Bruker Daltonics Technical Note No. 008, Molecular formula determinationunder automation.
[27] G. Bringmann, I. Kajahn, C. Neusüß, M. Pelzing, S. Laug, M. Unger, U. Holzgrabe,Electrophoresis 26 (2005) 1513.
[28] J.G.S. Filho, J.M. Duringer, D.E.A. Uchoa, H.S. Xavier, J.M.B. Filho, R.B. Filho, Nat.Prod. Commun. 2 (2007) 715.
[29] L. Ren, X. Xue, F. Zhang, Y. Wang, Y. Liu, C. Li, X. Liang, Rapid Commun. MassSpectrom. 21 (2007) 3039.
[30] T. Hennebelle, S. Sahpaz, B. Gressier, H. Joseph, F. Bailleul, Phytother. Res. 22(2008) 256.
[31] L.J. El-Naggar, J.L. Beal, J. Nat. Prod. 43 (1980) 649.[32] F.G. Barbosa, M.A.S. Lima, R. Braz-Filho, E.R. Silveira, Biochem. Syst. Ecol. 34
(2006) 819.[33] M. Ono, E. Oda, T. Tanaka, Y. Iida, T. Yamasaki, C. Masuoka, T. Ikeda, T. Nohara, J.
Nat. Med. 62 (2008) 101.[34] T. Hennebelle, S. Sahpaz, H. Joseph, F. Bailleul, Nat. Prod. Commun. 1 (2006) 727.[35] M. Yoshikawa, H. Matsuda, T. Morikawa, H. Xie, S. Nakamura, O. Muraoka, Boor-
gan Med. Chem. 14 (2006) 7468.[36] A.R. Bilia, M. Giomi, M. Innocenti, S. Gallori, F.F. Vincieri, J. Pharm. Biomed. Anal.
46 (2008) 463.[37] K. Robards, P.D. Prenzler, G. Tucker, P. Swatsitang, W. Glover, Food Chem. 66
(1999) 401.[38] K.H. Kang, S.K. Jang, B.K. Kim, M.K. Park, Arch. Pharm. Res. 17 (1994) 470.[39] J. Han, M. Ye, H. Guo, M. Yang, B. Wang, D.A. Guo, J. Pharm. Biomed. Anal. 44
(2007) 430.[40] H. Guo, A.H. Liu, M. Ye, M. Yang, D. Guo, Rapid Commun. Mass Spectrom. 21
(2007) 715.[41] H. Qu, B. Li, X. Li, G. Tu, J. Lü, W. Sun, Microchem. J. 89 (2008) 159.[42] V.A. Kurkin, Chem. Nat. Comp. 39 (2003) 123.
______Capítulo 1_______
Characterization of phenolic and other polar compounds in a
lemon verbena extract by capillary electrophoresis electrospray
ionization mass spectrometry
Caracterización de los compuestos fenólicos y otros
compuestos polares de un extracto de Lippia citriodora
mediante CE ESI MS
Capítulo 2
Research Article
Characterization of phenolic and other polarcompounds in a lemon verbena extract bycapillary electrophoresis-electrosprayionization-mass spectrometry
In this study, a CE-MS method has been developed to carry out the qualitative charac-
terization of a lemon verbena (Lippia citriodora) extract for the first time. The CE and ESI-
MS parameters were optimized with respect to resolution, analysis time and peak shape in
order to maximize the number of compounds detected and the sensitivity of their
determination. The use of two different MS analyzers, TOF-MS and IT-MS, enabled the
tentative identification of the major components of this extract. Thus, using this method,
16 compounds were determined. Some of them have been previously identified by HPLC
methods, although four compounds were also found for the first time in lemon verbena
such as asperuloside, tuberonic acid glucoside or 50-hydroxyjasmonic acid 50-O-glucoside,shanzhiside and ixoside. These results demonstrate that CE-MS generates data comple-
mentary to those obtainable by HPLC-MS and it is particularly suited to the analysis of
plant metabolites.
Keywords: CE / ESI / Lemon verbena / MS / Phenolic compoundsDOI 10.1002/jssc.201000228
1 Introduction
Lippia citriodora, commonly known as lemon verbena, is a
shrub from genus Lippia native of South America although
it is also cultivated in Southern Europe and North Africa.
Lemon verbena leaves are used to add a lemony flavor to
foods and beverages, as well as in folk medicine. In this way,
they have traditionally been used for the treatment of
asthma, fever, gastrointestinal disorders and skin diseases
[1]. Due to these pharmacological properties, this plant has
been the subject of a large number of studies. Several
researchers have demonstrated that lemon verbena
possesses digestive [2], antispasmodic [3], diuretic [4], anti-
inflammatory [5] and analgesic [6] activities. The chemical
composition of lemon verbena leaves has also been widely
studied [7–13]. They are rich in an essential oil whose main
components are neral, citral, geranial and limonene. More-
over, they contain a large number of polar compounds such
as phenylpropanoids, flavonoids, phenolic acids and iridoid
glycosides. The most abundant compound in lemon verbena
is verbascoside, which has been previously reported to have
relevant biological activity [14–17].
The medicinal use of extracts from this plant makes
essential the comprehensive knowledge of their composi-
tion. In this sense, the characterization of polyphenols and
other polar compounds from lemon verbena have been
successfully carried out by RP-HPLC with spectro-
photometric detection [7–9] and coupled with MS [10, 12,
13]. Nevertheless, to our knowledge, CE has not been
previously applied to lemon verbena analysis.
In the last decade, CE has gained widespread interest as
a valuable tool for the determination of bioactive
compounds in plant matrices [18–24]. This increasing
interest is due to its attractive advantages such as high speed
of analysis, the small sample amounts required and extre-
mely limited solvent waste [25]. However, CE can be not
only a worthy alternative to chromatographic techniques but
also a powerful complement. Separation in CZE is based on
the differences in mass to charge ratios of the compounds,
whereas separation in RP-HPLC is based on the differences
in polarity of the compounds. Therefore, since both
separation techniques are based on different principles, they
can provide complementary information. For this reason, as
a continuation of our studies on lemon verbena extracts by
HPLC [13], now we have developed a new method for the
characterization of these extracts using CZE coupled to MS.
Rosa Quirantes-Pine�
David Arraez-RomanAntonio Segura-CarreteroAlberto Fernandez-Gutierrez
Department of AnalyticalChemistry, Faculty of Sciences,University of Granada,Granada, Spain
Received April 6, 2010Revised June 24, 2010Accepted June 25, 2010
Abbreviations: BPE, base peak electropherogram; EIE,
extracted ion electropherogram; TEA, triethylamine; IT, iontrap
�Additional correspondence: Rosa Quirantes-Pine
E-mail: [email protected]
Correspondence: Dr. Antonio Segura Carretero, Research GroupFQM-297, Department of Analytical Chemistry, Faculty ofSciences, University of Granada, C/Fuentenueva s/n, E-18071Granada, SpainE-mail: [email protected]: 134958249510
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2010, 33, 2818–28272818
Capítulo 2_______
In general, if a separation technique is coupled with MS,
the interpretation of the analytical results can be more
straightforward [26–28]. TOF-MS can provide excellent mass
accuracy over a wide dynamic range and allow measure-
ments of the isotopic pattern, providing important additional
information for the determination of the elemental compo-
sition. Furthermore, an IT mass analyzer (IT-MS) can be
used to obtain fragmentation patterns which enable the
determination of compound structures in complex matrices.
Thus, the combined use of both analyzers provides enough
structural information which enables to identify the likeliest
compound when standards are not available.
The aim of this study has been to develop a CE-ESI-MS
method to determine phenolic and other related polar
compounds in a lemon verbena extract. This method has
enabled to determine new compounds, which our previous
HPLC method could not find in the same extract.
2 Materials and methods
2.1 Chemicals
All chemicals were of analytical reagent grade and used as
received. Ammonium acetate was purchased from Sigma
(St. Louis, MO, USA), ammonium hydrogen carbonate from
Panreac (Barcelona, Spain) and ammonia from Merck
(Darmstadt, Germany) were used to prepare CE running
buffers at different concentrations and pH values. Buffers
were prepared weighing the appropriate amount of ammo-
nium acetate or ammonium carbonate at the concentrations
indicated and adding ammonia to adjust the pH. The
buffers were prepared with doubly deionized water, stored at
41C and brought to room temperature before use.
Doubly deionized water was obtained with a Milli-Q
water purification system (Millipore, Bedford, MA, USA).
Triethylamine (TEA) used in the sheath flow liquid was
from Aldrich (Steinheim, Germany) and sodium hydroxide
used for capillary conditioning was obtained from Panreac.
2-Propanol and methanol used in the sheath flow liquids
and on the buffer were of HPLC grade and purchased from
Labscan (Dublin, Ireland). All solutions were filtered
through a 0.45 mm Millipore membrane filter before injec-
tion into the capillary.
2.2 Sample
Lemon verbena commercial extract was kindly provided by
Monteloeder (Elche, Spain). Briefly, the manufacture
procedure consisted of lemon verbena aerial part drying
and subsequent extraction in an overheated water extractor
(PILZ, Heraus-Witmann, Heidelberg, Germany) through
maceration/percolation by exhaustive recirculation of water
(r801C) to solvent saturation. Then, the aqueous solution
was concentrated by high vacuum at low temperature up to
201C Brix and filtered through a silica-cellulose press filter
(1 mm) to eliminate insoluble materials. Concentration
process was continued by vacuum up to 701C Brix and the
final syrup was dried by using an industrial hot plate
vacuum oven system. Final ratio of extract to raw material
was approximately 1:7.
The commercial extract manufactured according to the
above-described procedure was dissolved in methanol/water
(50:50, v/v) to obtain a final concentration of 20 mg/mL.
This solution was filtered through a 0.25 mm filter before the
CE analysis.
2.3 CE
The analyses were made in a Prince CE system (Prince
Technologies, Emmen, The Netherlands). Bare fused silica
capillary of 50 mm id and 100 cm total length (corresponding
to the MS detection length) was purchased from Beckman
Coulter (Fullerton, CA, USA).
Before first use, the bare capillaries were conditioned
with 0.1 M sodium hydroxide during 20 min followed by a
water rinse for other 10 min. At the end of the day, the
capillary was flushed with water for 10 min.
Capillary conditioning of the columns was done by
flushing for 2 min sodium hydroxide, 4 min with water and
then for 10 min with the separation buffer (a pressure of
1000 mbar was used during all the capillary conditioning).
After thorough optimization (Section 3), a running
buffer of 60 mM ammonium acetate at pH 9.25 and 5%v/v
2-propanol was used. The separation voltage was set to
30 kV at the inlet of the capillary. Injection was performed
hydrodynamically using a N2 pressure at 65 mbar during
5 s, corresponding to about 15 nL injected (0.76% of the
capillary length). Every analysis was run at room tempera-
ture.
2.4 ESI-TOF-MS
The CE system was coupled to a micrOTOF (Bruker
Daltonics, Bremen, Germany), an orthogonal-accelerated
TOF mass spectrometer, equipped with an orthogonal
coaxial sheath-flow ESI interface (Agilent Technologies,
Palo Alto, CA, USA). For the connection between the CE
system and the electrospray ion source of the mass
spectrometer, the outlet of the separation capillary was
fitted into the electrospray needle of the ion source and a
flow of conductive sheath liquid established the electrical
contact between capillary effluent and the electrospray
needle. 2-Propanol/water (60:40, v/v) with 0.01% v/v TEA
was applied as sheath liquid at a flow rate of 0.24 mL/h
delivered by a 5 mL gas-tight syringe (Hamilton, Reno, NV,
USA) using a 74900-00-05 syringe pump Cole-Parmer
(Vernon Hill, IL, USA).
The ESI-voltage of the TOF was applied at the end cap
of the transfer capillary to the MS with the spray needle
being grounded. An electrospray potential of 14.5 kV was
J. Sep. Sci. 2010, 33, 2818–2827 Electrodriven Separations 2819
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Bloque I_______
applied at the inlet of the MS (negative mode). The opti-
mum ESI conditions (Section 3) were a nebulizer gas (N2)
pressure of 5 psi applied to assist the spraying, a dry gas
temperature set to 1901C at a dry gas flow of 6 L/min.
The accurate mass data of the molecular ions were
processed through the software DataAnalysis 3.4 (Bruker
Daltonik GmgH, Bremen, Germany), which provided a list
of possible elemental formulas by using the Generate-
MolecularFormulaTM Editor. The GenerateMolecular-
FormulaTM Editor uses a CHNO algorithm, which provides
standard functionalities such as minimum/maximum
elemental range, electron configuration and ring-plus
double bonds equivalents, as well as a sophisticated
comparison of the theoretical with the measured isotopic
pattern (Sigma-ValueTM) for increased confidence in the
suggested molecular formula (Bruker Daltonics Technical
Note 008, Molecular formula determination under auto-
mation). Even with very high mass accuracy (o1 ppm),
many chemically possible formulas are obtained depending
on the mass regions considered. Hence, high mass accuracy
(o1 ppm) alone is not enough to exclude enough candi-
dates with complex elemental compositions. The use of
isotopic abundance patterns as a single further constraint
removes 495% of false candidates. This orthogonal filter
can condense several thousand candidates down to only a
small number of molecular formulas.
During the development of the CE method, external
instrument calibration was performed using a Cole Palmer
syringe pump directly connected to the interface, passing a
solution of sodium formate cluster, in a 5 mL gas-tight
syringe (Hamilton), containing 5 mM sodium hydroxide
and 0.2% v/v formic acid in 2-propanol/water (1:1, v/v).
Using this method, an exact calibration curve based on
numerous cluster masses each differing by 68 Da (NaCHO2)
was obtained. Due to the compensation of temperature drift
in the micrOTOF, this external calibration provided accurate
mass values (better than 5 ppm) for a complete run without
the need for a dual sprayer setup for internal mass cali-
bration.
2.5 ESI-IT-MS
MS and MS/MS experiments were performed on a Bruker
Daltonics Esquire 2000TM IT mass spectrometer (Bruker
Daltonik GmgH) equipped with an orthogonal coaxial
sheath flow ESI interface (Agilent Technologies). In this
case 2-propanol/water (50:50, v/v) with 0.01% v/v TEA was
applied as sheath liquid at a flow rate of 0.24 mL/h delivered
by a 5 mL gas-tight syringe (Hamilton) using a 74900-00-05
Cole Palmer syringe pump.
The mass spectrometer was run in the negative ion
mode and the capillary voltage was set at 13.2 kV. The IT
scanned at m/z 50–1000 range at 13 000 m/s during the
separation and detection. The maximum accumulation time
for the IT was set at 110.00 ms, the target count at 20 000
and the compound stability was set at 25%. ESI operating
conditions (described in Section 3) were a nebulizing gas
pressure (N2) of 5 psi, drying gas temperature was set to
2501C at a drying gas flow of 6.0 L/min. MS/MS spectra
were acquired on the most intense ions using selected ions
mode. The instrument was controlled by a PC running the
Esquire NT software from Bruker Daltonics.
3 Results and discussion
3.1 Development of CE-ESI-MS method
Optimization of the electrophoretic conditions was carried
out with IT-MS detection, using the methanol/water extract
of lemon verbena obtained as described in Section 2.2.
Initially, the separation parameters were optimized before
the MS conditions in order to obtain the best resolution,
sensitivity, analysis time and peak shape. Therefore, the
parameters tested were type, concentration and pH of the
buffer, as well as voltage and injection time. Temperature
cannot be optimized since in the coupling between CE
instrument and MS, the capillary is out of the cartridge and
exposed to the air. Therefore, every analysis was run at room
temperature.
First, different BGEs were studied. The commonly used
buffers for a conventional electrophoretic method in plant
extracts such as borate, phosphate and SDS are not suitable
for the CE coupled with ESI-MS because of their non-vola-
tility and the risk of MS source contamination. More
suitable buffers such as formic acid, acetic acid, ammonium
carbonate and ammonium acetate are compatible with a
CE-MS interface and/or with MS instrument [29]. Since
phenylpropanoids and flavonoids are weak acids with ioni-
zation constants (pKa) from 9 to 12 due to the presence of
phenolic hydroxyl group, an alkaline buffer was used to
ensure at least partial dissociation (negative net charge) of
these compounds. Therefore ammonium acetate and
ammonium carbonate at a concentration of 50 mM and pH
9.5 were evaluated. Best resolution, peak shape, baseline
and analysis time were obtained using ammonium acetate
as BGE.
The pH value and concentration of the BGE play an
important role in CE for its effect on zeta potential (x) andEOF, as well as the overall charge of every analyte, affecting
the migration time and the separation of the compounds.
Therefore, it is important to study their influences on CE to
obtain optimum separations. First, the effect of the BGE
ionic strength was studied, testing ammonium acetate
concentrations from 20 to 80 mM (in steps of 20). As
expected, the best resolution was obtained with 80 mM
although a broadening of peaks was observed. Furthermore,
the analysis time was quite shorter with 60 mM ammonium
acetate and the resolution was acceptable. Next, different pH
values adjusted with ammonia were evaluated from 8.5 to 10
(in steps of 0.25). Typical pKa value of phenols is about 9
and that of characteristic 4-carboxylated iridoids of Verbe-
naceae family is near to 4 [30]. Thus, the main analytes
J. Sep. Sci. 2010, 33, 2818–28272820 R. Quirantes-Pine et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Capítulo 2_______
migrated in the dissociated forms in the pH range tested,
whereas EOF increased as pH became higher, which
resulted in the gradual decrease of migration times with
increasing pH values (Fig. 1). At pH 8.5, an excessive peak
broadening was observed causing decreasing sensitivity and
loss of resolution. A good separation was achieved at pH 9
and 9.25, whereas higher values led to peaks overlaps.
Therefore, the best compromise in terms of resolution and
analysis time was found for pH 9.25 which also provided
good peak shapes (Fig. 1C).
Addition of organic modifiers into BGE slowed down
the EOF by increasing the viscosity of the buffer system,
improving resolution in this way. Thus, the presence of
2-propanol in the BGE was tested, adding concentrations in
the range of 0–10% v/v in 2.5% intervals. The addition of
2-propanol as organic modifier improved the resolution
although the migration time increased. When 5%v/v of
2-propanol is added, a better resolution was achieved;
however, using higher amounts of 2-propanol, the peak
shape got worse.
The voltage applied was varied between 15 and 30 kV,
and a voltage of 30 kV was finally chosen to achieve
the shortest analysis time, good resolution together
with satisfactory current, below 40 mA. The injections
were made at the anodic end using a N2 pressure of 65 mbar
for 5, 10 and 20 s, selecting 5 s as optimum injection time
due to the improvement of resolution with little loss of
sensitivity.
The ESI-IT-MS operating conditions were optimized by
adjusting the sheath liquid composition and flow rate,
nebulizer pressure, dry gas flow rate and ESI chamber
temperature. For the optimization of these parameters,
signals corresponding to the main peaks were used as
optimization criterion.
According to the previous studies [31, 32], the nature,
composition and flow rate of the sheath liquid have a critical
effect on the performance of CE-ESI interface [33]. The
organic solvents investigated were methanol and 2-propa-
nol, with 2-propanol giving the most stable and highest MS
signal. In addition, different 2-propanol/water compositions
were tested for the sensitivity and stability of the IT-MS
signals: 2-propanol/water 80:20, 60:40 and 50:50 v/v.
Generally, small amounts of volatile TEA or ammonium
hydroxide can be used to improve ionization for ESI-nega-
tive detection [34]. For this reason, different percentages of
TEA were added, from 0.01 to 0.1% v/v. Finally, the use of a
sheath liquid consisting of 2-propanol/water (50:50, v/v)
plus 0.01% v/v TEA provided higher current stability and
MS signal for IT. A value of 0.24 mL/h was selected as the
optimum sheath liquid flow because lower flows reduced
the ionization yield due to the instability of the spray,
whereas at higher flow rates dilution of the electrophoretic
bands was too high and the intensity of the MS signal for
these compounds was reduced.
Next, other ESI-IT-MS parameters were optimized, such
as nebulizing gas pressure as well as drying gas flow rate
and temperature, studying the signal-to-noise ratio. The
selected values for each parameter were nebulizing gas
pressure at 5 psi and a drying gas flow of 6 L/min at 2501C,providing good MS signals.
Figure 1. CE-ESI-IT-MS condi-tions using a bare fused silicacapillary of 50 mm id and100 cm total length and asBGE 60mM ammonium acet-ate at different pH values: (A)8.5, (B) 9.0, (C) 9.25, (D) 9.5, (E)9.75, (F) 10. The rest of CEconditions: voltage, 25 kV;injection, time 12 s; roomtemperature.
J. Sep. Sci. 2010, 33, 2818–2827 Electrodriven Separations 2821
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Bloque I_______
Under the optimized conditions, CE-ESI-IT-MS
separations such as the one shown in Fig. 2 were obtained
for the lemon verbena extract. The prolonged analysis time
with respect to CE-UV separations is a consequence of the
long capillary lengths which are needed to couple a CE
instrument to MS. This is not a problem as such, but it
counteracts one of the main advantages of CE, namely its
speed.
After carrying out the optimization of this CE-ESI-IT-
MS method, CE system was coupled to TOF-MS analyzer in
Figure 2. Base peak electropherogram obtained by CE-ESI-IT-MS for lemon verbena extract under optimized conditions, and extractedion electropherograms of found compounds. Optimized conditions: running buffer 60mM ammonium acetate15%v/v 2-propanol, pH9.25; voltage, 30 kV; injection time, 5 s; sheath liquid 2-propanol/water (50:50, v/v)10.01%v/v TEA at a flow rate of 0.24mL/h, drying flowrate of 6 L/min, at 2501C using a nebulizing gas pressure of 5 psi.
J. Sep. Sci. 2010, 33, 2818–28272822 R. Quirantes-Pine et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Capítulo 2_______
Table
1.Compoundsidentifiedin
thelemonverbenaextract
Peak
Migration
time
(min)
m/z
Expe
rimen
tal
m/z
Calculated
Error
(ppm
)
Sigma
value
Molec
ular
form
ula
Classifica
tion
orde
r
considering
othe
r
possibilities
Tolerance
(ppm
)
MS/M
S
frag
men
ts
Proposedco
mpoun
d
111.4
461.1656
461.1664
1.8
0.02
C20H29O12
1st(2)
10401,315,161,135
Verba
soside
211.8
651.2293
651.2294
0.2
0.03
C31H39O15
1st(2)
5505,475
Martyno
side
312.5
487.1449
487.1457
1.7
0.02
C21H27O13
1st(3)
10179
Cistano
side
F
412.6
637.2125
637.2138
2.1
0.05
C30H37O15
1st(2)
5621,487,461,322
Eukovoside
512.9
623.1951
623.1981
4.8
0.02
C29H35O15
1st(3)
5461,315
Verba
scoside
613.8
413.1098
413.1089
�2.1
0.02
C18H21O11
1st(2)
15411,395,353,251,161
Asperuloside
714.0
387.1664
387.1661
�0.9
0.03
C18H27O9
1st(2)
15369,225,207,163
Tube
ronicac
idgluc
oside/
50-Hydroxyjasm
onicac
id
50-O-gluco
side
814.1
391.1244
391.1246
0.4
0.02
C16H23O11
1st(2)
15331,229,211,185,167,149,123
Shanzhiside
914.2
373.1139
373.1140
0.4
0.04
C16H21O10
1st(2)
20211,167,149,123
Gardoside
1017.1
635.1270
635.1254
�2.6
0.04
C28H27O17
1st(3)
5501,351,193
Aca
cetin-7-diglucu
ronide
1117.6
195.0520
195.0510
�5.0
0.01
C6H
11O7
1st(1)
20177,159,129
Unknown
1218.1
651.1181
651.1203
3.3
0.04
C28H27O18
1st(3)
5395,351
Chrysoe
riol-7-diglucu
ro-
nide
1318.9
329.0665
329.0667
0.6
0.03
C17H13O7
1st(3)
20313,293
Jaceosidin
1420.0
389.1093
389.1089
�0.8
0.01
C16H21O11
1st(2)
15245,209,165
Theveside
1520.7
387.0943
387.0933
�2.7
0.04
C16H19O11
1st(2)
15343,223,205,181,161,137
Ixoside
1621.4
637.1067
637.1046
�3.2
0.04
C27H25O18
1st(3)
5381,351,285,193
Luteolin-7-diglucu
ronide
J. Sep. Sci. 2010, 33, 2818–2827 Electrodriven Separations 2823
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Bloque I_______
order to obtain molecular formulas of the compounds found
in the lemon verbena extract. Hence, ESI-MS parameters
previously selected for IT-MS were checked with TOF-MS.
As a result, the sheath liquid composition was changed to
60:40 v/v 2-propanol/water with 0.01% v/v TEA since TOF-
MS cannot raise as high temperature as IT-MS so a greater
percentage of organic solvent is needed. Drying gas
temperature was also modified to 1901C. The rest of ESI-MS
conditions were the same.
3.2 Characterization of polar compounds from
lemon verbena by CE-ESI-TOF/IT-MS
The optimum conditions were applied to the separation and
determination of the phenolic compounds and other polar
components in the lemon verbena extract. The base peak
electropherogram obtained from CE-ESI-IT-MS analysis of
lemon verbena extract is shown in Fig. 2, as well as the
extracted ion electropherogram of the 16 compounds found.
All the compounds were identified by the interpretation
of their mass spectra obtained by the TOF-MS and the
MS/MS spectra acquired with the IT-MS. In this way,
generated molecular formulas by TOF analysis were
checked and MS/MS fragments were also studied to corro-
borate structures, bearing in mind the data provided by the
literature. Data corresponding to these compounds are
summarized in Table 1. This table includes migration time,
calculated and experimental m/z, error, sigma value, mole-
cular formula, classification order considering other possi-
bilities (sorted with respect to sigma value), tolerance, MS/
MS fragments and proposed compounds.
Most of the compounds found in this study have been
previously described in lemon verbena leaves. Peaks 5 and
16 corresponded with verbascoside and luteolin-7-diglucuro-
nide, respectively, which are well-known compounds of this
plant [8, 10, 35]. The phenylpropanoids martynoside and
eukovoside (peaks 2 and 4) have also been identified in
lemon verbena by other authors [10, 11]. Furthermore,
verbasoside, cistanoside F, gardoside, acacetin-7-diglucuro-
nide, chrysoeriol-7-diglucuronide and theveside, corres-
ponding to peaks 1, 3, 9, 10, 12 and 14, respectively, have
been found in our previous study about this lemon verbena
extract by HPLC-DAD-ESI-MS [13]. However, some
compounds not found by HPLC have been identified by CE,
mainly iridoid glycosides, showing that both techniques can
provide complementary information. Figure 3 shows the
structures of these proposed compounds.
Peak 6, at m/z 413.1098, corresponded to the formula
[C18H22O11�H]�. Its MS/MS spectrum showed a main
fragment at 353, which was consistent with the loss of acetic
acid. Other minor fragments found at m/z 395, 251 and 161
were yielded by elimination of water, loss of glucose and
dehydration of glucose moiety, respectively. Asperuloside
Figure 3. Structure of compoundsfound for the first time in lemonverbena.
J. Sep. Sci. 2010, 33, 2818–28272824 R. Quirantes-Pine et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Capítulo 2_______
was proposed as compound taking into account the mole-
cular formula provided by TOF-MS, the presence of glucose
and one acetoxy group in the structure and the natural
occurrence of this compound in plants belonging to the
order Lamiales like lemon verbena which has been reported
[36–38].
The MS/MS spectrum of compound 7 showed a mole-
cular ion [M�H]� at m/z 387 and fragments at 369, 225, 207
and 163. Fragment 369 corresponded to [M�H�H2O]�,
whereas ions at m/z 225, 207 and 163 were consistent with
the loss of glucose moiety followed by successive dehydra-
tion and decarboxylation, respectively. According to these
data, it was proposed as tuberonic acid glucoside or 50-hydroxyjasmonic acid 50-O-glucoside, since it was not
possible to distinguish between these diastereomers. They
have been previously reported in Elsholtzia rugulosa [39] and
thyme leaf [40], respectively.
Compound 8 was identified as shanzhiside, taking
into account the molecular formula provided for its
accurate mass. MS/MS spectrum of this compound
showed fragments at m/z 229, 211, 185, 167 and 149
corresponding to [M�H�glucose]� (229) and the successive
losses of water (211) and CO2 (185), as well as subsequent
decarboxylation (167) and dehydration (149) from fragment
211. Another fragment was found at m/z 141, correspond-
ing to [M�glucose�88]�, which was obtained by the loss
of the 3-oxopropanic acid molecule according to the frag-
mentation pathway shown in Fig. 4. This fragmentation
pattern is consistent with data provided by the literature
[41, 42]. This compound has been previously reported in
other species from genus Lippia as shanzhiside methyl
ester [43], as well as in Eremostachys laciniata, from order
Lamiales [44].
Compound found at 17.6 min (peak 11) could not be
identified since the provided molecular formula did not
match any common structure in plant matrices. It presented
fragments at m/z 177 and 159 characteristic of two succes-
sive dehydrations.
Peak 13 corresponded to jaceosidin, which presented a
fragment at 313 due to the loss of a methyl group. This
flavone has been previously identified in lemon verbena by
other authors [9, 11], although our previous HPLC method
could not find it in the same extract.
Compound 15 was proposed as another iridoid
glycoside, ixoside. MS/MS fragmentation presented
fragments at m/z 343, 181 and 137 consistent with a
decarboxylation (343) followed by a loss of a glucose
moiety (181) and further elimination of another CO2
molecule. In this way, the presence of two carboxylic groups
and glucose in the molecule is confirmed. This compound
has been reported in other species from order Lamiales
[45, 46].
4 Concluding remarks
This study presents the first application of CE-ESI-MS to the
qualitative characterization of a lemon verbena extract. This
method enables the tentative identification of 16
compounds including phenylpropanoid glycosides, flavo-
noid glycosides and iridoid glycosides. Furthermore, to our
knowledge, asperuloside, tuberonic acid glucoside or
Figure 4. Proposed fragmentation pathway for shanzhiside.
J. Sep. Sci. 2010, 33, 2818–2827 Electrodriven Separations 2825
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Bloque I_______
50-hydroxyjasmonic acid 50-O-glucoside, shanziside and
ixoside have been described for the first time in lemon
verbena. We can conclude that CE-ESI-MS is a valuable tool
in the study of polar constituents in plant matrices and a
complementary technique to HPLC.
The authors are grateful to the Spanish Ministry ofEducation and Science for the project AGL2008-05108-C03-03and a grant (FPU, AP2007-03246) and to Andalusian Regio-nal Government Council of Innovation and Science for theprojects P07-AGR-02619 and P09-CTS-4564. The authors alsothank Monteloeder, S. L. for providing the lemon verbenaextract.
The authors have declared no conflict of interest.
5 References
[1] Newall, C. A., Anderson, L. A., Phillipson, J. D., HerbalMedicines – A Guide For Health Care Professionals, ThePharmaceutical Press, London 1996.
[2] Devesa-Jorda, F., Pellicer-Bataller, J., Ferrando-Gines-tar, J., Borghol-Hariri, A., Bustamante-Balen, M., Ortu-no-Cortes, J., Ferrando-Marrades, I., Llobera-Bertran, C.,Sala-Lajo, A., Minana-Morell, M., Nolasco-Bonmatı, A.,Fresquet-Febrer, J. L., Gastroenterol. Hepatol. 2004, 27,244–249.
[3] Ragone, M. I., Sella, M., Conforti, P., Volonte, M. G.,Consolini, A. E., J. Ethnopharmacol. 2007, 113, 258–266.
[4] Pascual, M. E., Slowing, K., Carretero, E., Sanchez-Mata,D., Villar, A., J. Ethnopharmacol. 2001, 76, 201–214.
[5] Deepak, M., Handa, S. S., Phytother. Res. 2000, 14,463–465.
[6] Nakamura, T., Okuyama, E., Tsukada, A., Yamazaki, M.,Satake, M., Nishibe, S., Deyama, T., Moriya, A., Maruno,M., Nishimura, H., Chem. Pharm. Bull. 1997, 45,499–504.
[7] Andrade, P. B., Seabra, R. M., Valentao, P., Areias, F.,J. Liq. Chromatogr. Rel. Technol. 1998, 21, 2813–2820.
[8] Carnat, A., Carnat, A. P., Fraisse, D., Lamaison, J. L.,Fitoterapia 1999, 70, 44–49.
[9] Valentao, P., Andrade, P. B., Areias, F., Ferreres, F.,Seabra, R. M., J. Agric. Food Chem. 1999, 47, 4579–4582.
[10] Bilia, A. R., Giomi, M., Innocenti, M., Gallori, S., Vincieri,F. F., J. Pharm. Biomed. Anal. 2008, 46, 463–470.
[11] Ono, M., Oda, E., Tanaka, T., Iida, Y., Yamasaki, T.,Masuoka, C., Ikeda, T., Nohara, T., J. Nat. Med. 2008, 62,101–106.
[12] Funes, L., Fernandez-Arroyo, S., Laporta, O., Pons, A.,Roche, E., Segura-Carretero, A., Fernandez-Gutierrez,A., Micol, V., Food Chem. 2009, 117, 589–598.
[13] Quirantes-Pine, R., Funes, L., Micol, V., Segura-Carre-tero, A., Fernandez-Gutierrez, A., J. Chromatogr. A 2009,1216, 5391–5397.
[14] Wang, P., Kang, J., Zheng, R., Yang, Z., Lu, J., Gao, J.,Jia, Z., Biochem. Pharmacol. 1996, 51, 687–691.
[15] Akbay, P., Calis, I., Undeger, U., Basaran, N., AhmetBasaran, A., Phytother. Res. 2002, 16, 593–595.
[16] Ohno, T., Inoue, M., Ogihara, Y., Saracoglu, I., Biol.Pharm. Bull. 2002, 25, 666–668.
[17] Dıaz, A. M., Abad, M. J., Fernandez, L., Silvan, A. M., DeSantos, J., Bermejo, P., Life Sci. 2004, 74, 2515–2526.
[18] Huck, C. W., Stecher, G., Ahrer, W., Stoggl, W. M.,Buchberger, W., Bonn, G. K., J. Sep. Sci. 2002, 25,904–908.
[19] Arias, M., Simo, C., Ortiz, L. T., Mozos-Pascual, M.,Barbas, C., Cifuentes, A., Electrophoresis 2005, 26,2351–2359.
[20] Chen, J., Li, S. L., Li, P., Song, Y., Chai, X. Y., Ma, D. Y.,J. Sep. Sci. 2005, 28, 365–372.
[21] Ben Hameda, A., Gajdosova, D., Havel, J., J. Sep. Sci.2006, 29, 1188–1192.
[22] Segura-Carretero, A., Puertas-Mejıa, M. A., Cortacero-Ramırez, S., Beltran, R., Alonso-Villaverde, C., Joven, J.,Dinelli, G., Fernandez-Gutierrez, A., Electrophoresis2008, 29, 2852–2861.
[23] Arraez-Roman, D., Zurek, G., BaXmann, C., Segura-Carretero, A., Fernandez-Gutierrez, A., Electrophoresis2008, 29, 2112–2116.
[24] Citova, I., Ganzera, M., Stuppner, H., Solich, P., J. Sep.Sci. 2008, 31, 195–200.
[25] Simo, C., Barbas, C., Cifuentes, A., Electrophoresis2005, 26, 1306–1318.
[26] Schmitt-Kopplin, P., Frommberger, M., Electrophoresis2003, 24, 3837–3867.
[27] Brocke, A., Nicholson, G., Bayer, E., Electrophoresis2001, 22, 1251–1266.
[28] Macia, A., Borrull, F., Calull, M., Aguilar, C., Electro-phoresis 2004, 25, 3441–3449.
[29] Tanaka, Y., Kishimoto, Y., Otsuka, K., Terabe, S.,J. Chromatogr. A 1998, 817, 49–57.
[30] Von Poser, J. L., Moulis, C., Sobral, M., Henriques, A. T.,Pl. Syst. Evol. 1995, 198, 287–290.
[31] Mercier, J. P., Chimbault, P., Morin, P., Dreux, M.,Tambutez, A., J. Chromatogr. A 1998, 825, 71–80.
[32] Bruins, A. P., J. Chromatogr. A 1997, 794, 345–357.
[33] Mateus, L., Cherkaoui, S., Christen, P., Veuthey, J. L.,Electrophoresis 1999, 20, 3402–3409.
[34] Voyksner, R. D., in: Cole, R. B. (Ed.), Electrospray Ioni-zation Mass Spectrometry, Wiley, New York 1997,p. 323.
[35] Carnat, A., Carnat, A. P., Chavignon, O., Heitz, A., Wylde,R., Lamaison, J. L., Planta Med. 1995, 61, 490.
[36] Bianco, A., Guiso, M., Passacantilli, P., J. Nat. Prod.1984, 47, 901–902.
[37] Calis, I., Kirmizibekmez, H., Sticher, O., J. Nat. Prod.2001, 64, 60–64.
[38] Suomi, J., Wiedmer, S. K., Jussila, M., Riekkola, M. L.,J. Chromatogr. A 2002, 970, 287–296.
[39] Li, H., Nakashima, T., Tanaka, T., Zhang, Y. J., Yang, C.R., Kouno, I., J. Nat. Med. 2008, 62, 75–78.
[40] Kitajima, J., Ishikawa, T., Urabe, A., Chem. Pharm. Bull.2004, 52, 1013–1014.
J. Sep. Sci. 2010, 33, 2818–28272826 R. Quirantes-Pine et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Capítulo 2_______
[41] Ren, L., Xue, X., Zhang, F., Wang, Y., Liu, Y., Li, C., Liang,X., Rapid Commun. Mass Spectrom. 2007, 21, 3039–3050.
[42] Li, C. M., Zhang, X. L., Xue, X. Y., Zhang, F. F., Xu, Q.,Liang, X. M., Rapid Commun. Mass Spectrom. 2008, 22,1941–1954.
[43] Barbosa, F. G., Lima, M. A. S., Braz-Filho, R., Silveira,E. R., Biochem. Syst. Ecol. 2006, 34, 819–821.
[44] Calis, I., Guvenc, A., Armagan, M., Koyuncu, M.,Gotfredsen, C. H., Jensen, S. R., Nat. Prod. Commun.2008, 3, 117–124.
[45] Takeda, T., Narukawa, Y., Hada, N., Chem. Pharm. Bull.1999, 47, 284–286.
[46] Sinaphet, B., Noiarsa, P., Rujirawat, S., Otsuka, H.,Kanchanapoom, T., J. Nat. Med. 2006, 60, 251–254.
J. Sep. Sci. 2010, 33, 2818–2827 Electrodriven Separations 2827
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
Bloque I_______
HPLC ESI QTOF MS as a powerful analytical tool for
characterizing phenolic compounds in olive leaf extracts
HPLC ESI QTOF MS como una potente herramienta analítica
para caracterizar los compuestos fenólicos de extractos de hoja
de olivo
CAPÍTULO 3
Capítulo 3
155
HPLC-ESI-QTOF-MS as a powerful analytical tool for characterizing phenolic
compounds in olive leaf extracts
Rosa Quirantes-Piné1,2, Jesús Lozano-Sánchez1,2, Miguel Herrero3, Elena Ibáñez3,
Antonio Segura-Carretero1,2, Alberto Fernández-Gutiérrez1,2.
1Department of Analytical Chemistry, Faculty of Sciences, University of Granada, c/
Fuentenueva s/n, 18071 Granada, Spain.
2Functional Food Research and Development Center, Health-Science Technological
Park, Avd. del Conocimiento, 3, 18100 Granada, Spain.
3Department of Bioactivity and Food Analysis, Institute of Food Science Research
(CIAL-CSIC), Nicolás Cabrera 9, Campus Cantoblanco, 28049, Madrid, Spain.
Author for correspondence: Dr. A. Segura Carretero, Department of Analytical
Chemistry, Faculty of Sciences, University of Granada, c/ Fuentenueva s/n, 18071
Granada, Spain.
E-mail: [email protected]
Fax: +34 958 637083
Keywords: olive leaves; phenolic compounds; high-performance liquid
chromatography; electrospray ionization; quadrupole time of flight.
Bloque I
156
SHORT ABSTRACT
HPLC-ESI-QTOF was used to characterize the phenolic compounds from two olive
leaves extracts obtained by pressurized liquid extraction using water and ethanol as
extracting solvents. The information provided by QTOF mass spectrometer enabled the
in-depth characterization of both olive leaf extracts, allowing the tentative identification
of 31 different phenolic compounds in these extracts including secoiridoids, simple
phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. A new compound in
olive leaves, lucidumoside C, was also identified.
Capítulo 3
157
ABSTRACT
Introduction – Olea europaea L. leaves may be considered a cheap, easily available
natural source of phenolic compounds. In a previous study we evaluated the possibility
of obtaining bioactive phenolic compounds from olive leaves by pressurized liquid
extraction (PLE) for their use as natural antioxidants. The alimentary use of these kinds
of extract makes a comprehensive knowledge of their composition essential.
Objective – To undertake a comprehensive characterization of two olive leaf extracts
obtained by PLE using high-performance liquid chromatography coupled to
electrospray ionization-quadrupole time of flight mass spectrometry (HPLC-ESI-
QTOF-MS).
Methodology – Olive leaves were extracted by PLE using ethanol and water as
extraction solvents at 150ºC and 200°C respectively. Separation was carried out in a
HPLC system equipped with a C18 column working in a gradient elution program
coupled to ESI-QTOF-MS operating in negative ion mode.
Results – This analytical platform was able to detect 48 compounds and tentatively
identify 31 different phenolic ones in these extracts, including secoiridoids, simple
phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C, a
new compound in olive leaves, was also identified.
Conclusion – The coupling HPLC-ESI-QTOF-MS led to the in-depth characterization
of the olive leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem
mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical
tool is very valuable in the study of phenolic compounds in plant matrices.
Bloque I
158
INTRODUCTION
The last decade has witnessed an ever increasing interest in phenolic compounds
because of their health-giving properties with regard to the prevention of degenerative
diseases. Phenolic compounds in general show a broad spectrum of bioactive properties,
including antioxidant, anti-inflammatory, antimicrobial, antiproliferative, anti-
arrhythmic, platelet anti-aggregant and vasodilatory effects (Scalbert et al., 2005). As a
result there has been growing interest in the use of these phytochemicals as natural
antioxidants in the food industry as a way of providing additional value to common
foodstuffs.
Olea europaea L. leaves are a significant by-product in olive oil production, containing
as they do high quantities of phenolic compounds, and may be considered a cheap,
easily available natural source of these phytochemicals. As far as their antioxidant
activity is concerned, several studies have described how exert hypoglycaemic,
antihypertensive, antimicrobial, antiviral and anti-atherosclerotic effects (El et al.,
2009).
In a previous study we evaluated the possibility of obtaining bioactive phenolic
compounds from olive leaves by PLE for their use as natural antioxidants (Herrero et
al., 2011). The alimentary use of these kinds of extract makes a comprehensive
knowledge of their composition essential. Therefore, as a continuation of our previous
study, we have undertaken a further, more detailed characterization of the most
promising olive leaf extracts obtained.
The precise identification of phenolic compounds can be a complex task as they contain
a wide variety of structures. Within this context, HPLC–MS has proved to be a very
Capítulo 3
159
useful tool in the characterization of natural products (Careri et al., 1998; Xing et al.,
2007). ESI in particular, has been widely applied as it is a mild ionization technique
resulting in both protonated and deprotonated molecules. Accurate mass measurement
of small molecules is used to determine elemental formulas, thus enabling the
identification of unknown substances. Sometimes, because compounds in real samples
co-elute or MS is unable to distinguish between isobaric substances, structural
information may be needed and this can be obtained via MS/MS by means of collision-
induced dissociation (CID). QTOF-MS combines high sensitivity and mass accuracy for
both precursor and product ions, providing the elemental composition of the parent and
fragment ions. This feature helps to identify compounds thoroughly and to differentiate
between isobaric compounds. The potential of HPLC-ESI-QTOF-MS for qualitative
purposes has been highlighted in several studies (Rodríguez-Medina et al. 2009;
Gómez-Romero et al., 2011).
The aim of our work here was to undertake a comprehensive characterization of two
olive leaf extracts by HPLC-ESI-QTOF-MS and thus arrive at an in-depth knowledge of
their composition.
EXPERIMENTAL
Chemicals
All chemicals were of analytical reagent grade and used as received. Acetic acid and
acetonitrile for HPLC were from Fluka, Sigma-Aldrich (Steinheim, Germany) and Lab-
Scan (Gliwice, Sowinskiego, Poland) respectively. Water was purified by a Milli-Q
system from Millipore (Bedford, MA, USA). All the standard compounds were from
Sigma-Aldrich (St. Louis, MO). The stock solutions containing these analytes were
Bloque I
160
prepared in methanol (Lab-Scan, Gliwice, Sowinskiego, Poland) and stored at -20ºC
until use.
Samples
Dried olive leaves (Olea europaea L., variety Hojiblanca), generated as by-products
from the olive oil industry, were provided by “Cooperativa Sor Angela de la Cruz”
(Sevilla, Spain). The leaves were dried following a traditional procedure: once the
leaves were separated from the rest of plant materials they were covered to avoid direct
light and left ventilated at ambient temperature to remove humidity for about 50 days,
depending upon the relative humidity of the air. The dried leaves were then ground up
under liquid nitrogen and stored in darkness at 4ºC until use.
Pressurized Liquid Extraction (PLE)
The phenolic compounds were extracted from olive leaves as described by Herrero et
al., 2011. In brief, an accelerated solvent extractor (ASE 200, Dionex, Sunnyvale, CA,
USA) was used with ethanol or water as extracting solvents. The ethanol and water
extracts were obtained at 150ºC and 200°C respectively, with a static time equal to 20
min.
HPLC-ESI-QTOF-MS analyses
Analyses were made using an Agilent 1100 Liquid Chromatography system (Agilent
Technologies, Palo Alto, CA, USA) equipped with a standard autosampler. The HPLC
column was a Phenomenex Gemini C18 (3 μm, 2 x 150 mm). Separation was carried
out at 25ºC with a gradient elution program at a flow rate of 0.2 ml/min. The mobile
phases consisted of water plus 0.5% acetic acid (A) and acetonitrile (B). The following
Capítulo 3
161
multi-step linear gradient was applied: 0 min, 5% B; 5 min, 15% B; 25 min, 30% B; 35
min, 95% B; 40 min, 5% B. The initial conditions were maintained for 5 min. The
injection volume in the HPLC system was 1 μl.
The HPLC system was coupled to a micrOTOF-Q II mass spectrometer (Bruker
Daltoniks, Bremen, Germany) equipped with an ESI interface (Bruker Daltoniks,
Bremen, Germany) operating in negative ion mode using a capillary voltage of +4 kV.
The other optimum values of the ESI-QTOF-MS parameters were drying gas
temperature, 210ºC; drying gas flow, 8 l/min; and nebulizing gas pressure, 2 bar.
Detection was carried out within a mass range of 50-1100 m/z. Collision energy values
for MS/MS experiments were adjusted as follows: m/z 100, 20 eV; m/z 500, 30 eV; m/z
1000, 35 eV. Nitrogen was used as drying, nebulizing and collision gas.
The accurate mass data of the molecular ions were processed using DataAnalysis 4.0
software (Bruker Daltoniks), which provided a list of possible elemental formulas via
the GenerateMolecularFormula Editor. The GenerateMolecularFormula Editor uses a
CHNO algorithm, which provides standard functionalities such as minimum/maximum
elemental range, electron configuration and ring-plus double-bond equivalents, as well
as a sophisticated comparison of the theoretical with the measured isotope pattern
(Sigma Value) for increased confidence in the suggested molecular formula. The widely
accepted accuracy threshold for confirmation of elemental compositions was established
at 5 ppm (Bringmann et al., 2005). It is important to point out that even with very high
mass accuracy (<1ppm) many chemically possible formulas may be obtained,
depending upon the mass regions considered and so high mass accuracy alone is not
enough to exclude enough candidates with complex elemental compositions. The use of
isotopic abundance patterns as a single further constraint, however, removes >95% of
Bloque I
162
the false candidates. This orthogonal filter can reduce several thousand candidates down
to only a small number of molecular formulas.
During the development of the HPLC method, the instrument was calibrated externally
with a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois, USA) directly
connected to the interface and injected with a sodium acetate cluster solution containing
5 mM sodium hydroxide and 0.2% acetic acid in water:isopropanol (1:1, v/v). The
calibration solution was injected at the beginning of each run and all the spectra were
calibrated prior to compound identification. By using this method, an exact calibration
curve based on numerous cluster masses, each differing by 82 Da (NaC2H3O2), was
obtained. Due to the compensation of temperature drift in the micrOTOF-Q II, this
external calibration provided accurate mass values of better than 5 ppm for a complete
run without the need for a dual sprayer setup for internal mass calibration.
RESULTS AND DISCUSSION
Figure 1 shows the base peak chromatograms (BPC) of both olive leaf extracts obtained
by PLE using ethanol at 150ºC (OL150ET) and water at 200ºC (OL200W) as extracting
solvents.
The compounds were identified by comparing their retention times and MS/MS spectra
provided by QTOF-MS with those of authentic standards whenever available. The
remaining compounds were identified by interpretation of their MS and MS/MS spectra
obtained by QTOF-MS combined with the data provided in the literature. Table 1
summarizes the MS data of the identified compounds, including experimental and
calculated m/z for the molecular formulas provided, error, sigma value and the main
Capítulo 3
163
fragments obtained by MS/MS, as well as the proposed compound for each peak. As
can be seen from these data, the phenolic compounds identified belong to different
classes such as secoiridoids, simple phenols, flavonoids, cinnamic acid derivatives and
benzoic acids.
Figure 1. BPC (50–1100 m/z) of OL150ET extract (a) and OL200W extract (b), in which the
peaks are identified with numbers 1-48 according to the order of elution.
Tab
le 1
. Com
poun
ds id
entif
ied
in o
live
leaf
ext
ract
s
Peak
R
T
(min
) M
easu
red
m/z
Fo
rmul
a T
heor
etic
al
m/z
E
rror
(p
pm)
mSi
gma
Frag
men
ts
Prop
osed
com
poun
d E
xtra
ct
1 1.
84
181.
0717
C
6 H
13
O 6
18
1.07
18
0.1
38.1
71
.010
5 (1
00),
89.0
261
(50.
2), 1
01.0
239
(98.
3),
113.
0282
(54)
su
gar
OL1
50ET
O
L200
W
2 2.
58
195.
0516
C
6 H
11
O 7
19
5.05
10
-2.8
1.
3 75
.006
5 (1
00.0
), 99
.007
5 (2
4.9)
, 129
.019
0 (7
9.5)
, 17
7.03
78 (3
4.3)
, 192
.062
4 (4
5.6)
gl
ucon
ic a
cid
OL2
00W
3 2.
6 19
1.05
59
C 7
H 1
1 O
6
191.
0561
1.
1 44
.3
85.0
274
(11.
9), 9
3.03
20 (5
.6),
127.
0387
(10.
6)
quin
ic a
cid
OL1
50ET
6 6.
61
389.
1083
C
16
H 2
1 O
11
389.
1089
1.
6 18
.9
89.0
216
(20.
8), 1
13.0
211
(20.
8), 1
19.0
365
(25)
, 12
1.06
45 (1
00),
165.
0547
(32.
4), 1
83.0
641
(61.
4)
oleo
side
/sec
olog
anos
ide
OL1
50ET
O
L200
W
7 6.
73
153.
0553
C
8 H
9 O
3
153.
0557
3
50.1
12
3.04
46 (1
00)
hydr
oxyt
yros
ola
OL1
50ET
O
L200
W
9 9.
09
137.
0276
C
7H
5 O
3
137.
0244
-2
3.6
11.9
10
8.02
32 (1
00.0
), 11
9.03
18 (8
7.9)
p-
hydr
oxyb
enzo
ic a
cid
OL2
00W
10
9.
11
565.
1753
C
23
H 3
3 O
16
565.
1774
3.
7 37
.2
el
enol
ic a
cid
digl
ucos
ide
OL1
50ET
11
9.56
16
3.04
01
C 9
H 7
O 3
16
3.04
01
-0.1
41
.6
107.
0483
(55.
7), 1
19.0
497
(100
.0),
121.
0279
(65.
2),
135.
0381
(52.
4)
p-co
umar
ic a
cid
OL2
00W
12
9.58
15
1.03
93
C 8
H 7
O 3
15
1.04
01
4.8
779.
3 12
3.04
489
(100
) va
nilli
n O
L150
ET
OL2
00W
14
10
.79
403.
1234
C
17
H 2
3 O
11
403.
1246
3
20.9
89
.023
1 (8
6.6)
, 101
.022
2 (1
00),
113.
0225
(59.
9),
119.
0345
(70.
6)
oleo
side
met
hyl e
ster
O
L150
ET
17
11.7
4 38
9.14
36
C 1
7 H
25
O 1
0 38
9.14
53
4.3
23.5
10
1.02
26
(21.
4), 1
13.0
239
(25.
6), 1
51.0
760
(100
.0),
161.
0479
(12.
9), 1
69.0
885
(16.
7), 1
83.1
028
(29.
2), 3
13.1
331
(14.
8), 3
57.1
194
(23.
7)
7-ep
iloga
nin
OL1
50ET
O
L200
W
18
12.5
6 40
3.12
33
C 1
7 H
23
O 1
1 40
3.12
46
3.3
20.4
89.0
235
(13.
6), 1
01.0
229
(16.
9), 1
13.0
239
(22.
9),
119.
0347
(16.
2), 1
21.0
287
(100
), 12
7.03
73 (2
3.8)
, 13
9.00
51 (2
4.7)
, 165
.054
2 (6
8.9)
, 181
.049
0 (1
9.1)
, 22
3.06
07 (1
3.8)
, 371
.096
7 (1
3.7)
elen
olic
aci
d gl
ucos
ide
OL1
50ET
O
L200
W
19
12.6
2 60
9.14
48
C 2
7 H
29
O 1
6 60
9.14
61
2.1
38.5
28
5.03
83 (1
0.3)
, 447
.092
0 (1
00)
lute
olin
-7,4
-O-d
iglu
cosi
de
OL1
50ET
23
15.1
6 55
5.17
18
C 2
5 H
31
O 1
4 55
5.17
19
0.2
33.5
15
1.03
92 (1
00),
223.
0619
(12.
6), 3
23.0
792
(6.7
) hy
drox
yole
urop
ein
OL1
50ET
O
L200
W
25
16.1
6 59
3.15
14
C 2
7 H
29
O 1
5 59
3.15
12
-0.3
37
.7
285.
0397
(60.
8)
lute
olin
-7-O
-rut
inos
ide
OL1
50ET
O
L200
W
26
16.3
3 60
9.14
64
C 2
7 H
29
O 1
6 60
9.14
61
-0.5
38
.5
151.
0030
(4.3
), 17
8.99
64 (4
.5),
300.
0264
(100
.0)
rutin
O
L150
ET
27
16.6
2 62
3.19
81
C 2
9 H
35
O 1
5 62
3.19
81
0.1
42
161.
0241
(100
.0),
179.
0341
(14.
8), 4
61.1
626
(14.
4)
verb
asco
side
a O
L150
ET
28
16.8
5 19
5.06
64
C 1
0 H
11
O 4
19
5.06
63
-0.7
9.
6 13
5.05
27 (1
00)
hydr
oxyt
yros
ol a
ceta
te
OL1
50ET
O
L200
W
Bloque I_______
29
17.1
5 44
7.09
31
C 2
1 H
19
O 1
1 44
7.09
33
0.3
25
285.
0392
(100
) lu
teol
in-7
-O-g
luco
side
a O
L150
ET
OL2
00W
30
18.1
6 70
1.22
94
C 3
1 H
41
O 1
8 70
1.22
98
0.7
10.7
14
9.02
45 (3
1.2)
, 179
.059
2 (2
8.7)
, 275
.090
7 (1
00.0
), 30
7.08
10 (7
5.4)
, 377
.121
6 (6
4.7)
, 539
.174
2 (6
9.1)
ol
euro
pein
dig
luco
side
(is
omer
1)
OL1
50ET
31
18.6
3 70
1.22
94
C 3
1 H
41
O 1
8 70
1.22
98
0.7
48.9
17
9.05
47 (1
1.7)
, 275
.087
0 (1
5.3)
, 307
.084
1 (1
5.2)
, 37
7.11
75 (1
8.1)
, 539
.178
2 (1
00)
oleu
rope
in d
iglu
cosi
de
(isom
er 2
) O
L150
ET
OL2
00W
32
18.8
6 57
7.15
61
C 2
7 H
29
O 1
4 57
7.15
63
0.2
37.2
26
9.04
4 (1
00)
apig
enin
-7-O
-rut
inos
ide
OL1
50ET
O
L200
W
35
20.2
5 43
1.09
84
C 2
1 H
19
O 1
0 43
1.09
84
-0.2
23
.1
268.
0371
(41.
3)
apig
enin
7-O
-glu
cosi
de
OL1
50ET
O
L200
W
36
20.6
7 44
7.09
36
C 2
1 H
19
O 1
1 44
7.09
33
-0.7
25
.1
285.
0396
(100
) lu
teol
in 4
-O-g
luco
side
/ lu
teol
in 3
-O-g
luco
side
O
L150
ET
OL2
00W
37
21.3
7 70
1.22
97
C 3
1 H
41
O 1
8 70
1.22
98
0.1
49.3
11
3.02
21 (2
5.8)
, 149
.023
2 (2
5.1)
, 161
.045
1 (3
1.3)
, 17
9.05
61 (1
6.8)
, 275
.093
1 (1
00),
307.
0829
(66.
4),
345.
0988
(23.
1), 3
77.1
246
(96.
6)
oleu
rope
in d
iglu
cosi
de
(isom
er 3
) O
L150
ET
OL2
00W
38
21.9
2 53
9.17
68
C 2
5 H
31
O 1
3 53
9.17
70
0.3
32.5
13
9.03
69 (2
5.3)
, 149
.024
2 (4
4.5)
, 179
.056
2 (1
9.7)
, 22
3.06
11 (4
5.7)
, 275
.087
8 (1
00.0
), 30
7.08
14 (8
9.0)
, 37
7.12
38 (1
9.8)
oleu
rope
ina
OL1
50ET
O
L200
W
39
22.8
4 53
9.17
76
C 2
5 H
31
O 1
3 53
9.17
70
-1.1
32
.4
139.
0382
(45.
7), 1
49.0
243
(78.
3), 2
23.0
617
(45.
6),
275.
0871
(100
.0),
307.
0833
(95.
3), 3
77.1
282
(30.
9)
ol
euro
pein
isom
er
OL1
50ET
41
23.6
3 53
9.17
76
C 2
5 H
31
O 1
3 53
9.17
70
-1.1
32
.5
149.
0247
(26.
9), 1
65.0
545
(33.
3), 1
79.0
577
(42.
3),
223.
0602
(68.
1), 2
75.0
830
(61.
2), 3
07.0
816
(100
.0),
371.
0953
(62.
2), 4
03.1
236
(37.
2)
oleu
rosi
de
OL1
50ET
O
L200
W
42
24.3
3 58
3.20
36
C 2
7 H
35
O 1
4 58
3.20
32
-0.6
36
.8
151.
0396
(100
), 17
9.05
55 (1
9.9)
, 223
.060
3 (4
3.4)
, 40
3.12
10 (1
3.4)
lu
cidu
mos
ide
C (i
som
er 1
) O
L150
ET
43
24.6
5 58
3.20
38
C 2
7 H
35
O 1
4 58
3.20
32
-1
36.8
15
1.03
96 (1
00),
179.
0561
(19.
5), 2
23.0
596
(40)
, 40
3.12
40 (1
1.7)
, 537
.158
5 (8
.2)
luci
dum
osid
e C
(iso
mer
2)
OL1
50ET
44
24.7
9 55
7.22
18
C 2
6 H
37
O 1
3 55
7.22
40
3.8
17.5
12
1.06
38
(34.
6), 1
83.0
664
(85.
3), 1
85.1
184
(40.
0),
227.
0573
(44.
0), 2
27.1
279
(52.
6)
6´
-O-[
2,6-
dim
ethy
l-8-h
ydro
xy-2
-oc
teno
ylox
y]se
colo
gano
side
O
L200
W
45
25.8
2 52
3.18
32
C 2
5 H
31
O 1
2 52
3.18
21
-2
31.5
10
1.02
34 (1
1.5)
, 127
.039
2 (1
4), 2
59.0
953
(20.
4),
291.
0862
(100
), 36
1.12
82 (8
.1)
ligst
rosi
de
OL1
50ET
O
L200
W
46
26.2
1 58
3.20
40
C 2
7 H
35
O 1
4 58
3.20
32
-1.3
36
.9
151.
0393
(100
), 17
9.05
64 (6
8.1)
, 223
.059
1 (7
9),
351.
1042
(18.
9), 3
71.0
943
(71.
2), 4
03.1
223
(56.
9),
537.
1616
(22.
7)
luci
dum
osid
e C
(iso
mer
3)
OL1
50ET
48
28.3
2 28
5.04
11
C 1
5 H
9 O
6
285.
0405
-2
.4
14.4
13
3.02
95 (6
.1),
151.
0013
(7.5
), 17
5.03
87 (4
.5),
255.
0299
(6.9
) lu
teol
ina
OL1
50ET
O
L200
W
a Iden
tific
atio
n co
nfirm
ed u
sing
com
mer
cial
stan
dard
s.
Capítulo 3_______
Bloque I
166
Secoiridoids
Olea europaea L. is rich in secoiridoids, especially in oleosides, which are oleaceae-
specific secoiridoids commonly esterified to a phenolic moiety. In fact one of the major
compounds found in both extracts was oleuropein (compound 38), which was
confirmed by comparison with the authentic standard. This compound has been
described previously as being the main component of olive leaves (Benavente-García et
al., 2000; Briante et al., 2002; Chiou et al., 2007; Pererira et al., 2007; Salta et al., 2007;
Altiok et al., 2008; Mylonaki et al., 2008; Goulas et al., 2009, 2010; Laguerre et al.,
2009; Fu et al., 2010). Two other oleuropein isomers with a similar fragmentation
pattern (39 and 41) were present in the extracts. Oleuropein has traditionally been found
in olive leaves together with its isomer oleuroside, which has been proposed as
compound 41 on the basis of its elution order. Other oleuropein derivatives such as
hydroxyoleuropein (23) and several oleuropein diglucoside isomers (30, 31 and 37),
were also found in both extracts.
Peak 6 has been identified as oleoside or secologanoside, both of which have been
reported before in olive leaves (Di Donna et al., 2010; Kiritsakis et al., 2010; Poudyal et
al., 2010). Because of the identical fragmentation pattern of these analytes (Fu et al.,
2010), it was impossible to distinguish between them.
Oleoside methyl ester has been proposed as compound 14. The presence of this oleoside
derivative in the olive fruit has been reported before, together with its main fragments at
m/z 119.0345 and 89.0231 (Bianco et al., 2003; Di Donna et al., 2007), and it has
recently been identified in olive leaves by Di Donna et al., 2010. Peak 18 showed the
same molecular formula as oleoside methyl ester but its MS/MS spectrum presented a
fragment at m/z 223.0607, which corresponds to dehydrated elenolic acid and so this
Capítulo 3
167
compound was proposed as being elenolic acid glucoside, which has been identified in
the same matrix by Fu et al., 2010. The diglucosidic form of elenolic acid has also been
found in the ethanolic extract (10).
Compound 17 has been characterized as 7-epiloganin, an intermediate in the
biosynthesis of oleoside-type secoiridoids (Jensen et al., 2002) that has been reported
previously in olive leaves (Rovellini and Cortesi, 1998). This assignment was consistent
with the presence in the MS/MS spectrum of a fragment at 169.0885, representing the
loss of the glucose moiety and the methylester group, as shown in Figure 2. Subsequent
dehydration was responsible for producing the major product ion at 151.0760. Another
fragment was detected at 357.1194, corresponding to the elimination of the methoxyl
group from the precursor ion.
Figure 2. Proposed fragmentation pathway for 7-epiloganin (compound 17).
The software provided the same molecular formula for peaks 42, 43 and 46 and they
also showed a similar fragmentation pattern. They were tentatively identified as
lucidumoside C and its isomers although it was impossible to assign any specific peak
Bloque I
168
to lucidumoside C itself. The structure of this secoiridoid closely resembles that of
oleuropein and it has been reported previously in other species belonging to the
Oleaceae family, such as Ligustrum lucidum (He et al., 2001; Guo et al., 2011). The
proposed fragmentation pathway is depicted in Figure 3. The intense fragment at
403.1235 was due to cleavage of the phenolic moiety, after which it could undergo
elimination of the methoxyl group (corresponding to fragment 371.0973) or glucose
moiety (fragment 223.0601). The ion at 537.1603 could be attributed to the loss of the
ethoxyl group from the precursor ion whilst the fragment at 351.1085 arose from the
cleavage of the elenolic ring as described for oleuropein (Japón-Luján et al., 2008). The
main fragment at 151.0389 matched with the elimination of the ethoxyl group from the
phenolic moiety.
Compound 44 corresponded to 6´-O-[2,6-dimethyl-8-hydroxy-2-
octenoyloxy]secologanoside, the structure and fragmentation pathway of which are
shown in Figure 4. Its assignment was consistent with the presence of fragments found
at m/z 227.0550 and 183.0652, representing the loss of the glucose moiety and its
subsequent decarboxylation. This compound has been described in the past in olive
leaves with boron deficiency (Karioti et al., 2006).
Compound 45 was proposed as being ligstroside since its molecular formula and
fragmentation pattern agreed with the data reported in the literature and it has also been
widely described in olive leaves (Briante et al., 2002; Laguerre et al., 2009; Fu et al.,
2010).
Figu
re 3
. Pro
pose
d fr
agm
enta
tion
path
way
for l
ucid
umos
ide
C (c
ompo
und
42, 4
3 or
46)
.
Capítulo 3_______
Bloque I
170
Figure 4. Structure and fragmentation pathway of 6´-O-[2,6-Dimethyl-8-hydroxy-2-
octenoyloxy]secologanoside (compound 44).
Simple phenols
Hydroxytyrosol has been widely described as one of the main components of olive
leaves (Benavente-García et al. 2000; Briante et al., 2002; Chiou et al., 2007; Salta et al.;
Altiok et al., 2008; Goulas et al., 2009, 2010; Fu et al., 2010) and it has been found in both
extracts (7), identifying it by comparison with commercial standards. Its acetate
derivative has also been identified (28).
Flavonoids
Flavonoids are another important group of phenolic compounds widely represented in
olive leaves. Among these, luteolin-7-O-glucoside (29), rutin (26), apigenin-7-O-
glucoside (35) and luteolin (48) are the most cited in the literature (Benavente-García et
al. 2000; Meirinhos et al., 2005; Pereira et al., 2007; Altiok et al., 2008; Mylonaki et al.,
2008; Goulas et al., 2009, 2010; Laguerre et al., 2009; Fu et al., 2010) and all of them were
identified in the extracts by comparing their molecular formulas and fragmentation
patterns with those reported in the literature and databases.
Capítulo 3
171
Other flavonoids found in the extracts were luteolin-7,4-O-diglucoside (19), luteolin-7-
O-rutinoside (25), apigenin-7-O-rutinoside (32) and luteolin-4-O-glucoside or luteolin-
3-O-glucoside (36). The latter compounds have also been reported in previous studies
although with a narrower spread (Meirinhos et al., 2005; Pereira et al., 2007; Mylonaki et
al., 2008; Goulas et al., 2009, 2010; Laguerre et al., 2009).
Cinnamic acids and derivatives
Compound 27 was identified as verbascoside according to the MS data and by
comparison with the retention time and MS/MS spectrum of the standard. This cinnamic
acid derivative is commonly present in all the derivates of the olive tree (Benavente-
García et al. 2000; Pereira et al., 2007; Altiok et al., 2008; Laguerre et al., 2009; Fu et al.,
2010).
The aqueous extract also contained p-coumaric acid (11). The molecular ion of p-
coumaric acid (m/z 163.0401) produced the major fragment ion at m/z 119.0497,
corresponding to the loss of carbon dioxide from the precursor ion.
Benzoic acids
As far as benzoic acids are concerned, vanillin (12) and p-hydroxybezoic acid (9) were
identified in the extracts. These compounds have been described previously in the
literature (Benavente-García et al., 2000; Chiou et al., 2007; Salta et al., 2007; Altiok et
al., 2008). These assignments were supported by the fragment ions produced in MS/MS
spectra. In the case of vanillin, just one ion, at m/z 123.0449, was yielded from the loss
of the carbonyl group, whilst the p-hydroxybenzoic acid spectrum showed an ion at
119.0318, resulting from dehydration of the parent ion.
Bloque I
172
Unknown compounds
Table 2 shows a list of compounds for which it was impossible to elucidate a structure
due to a lack of sufficient evidence. The table includes retention times, experimental
m/z, molecular formulas, errors, sigma values and MS/MS fragments.
In summary, a powerful analytical method has been used to characterize
comprehensively two olive leaf extracts obtained by PLE using ethanol and water as
solvents. The coupling HPLC-ESI-QTOF-MS enabled us to characterize tentatively
more than 30 different phenolic compounds, including secoiridoids, simple phenols,
flavonoids, cinnamic acid derivatives and benzoic acids. We may conclude therefore
that this analytical tool is very valuable in the study of phenolic compounds in plant
matrices. It is also important to highlight that, to the best of our knowledge, this is the
first time that lucidumoside C has been detected in olive leaves.
ACKNOWLEDGEMENTS
This work was supported by the projects AGL2008-05108-C03-03/01 and AGL2011-
29857-C03-02 of the Spanish Ministry of Science and Innovation; P09-CTS-4564, P10-
FQM-6563 and P11-CTS-7625 of the Andalusian Regional Government Council of
Innovation and Science, and GREIB.PT.2011.18. The authors are grateful to the
Spanish Ministry of Science and Innovation for FPU grant AP2007-03246 and a
“Ramón y Cajal” research contract. The authors thank “Cooperativa Sor Angela de la
Cruz” for providing the olive leaves.
Tab
le 2
. Unk
now
n co
mpo
unds
from
oliv
e le
ave
extra
cts
Peak
R
T (m
in)
Mea
sure
d m
/z
Form
ula
Theo
retic
al
m/z
Er
ror
(ppm
) m
Sigm
aFr
agm
ents
Ex
trac
t
4 2.
87
128.
0354
C
5 H
6 N
O 3
12
8.03
53
-0.3
2.
8
OL2
00W
5 3.
48
217.
0712
C
9 H
13
O 6
21
7.07
18
2.7
4.3
111.
0063
(26.
2), 1
29.0
556
(100
.0),
155.
0634
(20.
1),
173.
0436
(33.
8)
OL2
00W
8 8.
32
199.
0614
C
9 H
11
O 5
19
9.06
12
-0,8
16
,8
69.0
327
(49.
3), 8
5.03
10 (5
7.3)
, 95.
0472
(68.
3),
111.
0822
(100
.0)
OL1
50ET
O
L200
W
13
10.2
2 48
9.16
02
C 2
1 H
29
O 1
3 48
9.16
14
2.4
27
145.
0293
(100
), 16
3.04
04 (9
2.3)
, 205
.050
4 (2
2.6)
, 23
5.06
21 (1
5.2)
, 265
.069
5 (2
0.9)
O
L150
ET
OL2
00W
15
10.8
2 44
5.20
60
C 2
1 H
33
O 1
0 44
5.20
80
4.3
21.0
OL2
00W
16
11.3
5 37
7.14
44
C 1
6 H
25
O 1
0 37
7.14
53
2.5
17.7
15
3.09
25 (6
5.9)
, 197
.081
0 (1
00)
OL1
50ET
O
L200
W
20
13.0
7 24
5.10
28
C 1
1 H
17
O 6
24
5.10
31
1.2
11.8
11
1.08
11 (1
00),
155.
0709
(51.
7), 2
01.1
113
(68.
7)
OL1
50ET
21
13.6
1 19
1.03
47
C 1
0 H
7 O
4
191.
0350
1.
3 10
.5
108.
0203
(77.
8), 1
19.0
533
(29.
5), 1
21.0
298
(35.
9),
135.
0492
(45.
9), 1
47.0
426
(18.
6), 1
63.0
410
(100
.0)
OL2
00W
22
14.4
4 51
1.23
76
C 2
2 H
39
O 1
3 51
1.23
96
4 29
.2
185.
1158
(100
), 19
9.13
41 (6
0.2)
, 227
.128
4 (4
7),
343.
1228
(38.
1)
OL1
50ET
O
L200
W
24
15.3
3 24
5.10
32
C 1
1 H
17
O 6
24
5.10
31
-0.5
10
.9
111.
0818
(100
), 13
7.06
06 (7
), 15
5.07
20 (4
8.5)
, 20
1.11
27 (5
3.4)
, 227
.088
4 (6
.6)
OL1
50ET
33
19.4
9 49
1.17
66
C 2
1 H
31
O 1
3 49
1.17
70
0.7
26.9
10
1.02
32 (1
0.9)
, 167
.070
3 (1
4.5)
, 199
.060
3 (1
00),
269.
1010
(13.
2)
OL1
50ET
34
19.8
5 18
7.09
72
C 9
H 1
5 O
4
187.
0976
2.
1 12
.2
97.0
658
(6.8
), 12
3.07
88 (8
.8),
125.
0964
(1
00.0
),169
.089
8 (5
.0)
OL1
50ET
O
L200
W
40
23.2
7 53
7.16
14
C 2
5 H
29
O 1
3 53
7.16
14
-0.1
31
.9
151.
0399
(100
), 22
3.06
15 (1
7.4)
, 275
.090
0 (1
5.6)
O
L150
ET
47
27.0
7 62
3.14
16
C 3
1 H
27
O 1
4 62
3.14
06
-1.5
43
.6
161.
0225
(6.2
), 28
5.03
83 (8
1.1)
, 299
.055
8 (1
6),
323.
0772
(22.
8)
OL1
50ET
Capítulo 3_______
Bloque I
174
REFERENCES
Altiok E, Bayçin D, Bayraktar O, Ülkü S. 2008. Isolation of polyphenols from the
extracts of olive leaves (Olea europaea L.) by adsorption on silk fibroin. Sep
Purif Tech 62: 342–348.
Benavente-García O, Castillo J, Lorente J, Ortuño A, del Rio JA. 2000. Antioxidant
activity of phenolics extracted from Olea europaea L. leaves. Food Chem 68:
457-462.
Bianco A, Buiarelli F, Cartoni G, Coccioli F, Jasionowska R, Margherita P. 2003.
Analysis by liquid chromatography-tandem mass spectrometry of biophenolic
compounds in olives and vegetation waters, Part I. J Sep Sci 26: 409–416.
Briante R, Patumi M, Terenziani S, Bismuto E, Febbraio F, Nucci R. 2002. Olea
europaea L. leaf extract and derivatives: antioxidant properties. J Agric Food
Chem 50: 4934-4940.
Bringmann G, Kajahn I, Neusüß C, Pelzing M, Laug S, Unger M, Holzgrabe U. 2005.
Analysis of the glucosinolate pattern of Arabidopsis thaliana seeds by capillary
zone electrophoresis coupled to electrospray ionization-mass spectrometry.
Electrophoresis 26: 1513-1522.
Careri M, Mangia A, Musci M. 1998. Overview of the applications of liquid
chromatography mass spectrometry interfacing systems in food analysis:
naturally occurring substances in food. J Chromatogr A 794: 263–297.
Chiou A, Salta FN, Kalageropoulos N, Mylona A, Ntalla I, Andrikopoulos NK. 2007.
Retention and distribution of polyphenols after pan-frying of french fries in oils
enriched with olive leaf extract. J Food Sci 72: S574-S583.
Capítulo 3
175
Di Donna L, Mazzotti F, Napoli A, Salerno R, Sajjad A, Sindona G. 2007. Secondary
metabolism of olive secoiridoids. New microcomponents detected in drupes by
electrospray ionization and high-resolution tandem mass spectrometry. Rapid
Commun Mass Spectrom 21: 273–278.
Di Donna L, Mazzotti F, Naccarato A, Salerno R, Tagarelli A, Taverna D, Sindona G.
2010. Secondary metabolites of Olea europaea leaves as markers for the
discrimination of cultivars and cultivation zones by multivariate analysis. Food
Chem 121: 492–496.
El SN, Karakaya S. 2009. Olive tree (Olea europaea) leaves: potential beneficial effects
on human health Nutr Rev 67: 632–638.
Fu S, Arráez-Román D, Segura-Carretero A, Menéndez JA, Menéndez-Gutiérrez MP,
Micol V, Fernández-Gutiérrez A. 2010. Qualitative screening of phenolic
compounds in olive leaf extracts by hyphenated liquid chromatography and
preliminary evaluation of cytotoxic activity against human breast cancer cells.
Anal Bioanal Chem 397: 643–654.
Gómez-Romero M, Zurek G, Schneider B, Baessmann C, Segura-Carretero A,
Fernández-Gutiérrez A. 2011. Automated identification of phenolics in plant-
derived foods by using library search approach. Food Chem 124: 379–386.
Goulas V, Exarchou V, Troganis AN, Psomiadou E, Fotsis T, Briasoulis E,
Gerothanassis IP. 2009. Phytochemicals in olive-leaf extracts and their
antiproliferative activity against cancer and endothelial cells. Mol Nutr Food Res
53: 600 – 608.
Bloque I
176
Goulas V, Papoti VT, Exarchou V, Tsimidou MZ, Gerothanassis IP. 2010. Contribution
of flavonoids to the overall radical scavenging activity of olive (Olea europaea
L.) leaf polar extracts. J Agric Food Chem 58: 3303–3308.
Guo N, Yu Y, Ablajan K, Li L, Fan B, Peng J, Yan H, Ma F, Nie Y. 2011. Seasonal
variations in metabolite profiling of the fruits of Ligustrum lucidum Ait. Rapid
Commun Mass Spectrom 25: 1701-1714.
He ZD, But PPH, Chan TWD, Dong H, Xu HX, Lau CP, Sun HD. 2001. Antioxidative
glucosides from the fruits of Ligustrum lucidum. Chem Pharm Bull 49: 780-784.
Herrero M, Temirzoda TN, Segura-Carretero A, Quirantes R, Plaza M, Ibáñez E. 2011.
New possibilities for the valorization of olive oil by-products J Chromatogr A
1218: 7511-7520.
Japón-Luján R, Priego-Capote F, Marinas A, Luque de Castro MD. 2008. Liquid
chromatography/triple quadrupole tandem mass spectrometry with multiple
reaction monitoring for optimal selection of transitions to evaluate nutraceuticals
from olive-tree materials. Rapid Commun Mass Spectrom 22: 855–864.
Jensen SR, Franzyk H, Wallander E. 2002. Chemotaxonomy of the Oleaceae: iridoids
as taxonomic markers. Phytochem 60: 213–231.
Karioti A, Chatzopoulou A, Bilia AR, Liakopoulos G, Stavrianakou S, Skaltsa H. 2006.
Novel secoiridoid glucosides in Olea europaea leaves suffering from boron
deficiency. Biosci Biotechnol Biochem 70: 1898–1903.
Kiritsakis K, Kontominas MG, Kontogiorgis C, Hadjipavlou-Litina D, Moustakas A,
Kiritsakis A. 2010. Composition and antioxidant activity of olive leaf extracts
from Greek olive cultivars. J Am Oil Chem Soc 87: 369–376.
Capítulo 3
177
Laguerre M, López-Giraldo LJ, Piombo G, Figueroa-Espinoza MC, Pina M, Benaissa
M, Combe A, Rossignol-Castera A, Lecomte J, Villeneuve P. 2009.
Characterization of olive-leaf phenolics by ESI-MS and evaluation of their
antioxidant capacities by the CAT assay. J Am Oil Chem Soc 86: 1215–1225.
Meirinhos J, Silva JM, Valentão P, Seabra RM, Pereira JA, Dias A, Andrade PB,
Ferreres F. 2005. Analysis and quantification of flavonoidic compounds from
Portuguese olive (Olea Europaea L.) leaf cultivars. Nat Prod Res 19: 189–195.
Mylonaki S, Kiassos E, Makris DP, Kefalas P. 2008. Optimisation of the extraction of
olive (Olea europaea) leaf phenolics using water/ethanol-based solvent systems
and response surface methodology. Anal Bioanal Chem 392: 977–985.
Pereira AP, Ferreira ICFR, Marcelino F, Valentão P, Andrade PB, Seabra R, Estevinho
L, Bento A, Pereira JA. 2007. Phenolic compounds and antimicrobial activity of
olive (Olea europaea L. Cv. Cobrançosa) leaves. Molecules 12: 1153-1162.
Poudyal H, Campbell F, Brown L. 2010. Olive leaf extract attenuates cardiac, hepatic,
and metabolic changes in high carbohydrate–, high fat–fed rats. J Nutr 140: 946-
953.
Rodríguez-Medina IC, Segura-Carretero A, Fernández-Gutiérrez A. 2009. Use of high-
performance liquid chromatography with diode array detection coupled to
electrospray-Qq-time-of-flight mass spectrometry for the direct characterization of
the phenolic fraction in organic commercial juices. J Chromatogr A 1216: 4736–
4744.
Rovellini P, Cortesi N. 1998. Identification of three intermediates compounds in the
biosynthesis of secoiridoids as oleoside-type in different anatomic parts of Olea
Bloque I
178
europea L. by HPLC-electrospray-mass spectrometry. Riv Ital Sostanze Gr 75:
543-550.
Salta FN, Mylona A, Chiou A, Boskou G, Andrikopoulos NK. 2007. Oxidative stability
of edible vegetable oils enriched in polyphenols with olive leaf extract. Food Sci
Tech Int 13: 413-421.
Scalbert A, Manach C, Morand C, Rémésy C. 2005. Dietary polyphenols and the
prevention of diseases. Crit Rev Food Sci Nutr 45: 287-306.
Xing J, Xie C, Lou H. 2007. Recent applications of liquid chromatography mass
spectrometry in natural products bioanalysis. J Pharm Biomed Anal 44: 368–378.
BLOQUE II. ESTUDIOS
METABOLÓMICOS DE COMPUESTOS
FENÓLICOS BIOACTIVOS
Evaluation of different extraction approaches for the
determination of phenolic compounds and their metabolites in
plasma by nanoLC ESI TOF MS
Evaluación de diferentes estrategias de extracción para la
determinación de compuestos fenólicos y sus metabolitos en
plasma mediante nanoLC ESI TOF MS
CAPÍTULO 4
Capítulo 4
183
Evaluation of different extraction approaches for the determination of phenolic
compounds and their metabolites in plasma by nanoLC-ESI-TOF-MS
R. Quirantes-Piné1,2, V. Verardo3,4, D. Arráez-Román1,2, S. Fernández-Arroyo1,2, V.
Micol5, M. F. Caboni3,4, A. Segura-Carretero1,2, A. Fernández-Gutiérrez1,2
1Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avd.
Fuentenueva s/n, 18071, Granada, Spain.
2Functional Food Research and Development Center, Health Science Technological
Park, Avd. del Conocimiento, 3, 18100, Granada, Spain.
3Department of Food Science, University of Bologna, Piazza Goidanich 60, 47521
Cesena (FC), Italy.
4Inter-departmental Centre for Agri-Food Industrial Research (CIRI Agroalimentare),
University of Bologna, Piazza Goidanich 60, 47521 Cesena (FC), Italy.
5Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avenida de
la Universidad s/n, 03202 Elche, Alicante, Spain
Corresponding author: Dr. A. Segura Carretero, Department of Analytical Chemistry,
Faculty of Sciences, University of Granada, c/ Fuentenueva s/n, 18071 Granada, Spain.
E-mail: [email protected]
Fax: +34 958 637083
Bloque II
184
ABSTRACT
Sample preparation is an important step for the determination of phenolic compounds in
biological samples. Different extraction methods have been tested to determine phenolic
compounds and their metabolites in plasma by nano-liquid chromatography coupled to
electrospray ionization-time of flight mass spectrometry (nanoLC-ESI-TOF-MS). The
sample treatment optimisation was performed using commercial foetal bovine serum
spiked with representative phenolic standards, namely naringenin, luteolin,
verbascoside, apigenin, rutin, syringic acid and catechin. Different protein-precipitation
conditions were evaluated as well as enzymatic digestion with trypsin and solid-phase
extraction using different phases such as C-18, ABN and ENV+, working at different
pH values. The optimum extraction procedure consisted of a previous protein-
precipitation step using HCl 200 mM in methanol for 2.5 h at 50ºC followed by a solid-
phase extraction using C-18 cartridges at pH 2.5. This procedure was finally applied to
the plasma of rats overfed with a phenolic-rich Lippia citriodora extract. These samples
were analysed by nanoLC-ESI-TOF-MS, enabling the identification of five compounds
previously found in the administered Lippia citriodora extract and one metabolite.
Keywords: phenolic compounds, extraction procedure, plasma, nanoLC-ESI-TOF-MS.
Capítulo 4
185
1. Introduction
Over the last decade interest surged in phenolic compounds, mainly for the healthy
properties of these compounds related to the prevention of degenerative diseases. Plant
phenolic compounds have been associated with numerous beneficial properties
including antioxidant, anti-inflammatory, anticancer, and anti-atherosclerotic activities
[1]. In this sense, the study of the bioavailability, pharmacokinetic and metabolism of
phenolic compounds is necessary to understand their effects on health. However, the
information on the bioavailability and metabolism of polyphenols is scarce and
contradictory. This scarcity of data has resulted from a lack of sensitive, convenient
methods for measuring an extended spectrum of polyphenols in plasma and body fluids,
compared to the number of methods developed for their investigation in food [2, 3].
Phenolic compounds in biological samples have been studied primarily by conventional
liquid chromatography followed by DAD [4, 5], MS [6, 7] or NMR [8]. Meanwhile,
nanoLC has been less often applied to analyse phenolic compounds in biological
samples [9], despite that a large number of applications have been reported in
proteomics [10, 11] and other fields such as pharmaceutical [12], environmental [13],
enantiomeric [14], and food analysis [15, 16]. From a theoretical standpoint, it has been
demonstrated that the sensitivity heightens on shrinking the column internal diameter
(i.d.). This effect can be ascribed to both a lower analyte chromatographic dilution [17]
and higher efficiency [18]. A complementary approach used to intensify the sensitivity
in nanoLC, involves coupling with mass spectrometry. Hyphenation is easy to achieve
due to the relatively low flow rate involved in the separation process. Indeed, when the
electrospray ionization (ESI) is used as the continuous-flow ionization technique, a
Bloque II
186
slower flow rate will boost the number of ions in the gas phase, heightening sensitivity
[19]. Due to these features, nanoLC-MS may be considered a potential tool in
applications which demand high sensitivity, such as metabolomics.
Sample preparation, a crucial step in any analytical procedure, usually serves several
purposes, such as the extraction of the analytes from the matrix, bringing them into a
compatible format with the analytical technique used, and the removal of matrix
components, which can interfere with the analysis. This extraction procedure can
include a pre-concentration step to extend the detection limits of the analytical
technique applied. Sample preparation for the analysis of phenolic compounds in
plasma can include, but is not limited to, protein precipitation [20], liquid-liquid
extraction (LLE) [6], or solid-phase extraction (SPE) [21]. The choice of a particular
sample-treatment procedure is determined by the analytical technique. Despite the
availability of many sample-preparation protocols for analysing phenolic compounds in
plasma by LC, none have been devised for use at the nano-scale.
The aim of the present work was to develop an efficient procedure for extracting a
broad range of phenolic compounds from complex biological matrices for subsequence
analysis by nanoLC-ESI-TOF-MS. The procedure developed was applied for the
identification of phenolic compounds in the plasma of rats overfed with a phenolic-rich
Lippia citriodora extract previously characterized by HPLC-MS and CE-MS [22, 23].
2. Materials and methods
2.1. Chemicals
All chemicals were of analytical reagent grade and used as received. (±)-Naringenin,
luteolin, verbascoside, apigenin, rutin, syringic acid, and (+)-catechin were from Sigma
Capítulo 4
187
Aldrich (St. Louis, MO, USA). The stock solution containing these analytes was
prepared in ethanol at a concentration of 2 mg/mL of each compound and stored at -
20°C until used. Foetal bovine serum used as simulated sample was also from Sigma
Aldrich.
For the extraction procedures, methanol, ethanol, and ammonia were from Panreac
(Barcelona, Spain), hydrochloric acid from Scharlau (Barcelona, Spain) and acetic acid,
ammonium bicarbonate and trypsin from Sigma Aldrich.
Formic acid and acetonitrile used as nano-LC mobiles phases were from Sigma Aldrich
and Lab-Scan (Gliwice, Sowinskiego, Poland), respectively. Water was purified by a
Milli-Q system from Millipore (Bedford, MA, USA).
2.2. NanoLC system
NanoLC experiments were performed in a commercially available instrument EASY-
nLC (Bruker Daltonik, Bremen, Germany). The chromatographic separation was
performed in a C18 BioSphere capillary column (100 mm x 75 μm i.d., particle size 5
μm). An online C18 BioSphere trapping column (20 mm x 100 μm i.d., particle size 5
μm) was used before the analytical column.
The separation was carried out at room temperature with a gradient elution program at a
flow rate of 300 nl/min. The mobile phases consisted of water:acetonitrile (90:10, v/v)
with formic acid 0.1% (A) and acetonitrile (B). The following multi-step linear gradient
was applied: 0 min, 5% B; 35 min, 20% B; 45 min, 40% B; 70 min, 95% B; 75 min, 5%
B. The initial conditions were maintained for 10min. The injection volume was 1μl.
All nanoLC components were controlled by Hystar 3.1 software (Bruker Daltonik,
Bremen, Germany).
Bloque II
188
2.3. ESI-TOF-MS detection
The nanoLC column was interfaced to MS using a commercial sheathless nano-spray
interface with a tapered fused silica sprayer tip. The key parameters of this nano-ESI
operating in negative mode were adjusted for the flow rate used (300 nL/min) to achieve
stable spray across the entire gradient range: nebulising gas pressure 0.4 bar, dry gas
flow 4 L/min and dry gas temperature 140ºC. The detection was conducted considering
a mass range of 50-1000 m/z.
The accurate mass data of the molecular ions were processed through the software Data
Analysis 4.0 (Bruker Daltonik, Bremen, Germany), which provided a list of possible
elemental formulas by using the Smart Formula Editor. The Editor uses a CHNO
algorithm, which provides standard functionalities such as minimum/maximum
elemental range, electron configuration, and ring-plus double-bond equivalents, as well
as a sophisticated comparison of the theoretical with the measured isotope pattern
(sigma value) for increased confidence in the suggested molecular formula.
External instrument calibration was performed using a 74900-00-05 Cole Palmer
syringe pump (Vernon Hills, IL, USA) directly connected to the interface, with a
sodium formate cluster solution passing through containing 5 mM sodium hydroxide
and 0.2% formic acid in water:isopropanol (1/1, v/v). The calibration solution was
injected before each run and the instrument was calibrated prior to each analysis. This
method gave an exact calibration curve based on numerous cluster masses each
differing by 68 Da (NaCHO2). Due to the compensation of temperature drift in the
TOF-MS, this external calibration provided accurate mass values for a complete run.
Capítulo 4
189
2.4. Extraction procedures tested
Seventeen extraction procedures were tested using spiked serum (SS) containing 100
μg/mL of each phenolic standard, as described in simple terms in Figure 1.
Figure 1. Simplified scheme of the extraction procedures tested.
P1 and P5 procedures: 100 l of SS were treated with 500 l of HCl 200 mM in
methanol, vortex-mixed, kept for 2.5 h at -20ºC (P1) or at 50ºC (P5) and centrifuged at
14800 g for 5 min. The supernatants were evaporated in a vacuum concentrator and then
dissolved in 100 l of mobile phase A.
P2 and P6 procedures: P2 was like P1 and P6 like P5 but the supernatants were
neutralized at pH 7 by ammonia addition before the evaporation step.
Bloque II
190
P3 and P7 procedures: P3 was like P2 and P7 like P6 but the dried residues after the
evaporation step were dissolved in 100 l of aqueous formic acid 0.1% (v/v) and then, a
SPE of phenolic compounds was carried out on a Discovery DSC-18 cartridge (50 mg,
1 ml) from Supelco (Sigma Aldrich, Bellefonte, PA, USA). Prior to use, the SPE
cartridge was conditioned with 200 l of methanol/formic acid 0.1% (v/v) followed by
equilibration with 200 l of water/formic acid 0.1% (v/v). The serum solution
previously prepared was loaded into the cartridge, followed by a washing with 100 l of
water/formic acid 0.1% (v/v). The phenolic fraction was eluted with 100 l of methanol,
dried in a vacuum concentrator and then resolved in 100 l of mobile phase A.
P4 and P8 procedures: P4 was like P2 and P8 like P6, but the dried residues after the
evaporation step were dissolved in 50 l of aqueous ammonium bicarbonate 100 mM.
Afterwards, for enzymatic digestion with trypsin, the solution was mixed with 5 l of
trypsin at 0.3 g/ l in 50 mM ammonium bicarbonate buffer and incubated at 37ºC
overnight. The next day, 5 l of 5 mM acetic acid were added to stop the enzymatic
digestion. The mixture was dried in a vacuum concentrator and then resolved in 100 l
of mobile phase A.
P9, P10, and P11 procedures: 100 l of SS were treated with 500 l of HCl 200 mM in
methanol, vortex-mixed, kept for 2.5 h at 50ºC and centrifuged at 14800 g for 5 min.
The supernatants were neutralized at pH 7 by ammonia addition, evaporated in a
vacuum concentrator and then dissolved in 100 l of aqueous formic acid 1% (v/v) at
pH 2. Subsequently, a SPE of phenolic compounds was performed on Discovery DSC-
18 cartridges, Evolute ABN cartridges (25 mg, 1 ml) from Biotage (Uppsala, Sweden)
and Isolute ENV+ cartridges (50 mg, 1 ml) from Biotage (Uppsala, Sweden) in P9, P10,
and P11 procedures, respectively. Prior to use, the cartridges were conditioned with 200
Capítulo 4
191
l of methanol/formic acid 1% (v/v) followed by equilibration with 200 l of
water/formic acid 1% (v/v). The serum solutions previously prepared were loaded into
the cartridges and washing with 100 l of water/formic acid 1% (v/v). Finally, the
phenolic fraction was eluted with 100 l of methanol, dried in a vacuum concentrator,
and then resolved in 100 l of mobile phase A.
P12, P13, and P14 procedures: P12 was like P9, P13 like P10, and P14 like P11, but the
SPE was performed using aqueous formic acid 0.5% (v/v) at pH 2.5 as equilibration and
washing solution as well as sample solvent prior to SPE.
P15, P16, and P17 procedures: P15 was like P9, P16 like P10, and P17 like P11, but the
SPE was performed using aqueous formic acid 0.05% (v/v) at pH 3 as equilibration and
washing solution as well as sample solvent prior to SPE.
2.5. Study design and sample collection
Nine male wistar rats with a mean weight of 250 g were housed in standard cages at
room temperature with free access to food and water for two weeks. Throughout the
experiments, animals were processed according to the suggested ethical guidelines for
the care of laboratory animals [24].
Six rats were orally treated with Lippia citriodora extract (1440 mg/kg) via gastric
gavage. For the administration, the extract was suspended in saline serum (3 ml). The
control group consisted of three rats which received only saline serum. Rats were
subjected to ketamine/xylazine anaesthesia and the blood samples were withdrawn via
cardiac puncture into heparinized tubes at 20 min post dosing. All blood samples were
centrifuged at 1000g for 15 min at, 4ºC and then plasma was stored at -80ºC.
Bloque II
192
2.6. Statistical analysis
The significance of the differences (p<0.05) were evaluated with a one-way ANOVA
coupled with Tukey’s honest significant difference, using the Statistica 6.0 software
(StatSoft, Tulsa, OK, USA).
3. Results and discussion
3.1. Evaluation of extraction procedures of phenolic compounds from plasma
The prior extraction of phenolic compounds from plasma is a critical step due to the
complexity of this biological matrix. Therefore, different cleanup procedures were
tested for simulated samples similar to plasma. Samples of commercial foetal bovine
serum spiked with representative phenolic standards, namely naringenin, luteolin,
verbascoside, apigenin, rutin, syringic acid, and catechin, were used to evaluate the
recovery of the different extraction procedures and their applicability for their
subsequent nanoLC-ESI-TOF-MS analysis.
Plasma and serum are characterized by the presence of large amounts of proteins, which
may interfere in the final nanoLC-MS analysis. Therefore, protein removal is an
unavoidable step. However, phenolic compounds can be non-covalently bound to
proteins [25], so the development of efficient procedures for deproteinization with an
acceptable recovery of phenolic compounds is a challenging task. Different methods to
remove the proteins from plasma samples have been previously reported. Basically,
these methods consist of precipitating proteins by treating plasma with hydrochloric or
phosphoric acids [26-28]. Other authors combine the use of enzymes with ethyl-acetate
to remove the proteins [20, 29]. Due to the high variability of the methods, different
Capítulo 4
193
procedures with hydrochloric and phosphoric acid at different concentrations and
serum:solvent ratio were conducted in a preliminary experiment. The methanolic
solution of hydrochloric acid at 200 mM showed the highest recovery at a ratio of 1:5
for the serum:precipitation solution. Moreover, the amount of precipitated protein
increased with the time, so that different precipitation times were tested (30, 60, 90,
120, 150, 180, and 240 min). Differences in the amount of precipitated protein were
appreciable up to 150 min, but no substantial change was noted between 150 and 240
min.
Once the appropriate deproteinization method was established, several procedures using
the methanolic solution of hydrochloric acid were performed to evaluate the highest
recovery, as summarized in Figure 1A. Briefly, procedures P1-P4 consisted of a prior
step of protein-precipitation, adding 500 l of HCl 200 mM in methanol to 100 l of SS,
vortex-mixing and keeping for 2.5 h at -20ºC and later centrifugation (14800 g for 5
min) followed by supernatant collection. Subsequently, evaporation of the supernatants
with or without a previous neutralization step using ammonia (P1 and P2, respectively)
was performed, as well as a cleaning step using SPE C18 cartridges (P3) or an
enzymatic digestion with trypsin to break up the possible remains of proteins (P4). On
the other hand, P5-P8 procedures were carried out following the same procedures P1-
P4, where only the precipitation temperature was changed to 50°C. The recovery results
(Figure 2) showed that procedures P2 and P6 provided higher recovery values than did
P1 and P5, presumably due to the degradation of phenolic compounds during the
evaporation step because of the progressive rise in pH when methanol evaporates and
the hydrochloric acid became more concentrated. Accordingly, the rest of the
procedures included a neutralization step before the supernatant evaporation.
Bloque II
194
Figure 2. Recovery data corresponding to nanoLC-ESI-TOF-MS of phenolic compounds in
spiked serum for extraction procedures from P1 to P8 where di erent letters in the same column
indicate signi cantly di erent values (p < 0.05).
The methods in which trypsin digestion was performed (P4 and P8) registered poor
recovery values. Despite that the trypsin digestion guarantees the absence of large
proteins that otherwise could damage the column or block thin tubes used in the nanoLC
system, the temperature and long incubation time may assist in polyphenol degradation.
Therefore, the highest recovery rates were attained with procedures P2, P3, P6, and P7.
Procedures P2, P3, and P6 showed no significant differences (p<0.05), but displayed
different selectivity for single phenolics. In fact, P6 showed the highest recovery of
naringenin and luteolin but lower recovery of catechin and the absence of syringic acid
compared to P2 and P3.
Procedure P7 registered the best recovery data; indeed, it allowed the highest extraction
of total phenolics and the highest values of syringic acid, verbascoside, rutin, apigenin
Capítulo 4
195
and naringenin content. Moreover, it showed a recovery of luteolin and catechin
comparable to that of P2 and P3 and higher than that of all the other procedures. This
improvement in recovery could be due to a reduction of matrix components in the final
extract leading to minimum ion-suppression effects during nanoLC-ESI-TOF-MS
analysis.
These preliminary results highlighted that the protein-precipitation with methanolic
solution of hydrochloric acid (200 mM) at 50°C for 2.5 h followed by the C18-SPE
purification gave the best results.
Given these results, the next step was the study of different solid phases and the sample
pH, since it is well known that SPE efficiency depends on such factors as: the nature of
stationary phase, solvent and sample volume, solvent pH, and modifier content if
present. Figure 1B shows the different extraction procedures tested with different
stationary phases: C18, ABN (a water-wettable polymer-based sorbent) and ENV+ (a
hydroxylated polystyrene divinylbenzene co-polymer). In addition, the acidity of the
sample solution as well as conditioning and washing solutions was changed from pH
2.0 to 3.0 (with increments of 0.5).
Table 1 lists the recovery data with the solid phases at the different pH values.
Procedures P10, P11, P12, and P16 showed the highest recovery values of the phenolic
compounds. Therefore, all the stationary phases showed similar affinity for the analytes
and the pH was the deciding factor in the recovery percentages. In this sense, C18
cartridges showed an optimum selectivity for phenolic compounds at pH 2.5 while
ENV+ presented better recovery at pH 2.0. Regarding ABN cartridges, the pH value did
not appreciably affect the recovery of the analytes under study.
Tabl
e 1.
Rec
over
y da
ta (%
) obt
aine
d by
nan
oLC
-ESI
-TO
F-M
S of
phe
nolic
com
poun
ds in
spi
ked
seru
m. D
iffer
ent l
ette
rs in
the
sam
e co
lum
n in
dica
te
sign
ifica
ntly
diff
eren
t val
ues (
p <
0.05
).
C
atec
hin
Syri
ngic
ac
id
Ver
basc
osid
e R
utin
p-
coum
aric
ac
id
Lute
olin
N
arin
geni
n A
pige
nin
P9
%
66.8
(a)
56.5
(a,b
,c)
38.4
(b)
69.0
(a,b
) 56
.6 (a
,b)
45.7
(c,d
,e,f)
78
.6 (a
,b,c
) 47
.3 (d
,e)
RSD
1.
53
6.63
12
.16
5.18
4.
22
3.14
6.
80
6.47
P10
%
66.6
(a)
61.8
(a,b
) 37
.8 (b
) 70
.1 (a
,b)
59.4
(a,b
) 44
.3 (d
,e,f)
75
.4 (a
,b,c
,d)
69.6
(a,b
,c)
RSD
6.
93
2.33
13
.02
2.12
11
.42
4.32
8.
50
7.47
P11
%
70.1
(a)
62.7
(a,b
) 40
.0 (a
,b)
60.9
(a,b
,c)
64.6
(a)
52.4
(c,d
) 65
.6 (d
,e,f)
68
.9 (a
,b,c
) R
SD
6.47
9.
59
6.38
10
.38
8.96
2.
58
5.55
3.
72
P12
%
73.5
(a)
64.4
(a)
45.3
(a)
70.4
(a,b
) 65
.0 (a
) 55
.9 (c
) 86
.4 (a
) 52
.2 (c
,d)
RSD
4.
67
4.72
0.
61
2.57
3.
90
4.76
2.
49
4.74
P13
%
69.2
(a)
60.9
(a,b
) 17
.3 (d
) 70
.5 (a
) 65
.2 (a
) 80
.1 (b
) 73
.3 (b
,c,d
) 78
.3 (a
) R
SD
3.76
2.
44
16.3
9 8.
49
5.39
0.
11
6.79
4.
72
P14
%
65.1
(a)
52.7
(b,c
) 13
.2 (d
) 60
.2 (b
,c)
50.9
(b)
43.7
(d,e
,f)
54.3
(f)
44.5
(d,e
) R
SD
11.0
4 12
.44
7.41
7.
58
11.8
3 12
.28
11.5
2 18
.28
P15
%
65.6
(a)
62.1
(a,b
) 25
.9 (c
) 65
.4 (a
,b)
58.8
(a,b
) 50
.8 (c
,d,e
) 82
.4 (a
,b)
44.4
(d,e
) R
SD
9.88
1.
02
12.7
5 4.
86
3.14
6.
78
4.66
6.
55
P16
%
69.4
(a)
61.5
(a,b
) 39
.6 (a
,b)
61.7
(a,b
) 57
.0 (a
,b)
102.
1 (a
) 68
.7 (c
,d,e
) 57
.7 (b
,c,d
) R
SD
2.12
2.
34
2.33
2.
23
6.21
6.
74
0.74
27
.33
P17
%
64.2
(a)
49.1
(c)
30.2
(c)
50.8
(c)
48.3
(b)
36.9
(f)
55.8
(f)
30.3
(e)
RSD
6.
87
4.13
1.
845.
483.
25
9.43
2.91
2.87
Bloque II_______
Capítulo 4
197
However, procedure P12 was the only one that registered RSDs lower than 5%.
Therefore, this procedure was selected as the best to extract phenolic compounds in
serum for subsequent analysis by nanoLC-ESI-TOF-MS.
3.2. Plasma analysis
The plasma of nine rats was analysed 20 min after oral administration of a phenol-rich
Lippia citriodora extract or saline serum in the case of control group in order to
demonstrate the applicability of the proposed extraction procedure in real samples. This
method enabled the detection, in the treated group, of the compounds shown in Table 2
together with their retention time and MS data, including experimental and calculated
m/z for the molecular formulas provided, error and sigma value.
Table 2. Compounds identified in plasma of rats after oral administration of a phenol-
rich Lippia citriodora extract.
Peak tR
(min) m/z exp.
Molecular
formula m/z cal.
Error
(ppm) mSigma Proposed compound
1 16.84 389.1131 C16H21O11 389.1089 -10.8 9.9 Theveside
2 18.76 387.1865 C18H27O9 387.1661 -11.2 36.4
Tuberonic acid glucoside/
5´-hydroxyjasmonic acid 5´-O-glucoside
3 24.06 623.2049 C29H35O15 623.1981 -10.9 7.3 Verbascoside
4 24.21 193.0523 C10H9O4 193.0506 -8.7 48.9 Ferulic acid
5 25.69 623.2047 C29H35O15 623.1981 -10.6 10.4 Isoverbascoside
6 34.70 487.1500 C21H27O13 487.1457 -37.4 82.0 Cistanoside F
Bloque II
198
The compounds were identified by comparison of retention times from plasma samples
and Lippia citriodora extract, in the case of the compounds previously detected in the
extract, as well as by using molecular formulas provided by the software according to
the accurate mass and isotopic pattern. The extracted ion chromatograms of these
compounds are shown in Figure 3.
Figure 3. Extracted ion chromatograms of the compounds found in the plasma of rats after oral
administration of a phenol-rich Lippia citriodora extract.
Most of the compounds detected in plasma samples have been previously identified in
the extract administered to the rats [22, 23]. Thus, verbascoside and isoverbascoside, the
main compounds from the extract, were found in most of the plasma samples of the
Capítulo 4
199
treated group, together with cistanoside F and the iridoid derivative theveside.
Furthermore, tuberonic acid glucoside or its diastereomer 5´-hydroxyjasmonic acid 5´-
O-glucoside was detected only in two plasma samples.
Apart from these intact compounds, ferulic acid was also found in the treated group,
probably formed by methylation of caffeic acid after hydrolysis of the glucosidic bond
of verbascoside and isoverbascoside. Polyphenol glycosides are absorbed mainly
through the gut barrier after deglycosylation [30] and the catechol-like structure of
caffeic acid makes it predictably prone to O-methylation by soluble catechol-O-
methyltransferase (COMT), resulting in ferulic acid structure. Indeed, ferulic acid has
been described as a metabolite of caffeic acid after coffee ingestion by humans [31].
Conclusions
This work reports the first approach to the analysis of phenolic compounds in plasma by
nanoLC-ESI-TOF-MS. For this purpose, different extraction procedures have been
tested using commercial foetal bovine serum spiked with representative phenolic
compounds, including different protein precipitation agents, enzymatic digestion and
SPE using different solid phases and pH values. The best recoveries were found when a
previous protein-precipitation step with HCl 200 mM in methanol for 2.5 h at 50ºC was
followed by a SPE using C-18 cartridges at pH 2.5. This extraction procedure was
verified for the extraction of phenolic compounds from rat plasma after oral
administration of a Lippia citriodora extract. The subsequent analysis of those plasma
samples by nanoLC-ESI-TOF-MS allowed the identification of five compounds
previously found in the administered Lippia citriodora extract and one metabolite
Bloque II
200
coming from verbascoside and isoverbascoside. In this way, the sample-treatment
procedure developed has proved to be a good solution to analyse the phenolic
compounds in a complex biological matrix as plasma with nanoLC-ESI-TOF-MS
equipment, without problems for the life of the capillary column.
4. Acknowledgements
This work was supported by the project AGL2011-29857-C03-02 (Spanish Ministry of
Science and Innovation), as well as P09-CTS-4564, P10-FQM-6563 and P11-CTS-7625
(Andalusian Regional Government Council of Innovation and Science). The authors are
grateful to the Spanish Ministry of Science and Innovation for a grant FPU (AP2007-
03246).
5. References
[1] Scalbert A, Manach C, Morand C, Rémésy C (2005) Dietary polyphenols and the
prevention of diseases. Crit Rev Food Sci Nutr 45: 287-306.
[2] Naczk M, Shahidi F (2006) Phenolics in cereals, fruits and vegetables: Occurrence,
extraction and analysis. J Pharm Biomed Anal 41: 1523–1542.
[3] Harnly JM, Bhagwat S, Lin LZ (2007) Profiling methods for the determination of
phenolic compounds in foods and dietary supplements. Anal Bioanal Chem 389: 47–61.
[4] Hosoda K, Furuta T, Ishii K (2010) Simultaneous determination of glucuronic acid
and sulfuric acid conjugated metabolites of daidzein and genistein in human plasma by
high-performance liquid chromatography. J Chromatogr B 878: 628–636.
Capítulo 4
201
[5] Ruiz-Gutiérrez V, Juan ME, Cert A, Planas JM (2000) Determination of
hydroxytyrosol in plasma by HPLC. Anal Chem 72: 4458-4461.
[6] Azzini E, Vitaglione P, Intorre F, Napolitano A, Durazzo A, Foddai MS, Fumagalli
A, Catasta G, Rossi L, Venneria E, Raguzzini A, Palomba L, Fogliano V, Maiani G
(2010) Bioavailability of strawberry antioxidants in human subjects. Br J Nutr 104:
1165–1173.
[7] Suárez M, Romero MP, Macià A, Valls RM, Fernández S, Solà R, Motilva MJ
(2009) Improved method for identifying and quantifying olive oil phenolic compounds
and their metabolites in human plasma by microelution solid-phase extraction plate and
liquid chromatography–tandem mass spectrometry. J Chromatogr B 877: 4097–4106.
[8] Kida K, Suzuki M, Matsumoto N, Nanjo F, Hara Y (2000) Identification of biliary
metabolites of (-)-epigallocatechin gallate in rats. J Agric Food Chem 48: 4151-4155.
[9] Fernández-Arroyo S, Gómez-Martínez A, Rocamora-Reverte L, Quirantes-Piné R,
Segura-Carretero A, Fernández-Gutiérrez A, Ferragut JA (2012) Application of
nanoLC-ESI-TOF-MS for the metabolomic analysis of phenolic compounds from extra-
virgin olive oil in treated colon-cancer cells. J Pharm Biomed Anal. Doi:
10.1016/j.jpba.2012.01.033.
[10] Shen Y, Smith RD (2005) Advanced nanoscale separations and mass spectrometry
for sensitive high-throughput proteomics. Expert Rev Proteomic 2: 431-447.
[11] Tao D, Zhang L, Shan Y, Liang Z, Zhang Y (2011) Recent advances in micro-scale
and nano-scale high-performance liquid-phase chromatography for proteome research.
Anal Bioanal Chem 399:229–241.
Bloque II
202
[12] Fanali S, Aturki Z, D’Orazio G, Rocco A (2007) Separation of basic compounds of
pharmaceutical interest by using nano-liquid chromatography coupled with mass
spectrometry. J Chromatogr A 1150: 252–258.
[13] Cappiello A, Famiglini G, Mangani F, Palma P, Siviero A (2003) Nano-high-
performance liquid chromatography–electron ionization mass spectrometry approach
for environmental analysis. Anal Chim Acta 493: 125–136.
[14] D’Orazio G, Cifuentes A, Fanali S (2008) Chiral nano-liquid chromatography–
mass spectrometry applied to amino acids analysis for orange juice profiling. Food
Chem 108: 1114–1121.
[15] Rocco A, Fanali S (2009) Analysis of phytosterols in extra-virgin olive oil by
nano-liquid chromatography. J Chromatogr A 1216: 7173–7178.
[16] García-Villalba R, Carrasco-Pancorbo A, Zurek G, Behrens M, Bäßmann C,
Segura-Carretero A, Fernández-Gutiérrez A (2010) Nano and rapid resolution liquid
chromatography–electrospray ionization–time of flight mass spectrometry to identify
and quantify phenolic compounds in olive oil. J Sep Sci 33: 2069–2078.
[17] Vissers JPC (1999) Recent developments in microcolumn liquid chromatography. J
Chromatogr A 856: 117–143.
[18] Hsieh S, Jorgenson JW (1996) Preparation and evaluation of slurrypacked liquid
chromatography microcolumns with inner diameters from 12 to 33 m. Anal Chem 68:
1212–1217.
[19] Legido-Quigley C, Smith NW, Mallet D (2002) Quantification of the sensitivity
increase of a micro-high-performance liquid chromatography ionization mass
spectrometry system with decreasing column diameter. J Chromatogr A 976: 11–18.
Capítulo 4
203
[20] Funes L, Fernández-Arroyo S, Laporta O, Pons A, Roche E, Segura-Carretero A,
Fernández-Gutiérrez A, Micol V (2009) Correlation between plasma antioxidant
capacity and verbascoside levels in rats after oral administration of lemon verbena
extract. Food Chem 117: 589–598.
[21] Ritter C, Zimmermann BF, Galensa R (2010) Chiral separation of (+)/( )-catechin
from sulfated and glucuronidated metabolites in human plasma after cocoa
consumption. Anal Bioanal Chem 397:723–730.
[22] Quirantes-Piné R, Funes L, Micol V, Segura-Carretero A, Fernández-Gutiérrez A
(2009) High- performance liquid chromatography with diode array detection coupled to
electrospray time-of-flight and ion-trap tandem mass spectrometry to identify phenolic
compounds from a lemon verbena extract. J Chromatogr A 1216: 5391–5397.
[23] Quirantes-Piné R, Arráez-Román D, Segura-Carretero A, Fernández-Gutiérrez A
(2010) Characterization of phenolic and other polar compounds in a lemon verbena
extract by capillary electrophoresis-electrospray ionization-mass spectrometry. J Sep
Sci 33: 2818–2827.
[24] Morton DB, Jennings M, Buckwell A, Ewbank R, Godfrey C, Holgate B, Inglis I,
James R, Page C, Sharman I, Verschoyle R, Westall L, Wilson AB (2001) Refining
procedures for the administration of substances. Report of the
BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. British
Veterinary Association Animal Welfare Foundation/Fund for the Replacement of
Animals in Medical Experiments/Royal Society for the Prevention of Cruelty to
Animals/Universities Federation for Animal Welfare. Lab Anim 35: 1–41.
Bloque II
204
[25] Papadopoulou A, Frazier RA (2004) Characterization of protein–polyphenol
interactions. Trends Food Sci Tech 15: 186–190.
[26] Roura E, Andrés-Lacueva C, Jauregui O, Badia E, Estruch R, Izquierdo-Pulido M,
Lamuela-Raventos RM (2005) Rapid Liquid Chromatography Tandem Mass
Spectrometry Assay To Quantify Plasma (-)-Epicatechin Metabolites after Ingestion of
a Standard Portion of Cocoa Beverage in Humans. J Agric Food Chem 53: 6190-6194.
[27] Urpí-Sarda M, Jauregui O, Lamuela-Raventos RM, Jaeger W, Miksits M, Covas
MI, Andrés-Lacueva C (2005) Uptake of Diet Resveratrol into the Human Low-Density
Lipoprotein. Identification and Quantification of Resveratrol Metabolites by Liquid
Chromatography Coupled with Tandem Mass Spectrometry. Anal Chem 77: 3149-3155.
[28] Zhang ZC, Xu M, Sun SF, Qiao X, Wang BR, Han J, Guo DA (2008) Metabolic
analysis of four phenolic acids in rat by liquid chromatography–tandem mass
spectrometry. J Chromatogr B 871: 7–14.
[29] Renouf M, Guy P, Marmet C, Longet K, Fraering AL, Moulin J, Barron D, Dionisi
F, Cavin C, Steiling H, Williamson G (2010) Plasma appearance and correlation
between coffee and green tea metabolites in human subjects. Br J Nutr 104: 1635–1640.
[30] Scalbert A, Morand C, Manach C, Rémésy C (2002) Absorption and metabolism of
polyphenols in the gut and impact on health. Biomed Pharmacother 56: 276–282.
[31] Stalmach A, Mullen W, Barron D, Uchida K, Yokota T, Cavin C, Steiling H,
Williamson G, Crozier A (2009) Metabolite Profiling of Hydroxycinnamate Derivatives
in Plasma and Urine after the Ingestion of Coffee by Humans: Identification of
Biomarkers of Coffee Consumption. Drug Metab Dispos 37:1749–1758.
Phenylpropanoids and their metabolites are the major
compounds responsible for blood cell protection against
oxidative stress after lemon verbena administration in rats
Los fenilpropanoides y sus metabolitos son los principales
compuestos responsables de la protección de las células
sanguíneas contra el estrés oxidativo tras la administración de
un extracto de Lippia citriodora
CAPÍTULO 5
Capítulo 5
207
Phenylpropanoids and their metabolites are the major compounds responsible for
blood cell protection against oxidative stress after lemon verbena administration in
rats
R. Quirantes-Piné1,2, M. Herranz-López3, V. Micol3, A. Segura-Carretero1,2, A.
Fernández-Gutiérrez1,2
1Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avd.
Fuentenueva s/n, 18071, Granada, Spain.
2Functional Food Research and Development Center, Health Science Technological
Park, Avd. del Conocimiento, 3, 18100, Granada, Spain.
3Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avenida de
la Universidad s/n, 03202 Elche, Alicante, Spain
Corresponding author: Dr. A. Segura Carretero, Department of Analytical Chemistry,
Faculty of Sciences, University of Granada, c/ Fuentenueva s/n, 18071 Granada, Spain.
E-mail: [email protected]
Fax: +34 958 637083
Abbreviations: ROS, reactive oxygen species; SOD, superoxide dismutase; CAT,
catalase; GRed, glutathione reductase; GPx, glutathione peroxidise; MPO,
myeloperoxidase
Bloque II
208
ABSTRACT
Lemon verbena (Lippia citriodora) has been widely used in folk medicine for its
pharmacological properties, usually attributed to its main compound verbascoside. The
protective effects of this plant have been attributed, among several factors, to its
antioxidant activity. The purpose of this study was to test the effect of lemon verbena
extract intake on blood cells antioxidant response and to correlate it with the phenolic
metabolites found in plasma. For this purpose, catalase (CAT), glutathione peroxidase
(GPx), and glutathione reductase (GRed) activities were determined in lymphocytes,
erythrocytes and neutrophils of rats after acute intake of lemon verbena extract, and
phenolic metabolites were analysed in plasma by HPLC-ESI-TOF-MS.
Myeloperoxidase (MPO) activity in neutrophils, which has been proposed as a marker
for inflammatory vascular damage, was also determined. After lemon verbena
administration, the antioxidant enzymes activities significantly increased (p < 0.05)
while MPO activity decreased, indicating that the extract protects blood cells against
oxidative damage and it has potential antiinflammatory and antiatherogenic activities.
The main compounds found in plasma were verbascoside and isoverbascoside at a
concentration of 80 ± 10 and 57 ± 4 ng/ml, respectively. Five metabolites deriving from
verbascoside and isoverbacoside were also found in plasma due to the metabolism of
the rats, namely, hydroxytyrosol, caffeic acid, ferulic acid, ferulic acid glucuronide and
homoprotocatechuic acid, together with other eight phenolic compounds. Therefore, the
phenylpropanoids verbascoside and isoverbascoside, as well as their metabolites seem
to be the responsible for the above mentioned effects although the post-transcriptional
Capítulo 5
209
activation mechanism of blood cells antioxidant enzymes by these compounds should
be further investigated.
Keywords: Lemon verbena, antioxidant enzymes, phenolic compounds
Bloque II
210
1. INTRODUCTION
Lemon verbena (Lippia citriodora) is a shrub indigenous to South America that was
introduced into Europe at the end of the 17th century. It has been widely used in
infusion for its antispasmodic, antipyretic, sedative and digestive properties [1-3].
Lemon verbena leaves contain a large number of polar compounds such as
phenylpropanoids, flavonoids, phenolic acids and iridoid glycosides, being verbascoside
the most abundant one [4]. Several properties have been described for this compound
such as antiinflammatory [5, 6], antimicrobial [7] and antitumor [8]. These protective
effects have been attributed, among several factors, to its antioxidant activity [9, 10].
Reactive oxygen species (ROS) have been implicated in the mediations of several
pathological processes such as inflammatory diseases, cancer, and atherosclerosis.
Phenolic compounds can help to limit the oxidative damage caused by ROS either
acting directly on ROS or stimulating endogenous defence systems. These defence
systems include antioxidant enzymes, namely superoxide dismutase (SOD), catalase
(CAT), glutathione reductase (GRed), and glutathione peroxidase (GPx), that act as
scavengers of the ROS. SOD catalyzes the dismutation of superoxide to H2O2, CAT
catalyzes the conversion of H2O2 to water, preventing the generation of hydroxyl
radicals, GRed reduces glutathione disulfide to the sulfhydryl form, and GPx reduces
lipid hydroperoxides to their corresponding alcohols and free hydrogen peroxide to
water [11].
A previous study has reported an increased SOD activity in rat plasma after acute lemon
verbena administration [12]. This effect was attributed to verbascoside since it was the
only compound found in plasma. Other studies in humans have reported that
Capítulo 5
211
consumption of lemon verbena extract promote protection of blood cells by activation
of GRed and CAT in erythrocytes and lymphocytes and decrease sport-induced
oxidative damage in neutrophils [13, 14]. Nevertheless, these studies were not able to
find any metabolite deriving from lemon verbena in human volunteers’ plasma.
Therefore, there is a lack of knowledge about the effects of lemon verbena metabolites
on white and red blood cells’ antioxidant defences.
The in vivo antioxidant activity of lemon verbena, like every biological property,
depends on the absorption of its components in the gut and on their metabolism. The
pharmacokinetic of verbascoside has been previously investigated [12, 15], however,
little is known about its metabolism as well as the bioavailability of the other
compounds present in this plant.
Therefore, the aim of this study was to test the effect of lemon verbena extract intake on
lymphocytes, erythrocytes and neutrophils antioxidant response, and to correlate it with
the phenolic metabolites found in plasma. In this way, the compound or compounds
responsible of the blood cell protection against oxidative stress shown by lemon verbena
could be determined.
2. MATERIALS AND METHODS
2.1. Chemicals
All chemicals were of analytical reagent grade and used as received. Verbascoside and
taxifolin were from Sigma Aldrich (St. Louis, MO, USA). The stock solutions
containing these analytes were prepared in methanol at a concentration of 100 μg/mL
and stored at -20°C until used. Acetonitrile, methanol and ammonia were from Panreac
(Barcelona, Spain), hydrochloric acid from Scharlau (Barcelona, Spain) and formic acid
Bloque II
212
from Sigma Aldrich (St. Louis, MO, USA). The Ficoll reagent was obtained from GE
Healthcare, Sweden. All other reagents or chemicals were obtained from Sigma-
Aldrich. Water was purified by a Milli-Q system from Millipore (Bedford, MA, USA).
The lemon verbena extract (Lippia citriodora) (20% verbascoside, w/w) was kindly
provided by Monteloeder (Elche, Spain).
2.2. Animals and Experimental Design
Nine male Wistar rats (250–300 g) from ten to twelve-week-old were housed in
standard cages at room temperature with free access to food and water for two weeks.
Throughout the experiments, animals were processed according to the suggested ethical
guidelines for the care of laboratory animals [16].
Rats were orally treated with lemon verbena extract (1440 mg/kg, corresponding to 360
mg/kg of verbascoside) via gastric gavage (n=6). For the administration, the extract was
dissolved in saline serum (2.5 mL). The control group (n=3) received only saline serum.
Rats were subjected to ketamine/xylazine anaesthesia.
2.3. Erythrocytes, lymphocytes and neutrophils purification
Blood samples were withdrawn via cardiac puncture into heparinized tubes at 20 min
post dosing and were used to purify erythrocytes, lymphocytes and neutrophils
following an adaptation of the method described by Boyum [17], and plasma was stored
at -80 ºC for further analysis of metabolites.
2.4. Enzymatic determinations
CAT activity was measured by the spectrophotometric method of Aebi [18] based on
the decomposition of H2O2. GRed activity was measured by a modification of the
Capítulo 5
213
Goldberg and Spooner spectrophotometric method [19]. This assay required oxidized
glutathione as the substrate. GPx activity was measured by an adaptation of the
spectrophotometric method of Flohé and Gunzler using H2O2 as the substrate [20].
Myeloperoxidase (MPO) activity of neutrophils was measured by guaiacol oxidation
[21]. All activities were determined with a SPECTROstar Omega microplate reader at
37ºC.
2.5. Plasma treatment for HPLC-ESI-TOF-MS analysis
1 ml of plasma was spiked with 10 μl of the taxifolin stock solution used as internal
standard. Afterwards, the plasma was treated with 5 ml of HCl 200 mmol/L in
methanol, vortex-mixed, kept for 2.5 hours at 50 ºC and centrifuged at 14800 g for 5
min. The supernatant was neutralized at pH 7 by ammonia addition, evaporated in a
vacuum concentrator and then dissolved in 1 ml of aqueous formic acid 0.5% (v/v) at
pH 2.5. Subsequently, a solid phase extraction of phenolic compounds was performed
on Discovery DSC-18 cartridges (50 mg, 1 ml) Supelco, Sigma Aldrich (Bellefonte,
PA, USA). Prior to use, the cartridge was conditioned with 2 ml of methanol/formic
acid 0.5% (v/v) followed by equilibration with 2 ml of water/formic acid 0.5% (v/v).
The plasma solution previously prepared was loaded into the cartridge, followed by a
washing with 1 ml of water/formic acid 0.5% (v/v). Finally, the phenolic fraction was
eluted with 1 ml of methanol, dried in a vacuum concentrator, and then, resolved in 100
l of mobile phase A.
2.6. HPLC-ESI-TOF-MS analyses
Analyses were performed out using an Agilent 1200 Series Rapid Resolution Liquid
Chromatography system (Agilent Technologies, Palo Alto, CA, USA), including a
Bloque II
214
standard autosampler and a diode array detector. The HPLC column used was a
Phenomenex Gemini C18 (5 μm, 4.6 x 250 mm). The separation was carried out at
room temperature with a gradient elution program at a flow rate of 0.2 ml/min. The
mobile phases consisted in water:acetonitrile (90:10, v/v) with 1% of formic acid (A)
and acetonitrile (B). The following multi-step linear gradient was applied: 0 min, 5% B;
35 min, 20% B; 45 min, 40% B; 50 min, 5% B. The initial conditions were held for 10
min. The injection volume in the HPLC system was 10 μl.
The HPLC system was coupled to a microTOF mass spectrometer (Bruker Daltonik,
Bremen, Germany) equipped with an ESI interface operating in negative ion mode
using a capillary voltage of +4 kV. The other optimum values of the ESI-TOF-MS
parameters were drying gas temperature, 190 ºC; drying gas flow, 7 l/min, and
nebulizing gas pressure, 1.5 bar. The detection was conducted considering a mass range
of 50-1000 m/z.
The accurate mass data of the molecular ions were processed through the software Data
Analysis 4.0 (Bruker Daltonik, Bremen, Germany), which provided a list of possible
elemental formulas by using the Smart Formula Editor. This editor uses a CHNO
algorithm, which provides standard functionalities such as minimum/maximum
elemental range, electron configuration, and ring-plus double-bond equivalents, as well
as a sophisticated comparison of the theoretical with the measured isotope pattern
(sigma value) for increased confidence in the suggested molecular formula.
During the development of the HPLC method, the instrument was externally calibrated
with a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, IL, USA) directly
connected to the interface and injected with a sodium formate cluster solution
containing 5 mM sodium hydroxide and 0.2% formic acid in water:isopropanol (1:1,
Capítulo 5
215
v/v). The calibration solution was injected at the beginning of each run and all the
spectra were calibrated prior to compound identification. This method gave an exact
calibration curve based on numerous cluster masses each differing by 68 Da (NaCHO2).
Due to the compensation of temperature drift in the TOF-MS, this external calibration
provided accurate mass values for a complete run.
2.7. Validation of HPLC-ESI-TOF-MS method
The accuracy of the method was further assessed with recovery studies by spiking
verbascoside into control plasma in triplicates. The linearity range of the method was
determined on five concentration levels from 0.5 to 10 μg/ml with three injections for
each level. Limits of detection (LOD) and quantification (LOQ) were respectively set at
S/N = 3 and S/N = 10 where S/N is the signal-to-noise ratio. Repeatability of the
method was measured as relative standard deviation (RSD %) in terms of concentration.
A plasma sample was injected (n=3) on the same day (intraday precision) and 3 times
on the 2 consecutive days (interday precision, n=9).
2.8. Statistical analysis
Statistical analysis was performed using the software OriginPro v 7.5. The results were
expressed as the mean and standard deviation (Mean ± SD). The means of quantitative
variables were analyzed using one way ANOVA test. All determinations were
performed in triplicate. Differences between the groups were compared using non-
parametric tests and were considered statistically significant when p < 0.05.
Bloque II
216
3. RESULTS AND DISCUSSION
3.1. Blood cells antioxidant response in rats after oral administration of lemon
verbena extract
The ex vivo activity of several antioxidant enzymes (CAT, GPx and GRed) was
determined in erythrocytes, lymphocytes and neutrophils of rats 20 min after the acute
intake of lemon verbena extract since previous studies in rats showed the maximum
plasma antioxidant capacity at 20 min [12]. Myeloperoxidase activity, which has been
proposed as a marker for inflammatory vascular damage [22, 23], was also determined
in neutrophils of the same animals. Mean values of antioxidant enzyme activities and
MPO in different circulating cell types are shown in Figure 1.
Figure 1. Effect of lemon verbena administration on the activity of antioxidant enzymes of several blood cells and myeloperoxidase activity in neutrophyls. *Significant difference between control group and lemon verbena administered rats (p < 0.05). As shown, CAT activity was enhanced in lymphocytes and erythrocytes, although no
significant effect was observed in neutrophils. GPx activity underwent a considerably
increase in all three cell fractions studied. The results also showed a significant
Capítulo 5
217
activation of GRed in both lymphocytes and neutrophils, being this increase especially
significant in neutrophils. Finally, a significant decrease of the MPO activity was
observed in neutrophils, which indicates a reduction in the release of damaging ROS in
this cell type [23].
3.2. Characterization of metabolites by HPLC-ESI-TOF-MS
In order to understand how the phenolic compounds from the lemon verbena extract
affect the antioxidant and antiinflammatory processes, it is necessary to determine
which compounds from the extract effectively reach the target tissues and whether they
do in their native form or as metabolites. Therefore, plasma samples were analysed by
HPLC-ESI-TOF-MS to determine the phenolic compounds and their metabolites
present in plasma.
The applied method was previously validated and its analytical parameters are shown in
Table 1. The verbascoside recovery was established at 76 ± 8%, and LOD and LOQ
were 7.5 and 25 ng/ml, respectively. Intraday repeatability of the developed method was
1.95%, whereas the interday repeatability was 4.04%.
Table 1. Analytical parameters of the HPLC-ESI-TOF-MS method
Analyte Verbascoside
Recovery 76 ± 8 %
Calibration range (ng/ml) 500 - 10000
Calibration equation y = 0.062x – 0.030
r2 0.991
LOD (ng/ml) 7.5
LOQ (ng/ml) 25
RSD intraday (n=3) 1.95%
RSD interday (n=9) 4.04%
Bloque II
218
This validated method enabled the identification in plasma samples of the compounds
shown in Table 2 together with their retention time and MS data, including
experimental and calculated m/z for the molecular formulas provided, error and sigma
value. The compounds were identified by comparison of retention times from plasma
samples and lemon verbena extract, in the case of the compounds previously detected in
the extract, as well as by using molecular formulas provided by the software according
to the accurate mass and isotopic pattern. Figure 2 shows the extracted ion
chromatograms (EIC) of some of the compounds detected in plasma.
Figure 2. Extracted ion chromatograms of the main compounds found in rat plasma 20 min
after lemon verbena acute intake.
Tabl
e 2.
Com
poun
ds id
entif
ied
in p
lasm
a of
rats
afte
r ora
l adm
inis
tratio
n of
the
lem
on v
erbe
na e
xtra
ct b
y H
PLC
-ESI
-TO
F-M
S.
t r (m
in)
m/z
ex
peri
men
tal
Mol
ecul
ar
form
ula
m/z
ca
lcul
ated
Er
ror
(ppm
) m
sigm
a Pr
opos
ed c
ompo
und
10.3
37
3.11
33
C16
H21
O10
37
3.11
40
2.0
90.2
G
ardo
side
10.9
46
1.16
65
C20
H29
O12
46
1.16
64
-0.2
25
.1
Ver
baso
side
12.5
48
7.14
74
C21
H27
O13
48
7.14
57
-3.4
34
.1
Cis
tano
side
F
12.9
36
7.08
93
C20
H15
O7
367.
0823
-1
8.9
58.0
A
cace
tin d
iace
tate
14.4
15
3.05
51
C8H
9O3
153.
0557
4.
0 50
.1
Hyd
roxy
tyro
sol
14.8
16
7.03
31
C8H
7O4
167.
0350
11
.1
95.4
H
omop
roto
cate
chui
c ac
id
15.5
38
9.10
60
C16
H21
O11
38
9.10
894
7.6
90.6
Th
eves
ide
17.8
0 19
3.04
82
C10
H9O
4 19
3.05
06
12.8
62
.9
Feru
lic a
cid
19.9
17
9.03
21
C9H
7O4
179.
0350
16
.3
56.7
C
affe
ic a
cid
27.6
63
7.10
76
C27
H25
O18
63
7.10
46
-4.7
15
4.9
Lute
olin
dig
lucu
roni
de
28.1
36
9.08
36
C16
H17
O10
36
9.08
27
-2.3
90
.2
Feru
lic a
cid
gluc
uron
ide
33.1
62
3.19
50
C29
H35
O15
62
3.19
81
5.1
10.5
V
erba
scos
ide
34.7
65
1.11
47
C28
H27
O18
65
1.12
03
8.5
55.6
C
hrys
oerio
l dig
lucu
roni
de
36.4
62
3.19
28
C29
H35
O15
62
3.19
81
8.5
31.5
Is
over
basc
osid
e
39.1
63
7.21
04
C30
H37
O15
63
7.21
38
5.3
110.
2 Eu
kovo
side
46.7
65
1.22
30
C31
H39
O15
65
1.22
94
9.8
178.
5 M
arty
nosi
de
Capítulo 5_______
Bloque II
220
The main compounds found in plasma were verbascoside and isoverbascoside. These
compounds were quantified showing a mean concentration of 80 ± 10 and 57 ± 4 ng/ml,
respectively. The detection of high levels of intact verbascoside and isoverbascoside in
plasma proved that they may be absorbed in their native forms which is in agreement
with previous studies where verbascoside was found in plasma after oral administration
of verbascoside-rich extracts [12, 15].
Five metabolites deriving from verbascoside and isoverbacoside were also found in
plasma due to the metabolism of the rats, namely, hydroxytyrosol, caffeic acid, ferulic
acid, ferulic acid glucuronide and homoprotocatechuic acid. Polyphenol glycosides are
mainly absorbed through the gut barrier after deglycosilation by action of lactase
phloridzin hydrolase in the brush-border of the small intestine epithelial cells, cytosolic
ß-glucosidase within the epithelial cells or colonic microbiota in the large intestine [24].
Therefore, hydroxytyrosol and caffeic acid may be formed by hydrolysis of the
glycosidic bonds of verbascoside and isoverbascoside. Caffeic acid may come from the
hydrolysis of other complex polyphenols from the extract such as cistanoside F,
campneoside I and forsythoside A, which have in their structures a caffeoyl group
bound to glycoside moieties. In the same way, hydroxytyrosol may be formed by
hydrolysis of verbasoside, forsythoside A and eukovoside.
The catechol-like structure of caffeic acid makes it predictably prone to O-methylation
by soluble catechol-O-methyltransferase, resulting in ferulic acid structure, which may
suffer subsequent glucuronidation due to the action of uridine-5´-diphosphate
glucuronosyltransferases. Indeed, ferulic acid has been described as a metabolite of
caffeic acid after the ingestion of coffee by humans [25]. Furthermore, the hydrolysis of
eukovoside and martynoside may contribute to the formation of ferulic acid.
Capítulo 5
221
Homoprotocatechuic acid has been also described as a metabolite of caffeic acid [26,
27].
Furthermore, flavone derivatives have been detected in the plasma samples, mainly,
acacetin diacetate, luteolin diglucuronide and chrysoeriol diglucuronide. Acacetin
diacetate probably arises from the metabolism of acacetin diglucuronide, which may
cleavage the glucuronic bond and then, conjugate with two acetate groups as a result of
the phase II metabolism. Whereas luteolin diglucuronide and chrysoeriol diglucuronide
may come from the absorption of the intact compounds from the extract as well as the
further conjugation with two glucuronic moieties after absorption of the free flavones in
the gut.
Other compounds from the extract have been detected intact in plasma, such as
gardoside, cistanoside F, theveside, eukovoside and martynoside. As above mentioned,
polyphenol glycosides are mainly absorbed through the gut barrier after deglycosilation
and subsequently, they are conjugated to form O-glucuronides, sulphate esters and O-
methyl ethers [28]. However, some studies have suggested that intact glycosides of
quercetin may be absorbed from the small intestine by a mechanism involving the
glucose transport pathway [29, 30] and non-conjugated polyphenols have been also
detected in studies in which pharmacological doses were administered, indicating a
possible saturation of the conjugation pathways [31].
3.3. Correlation between plasmatic phenolic metabolites and antioxidant enzymes
modulation
The major metabolites found in the plasma of rats were the phenylpropanoids
verbascoside and isoverbascoside. Some other metabolites, probably deriving from
verbascoside and/or isoverbascoside metabolism, were also found in plasma but at much
Bloque II
222
lower concentration, mainly hydroxytyrosol and phenolic acids derivatives such as
caffeic, ferulic and homoprotocatechuic acids. In previous studies, the antioxidant effect
of lemon verbena extracts has been totally attributed to verbascoside. However, isolated
phenolic acids such as gentisic, gallic, ferulic, and p-coumaric acids, as well as coffee
rich in caffeic, ferulic, and p-coumaric acids have shown to increase hepatic SOD, GPx,
and CAT activities [32, 33]. Likewise, other study has reported increased CAT and
SOD activities in the liver after hydroxytyrosol supplementation in rats [34]. Therefore,
the enhancement of the antioxidant defences may not be exclusively due to the direct
modulation of enzymes activity by verbascoside but the combined action of its
metabolites.
Moreover, low concentrations of flavone derivatives (acacetin, luteolin and chrysoeriol)
and phenylpropanoids different from verbascoside were also found. Many flavonoids,
including flavones, have been shown to modulate CAT activity by binding to the heme
group or a protein region of CAT structure contributing to enhancement of activity [35].
However little is known about the effect of the other phenylpropanoids found in plasma
on antioxidant enzymes.
Therefore, verbascoside, isoverbascoside and their metabolites seem to be the best
candidate compounds to be responsible for the observed enzymatic activation in the
different blood cells. At this moment, the precise mechanism by which antioxidant
enzymes are activated after the administration of the extract is unknown, although the
gene expression regulation must be discarded due to the short time of the observed
effect.
Capítulo 5
223
4. CONCLUSIONS
In conclusion, these findings demonstrate that the consumption of lemon verbena
extract protects blood cells by powering endogenous antioxidant defences of the
different cell types, especially in lymphocytes, and shows potential antiinflammatory
and antiatherogenic activities through the inhibition of MPO in neutrophils. As derived
from the metabolites detected in plasma by HPLC-ESI-TOF-MS, the phenylpropanoids
verbascoside and isoverbascoside, as well as their metabolites seem to be the
responsible for the above mentioned effects. Anyhow, further research may be required
to elucidate the post-transcriptional activation mechanism of blood cells antioxidant
enzymes.
5. ACKNOWLEDGEMENTS
This work was supported by the project AGL2011-29857-C03-02 (Spanish Ministry of
Science and Innovation), as well as P09-CTS-4564, P10-FQM-6563 and P11-CTS-7625
(Andalusian Regional Government Council of Innovation and Science). The authors are
grateful to the Spanish Ministry of Science and Innovation for a grant FPU (AP2007-
03246).
Bloque II
224
6. REFERENCES
[1] Carnat A, Carnat AP, Fraisse D, Lamaison JL (1999) The aromatic and polyphenolic
composition of lemon verbena tea. Fitoterapia 70: 44–49.
[2] Pascual ME, Slowing K, Carretero E, Sánchez Mata D, Villar A (2001) Lippia:
Traditional uses, chemistry and pharmacology: A review. J Ethnopharmacol 76: 201–
214.
[3] Valentão P, Andrade PB, Areias F, Ferreres F, Seabra RM (1999) Analysis of
vervain flavonoids by HPLC/diode array detector method. Its application to quality
control. J Agr Food Chem 47: 4579–4582.
[4] Quirantes-Pine R, Funes L, Micol V, Segura-Carretero A, Fernandez-Gutierrez A
(2009) High-performance liquid chromatography with diode array detection coupled to
electrospray time-of-flight and ion-trap tandem mass spectrometry to identify phenolic
compounds from a lemon verbena extract. J Chromatogr A 1216: 5391–5397.
[5] Deepak M, Handa SS (2000) Antiinflammatory activity and chemical composition
of extracts of Verbena officinalis. Phytother Res 14: 463–465.
[6] Díaz AM, Abad MJ, Fernández L, Silván AM, De Santos J, Bermejo P (2004)
Phenylpropanoid glycosides from Scrophularia scorodonia: In vitro anti-inflammatory
activity. Life Sci 74: 2515–2526.
[7] Avila J, de Liverant J, Martinez A, Martinez G, Munoz J, Arciniegas A, de Vivar A
(1999) Mode of action of Buddleja cordata verbascoside against Staphylococcus
aureus. J Ethnopharmacol 66: 75–78.
Capítulo 5
225
[8] Ohno T, Inoue M, Ogihara Y, Saracoglu I (2002) Antimetastatic activity of
acteoside, a phenylethanoid glycoside. Biol Pharm Bull 25: 666–668.
[9] Valentão P, Fernandes E, Carvalho F, Andrade P, Seabra R, Bastos M (2002)
Studies on the antioxidant activity of Lippia citriodora infusion: Scavenging effect on
superoxide radical, hydroxyl radical and hypochlorous acid. Biol Pharm Bull 25: 1324–
1327.
[10] Wong IYF, He Z, Huang Y, Chen Z (2001) Antioxidative activities of
phenylethanoid glycosides from Ligustrum purpurascens. J Agric Food Chem 49:
3113–3119.
[11] Aymoto Hassimotto NM, Pinto MDS, Lajolo FM (2008) Antioxidant Status in
Humans after Consumption of Blackberry (Rubus fruticosus L.) Juices With and
Without Defatted Milk. J Agr Food Chem 56: 11727–11733.
[12] Funes L, Fernandez-Arroyo S, Laporta O, Pons A, Roche E, Segura-Carretero A,
Fernandez-Gutierrez A, Micol V (2009) Correlation between plasma antioxidant
capacity and verbascoside levels in rats after oral administration of lemon verbena
extract. Food Chem 117: 589–598.
[13] Funes L, Carrera-Quintanar L, Cerdan-Calero M, Ferrer MD, Drobnic F, Pons A,
Roche E, Micol V (2011) Effect of lemon verbena supplementation on muscular
damage markers, proinflammatory cytokines release and neutrophils' oxidative stress in
chronic exercise. Eur J Appl Physiol 111: 695–705.
[14] Carrera-Quintanar L, Funes L, Viudes E, Tur J, Micol V, Roche E, Pons A (2010)
Effect of lemon verbena supplementation on muscular damage markers,
Bloque II
226
proinflammatory cytokines release and neutrophils’ oxidative stress in chronic exercise.
Scand J Med Sci Sports. Doi: 10.1111/j.1600-0838.2010.01244.x
[15] Wu Y, Lin L, Sung JS, Tsai T (2006) Determination of acteoside in Cistanche
deserticola and Boschniakia rossica and its pharmacokinetics in freely-moving rats
using LC-MS/MS. J Chromatogr B 844: 89–95.
[16] Morton D, Jennings M, Buckwell A, Ewbank R, Godfrey C, Holgate B, Inglis I,
James R, Page C, Sharman I, Verschoyle R, Westall L, Wilson A, BVAAWF FRAME
RSPCA UFAW (2001) Refining procedures for the administration of substances. Lab
Anim 35: 1–41.
[17] Boyum A (1964) Separation of White Blood Cells. Nature 204: 793–794.
[18] Aebi H (1984) Catalase In vitro. Meth Enzymol 105: 121–126.
[19] Goldberg DM, Spooner RJ (1983) in Bergmeyer HU, Bergmeyer J, Grabl M (eds)
Methods of enzymatic analysis, Verlag Chemie, Germany.
[20] Flohe L, Gunzler W (1984) Assays of Glutathione-Peroxidase. Meth Enzymol 105:
114–121.
[21] Capeillere-Blandin C (1998) Oxidation of guaiacol by myeloperoxidase: a two-
electron-oxidized guaiacol transient species as a mediator of NADPH oxidation.
Biochem J 336: 395–404.
[22] Koelsch M, Mallak R, Graham GG, Kajer T, Milligan MK, Nguyen LQ, Newsham
DW, Keh JS, Kettle AJ, Scott KF, Ziegler JB, Pattison DI, Fu S, Hawkins CL, Rees
MD, Davies MJ (2010) Acetaminophen (paracetamol) inhibits myeloperoxidase-
catalyzed oxidant production and biological damage at therapeutically achievable
concentrations. Biochem Pharmacol 79: 1156–1164.
Capítulo 5
227
[23] Faith M, Sukumaran A, Pulimood AB, Jacob M (2008) How reliable an indicator
of inflammation is myeloperoxidase activity? Clinica Chimica Acta 396: 23–25.
[24] Crozier A, Del Rio D, Clifford MN (2010) Bioavailability of dietary flavonoids
and phenolic compounds. Mol Aspects Med 31: 446– 467.
[25] Stalmach A, Mullen W, Barron D, Uchida K, Yokota T, Cavin C, Steiling H,
Williamson G, Crozier A (2009) Metabolite Profiling of Hydroxycinnamate Derivatives
in Plasma and Urine after the Ingestion of Coffee by Humans: Identification of
Biomarkers of Coffee Consumption. Drug Metab Disposition 37: 1749–1758.
[26] Gonthier M, Verny M, Besson C, Remesy C, Scalbert A (2003) Chlorogenic acid
bioavailability largely depends on its metabolism by the gut microflora in rats. J Nutr
133: 1853–1859.
[27] Olthof M, Hollman P, Buijsman M, van Amelsvoort J, Katan M (2003)
Chlorogenic acid, quercetin-3-rutinoside and black tea phenols are extensively
metabolized in humans. J Nutr 133: 1806–1814.
[28] Scalbert A, Morand C, Manach C, Remesy C (2002) Absorption and metabolism of
polyphenols in the gut and impact on health. Biomed Pharmacother 56: 276–282.
[29] Hollman P, Devries J, Vanleeuwen S, Mengelers M, Katan M (1995) Absorption of
Dietary Quercetin Glycosides and Quercetin in Healthy Ileostomy Volunteers. Am J
Clin Nutr 62: 1276–1282.
[30] Gee J, DuPont M, Rhodes M, Johnson I (1998) Quercetin glucosides interact with
the intestinal glucose transport pathway. Free Radical Bio Med 25: 19–25.
[31] Das N (1971) Studies on Flavonoid Metabolism - Absorption and Metabolism of
(+)-Catechin in Man. Biochem Pharmacol 20: 3435–3445.
Bloque II
228
[32] Yeh C, Yen G (2006) Induction of hepatic antioxidant enzymes by phenolic acids
in rats is accompanied by increased levels of multidrug resistance-associated protein 3
mRNA expression. J Nutr 136: 11–15.
[33] Valadao Vicente SJ, Ishimoto EY, Cruz RJ, Seabra Pereira CD, Ferraz Da Silva
Torres, Elizabeth Aparecida (2011) Increase of the Activity of Phase II Antioxidant
Enzymes in Rats after a Single Dose of Coffee. J Agr Food Chem 59: 10887–10892.
[34] Jemai H, Fki I, Bouaziz M, Bouallagui Z, El Feki A, Isoda H, Sayadi S (2008)
Lipid-lowering and antioxidant effects of hydroxytyrosol and its triacetylated derivative
recovered from olive tree leaves in cholesterol-fed rats. J Agr Food Chem 56: 2630–
2636.
[35] Doronicheva N, Yasui H, Sakurai H (2007) Chemical structure-dependent
differential effects of flavonoids on the catalase activity as evaluated by a
chemiluminescent method. Biol Pharm Bull 30: 213–217.
A metabolite profiling approach to assess the uptake and
metabolism of phenolic compounds from olive leaves in SKBR3
cells by HPLC ESI QTOF MS
Evaluación de la absorción y metabolismo de compuestos
fenólicos de hojas de olivo en células SKBR3 mediante perfilado
metabólico con HPLC ESI QTOF MS
Capítulo 6
Capítulo 6
231
A metabolite profiling approach to assess the uptake and metabolism of phenolic
compounds from olive leaves in SKBR3 cells by HPLC-ESI-QTOF-MS
R. Quirantes-Piné1,2, G. Zurek3, E. Barrajón-Catalán4, C. Bäßmann3, V. Micol4, A.
Segura-Carretero1,2, A. Fernández-Gutiérrez1,2
1Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avd.
Fuentenueva s/n, 18071, Granada, Spain.
2Functional Food Research and Development Center, Health Science Technological
Park, Avd. del Conocimiento, 3, 18100, Granada, Spain.
3Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany.
4Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avenida de
la Universidad s/n, 03202 Elche, Alicante, Spain
Corresponding author: Dr. A. Segura Carretero, Department of Analytical Chemistry,
Faculty of Sciences, University of Granada, c/ Fuentenueva s/n, 18071 Granada, Spain.
E-mail: [email protected]
Fax: +34 958 637083
Bloque II
232
ABSTRACT
Olive leaves, an easily available natural material of low cost, have shown an important
antitumor activity inhibiting cell proliferation in breast cancer cell lines MCF-7, SKBR3
and JIMT-1. In this work, a metabolite profiling approach has been used to assess the
uptake and metabolism of phenolic compounds from an olive-leaf extract in the breast
cancer cell line SKBR3 to evaluate the compound or compounds responsible for its
cytotoxic activity. For this aim, firstly, the olive-leaf extract under study was
quantitatively characterized by high-performance liquid chromatography coupled to
electrospray ionization-quadrupole time of flight mass spectrometry (HPLC-ESI-
QTOF-MS). Then, SKBR3 cells were incubated with 200 μg/ml of the olive-leaf extract
for different times (15 min, 1, 2, 24 and 48 h). A metabolite profiling approach based on
HPLC-ESI-QTOF-MS was used to determine the intracellular phenolic compounds,
enabling the identification of sixteen intact phenolic compounds from the extract and
four metabolites derived from these compounds in the cells cytoplasm. The major
compounds found within the cells were oleuropein, luteolin-7-O-glucoside and its
metabolites luteolin aglycone and methyl-luteolin glucoside, as well as apigenin, and
verbascoside. Neither hydroxytyrosol nor any of its metabolites were found within the
cells at any incubation time. These findings suggest that the major responsible
compounds for the cytotoxic activity of the olive-leaf extract in SKBR3 cells could be
oleuropein and the flavones luteolin and apigenin, since these compounds showed a
high uptake and they have previously reported antitumor activity.
Keywords: Olive leaves, phenolic compounds, breast cancer, SKBR3 cell line, HPLC-
MS
Capítulo 6
233
1. INTRODUCTION
Recently, Olea europea L. leaves have gained the rising interest of the scientific
community. In the cultivation of the olive tree, the pruning step generates a considerable
volume of leaves, therefore, they may be considered an abundant, easily available and
low cost by-product. Hence, important scientific and technical efforts have been made
to determine the value of this agricultural residue.
Historically, olive leaves have been used as a folk remedy for combating fevers and
other diseases, such as malaria. These medicinal properties have been mainly attributed
to oleuropein and related derivatives. Several studies have reported antioxidant,
hypoglycemic, antihypertensive, antimicrobial, antiatherosclerotic and antiviral,
including anti-HIV, properties of olive leaves [1-4]. Furthermore, recent studies have
highlighted the antitumor activity of olive-leaf extracts, inhibiting cell proliferation in
breast cancer cell lines MCF-7, SKBR3 and JIMT-1 [5-7]. However, the molecular
mechanism as well as which specific compound or mixture of compounds is responsible
for this bioactivity is not yet known in detail.
The aim of this study was to investigate the uptake and metabolism of phenolic
compounds from an olive-leaf extract by SKBR3 cells as a first approach to the
assessment of which compound or compounds may be accounted for the antitumor
activity of this extract. For this purpose, a metabolite profiling method based on high-
performance liquid chromatography coupled to electrospray ionization-quadrupole time
of flight mass spectrometry (HPLC-ESI-QTOF-MS) has been developed for the analysis
of cytoplasm from lysed cells incubated with an olive-leaf extract to determine which
Bloque II
234
compounds from the extract are able to enter through the cell membrane and which
metabolites they gave rise.
2. MATERIALS AND METHODS
2.1. Chemicals
All Chemicals were of analytical reagent grade and used as received. Acetic acid and
acetonitrile for HPLC were from Fluka, Sigma-Aldrich (Steinheim, Germany) and Lab-
Scan (Gliwice, Sowinskiego, Poland), respectively. Methanol and ethanol used for
protein precipitation were from Lab-Scan (Gliwice, Sowinskiego, Poland). Water was
purified by a Milli-Q system from Millipore (Bedford, MA, USA). Standards of
hydroxytyrosol, verbascoside, luteolin-7-O-glucoside, apigenin, and 3-
hydroxycoumarin were from Sigma-Aldrich (St. Louis, MO), and oleuropein was from
Extrasynthese (Lyon, France). Stock solutions containing these analytes were prepared
in methanol at a concentration of 50 μg/mL and stored at -20ºC until use.
2.2. Olive-leaf extract
Olive-leaf commercial extract powder was provided by New Developments in
Nutraceuticals (Spain). This extract was dissolved in dimethylsulfoxide in a stock
solution of 300 mg/ml.
2.3. Cell cultures
The human breast carcinoma cell line SKBR3 was obtained from the American Type
Culture Collection (ATCC, Manassas, VA). Cells were routinely grown in
Capítulo 6
235
DMEM+GlutaMAX medium supplemented with 10% of heat-inactivated fetal bovine
serum (GIBCO-Life) and 50 U/ml of penicillin and 50 mg/mL of streptomycin
(GIBCO-Life). Cells were incubated at 37ºC in a humidified 5% CO2 air atmosphere.
2.4. Cell treatment
SKBR3 cells were plated in 6-well plate at a density yielding 70-80% confluence,
followed by a serum starvation for 12 hours to synchronize the culture before starting
the assay. The olive-leaf extract stock solution was added to the cells at a final
concentration of 200 μg/ml and incubated for different times (15 min, 1, 2, 24 and 48 h)
in presence of 0.1% of fetal bovine serum. At the different times, firstly, cells were
washed with phosphate-buffered saline solution (Sigma-Aldrich, St. Louis, MO) and
sedimented. Then, the pellets were lysed with a lysis buffer containing 50 mM Tris pH
7.4, 1% Igepal CA-630, 150 mM NaCl, 5 mM EDTA and 10 mg/ml of protease
inhibitor cocktail (Sigma-Aldrich, Europe). Cells were kept on ice for 20 minutes and,
after a freezing/thawing cycle, they were centrifuged at 12000 rpm for 5 minutes at 4ºC.
The cell extracts (supernatant) were collected for analytical purposes and kept frozen at
-80ºC until use.
2.5. Cytoplasm treatment for HPLC-ESI-QTOF-MS analysis
50 l of cytoplasm were treated with 100 l of ethanol:methanol (50:50, v/v), vortex-
mixed, kept at -20 °C for 2 hours to allow protein precipitation, and centrifuged at
14800 g for 5 min. The supernatants were evaporated in a vacuum concentrator and then
dissolved in 50 l of methanol.
Bloque II
236
2.6. HPLC-ESI-QTOF-MS
Analyses were performed using an Agilent 1100 Liquid Chromatography system
(Agilent Technologies, Palo Alto, CA, USA), including a standard autosampler. The
HPLC column used was a Phenomenex Gemini C18 (3 μm, 2 x 150 mm). The
separation was carried out at 25ºC with a gradient elution program at a flow rate of 0.2
ml/min. The mobile phases consisted in water with 0.5% of acetic acid (A) and
acetonitrile (B). The following multi-step linear gradient was applied: 0 min, 5% B; 5
min, 15% B; 25 min, 30% B; 35 min, 95% B; 40 min, 5% B. The initial conditions were
held for 5 min. The injection volume in the HPLC system was 1 μl.
The HPLC system was coupled to a micrOTOF-Q II mass spectrometer (Bruker
Daltonik, Bremen, Germany) equipped with an ESI interface operating in negative ion
mode using a capillary voltage of +4 kV. The other optimum values of the ESI-QTOF
parameters were: drying gas temperature, 210ºC; drying gas flow, 8 l/min, and
nebulizing gas pressure, 2 bar. The detection was carried out considering a mass range
of 50-1100 m/z and the acquisition rate was 1 Hz. Nitrogen was used as drying and
nebulizing gas.
During the development of the method, external instrument calibration of the mass
spectrometer was performed using a 74900-00-05 Cole Palmer syringe pump (Vernon
Hills, Illinois, USA) directly connected to the interface and a sodium acetate cluster
solution (5 mM sodium hydroxide and 0.2% acetic acid in water:isopropanol (1:1, v/v)).
The calibration solution was injected at the beginning of each run and all the spectra
were calibrated prior to the compound identification. By using this method, an exact
calibration curve based on numerous cluster masses each differing by 82 Da
Capítulo 6
237
(NaC2H3O2) was obtained. Due to the compensation of temperature drift in the
micrOTOF-Q II, this external calibration provided accurate mass values (better than 5
ppm) for a complete run without the need for a dual sprayer setup for internal mass
calibration.
The accurate mass data of the molecular ions were processed through the software Data
Analysis 4.0 (Bruker Daltonik, Bremen, Germany), which provided a list of possible
elemental formulas by using the Smart Formula algorithm. The Smart Formula
algortihm assumes a CHNO distribution, if no other elements are specified, and
provides standard functionalities such as minimum/maximum elemental range, electron
configuration, and ring-plus double-bond equivalents, as well as a sophisticated
comparison of the theoretical with the measured isotope pattern (sigma value) for
increased confidence in the suggested molecular formula.
3. RESULTS AND DISCUSSION
3.1. Quantitative characterization of the olive-leaf extract
The olive-leaf extract under study had been qualitatively characterized in a previous
work [7], however, quantitative data were not reported. Therefore, the main compounds
of the extract were quantified before the uptake study. Figure 1 shows the base peak
chromatogram (BPC) of the extract obtained by the described method where the main
peaks have been numbered according to their elution order.
Bloque II
238
Figure 1. Base peak chromatogram (BPC) of the olive-leaf extract obtained by HPLC-ESI-
QTOF-MS where the main peaks have been numbered according to their elution order.
Standard calibration graphs of hydroxytyrosol, verbascoside, luteolin-7-O-glucoside,
oleuropein and apigenin were prepared using 3-hydroxycoumarin in a concentration of
50 μg/mL as internal standard. All calibration curves showed good linearity between
different concentrations depending on the analytes studied (Table 1). Limits of
detection (LODs) and limits of quantification (LOQs) for individual compounds in
standard solutions were also calculated as S/N = 3 and S/N = 10, respectively, where
S/N is the signal-to-noise ratio.
Capítulo 6
239
Table 1. Calibration data, where LOD is the limit of detection and LOQ is the limit of
quantification.
The compound concentrations were determined using the corrected area of each
individual compound (three replicates) and by interpolation in the corresponding
calibration curve. Hydroxytyrosol, verbascoside, luteolin-7-O-glucoside, oleuropein,
and apigenin were quantified by the calibration curves obtained from their respective
commercial standards. The other phenolic compounds were tentatively quantified on
basis of other compounds having similar structures. Secoiridoids as oleoside,
secologanoside, elenolic acid glucosides, ligstroside, and oleuropein derivatives were
quantified with oleuropein standard. Apigenin glucoside was expressed as its aglycone,
and isoverbascoside as verbascoside. It has to be taken into account that the response of
the standards can be different from that one of the analytes present in the extract, and
consequently the quantification of these compounds is only an estimation of their actual
concentrations. Anyway, they can be considered as a useful approximation to quantify
the olive-leaf extract.
AnalyteLOD
(μg/ml) LOQ
(μg/ml) Calibration
range (μg/ml) Calibration equations r2
Hydroxytyrosol 0.0446 0.1487 0.1-5 y = 0.1683x – 0.0108 0.9991
Verbascoside 0.0170 0.0566 0.1-5 y = 0.1710x – 0.0198 0.9982
Luteolin-7-O-glucoside 0.0079 0.0264 0.1-5 y = 0.3003x + 0.0327 0.9989
Oleuropein 0.0140 0.0466 0.05-10 y = 0.4105x + 0.0125 0.9995
Apigenin 0.0021 0.0069 0.001-0.5 y = 0.0008x + 0.0090 0.9964
Bloque II
240
Table 2 summarizes the quantitative results obtained by HPLC-ESI-QTOF-MS for the
extract. As expected, the most abundant compound was oleuropein, with a concentration
around 10-fold higher than the following compound, one of its isomers, the peak 14.
Although the other compounds were present in the extract at a concentration
significantly lower than oleuropein, the hydroxycinnamic acid derivative verbascoside
was more abundant than the other secoiridoids and the flavones. Oleoside,
hydroxytyrosol, luteolin-7-O-glucoside, isoverbascoside, ligstroside and oleuropein
aglycone showed a similiar concentration ranging from 10 to 17 mg/g.
The phenolic content of olive-leaf extracts strongly depends on several factors, mainly
the olive variety [8-10] and the extraction procedure [5]. The olive-leaf extract used in
this study showed a higher amount of oleuropein than the methanolic and aqueous
extracts reported by Goulas et al. [5], but a lower amount of hydroxytyrosol and
luteolin-7-O-glucoside. However, the oleuropein, verbascoside, hydroxytyrosol, and
luteolin-7-O-glucoside contents were higher than those reported for lyophilized extracts
[11] or extracts obtained by microwave-assisted extraction [12].
Capítulo 6
241
Table 2. Concentration of phenolic compounds in olive-leaf extract used in cultured SKBR3
breast cancer cells. Value = X ± SD
Peak Analyte Concentration (mg/g ext) 1 Oleoside 10.8 ± 0.3 2 Hydroxytyrosol 11.4 ± 0.5 3 Secologanoside 7.3 ± 0.5 4 Eleanolic acid glucoside isomer 1 5.6 ± 0.2 5 Eleanolic acid glucoside isomer 2 2.9 ± 0.2 6 Demethyloleuropein 2.3 ± 0.1 7 Verbascoside 29 ± 1 8 Luteolin-7-O-glucoside 10.5 ± 0.4 9 Isoverbascoside 17.2 ± 0.7 10 Oleuropein diglucoside 6.6 ± 0.5 11 Apigenin glucoside 0.44 ± 0.02 12 Oleuropein 441 ± 7 13 Oleuropein isomer 1 6.9 ± 0.3 14 Oleuropein isomer 2 58 ± 2 15 Ligstroside 12.4 ± 0.7 16 Apigenin 0.48 ± 0.04 17 Oleuropein aglycone 14.8 ± 0.8
3.2. Metabolite profiling of phenolic compounds from olive leaves in SKBR3 cells
In order to assess whether the ability of the olive-leaf extract to decrease breast cancer
cell viability may be associated to some specific compound or compounds, experiments
were performed to determine the intracellular phenolic compounds present at different
incubation times (15 min, 1, 2, 24 and 48 h). As shown in Table 3, several phenolic
compounds from the extract were detected intact in the cells cytoplasm. Furthermore,
some metabolites derived from these compounds were identified by the interpretation of
their mass spectra obtained by the QTOF–MS, elution order and data reported in the
literature, namely, methyl-luteolin glucoside, ligstroside glucuronide, methyloleuropein,
and luteolin. The identification of luteolin was confirmed by comparison with authentic
standard.
Tab
le 3
. Phe
nolic
com
poun
ds fr
om o
live-
leaf
ext
ract
and
thei
r met
abol
ites i
dent
ified
in c
ytop
lasm
of S
KB
R3
cell
line
at d
iffer
ent i
ncub
atio
n tim
es.
t r (m
in)
m/z
ex
peri
men
tal
Mol
ecul
ar
form
ula
m/z
ca
lcul
ated
E
rror
(p
pm)
mSi
gma
Prop
osed
com
poun
d 15
min
1
h 2
h 24
h
48 h
Inta
ct c
ompo
unds
6.57
38
9.10
89
C16
H21
O11
38
9.10
89
0.1
143.
8 O
leos
ide
X
9.51
38
9.11
13
C16
H21
O11
38
9.10
89
6.1
20.4
Se
colo
gano
side
X
X
X
X
X
10.7
8 40
3.12
52
C17
H23
O11
40
3.12
46
1.6
20.6
El
enol
ic a
cid
gluc
osid
e (is
omer
1)
X
X
X
X
X
12.5
5 40
3.12
41
C17
H23
O11
40
3.12
46
1.3
30.2
El
enol
ic a
cid
gluc
osid
e (is
omer
2)
X
X
X
15.0
1 52
5.15
90
C24
H29
O13
52
5.16
14
4.5
30.4
D
emet
hylo
leur
opei
n X
X
15.2
2 55
5.17
25
C25
H31
O14
55
5.17
19
1.0
38.0
H
ydro
xyol
euro
pein
X
X
16.6
6 62
3.19
83
C29
H35
O15
62
3.19
81
0.2
41.6
V
erba
scos
ide
X
X
X
17.1
7 44
7.09
36
C21
H19
O11
44
7.09
33
0.8
9.6
Lute
olin
-7-O
-glu
cosi
de
X
X
X
X
X
18.3
1 62
3.19
80
C29
H35
O15
62
3.19
81
0.3
43.8
Is
over
basc
osid
e X
X
X
18.7
0 70
1.23
21
C31
H41
O18
70
1.22
98
3.3
57.7
O
leur
opei
n di
gluc
osid
e X
X
X
21.9
9 53
9.17
79
C25
H31
O13
53
9.17
70
1.7
5.4
Ole
urop
ein
X
X
X
X
X
Bloque II_______
22.9
1 53
9.17
98
C25
H31
O13
53
9.17
70
5.1
33.5
O
leur
opei
n (is
omer
1)
X
X
X
23.6
7 53
9.17
94
C25
H31
O13
53
9.17
70
4.4
4.3
Ole
urop
ein
(isom
er 2
) X
X
X
25.8
6 52
3.18
27
C25
H31
O12
52
3.18
21
1.1
3.6
Ligs
trosi
de
X
X
X
X
X
31.6
2 26
9.04
67
C15
H9O
5 26
9.04
55
4.4
7.0
Api
geni
n X
X
X
31.9
6 37
7.12
60
C19
H21
O8
377.
1242
4.
8 25
.9
Ole
urop
ein
agly
cone
X
X
X
Met
abol
ites
20.6
1 46
1.10
95
C22
H21
O11
46
1.10
89
-1.2
12
3.7
Met
hyl-l
uteo
lin g
luco
side
X
X
X
23.3
6 53
7.15
98
C25
H29
O13
53
7.16
14
2.9
93.9
Li
gstro
side
glu
curo
nide
X
X
X
X
X
26.0
3 55
3.19
46
C26
H33
O13
55
3.19
27
-3.6
59
.6
Met
hylo
leur
opei
n
X
X
X
X
X
28.4
3 28
5.04
44
C15
H9O
6 28
5.04
05
-13.
8 94
.6
Lute
olin
X
X
X
Capítulo 6_______
Bloque II
244
Figure 2. Extracted ion chromatograms (EIC) of the main compounds found in the cytoplasm at
the different incubation times.
Figure 2 shows the extracted ion chromatograms (EIC) of the main compounds found
in the cytoplasm samples at the different incubation times. As can be observed, they
exhibit a time-dependent behaviour, with maximum content at 1 h except for apigenin
that presented a maximum uptake at 15 min and methyl-luteolin glucoside that showed
the maximum intensity at 2h. Most of the compounds detected in cytoplasm could not
be quantified since they were below LOQs, solely the main compounds at their
maximum uptake time could be quantified. Oleuropein was the main compound found
Capítulo 6
245
in cytoplasm, with a maximum concentration of 4.3 ± 0.2 μg/ml at 1 h while its
methylated metabolite was present only at trace level. Luteolin-7-O-glucoside also was
present at high intensity, reaching a maximum concentration of 0.26 ± 0.03 μg/ml, but
this compound was further metabolized since its metabolites luteolin aglycone and
methyl-luteolin glucoside showed high intensity, especially the latter, as shown in
Figure 2. Apigenin was one of the major compounds found the cytoplasm samples
despite the fact that it is a minor component of the olive-leaf extract, suggesting a high
uptake rate for this compound. The intensity of other compounds found in cytoplasm
was considerably lower. It is also noteworthy that neither hydroxytyrosol nor any of its
metabolites have been found in cytoplasm although the olive-leaf extract has a similar
concentration of hydroxytyrosol and luteolin-7-O-glucoside, indicating a limited uptake
of hydroxytyrosol by SKBR3 cells.
Several studies have reported that oleuropein and hydroxytyrosol decreased cell
viability, inhibited cell proliferation, and induced cell apoptosis in the breast cancer cell
line MCF-7 [5, 6, 13]. Therefore, the anticarcinogenic activity of olive leaves has been
mainly attributed to these compounds. In agreement with these studies, the main
compound found in cytoplasm was oleuropein; hence it may be considered the major
responsible for this activity. However, the limited uptake of hydroxytyrosol suggested
that this compound is not key to the decrease of cell viability in SKBR3 cell line.
As mentioned above, the anticancer activity of olive-leaf extracts has been usually
attributed to oleuropein and hydroxytyrosol, disregarding the role of flavones
derivatives. However, cytoplasm analysis has shown an important luteolin-7-O-
glucoside uptake and further metabolism, being found among other metabolites, luteolin
aglycone. Apigenin has also been detected with high intensity despite that this flavone
Bloque II
246
was at low concentration in the olive-leaf extract. Several studies have reported that
luteolin may delay or block the development of cancer cells in vitro and in vivo by
protection from carcinogenic stimuli, by inhibition of tumor cell proliferation, by
induction of cell cycle arrest and by induction of apoptosis via intrinsic and extrinsic
signaling pathways [14]. Furthermore, a recent study has shown that dietary flavones
and flavonones, including luteolin and apigenin, might regulate aromatase transcription
and inhibite the enzyme level in the breast cells MCF-7 [15], being the aromatase
inhibition a major strategy in treating breast cancer patients. Therefore, the cytotoxic
activity of olive-leaf extracts against the breast cancer cells SKBR3 could be partly due
to the action of the flavones luteolin and apigenin and their role in these antitumor
effects deserves further research.
4. CONCLUSIONS
This work reports the study of uptake and metabolism of phenolic compounds from an
olive-leaf extract by SKBR3 cells as a first approach to the assessment of which
compound or compounds may be responsible for the cytotoxic activity of this extract.
For this purpose, a metabolite profiling approach based on HPLC-ESI-QTOF-MS has
been used to identify the intracellular phenolic compounds at different incubation times
(15 min, 1, 2, 24 and 48 h). This strategy enabled the identification of sixteen intact
phenolic compounds from the extract and four metabolites derived from these
compounds in the cells cytoplasm. Oleuropein, the main compound from the extract,
was also the major compound found in cytoplasm, together with luteolin-7-O-glucoside
and its metabolites luteolin aglycone and methyl-luteolin glucoside, as well as apigenin.
Capítulo 6
247
Neither hydroxytyrosol nor any of its metabolites were found within the cells at any
incubation time. These findings suggest that the major responsible for the cytotoxic
activity of the olive-leaf extract could be oleuropein and the flavones luteolin and
apigenin, since these compounds showed a high uptake and they have previously
reported antitumor activity.
5. ACKNOWLEDGEMENTS
This work was supported by the project AGL2011-29857-C03-02 (Spanish Ministry of
Science and Innovation), as well as P09-CTS-4564, P10-FQM-6563 and P11-CTS-7625
(Andalusian Regional Government Council of Innovation and Science). The authors are
grateful to the Spanish Ministry of Science and Innovation for a grant FPU (AP2007-
03246).
6. REFERENCES
[1] S.N. El, S. Karakaya, Olive tree (Olea europaea) leaves: potential beneficial effects
on human health, Nutr. Rev. 67 (2009) 632-638.
[2] S. Lee-Huang, L. Zhang, P. Huang, Y. Chang, P. Huang, Anti-HIV activity of olive
leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and
OLE treatment, Biochem. Biophys. Res. Commun. 307 (2003) 1029-1037.
[3] L.I. Somova, F.O. Shode, P. Ramnanan, A. Nadar, Antihypertensive,
antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea
europaea, subspecies africana leaves, J. Ethnopharmacol. 84 (2003) 299-305.
Bloque II
248
[4] A.N. Sudjana, C. D'Orazio, V. Ryan, N. Rasool, J. Ng, N. Islam, T.V. Riley, K.A.
Hammer, Antimicrobial activity of commercial Olea europaea (olive) leaf extract, Int.
J. Antimicrob. Agents 33 (2009) 461-463.
[5] V. Goulas, V. Exarchou, A.N. Troganis, E. Psomiadou, T. Fotsis, E. Briasoulis, I.P.
Gerothanassis, Phytochemicals in olive-leaf extracts and their antiproliferative activity
against cancer and endothelial cells, Mol. Nutr. Food Res. 53 (2009) 600-608.
[6] J. Han, T.P.N. Talorete, P. Yamada, H. Isoda, Anti-proliferative and apoptotic
effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells,
Cytotechnology 59 (2009) 45-53.
[7] S. Fu, D. Arraez-Roman, A. Segura-Carretero, J.A. Menendez, M.P. Menendez-
Gutierrez, V. Micol, A. Fernandez-Gutierrez, Qualitative screening of phenolic
compounds in olive leaf extracts by hyphenated liquid chromatography and preliminary
evaluation of cytotoxic activity against human breast cancer cells, Anal. Bioanal. Chem.
397 (2010) 643-654.
[8] R. Japon-Lujan, M.D.L. De Castro, Small branches of olive tree: A source of
biophenols complementary to olive leaves, J. Agric. Food Chem. 55 (2007) 4584-4588.
[9] J. Meirinhos, B. Silva, P. Valentão, R. Seabra, J. Pereira, A. Dias, P. Andrade, F.
Ferreres, Analysis and quantification of flavonoidic compounds from Portuguese olive
(Olea europaea L.) leaf cultivars, Nat. Prod. Res. 19 (2005) 189-195.
[10] C. Savournin, B. Baghdikian, R. Elias, F. Dargouth-Kesraoui, K. Boukef, G.
Balansard, Rapid high-performance liquid chromatography analysis for the quantitative
Capítulo 6
249
determination of oleuropein in Olea europaea leaves, J. Agric. Food Chem. 49 (2001)
618-621.
[11] A.P. Pereira, I.C.F.R. Ferreira, F. Marcelino, P. Valentão, P.B. Andrade, R. Seabra,
L. Estevinho, A. Bento, J.A. Pereira, Phenolic compounds and antimicrobial activity of
olive (Olea europaea L. cv. Cobrancosa) leaves, Molecules 12 (2007) 1153-1162.
[12] R.J. Lujan, F.P. Capote, A. Marinas, M.D.L. de Castro, Liquid
chromatography/triple quadrupole tandem mass spectrometry with multiple reaction
monitoring for optimal selection of transitions to evaluate nutraceuticals from olive-tree
materials, Rapid Commun. Mass Sp. 22 (2008) 855-864.
[13] R. Sirianni, A. Chimento, A. De Luca, I. Casaburi, P. Rizza, A. Onofrio, D.
Iacopetta, F. Puoci, S. Ando, M. Maggiolini, V. Pezzi, Oleuropein and hydroxytyrosol
inhibit MCF-7 breast cancer cell proliferation interfering with ERK1/2 activation, Mol.
Nutr. Food Res. 54 (2010) 833-840.
[14] G. Seelinger, I. Merfort, U. Woelfle, C.M. Schempp, Anti-carcinogenic Effects of
the Flavonoid Luteolin, Molecules 13 (2008) 2628-2651.
[15] F. Li, L. Ye, S. Lin, L.K. Leung, Dietary flavones and flavonones display
differential effects on aromatase (CYP19) transcription in the breast cancer cells MCF-
7, Mol. Cell. Endocrinol. 344 (2011) 51-58.
CONCLUSIONES
Conclusiones
253
1. Se han caracterizado de forma cualitativa los compuestos bioactivos de un
extracto de Lippia citriodora de demostrada actividad antioxidante y
antiinflamatoria mediante HPLC DAD ESI TOF/IT MS, demostrando la
complementariedad de la información proporcionada por la espectroscopía
UV/visible y la espectrometría de masas con analizadores de tiempo de
vuelo y trampa de iones para la identificación de compuestos fenólicos en
Lippia citriodora. El método empleado permitió la separación simultánea de
una amplia gama de iridoides, flavonoides y fenilpropanoides glicosados en
menos de 30 min así como la identificación tentativa de los principales
compuestos de este extracto, incluyendo compuestos que no se habían
descritos previamente en Lippia citriodora, como gardósido, verbasósido,
cistanósido F, tevésido, campneósido I, crisoeriol 7 diglucurónido,
forsitósido A y acacetina 7 diglucurónido.
2. Se ha optimizado un método de CE ESI IT/TOF MS para la caracterización
cualitativa del extracto de Lippia citriodora. Para ello se estudiaron de forma
pormenorizada los principales parámetros electroforéticos así como los de
la transferencia al analizador de masas. El método desarrollado ha
permitido la separación e identificación tentativa de 16 compuestos,
incluyendo fenilpropanoides, flavonoides e iridoides glicosados en menos
de 25 min. Aunque algunos de los compuestos identificados eran comunes a
los caracterizados con el método cromatográfico previamente empleado, la
aplicación del método de CE ESI MS desarrollado ha permitido identificar
nuevos compuestos polares que no se habían descrito previamente en
Lippia citriodora, como asperulósido, ácido tuberónico o ácido 5´
hidroxijasmónico 5´ O glucósido, shanzísido e ixósido. De esta forma se ha
demostrado que la CE MS es una potente herramienta en el estudio de
Conclusiones
254
compuestos polares de las plantas y una técnica complementaria a HPLC
MS.
3. Se ha empleado un método analítico potente mediante el acoplamiento
HPLC ESI QTOF MS para caracterizar exhaustivamente dos extractos de hoja
de olivo obtenidos mediante PLE utilizando etanol y agua como disolventes
de extracción. El analizador de masas QTOF ha demostrado ser un sistema
de detección muy potente para la determinación de compuestos fenólicos
ya que proporciona exactitud de masas y distribución isotópica tanto en los
espectros de masas como de masas/masas. Gracias a este acoplamiento se
han identificado tentativamente más de 30 compuestos fenólicos
diferentes, incluyendo secoiridoides, fenoles simples, flavonoides, derivados
de ácidos cinámicos y ácidos benzoicos. Especialmente destacable es que se
ha identificado por primera vez lucidumósido C en hoja de olivo.
4. Se han evaluado diferentes procedimientos de extracción para el análisis de
compuestos fenólicos en plasma mediante nanoLC ESI TOF MS. Los
procedimientos examinados incluyeron diferentes agentes precipitantes de
proteínas así como condiciones de precipitación, digestión enzimática y SPE
utilizando diferentes fases sólidas y valores de pH. Las mayores
recuperaciones se obtuvieron con un paso previo de precipitación de
proteínas con HCl 200 mM en metanol durante 2,5 horas a 50 °C seguido de
SPE usando cartuchos C 18 a pH 2,5. Este procedimiento de extracción se
aplicó a muestras de plasma de rata tras la administración oral de un
extracto de Lippia citriodora para su posterior análisis mediante nanoLC ESI
TOF MS, que permitió la identificación de 5 compuestos procedentes del
extracto intactos y un metabolito común del verbascósido y/o del
isoverbascósido.
Conclusiones
255
5. Se ha evaluado el efecto del consumo de un extracto de Lippia citriodora en
la respuesta antioxidante de las células sanguíneas y se ha correlacionado
con los metabolitos fenólicos encontrados en plasma. Para ello, se han
determinado las actividades de las enzimas antioxidantes CAT, GPx y Gred
en linfocitos, eritrocitos y neutrófilos de ratas tras la ingesta aguda del
extracto, y se han analizado los metabolitos fenólicos en plasma mediante
HPLC ESI TOF MS. También se ha determinado la actividad de MPO en los
neutrófilos como marcador del daño vascular inflamatorio. Tras la
administración del extracto, las actividades de las enzimas antioxidantes
aumentaron significativamente mientras que disminuyó la actividad de
MPO, demostrando que el extracto de Lippia citriodora protege las células
sanguíneas mediante la activación de los sistemas de defensa antioxidante
endógenos de los diferentes tipos de células, especialmente en los
linfocitos, y que tiene una potencial actividad antiinflamatoria y
antiaterogénica mediante la inhibición de la MPO de los neutrófilos. Los
principales compuestos encontrados en plasma mediante HPLC ESI TOF MS
fueron verbascósido e isoverbascósido junto con otros 5 metabolitos
derivados de estos compuestos: hidroxitirosol, ácido cafeico, ácido
homoprotocatecuico y ácido ferúlico junto a su forma glucuronidada.
También se identificaron otros 8 compuestos fenólicos del extracto intactos.
Por lo tanto, los fenilpropanoides verbascósido e isoverbascósido, así como
sus metabolitos parecen ser los responsables de estos efectos protectores.
6. Se ha llevado a cabo un estudio de la absorción y metabolismo in vitro de
los compuestos fenólicos de un extracto de hoja de olivo en células de
cáncer de mama SKBR3 como una primera aproximación a la evaluación de
qué compuesto o compuestos pueden ser responsables de la actividad
Conclusiones
256
citotóxica previamente mostrada por este extracto. Para ello, en primer
lugar, el extracto de hoja de olivo en estudio se ha caracterizado
cuantitativamente mediante HPLC ESI QTOF MS, resultando el compuesto
más abundante la oleuropeína junto con uno de sus isómeros, seguido por
el verbascósido, que se encontró en una concentración significativamente
mayor que los demás secoiridoides y las flavonas. A continuación, se ha
analizado el perfil metabólico mediante HPLC ESI QTOF MS para identificar
los compuestos fenólicos intracelulares a diferentes tiempos de incubación
(15 min, 1, 2, 24 y 48 h). Esta estrategia ha permitido la identificación en el
citoplasma de 16 compuestos fenólicos intactos procedentes del extracto y
4 metabolitos derivados de estos compuestos. La oleuropeína, el
compuesto principal del extracto, fue también el principal compuesto
encontrado en el citoplasma, junto con luteolina 7 O glucósido y sus
metabolitos luteolina aglicona y metil luteolina glucósido, así como
apigenina. Ni el hidroxitirosol, ni ninguno de sus metabolitos se encontraron
en el citoplasma a ningún tiempo de incubación. Estos resultados sugieren
que los principales responsables de la actividad citotóxica del extracto de
hoja de olivo podrían ser la oleuropeína y las flavonas luteolina y apigenina.
FINAL CONCLUSIONS
Final conclusions
259
1. Bioactive compounds from a Lippia citriodora extract with proved
antioxidant and anti inflammatory activity have been qualitatively
characterized by HPLC DAD ESI TOF/IT MS, showing the complementarity of
the information provided by UV Vis spectroscopy and mass spectrometry
with time of flight and ion trap analyzers. The used method enabled the
simultaneous separation of a wide range of iridoids, flavonoids and
phenylpropanoids glycosides in less than 30 min, as well as the tentative
identification of the main compounds from this extract, including some of
them which had not been previously reported in Lippia citriodora, such as
gardoside, verbasoside, cistanoside F, theveside, campneoside I,
chrysoeriol 7 diglucuronide, forsythoside A and acacetin 7 diglucuronide.
2. A CE ESI IT/TOF MS method has been optimized to qualitatively
characterize the Lippia citriodora extract. Hence, the main electrophoretic
conditions as well as the ions transfer to the mass analyzer were carefully
studied. The developed method has enabled the separation and tentative
identification of 16 compounds, including phenylpropanoids, flavonoids and
iridoids glycosides in less than 25 min. Although some of the identified
compounds were previously characterized with the former chromatographic
method, the application of the developed CE ESI MS method has enabled to
identify new polar compounds which have not been previously described in
Lippia citriodora, such as asperuloside, tuberonic acid glucoside or 5´
hydroxyjasmonic acid 5´ O glucoside, shanziside, and ixoside. In this way,
CE MS has proved to be a valuable tool in the study of polar constituents in
plant matrices and a complementary technique to HPLC MS.
Final conclusions
260
3. A powerful analytical method by HPLC ESI QTOF MS has been used to
characterize comprehensively two olive leaf extracts obtained by PLE using
ethanol and water as solvents. The QTOF mass analyzer has proved to be a
valuable detection system for the identification of phenolic compounds,
since it provides mass accuracy and true isotopic pattern in both MS and
MS/MS spectra. This coupling has enabled to characterize tentatively more
than 30 different phenolic compounds, including secoiridoids, simple
phenols, flavonoids, cinnamic acid derivatives and benzoic acids. It is also
important to highlight that lucidumoside C has been identified in olive
leaves for the first time.
4. Different extraction procedures have been assessed to carry out the analysis
of phenolic compounds in plasma by nanoLC ESI TOF MS. The tested
procedures included different protein precipitation agents, enzymatic
digestion and SPE using different solid phases and pH values. The best
recoveries were found when a previous protein precipitation step with HCl
200 mM in methanol for 2.5 h at 50ºC was followed by a SPE using C 18
cartridges at pH 2.5. This extraction procedure was verified for the
extraction of phenolic compounds from rat plasma after oral administration
of a Lippia citriodora extract. The subsequent analysis of those plasma
samples by nanoLC ESI TOF MS allowed the identification of five
compounds previously found in the administered Lippia citriodora extract
and one metabolite coming from verbascoside and/or isoverbascoside.
5. The effect of a Lippia citriodora extract intake on blood cells antioxidant
response has been tested and correlated with the phenolic metabolites
found in plasma. For this purpose, CAT, GPx, and GRed activities were
Final conclusions
261
determined in lymphocytes, erythrocytes and neutrophils of rats after acute
intake of the extract, and phenolic metabolites were analysed in plasma by
HPLC ESI TOF MS. Myeloperoxidase (MPO) activity in neutrophils, which has
been proposed as a marker for inflammatory vascular damage, was also
determined. After extract administration, the antioxidant enzymes activities
significantly increased while MPO activity decreased, indicating that the
Lippia citriodora extract protects blood cells by powering endogenous
antioxidant defences of the different cell types, especially in lymphocytes,
and shows potential antiinflammatory and antiatherogenic activities
through the inhibition of MPO in neutrophils. The main compounds found in
plasma by HPLC ESI TOF MS were verbascoside and isoverbascoside
together with five metabolites deriving from them: hydroxytyrosol, caffeic
acid, ferulic acid, ferulic acid glucuronide and homoprotocatechuic acid.
Other eight phenolic compounds from the extract were also found intact in
plasma. Therefore, the phenylpropanoids verbascoside and isoverbascoside,
as well as their metabolites seem to be the responsible for these protective
effects.
6. A study of in vitro uptake and metabolism of phenolic compounds from an
olive leaf extract by breast cancer SKBR3 cells has been performed as a first
approach to the assessment of which compound or compounds may be
responsible for the cytotoxic activity of this extract. For this aim, firstly, the
olive leaf extract under study was quantitatively characterized by HPLC ESI
QTOF MS, resulting oleuropein the most abundant compound together with
one of its isomers, followed by verbascoside that was found at a significant
higher concentration than the other secoiridoids and the flavones. Then a
metabolite profiling approach based on HPLC ESI QTOF MS has been used
to identify the intracellular phenolic compounds at different incubation
Final conclusions
262
times (15 min, 1, 2, 24 and 48 h). This strategy enabled the identification of
16 intact phenolic compounds from the extract and 4 metabolites derived
from these compounds in the cells cytoplasm. Oleuropein, the main
compound from the extract, was also the major compound found in
cytoplasm, together with luteolin 7 O glucoside and its metabolites luteolin
aglycone and methyl luteolin glucoside, as well as apigenin. Neither
hydroxytyrosol nor any of its metabolites was found within the cells at any
incubation time. These findings suggest that the major responsible for the
cytotoxic activity of the olive leaf extract could be oleuropein and the
flavones luteolin and apigenin.
