Análisis de polifenoles en algas: implicación de estos compuestos en mecanismos para disminuir la toxicidad del cobre y regular la disponibilidad del hierro en microalgas

D/Dª JOSÉ ALBERTO HERRERA MELIÁN, SECRETARIO DEL DEPARTAMENTO DE QUÍMICA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA, CERTIFICA, Que el Consejo de Doctores del Departamento en su sesión de fecha 12 de noviembre de 2015 tomó el acuerdo de dar el consentimiento para su tramitación, a la tesis doctoral titulada “ANÁLISIS DE POLIFENOLES EN ALGAS. IMPLICACIÓN DE ESTOS COMPUESTOS EN MECANISMOS PARA DISMINUIR LA TOXICIDAD DEL COBRE Y REGULAR LA DISPONIBILIDAD DEL HIERRO EN MICROALGAS” presentada por la doctoranda Dª AROA LÓPEZ MONZÓN y dirigida por la Doctora MILAGROS RICO SANTOS. Y para que así conste, y a efectos de lo previsto en el Artº 6 del Reglamento para la elaboración, defensa, tribunal y evaluación de tesis doctorales de la Universidad de Las Palmas de Gran Canaria, firmo la presente en Las Palmas de Gran Canaria, a 12 de noviembre de dos mil quince. UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA Programa de Doctorado en Oceanografía Bienio 2004/2006 Título de la Tesis Doctoral “ANÁLISIS DE POLIFENOLES EN ALGAS. IMPLICACIÓN DE ESTOS COMPUESTOS EN MECANISMOS PARA DISMINUIR LA TOXICIDAD DEL COBRE Y REGULAR LA DISPONIBILIDAD DEL HIERRO EN MICROALGAS”. Tesis Doctoral presentada por Dª AROA LÓPEZ MONZÓN Dirigida por el Dra. Dª MILAGROS RICO SANTOS Codirigida por el Dr. D ARGIMIRO RIVERO ROSALES La Directora, El Codirector, La Doctoranda, Las Palmas de Gran Canaria a 6 de noviembre de 2015. D. ARGIMIRO RIVERO ROSALES, DIRECTOR DEL DEPARTAMENTO DE QUÍMICA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA, HACE CONSTAR: Que la presente memoria titulada “ANÁLISIS DE POLIFENOLES EN ALGAS. IMPLICACIÓN DE ESTOS COMPUESTOS EN MECANISMOS PARA DISMINUIR LA TOXICIDAD DEL COBRE Y REGULAR LA DISPONIBILIDAD DEL HIERRO EN MICROALGAS”, que presenta la doctoranda Dña. Aroa López Monzón para optar al grado de Doctor por esta Universidad, ha sido realizada en los laboratorios del Departamento de Química de la Universidad de Las Palmas de Gran Canaria. Tras su registro y trámite autorizado por el Departamento, autorizo con ésta fecha de presentación. Y para que así conste a los efectos oportunos, firmo la presente en Las Palmas de Gran Canaria, a 12 de noviembre de 2015. REGLAMENTO DE ESTUDIOS DE DOCTORADO DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA Aprobado por el Consejo de Gobierno el 17 de diciembre de 2012 (BOULPGC de 9 de enero de 2013) y modificado por el Consejo de Gobierno de 23 de octubre de 2013 (BOULPGC de 4 de noviembre de 2013) Artículo 12.- Tesis por compendio de publicaciones 1. Para la presentación de tesis por compendio de publicaciones será necesario: a. Un mínimo de tres publicaciones, con unidad temática, indexadas en el Journal Citations Reports, Arts and Humanities Citation Index o equivalentes, de las que el doctorando sea el primer autor o autor principal. Al menos una de ellas deberá haber sido publicada en una revista cuyo índice de impacto la sitúe dentro de la primera mitad en orden decreciente de índice de impacto entre las revistas del área. b. Para acreditar la condición de autor principal, esta deberá ser reconocida por el resto de los autores de las publicaciones presentadas como núcleo de la tesis doctoral, al mismo tiempo que estos deberán renunciar a utilizar estas publicaciones como núcleo principal de otras tesis doctorales, sin perjuicio de que dichas publicaciones puedan ser presentadas como méritos complementarios en las tesis doctorales que pudieran presentar los otros autores de dichas publicaciones. c. En áreas de especial incidencia tecnológica dos de estas publicaciones podrán ser sustituidas por patentes en explotación o publicaciones en congresos reconocidos por la ANEP en sus baremos para la obtención de sexenios. d. Que en las publicaciones o patentes conste la ULPGC a través de la filiación del director o del doctorando. A mi hermana María José, que me aguanta y con la que siempre puedo contar. A mi primo David, que escucha atentamente siempre que preparo una presentación. A mi ahijada Alejandra, a mis tíos y primos que ocupan un lugar especial en mi vida. A los amigos con los que comparto tantos recuerdos y buenos momentos, Montse, Ariadna, Eleazar, Yaiza, Dani, Elva y Davinia. Gracias por hacerme sentir acompañada. A mi marido, Miguel, quién más ha sufrido los efectos de este trabajo. Desde lo más profundo de mi corazón gracias por tu comprensión y tu cariño. Espero poder acompañarte en tus proyectos como tú lo has hecho conmigo. A todos gracias. ÍNDICE Capítulo 1. OBJETIVOS 25 1.1. Objetivos del trabajo 27 1.2. Líneas de trabajo futuras 32 Capítulo 2. INTRODUCCIÓN 35 2.1. Compuestos polifenólicos 37 2.2. Actividad fisiológica en los organismos vegetales 39 2.3. Efectos sobre la salud humana 42 2.4. Efectos en la oxidación y conservación de alimentos 45 2.5. Mecanismos de reacción 47 2.6. Polifenoles en algas 51 Capítulo 3. MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES 61 3.1. Polifenoles estudiados 63 3.2. Material vegetal 66 3.3. Cultivo de microalgas 69 3.4. Extracción 71 3.5. Preparación de muestras. Extracción en fase sólida 72 3.6. Contenido total de polifenoles 73 3.7. Determinación de la actividad antioxidante 75 3.7.1. Capacidad de inhibir radicales libres 75 3.7.2. Capacidad de reducir hierro 76 3.8. Análisis cualitativo y cuantitativo de polifenoles 77 Capítulo 4. DISCUSIÓN Y RESULTADOS 81 4.1. Stypocaulon scoparium Efecto de los disolventes de extracción sobre el contenido fenólico y la actividad antioxidante de extractos del alga parda Stypocaulon scoparium 83 4.2. Phaeodactylum tricornutum 4.2.1. Variación del perfil fenólico de la diatomea Phaeodactylum tricornutum cultivada en condiciones de estrés por hierro y cobre 95 4.2.2. Caracterización de los polifenoles exudados por la diatomea Phaeodactylum tricornutum y sus efectos en la química del Fe(II)-Fe(III) 111 4.3. Dunaliella tertiolecta Perfil fenólico de la microalga verde Dunaliella tertiolecta cultivada en presencia de altas concentraciones de hierro y cobre 123 4.4. Diferencias entre las dos especies de microalgas 139 4.5. Aplicación de la metodología desarrollada a extractos de distintos materiales vegetales terrestres y marinos 144 Capítulo 5. CONCLUSIONES 189 BIBLIOGRAFÍA 193 Capítulo 1: OBJETIVOS Capitulo 1: OBJETIVOS Este trabajo se centra en la optimización de métodos de identificación y cuantificación de polifenoles, principalmente en macroalgas recolectadas en las costas de las Islas Canarias y en microalgas cultivadas en presencia y en ausencia de altas concentraciones de metales a partir de cepas proporcionadas por el Banco Español de Algas. Un objetivo primordial del trabajo consistió en evaluar la influencia de las condiciones de cultivo en el perfil polifenólico de las microalgas y la interacción de dichos compuestos con el cobre (tóxico a altas concentraciones) y el hierro (fertilizante) disueltos en el agua de cultivo para determinar su posible papel en los mecanismos de detoxificación de metales y en los equilibrios de especiación. 1.1. Objetivos del trabajo Objetivo I: desarrollar y optimizar una metodología para la extracción, identificación y cuantificación de compuestos polifenólicos en macroalgas mediante cromatografía líquida de alta resolución en fase reversa (RP-HPLC) acoplada a un detector de Diodo Array (DAD). Optimizar la extracción de los compuestos polifenólicos de algas mediante el uso de diferentes disolventes. Realizar el análisis cromatográfico de los polifenoles extraídos con cada disolvente de manera rápida, sensible y con alta reproducibilidad y comparar así, la capacidad de 27 Capitulo 1: OBJETIVOS complejación. Se ha observado que, en presencia de determinados compuestos orgánicos como los polifenoles, aminoácidos o azúcares, el crecimiento de organismos eucariotas se ve favorecido. Investigar qué compuestos y mecanismos son los que determinan la presencia de Fe(II) en el medio marino y cómo se ven estos afectados por la acidificación y calentamiento oceánico es el objetivo principal de este proyecto. Para ello, se combinarán los estudios que se realizarán en la región Subártica Atlántica, una de las más afectadas por la acidificación oceánica, con los estudios en laboratorio usando compuestos orgánicos individuales y exudados procedentes de fitoplancton,en los que se trataran cada una de las variables de forma independiente, para poder definir así, la contribución de cada una de ellas al proceso. La cuantificación y caracterización de ligandos, polifenoles, sacáridos y aminoácidos, se realizará mediante cromatografía líquida de alta resolución (HPLC). 34 Capítulo 2: INTRODUCCIÓN Capitulo 2: INTRODUCCIÓN 2.1. Compuestos polifenólicos Durante el transcurso de la evolución, los organismos vegetales han desarrollado mecanismos para enfrentarse a los cambios producidos en las condiciones ambientales haciendo uso de la gran variedad estructural de compuestos químicos producidos por sus metabolismos secundarios. Entre estos grupos de compuestos, los polifenoles o fenoles, así como los alcaloides y los terpenoides, han demostrado su actividad protectora en las plantas, contribuyendo a mantener el equilibrio ecológico entre los productores primarios y los consumidores. Los polifenoles merecen especial atención si se tienen en cuenta los múltiples beneficios que ofrecen a las plantas y, por lo tanto, a otros organismos vivos; resultado, principalmente, de sus propiedades fisicoquímicas vinculadas al grupo funcional fenol [Quideau y colaboradores, 2011]. Constituyen un grupo muy amplio de compuestos cuya forma estructural más elemental es el fenol, que consiste en un anillo aromático plano y un grupo hidroxilo. Se clasifican en polifenoles y fenoles simples dependiendo del número de subunidades de fenol. Los fenoles simples incluyen los ácidos fenólicos y los fenilpropanoides o derivados del ácido cinámico [Ignat y colaboradores, 2011] (FIGURA 1). Los polifenoles que poseen dos subunidades de fenol son los flavonoides y estilbenos, y los compuestos con tres o más subunidades de fenol se conocen como taninos. Los flavonoides constituyen el grupo de 37 Capitulo 2: INTRODUCCIÓN POLIFENOLES SIMPLES FENILPROPANOIDES fenol ácido gálico ácido cinámico ácido sinápico OH COOH OH OH OH COOH COOH OCH 3 OH CH 3 O FLAVONOIDES flavonas flavonoles flavanoles O O OH O OH flavanonas isoflavonas flavanonoles O O O O O OH O chalconas antocianidinas estilbenos O O + OH OR 1 OR 2 OH R 3 FIGURA 1.- Compuestos fenólicos. 38 Capitulo 2: INTRODUCCIÓN compuestos polifenólicos más común y ampliamente distribuido en especies vegetales. Su estructura básica consiste en un sistema de dos anillos de benceno (A y C) unidos por un anillo de pirano (C) (ver flavonas en FIGURA 1). Las actividades de los polifenoles están relacionadas con su estructura y naturaleza química, que depende de su grado de hidroxilación, sustituciones, conjugaciones y grado de polimerización [Kelly y colaboradores, 2002; Wright y colaboradores, 2001]. 2.2. Actividad fisiológica en los organismos vegetales. Inicialmente, los polifenoles fueron considerados productos de desecho procedentes del metabolismo secundario de los vegetales. Actualmente, se sabe que estos metabolitos tienen una amplia variedad de funciones que han sido objeto de diversas revisiones bibliográficas [Harborne y Williams, 2000; Treutter, 2006; Quideau, 2011]: (1) Atracción de animales polinizadores: muchos flavonoides son componentes de pigmentos presentes en las flores y hojas, que les confieren coloraciones atrayentes de insectos polinizadores. Con frecuencia, el color azul de las flores se debe a la presencia de la antocianina delfinidina, presente en los pétalos, que normalmente requiere la presencia de un copigmento. El color azul es el más atractivo para las abejas polinizadoras, por lo que se observa una evolución hacia este color en la flora de clima templado, donde la abeja es el 39 Capitulo 2: INTRODUCCIÓN polinizador predominante [Gottlieb, 1982]. (2) Protección de la radiación ultravioleta (UV): algunos flavonoides suelen acumularse en las capas superficiales de las plantas y captan gran parte de las radiaciones UV, evitando sus efectos nocivos en el interior. Distintas investigaciones muestran que las plantas sometidas artificialmente a la radiación UV-B (rayos UV de longitud de onda entre 280 y 320 nanómetros) responden con cambios en las rutas metabólicas de la síntesis de flavonoides. Los cambios en los niveles de estos compuestos no solo se observan en las células epidérmicas de la superficie del hazde las hojas, también se producen en la cera y en las vellosidades [Olsson y colaboradores, 1998; Gitz y colaboradores, 1998; Cuadra y colaboradores, 1997]. (3) Actividad antimicrobiana y fungicida: una de las principales funciones de los polifenoles es la protección de las plantas contra la invasión microbiana. Esto no solo implica su presencia en plantas como constituyentes sino también su acumulación como fitoalexinas en respuesta a los microbios atacantes [Harborne, 1999; Dixon y colaboradores, 1983]. En general tienen capacidad de inhibir la germinación de las esporas de patógenos. (4) Aleloquímicos en interacciones entre plantas: a pesar de que aún no se conoce exactamente la manera en la que actúan, se ha observado actividad antimicrobiana de flavonoides 40 Capitulo 2: INTRODUCCIÓN en interacciones alelopáticas entre plantas [Chou, 1999; Inderjit y Gross, 2000]. La (+)-catequina se ha identificado como un aleloquímico fitotóxico que es exudado por la planta herbácea Centaurea maculosa a través de sus raíces inhibiendo la germinación y el crecimiento de Centaurea diffusa y Arabidopsis thaliana [Bais y colaboradores, 2003]. Por otra parte, un estudio de Gao y colaboradores (2011) demostró la interacción alelopática de 3 macroalgas, Elodea nuttallii (Planch), Hydrilla verticillata (L.f.) y Veitchia spiralis (L.) de la familia Hydrocharitaceae, para inhibir el crecimiento de especies tóxicas y no tóxicas de la cianobacteria Microcystis aeruginosa por excreción de compuestos fenólicos en el agua. (5) Interacción entre plantas y animales: algunos flavonoides protegen a las especies vegetales de los organismos herbívoros generando sabores o texturas desagradables. Muchos depredadores muestran sensibilidad frente a estos compuestos [Hoffmann-Campo y colaboradores, 2001; Haribal y Feeny, 2003; Thoison y colaboradores, 2004; Chen y colaboradores, 2004]. Sin embargo, no actúan solamente como mecanismos de defensa de amplio espectro [Forkner y colaboradores, 2004]. Algunos polifenoles confieren aromas y colores a los frutos que los hacen más apetecibles para los herbívoros y que estos actúen como dispersores de semillas. Plantas carnívoras poseen antocianinas en sus flores y hojas que atraen a los insectos que les sirven de alimento [Schaefer y Ruxton, 2008]. 41 Capitulo 2: INTRODUCCIÓN (6) Protección frente a condiciones climáticas extremas: en ocasiones se atribuye a los compuestos fenólicos la resistencia de las plantas a las heladas [Chalker-Scott y Krahmer, 1989] y a la sequía [Moore y colaboradores, 2005; Tattini y colaboradores, 2004] debido a las funciones que desempeñan en las membranas y paredes celulares. (7) Protección frente a contaminantes en el medio ambiente: protegen a las plantas que viven en suelos ricos en metales tóxicos como el aluminio [Barceló y Poschenrieder, 2002]. Roitto y colaboradores (2005) comprobaron que los árboles de pino silvestre (Pinus sylvestris L.) sometidos a los efectos de altas concentraciones de cobre (Cu) y níquel (Ni) presentaban concentraciones más altas de taninos que los árboles de zonas no sometidas a estos metales. 2.3. Efectos sobre la salud humana. La oxidación es un proceso natural que tiene lugar en las moléculas orgánicas con importantes implicaciones negativas en la salud humana. El metabolismo oxidativo es esencial para la supervivencia de las células con la producción de radicales libres y otras especies oxidantes. Cuando se produce un exceso de estas especies, las enzimas protectoras frente a la oxidación (catalasas, peroxidasas, superóxido dismutasa) pueden saturarse, lo que puede causar efectos destructivos y letales para las células, por oxidación de los lípidos de la membrana celular, de 42 Capitulo 2: INTRODUCCIÓN proteínas celulares, de ácido desoxirribonucleico (ADN) y enzimas, etc., parando la respiración celular [Winrow y colaboradores, 1993; Bauer y colaboradores, 1999]. Los radicales libres son moléculas altamente reactivas que poseen electrones desapareados. Se trata de especies químicas deficientes en electrones que, por ello, se combinan rápidamente con un electrón de otro átomo. “Atacan” a las moléculas más cercanas para “robarles” sus electrones. Cuando la molécula que ha sido atacada pierde su electrón, se transforma ella misma en otro radical y comienza entonces una “cadena de robos de electrones”. La oxidación causada por los radicales libres reduce la capacidad de combatir el envejecimiento y las enfermedades que lleva asociadas. Crece el número de investigaciones que confirman la relación entre una excesiva producción de radicales libres y algunos estados patológicos asociados al envejecimiento tales como cáncer, enfermedades cardiovasculares (arterosclerosis, obstrucción coronaria, etc.) o enfermedades neurodegenerativas como la enfermedad de Alzheimer y Parkinson. [Uttara y colaboradores, 2009; Nakabeppu y colaboradores, 2007; Halliwell, 1992]. La propiedad mejor descrita de los polifenoles es la capacidad antioxidante hacia los radicales libres y las especies reactivas de oxígeno (EROs) normalmente producidos por el metabolismo de las células o en respuesta a factores externos. Los compuestos polifenólicos tienen, por lo tanto, la capacidad 43 Capitulo 2: INTRODUCCIÓN de los radicales más reactivos, con un tiempo de vida media muy corto (10-9 segundos). La reacción de Fenton es responsable de la acumulación de radicales libres que inician procesos de degradación de biomoléculas. H2O2 + Mn+  OH + OH- + M(n+1)+ FIGURA 5.- Reacción de Fenton. Los polifenoles no solo presentan actividad de manera individual, también se ha de tener en cuenta su comportamiento en mezclas, ya que pueden actuar de manera aditiva, sinérgica o antagónica [Jacobo-Velazquez y Cisneros-Zevallos, 2009]. Como se ha señalado anteriormente, la dificultad de separar estos compuestos de matrices vegetales y la actividad total de la mezcla, muchas veces superior a la de compuestos aislados, hace que se recurra al uso de los extractos. Tafesh y colaboradores (2011) evaluaron la acción de inhibición del crecimiento de varias cepas de bacterias (gram positiva Streptococcus pyogenes y Staphylococcus aureus y gram-negativa Escherichia coli y Klebsiella pneumoniae) por hidroxitirosol y ácido gálico. Demostraron la actividad sinérgica positiva de mezclas de hidroxitirosol y ácido gálico que, en concentraciones de 100 y 200 μg mL−1 respectivamente, provocaron una inhibición completa de las cuatro cepas bacterianas. Cuando cada compuesto se utilizó por separado fueron necesarias concentraciones muy superiores, de 400 µg mL-1 de hidroxitirosol para la inhibición total de las 50 Capitulo 2: INTRODUCCIÓN cuatro cepas. El ácido gálico produjo la inhibición del crecimiento de las dos cepas gram positivas S. aureus and S. pyogenes a concentraciones de 200 y 400 µg mL-1 respectivamente, no inhibiendo el crecimiento de las cepas gram negativas. 2.6. Polifenoles en algas Las macroalgas son los principales productores primarios en los ecosistemas costeros y se emplean, principalmente, como alimento, pero también como fertilizante, en la medicina tradicional o en la industria cosmética, entre otras. Las algas se consideran una fuente de compuestos bioactivos, ricas en distintos tipos de antioxidantes [Lim y colaboradores, 2002; Yan y colaboradores, 1999]. Los extractos de varias especies de algas han mostrado actividades biológicas relacionadas con su contenido en florotaninos [Holdt y Kraan, 2011; Wijesinghe y Jeon, 2011]. Los roles y funciones de estos compuestos han sido objeto de muchos estudios, especialmente los relacionados con las interacciones con herbívoros y antifouling [Cronin, 2001; Amsler y Fairhead, 2006]. Arnold y Targett (2002) sugieren que la función de los polifenoles en las plantas marinas es osmorreguladora a nivel de la pared celular. Los taninos pueden afectar a la palatabilidad debido a su sabor, actúan como potenciales agentes antioxidantes y, además, interactúan en las reacciones redox de las plantas [Luck y 51 Capitulo 2: INTRODUCCIÓN colaboradores, 1994; Larson, 1997]. A pesar de las propiedades de los polifenoles y del potencial de las algas como fuente de esos compuestos [ChacónLee y González-Mariño, 2010], pocos estudios se han centrado en su identificación y cuantificación en algas y en su implicación en los mecanismos de defensa celular frente a especies reactivas de oxígeno [Cirulis y colaboradores, 2013]. En diferentes estudios se ha evaluado la capacidad antioxidante y el contenido total de polifenoles mediante el ensayo de Folin-Ciocaleau. Hajimahmoodi y colaboradores (2010) determinan la actividad antioxidante y el contenido total de polifenoles para 12 microalgas demostrando un importante papel de los compuestos polifenólicos como los mayores contribuyentes a la capacidad antioxidante. Goiris y colaboradores (2012) obtienen resultados similares al estudiar la capacidad antioxidante y el contenido total de polifenoles de 32 muestras de microalgas. En todos los estudios mencionados, el contenido total de polifenoles se evaluó a través del ensayo de Folin-Ciocaleau mediante espectrofotómetro de UV-visible. El reactivo de Folin-Ciocaleau es un reactivo no específico capaz de oxidar simultáneamente carbohidratos, aminoácidos y polifenoles [Apak y colaboradores, 2007; Huang y colaboradores 2005] y, por lo tanto, no refleja únicamente el contenido en polifenoles. Existen muchas evidencias de la estimulación del metabolismo polifenólico en plantas superiores que crecen en condiciones de estrés causado por la presencia en el medio de 52 Capitulo 2: INTRODUCCIÓN cultivo de altas concentraciones de metales pesados o por la ausencia de metales esenciales para la supervivencia [Michalak, 2006]. Wei y colaboradores (2007) realizaron un estudio con plantas de trébol rojo sometidas a deficiencia de hierro, observando que estas condiciones inducen la secreción de compuestos fenólicos exudados por la raíz. La toxicidad producida por la presencia de diferentes metales pesados en el medio de cultivo también aumenta la producción de compuestos polifenólicos en cultivos de manzanilla (Matricaria chamomilla L.), de trigo y de maíz [Díaz y colaboradores, 2001; WinkelShirley, 2002; Kovacik y Backor, 2007]. El importante papel de los compuestos polifenólicos también se ha observado en manglares (Aegiceras corniculatum L.) expuestos a altas concentraciones de metales pesados, aumentando notablemente los niveles de estos compuestos en las hojas [Guangqiu y colaboradores, 2007]. Lavid y colaboradores (2001) comprobaron en un estudio realizado con nenúfares (Nymphaeaceae) que son capaces de acumular altas concentraciones de metales pesados sin que se observe ningún síntoma de toxicidad en la planta, siendo los compuestos polifenólicos y las peroxidasas los responsables de la tolerancia e implicando, al menos, dos mecanismos: quelación directa de los metales por los polifenoles; y captura y precipitación de los metales durante el proceso de polimerización de polifenoles por peroxidasas. Jung y colaboradores (2003) han sugerido, en estudios 53 Capitulo 2: INTRODUCCIÓN realizados en plantas de altramuces (Lupinus albus L.), que los compuestos polifenólicos exudados por las raíces de las plantas o aquellos que se encontraron asociados a las paredes de las células de la raíz, producidos como respuesta a las altas concentraciones de cobre en el medio de cultivo, son responsables de la quelación del cobre y, por lo tanto, de restringir su toxicidad para la planta. Los autores del trabajo concluyen que la formación de polifenoles constituye un mecanismo físico-químico simple para la comprensión de las estrategias desarrolladas por los microorganismos en la lucha para controlar la toxicidad de los metales en la superficie de la célula, evitando su entrada masiva. Del mismo modo, la complejación de metales tóxicos por dichos polifenoles podría añadir nuevas dimensiones, no solo a la comprensión de los mecanismos moleculares desarrollados por microorganismos para contener la toxicidad del metal, sino también a la capacidad bioabsortiva de las paredes celulares. Suresh y colaboradores (1998) han publicado estudios bioquímicos y biofísicos realizados con hongos Neurospora crassa cultivados en presencia de altas concentraciones de cobre. Dichos autores sugieren que la defensa celular contra la toxicidad del cobre implica la unión del metal a polifenoles asociados a la pared celular. A pesar de que las algas planctónicas pueden regular la especiación y disponibilidad de metales traza a través de la producción de ligandos orgánicos [Koukal y colaboradores, 2007] 54 Capitulo 2: INTRODUCCIÓN y de que estos ligandos pueden complejar más del 99% del hierro y cobre total presente en aguas naturales [Gledhill y van den Berg, 1994; Wu y Luther, 1995], no se han realizado estudios centrados en la implicación de los polifenoles como componentes de esos posibles ligandos. En particular, los grupos hidroxilo y carboxilo de los compuestos polifenólicos pueden enlazar fuertemente Cu y Fe [Martell y Smith, 1989]. Este comportamiento quelante los convierte en candidatos idóneos para la prevención de la formación de radicales libres catalizada por metales y para combatir la toxicidad de los metales [Lopes y colaboradores, 1999]. Los conocimientos relativos a las algas en este aspecto se basan, principalmente, en observaciones y estudios realizados en plantas superiores. El principal mecanismo de toxicidad del cobre implica la reacción de Haber-Weiss [Hammond-Kosack, 1996], produciendo radical hidroxilo a través de la reacción de Fenton anteriormente mencionada (FIGURA 6). Se ha observado un incremento de la peroxidación de lípidos inducido por estrés oxidativo por Cu en plantas acuáticas [Nikookar y colaboradores, 2005]. Cu2+ + O2- → Cu1+ + O2 Cu1+ + H2O2 → Cu2+ + OH- + OH La reacción neta es: O2- + H2O2 → OH + OH- + O2 FIGURA 6.- Mecanismo de Haber-Weiss. 55 Capitulo 2: INTRODUCCIÓN Por lo tanto, los microorganismos han desarrollado diversos mecanismos de protección frente al envenenamiento o toxicidad de metales [Worms y colaboradores, 2006]: El secuestro intracelular de metales traza es un mecanismo eficaz utilizado por microorganismos (principalmente eucariotas) para atrapar los iones metálicos tóxicos, como el cobre, impidiendo que produzca radicales libres o para atrapar aquellos iones esenciales para la vida y menos biodisponibles. Wells y colaboradores (2005) demostraron que las diatomeas del género Pseudo-nitzschia tenían una inusual capacidad de adaptación a la limitación de hierro en medio de cultivo sintético a través de la producción de un fuerte ligando orgánico secuestrante de hierro, el ácido domoico. Los efectos del exceso de iones metálicos en el citosol también pueden reducirse por emanación o compartimentación. Los metales tóxicos que son complejados por fitoquelatinas, ácidos orgánicos, etc. pueden almacenarse en compartimentos internos como vacuolas o cloroplastos. Los microorganismos (principalmente procariotas) tienen mecanismos de eflujo eficaces para metales. Por ejemplo, la bacteria Cupriviadus metallidurans es capaz de activar la síntesis de una bomba de eflujo de níquel, una vez que el níquel ha entrado en el periplasma [Gras y colaboradores, 2005]. El alga unicelular verde Chlorella vulgaris excluye cobre mientras que 56 Capitulo 2: INTRODUCCIÓN algunas diatomeas y cianobacterias son capaces de excluir complejos de los metales [Foster, 1977]. Los microorganismos también pueden excretar compuestos capaces de complejar los iones metálicos en el medio extracelular con el fin de reducir su biodisponibilidad. Por ejemplo, la bioacumulación de Cd por bacterias gram negativas Rhodospirillum rubrum disminuyó gravemente por la producción de un ligando extracelular no identificado. Las algas son capaces de producir y exudar agentes secuestrantes como polisacáridos, proteínas, péptidos y ácidos orgánicos pequeños que disminuyen la concentración de metales biodisponibles en las inmediaciones de la célula [Gortzi y colaboradores, 2008]. Los metales traza esenciales son, a menudo, altamente regulados para evitar ambas situaciones de deficiencia del metal traza y de saturación. Las estrategias son variadas, según el organismo, el metal y la fisicoquímica del metal en disolución. Ninguno de los procesos es totalmente independiente. Los nuevos avances para entender los procesos biológicos, químicos y físicos que ocurren en la inter-fase biológica permitirán una mejor comprensión de la biodisponibilidad de los metales traza en el medio natural. Es bien sabido que el hierro actúa como fertilizante en el océano y, en especial, en áreas de alta concentración de clorofila y baja concentración de nutrientes, mientras que el cobre, un 57 Capitulo 2: INTRODUCCIÓN nutriente esencial en baja concentración, llega a ser tóxico en altas concentraciones [Stumm y Morgan, 1996; Franklin y colaboradores, 2002]. El hierro es necesario para la mayoría del fitoplancton [Ho y colabradores, 2003] y debido a sus propiedades redox, es un componente de muchas enzimas y de proteínas participantes en la fotosíntesis y en la respiración [Raven y colaboradores, 1999]. Los compuestos polifenólicos pueden formar complejos débiles con el hierro y modificar la especiación química y biodisponibilidad de este metal traza [Mira y colaboradores, 2002; Andjelković y colaboradores, 2006]. Algunos polifenoles también pueden formar complejos fuertes con hierro con constantes cuyo logaritmo, log K, va desde 18 a 22 [Hynes y O'Coinceanainn, 2001; 2004], aunque la afinidad de estos compuestos por Fe (III) es mucho menor que la de sideróforos naturales como enterobactina o deferoxamina que forman complejos hexadentados [Elhabiri y colaboradores, 2007].Los compuestos fenólicos pueden influir en la química redox del hierro y favorecer la persistencia del hierro (II) proporcionando los mecanismos posibles para que esté disponible para abastecer los requerimientos de la célula. Determinar cómo cambian las concentraciones de polifenoles en microalgas en respuesta a altos niveles de cobre y hierro (dos metales con efectos diferentes) podría demostrar el papel de los polifenoles como herramientas defensivas para evitar el envenenamiento celular y podría ser útil para ayudar a 58 Capitulo 2: INTRODUCCIÓN explicar la dinámica de esta importante clase de compuestos en el agua de mar [Santana-Casiano y colaboradores, 2014]. 59 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES ÁCIDO CLOROGÉNICO Ácido (1S,3R,4R,5R)-3-[[3-(3,4dihidroxifenil)-1-oxo-2-propenil]oxi]- 1,4,5trihidroxiciclohexanocarboxílico C16H18O9 OH COOH OH OH O O OH OH APIGENINA 5,7-dihidroxi-2-(4-hidroxifenil)-4H-1benzopiran-4-ona C15H10O5 OOH OH O OH FIGURA 7.- Compuestos fenólicos sometidos a estudio 3.2. Material vegetal Macroalga Stypocaulon scoparium (Linnaeus) Kützing (1843) [división: Heterokontophyta, clase: Phaeophyceae, orden: Sphacelariales, familia: Stypocaulaceae, género: Stypocaulon]. Es un alga parda filamentosa de talo arbuscular, con aspecto de escoba, de color marrón y de hasta 20 centímetros de alto. Se fija al sustrato por un disco basal fibroso-esponjoso. Con ramificación densa, irregularmente alterna y dística. Está distribuida por gran parte del océano Atlántico y el mar Mediterráneo y su presencia es muy frecuente en las Islas Canarias [Price y colaboradores, 1978], en las zonas rocosas y grietas batidas por las mareas y bien iluminadas, hasta 15 metros de profundidad. Se presenta en plataformas rocosas y charcos del eulitoral y en los primeros metros del sublitoral. 66 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES Las algas analizadas en el presente trabajo fueron recolectadas en Gran Canaria, entre 0 y 0,1 metros de profundidad. Las algas se enjuagaron con agua de mar y posteriormente se congelaron. Estas muestras congeladas se liofilizaron (liofilizador Cryodos 30, Telstar) y pulverizaron en una batidora (Moulinex 600W). Finalmente, se reservaron en el congelador a -20ºC en total oscuridad hasta el momento de hacer los análisis.Microalga Phaeodactylum tricornutum [división: Heterokontophyta, clase: Bacillariophyceae, orden: Naviculales, familia: Phaeodactylaceae, género: Phaeodactylum] Son algas unicelulares presentes en abundancia en el fitoplancton y se caracterizan por poseer una pared celular impregnada en sílice. La diatomea Phaeodactylum tricornutum es la única especie que compone el género Phaeodactylum. Se encuentra en distintas zonas costeras de todo el mundo con grandes variaciones de salinidad. Debido a que fue de las primeras diatomeas en la que se describió la secuencia genómica completa ha sido utilizada en multitud de ensayos. Puede existir en diferentes morfologías (trirradiada, fusiforme y ovalada) (FIGURA 8). Las condiciones ambientales influyen en los cambios en la morfología y el tamaño de estas algas [Martino y colaboradores, 2007], pudiendo producirse esos cambios en medios con altas concentraciones de cobre [Markina y Aizdaicher, 2006]. 67 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES FIGURA 8.- Distintas morfologías de P. Tricornutum: (1)trirradiada, (2)ovalada y (3)fusiforme Microalga Dunaliella tertiolecta [división: Chlorophyta, clase: Chlorophyceae, orden: Volvocales, familia: Dunaliellaceae, género: Dunaliella] Es un alga verde unicelular de forma normalmente ovalada y simetría radial, de tamaño entre 5 y 18 micrómetros (μm) y con flagelos que duplican su longitud [Butcher, 1959]. Presenta una alta tolerancia a la salinidad y su principal característica morfológica es que no posee pared celular de polisacáridos. Es una cepa de alto crecimiento, por lo que tiene una alta tasa de secuestro de dióxido de carbono [Nozaki y colaboradores, 2002]. 68 (1) (2) (3) Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES 3.3. Cultivo de microalgas Las microalgas utilizadas en los trabajos que se exponen en el presente documento fueron cultivadas a partir de cepas suministradas por el Banco Español de Algas. Para realizar los cultivos de microalgas (FIGURA 9) se toma agua de mar offshore enriquecida con los siguientes nutrientes de cultivo f/2 ([NO3-] = 883 µmol L-1, [HPO42-] = 29,3 µmol L-1, [SiO32-] = 142 µmol L-1) [Guillard, 1975]; tratada con luz UV y filtrada con un filtro de 0,45μm. Para garantizar la ausencia de polifenoles en el agua de cultivo, se realizó la identificación de los compuestos polifenólicos antes de cultivar en ella las distintas especies de microalgas. Los cultivos se mantuvieron en una cámara de cultivo (Friocell FC111) a temperatura constante de 24ºC con iluminación permanente, 24 horas de 8000 lux. La densidad inicial de células utilizada en todos los experimentos fue de 2·107 células por litro. Se cultivaron bajo condiciones experimentales diferentes: para el cultivo de referencia (control) se utilizó agua de mar con nutrientes f2; tres cultivos fueron preparados con agua de mar con nutrientes f2 y enriquecida con 315 nmol L-1 de Cu(II); 790 nmol·L-1 de Cu(II); y 900 nmol·L-1 de Fe(III). En los cultivos enriquecidos con cobre se seleccionaron estas cantidades de cobre porque están por debajo y por encima, respectivamente, de la concentración de 551 nmol L-1 que produce un 50% de inhibición del crecimiento celular en los trabajos realizados por Moreno-Garrido y colaboradores (2000) para la misma diatomea y 69 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES FIGURA 9.- Esquema de cultivo de microalgas utilizando la misma densidad celular inicial. En todos los casos, las células se contaron diariamente haciendo uso de un microscopio con hemocitómetro (Microbiotest, Inc.) y midiendo la absorbancia a 670 namómetros (nm) con un espectrofotómetro(USB4000). Los cultivosse filtraron por etapas: separación por gravedad con filtros de 1,2 μm para recoger las células y mediante filtración con vacío en filtros de 0,1 μm para evitar la rotura de las células. El agua de mar enriquecida con exudados orgánicos y las células que quedaron en los filtros, a las que se les extrajo el agua por liofilización (Cryodos 30, Telstar), se reservaron a -20ºC hasta su posterior análisis. 70 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES 3.4. Extracción Los polifenoles pueden degradarse por acción enzimática cuando el material vegetal permanece fresco; por lo que es recomendable utilizar muestras en seco, liofilizadas o congeladas. Cuando se utiliza material vegetal seco generalmente se tritura hasta conseguir polvo. La mayor parte de las extracciones de polifenoles del material vegetal se realizan por extracción directa con el disolvente y, generalmente, con agitación magnética o vibración para aumentar la velocidad del procedimiento [Marston y Hostettmann, 2006]. El rendimiento de la extracción química depende del tipo de disolvente y de su polaridad, del pH, del tiempo de extracción y de la temperatura, así como de la composición de la muestra. Si se mantienen las condiciones de tiempo y temperatura, los factores más determinantes son el tipo de disolvente y las propiedades químicas de las muestras [Hayouni y colaboradores, 2007]. El disolvente se elige en función del tipo de polifenol que se necesita extraer. Se ha estudiado el efecto de distintos disolventes en la extracción de polifenoles de materiales vegetales [Pinelo y colaboradores, 2004]. Algunos trabajos [Khokhar y Magnusdottír, 2002] indican que el etanol acuoso da mejores resultados en la extracción de flavonoides del té que el metanol y la acetona. Para la extracción de compuestos 71 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES polifenólicos de algas pardas y rojas se utilizan comúnmente metanol, etanol, acetona, cloroformo o agua [Duan y colaboradores, 2007; Ganesan y colaboradores, 2008; Chandini y colaboradores, 2008; Yuan y Walsh, 2006]. Los polifenoles menos polares (isoflavonas o flavonoles, por ejemplo) se extraen con cloroformo, diclorometano, éter dietílico o acetato de etilo; mientras los compuestos más polares, como los glicósidos, se extraen con alcoholes o mezclas de alcohol-agua donde es mayor su solubilidad. Con frecuencia catequinas, proantocianidinas y taninos condensados pueden extraerse directamente con agua [Hussein y colaboradores,1990]. Los disolventes utilizados en nuestros trabajos fueron metanol, agua, etanol y una mezcla de agua con metanol al 50%. Para la extracción de microalgas se utilizó metanol. 3.5. Preparación de muestras. Extracción en fase sólida La filtración y la extracción líquido-líquido se emplean con frecuencia en la preparación de las muestras de polifenoles [Tura y colaboradores, 2002]. Actualmente, también se usan otras técnicas como la extracción en fase sólida (SPE) que permite eliminar las sustancias interferentes de la matriz vegetal, reteniendo y concentrando los analitos de interés. Es una técnica rápida y sensible en la que se emplean distintos tipos de cartuchos con gran variedad de adsorbentes para sustancias polares, hidrofóbicas e iónicas. Con la SPE se consigue preparar y 72 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES concentrar las muestras en un único paso, con un bajo consumo de disolvente, ahorro de tiempo y un alto grado de automatización del proceso. Entre las numerosas aplicaciones de la SPE está la separación de ácidos fenólicos y flavonoides de vinos y zumos de frutas [Chen y colaboradores, 2001], la identificación de flavonoides antimutagénicos en extractos acuosos de espinacas, tras la extracción de sustancias lipófilas por SPE [Edenharder y colaboradores, 2001]. En el presente estudio, se utilizó como adsorbente un copolímero de divinilbenceno-poliestireno modificado especialmente recomendado para muestras acuosas: cartuchos Chromabon Easy (Macherey-Nagel, 500 mg, tamaño de partícula 93 m). 3.6. Contenido total de polifenoles Otro paso importante es la medida de la concentración total de polifenoles mediante el uso del reactivo de FolinCiocalteu [Julkunen-Tiitto, 1985], que consiste en una mezcla de los ácidos fosfomolíbdico y fosfotúngstico, en los que el molibdeno y el tungsteno se encuentran en el estado de oxidación 6+. Este método describe la capacidad antioxidante en base a la transferencia de un solo electrón desde el compuesto fenólico al Mo (VI) en el reactivo. En este proceso el molibdeno (VI) se reduce a molibdeno (V), de color azul y con un máximo de absorción a 765 nm (FIGURA 10). Se debe preparar una curva de calibrado, generalmente con ácido gálico, de manera que los 73 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES resultados se presentan como equivalentes de ácido gálico (EAG). FIGURA 10.- Ensayo Folin Ciocalteau El ensayo fue desarrollado en 1927 para la medición de la tirosina y de otros aminoácidos y péptidos y se ha utilizado durante muchos años por la industria alimentaria y agrícola para determinar el contenido fenólico de los productos vegetales. Sin embargo, este reactivo no solo mide los fenoles totales, sino que reaccionará con cualquier sustancia reductora y, en consecuencia, el reactivo mide la capacidad reductora total de una muestra, no solo el nivel de compuestos fenólicos. La medición de los polifenoles totales a través del método Folin-Ciocalteu no muestra una imagen completa de la cantidad o la calidad de los compuestos polifenólicos de los extractos, ya que se desconoce la química exacta y el potencial redox del reactivo Folin [Huang y colaboradores, 2005] que puede actuar como un reactivo no especifico oxidando otro tipo de compuestos como azúcares o aminas [Apak y colaboradores, 2007]. 74 Capitulo 3: MATERIALES Y MÉTODOS APLICADOS EN EL ANÁLISIS DE POLIFENOLES 3.7. Determinación de la actividad antioxidante La actividad antioxidante de los extractos no se puede medir directamente, por lo que es necesario recurrir a métodos indirectos en los que se miden los efectos del antioxidante en el control de la extensión de la oxidación. En este estudio se han seleccionado los métodos que se describen a continuación. 3.7.1. Capacidad de inhibir radicales libres La actividad antioxidante se puede evaluar midiendo el descenso de la concentración del radical DPPH [Chu y colaboradores, 2000]. El radical DPPH es un radical estable de coloración violeta que puede ser neutralizado por transferencia de átomos de hidrógeno y por transferencia de un electrón. La reducción de este radical puede ser medida por resonancia spin electrón o por decrecimiento de la absorbancia medida a una longitud de onda de 515 nm. El método de la decoloración propuesto por Bondet y colaboradores (1997) mide la actividad antioxidante de los compuestos a través de la pérdida de coloración del radical DPPH, tras la reacción con dichos compuestos (FIGURA 11). Los resultados se expresan en porcentaje de inhibición que se calcula según la siguiente ecuación: Inhibición del radical(%) = (1Abs muestra / Abs 0) × 100 (Ecuación 1) 75 Capitulo 4: DISCUSIÓN Y RESULTADOS 4.1. Stypocaulon scoparium. Efecto de los disolventes de extracción sobre el contenido fenólico y la actividad antioxidante de extractos del alga parda Stypocaulon scoparium. Se prepararon extractos de alga parda Stypocaulon scoparium utilizando diferentes disolventes (agua, metanol, etanol y una mezcla de agua y metanol al 50%) para determinar el contenido total de polifenoles mediante el ensayo de FolinCiocalteu [Julkunen-Tiitto, 1985]. También se realizó el análisis de la actividad antioxidante de los extractos a través del ensayo de inhibición del radical DPPH [Chu y colaboradores, 2000]. Los resultados mostraron correlación significativa entre la actividad antioxidante y el contenido total de polifenoles, donde la mayor actividad antioxidante y el contenido fenólico más alto correspondieron al extracto acuoso. Se desarrolló, además, una metodología para la identificación y cuantificación de 14 polifenoles de manera rápida y precisa en todos los extractos anteriormente mencionados para comparar la capacidad de cada disolvente para extraer un grupo seleccionado de polifenoles. La presencia de polifenoles en los extractos se confirmó por comparación de los tiempos de retención y por solapamiento de los espectros de ultravioleta de los compuestos estándar. Cada extracto mostró la presencia de los catorce polifenoles, entre los que la quercetina, el ácido cafeico y el ácido cumárico aparecen en menor cantidad que el resto. El ácido gálico fue el compuesto 83 Capitulo 4: DISCUSIÓN Y RESULTADOS predominante en cada extracto analizado. También se encontraron cantidades significativas de (+)-catequina y (-)- epicatequina en todos los extractos. Los cambios de polaridad del disolvente alteran su capacidad para extraer un grupo seleccionado de compuestos antioxidantes. Un aumento en la polaridad del disolvente dio un aumento en la cantidad de ácido gálico en los extractos. Teniendo en cuenta la suma total del resto de polifenoles identificados: 32,2 mg en el extracto acuoso; 24,2 mg en la mezcla de agua y metanol al 50%; 25,2 mg en metanol y 23,3 mg en etanol (todas las cantidades referidas a 100 gramos de alga seca), parece que la actividad antioxidante se correlaciona con el contenido de ácido gálico, el antioxidante que presentó mayor actividad en inhibir el radical DPPH de los ensayados (FIGURA 12). FIGURA 12.- Inhibición del radical DPPH (%) por los compuestos estándar 84 Capitulo 4: DISCUSIÓN Y RESULTADOS Es difícil describir la correlación entre rendimiento, contenido fenólico total y actividad antioxidante de los extractos, ya que cada extracto puede tener diferentes clases de compuestos con diferentes actividades antioxidantes (FIGURA 12) y que dan diferente repuesta al ensayo Folin Ciocalteau (FIGURA 13). Algunos autores han señalado que existe una estrecha relación entre la actividad antioxidante y el contenido total de polifenoles [Duan y colaboradores, 2007; Zhao y colaboradores, 2006]. Otros autores han indicado en sus trabajos que no han encontrado tal correlación y que, por lo tanto, en esos casos los polifenoles no son los responsables de la actividad antioxidante de las muestras [Kähkönen y colaboradores, 1999; Patthamakanokporn y colaboradores, 2008; Rapisarda y colaboradores, 2008]. Aunque estas interpretaciones son válidas en algunos casos, no consideran factores tales como las diferencias en el perfil fenólico entre las distintas muestras y las interacciones entre sus componentes (actividad sinérgica, aditiva o antagónica) [Jacobo-Velázquez y Cisneros-Zevallos, 2009]. 85 Capitulo 4: DISCUSIÓN Y RESULTADOS FIGURA 13.- Respuesta de los compuestos estándar al ensayo de FolinCiocalteau 86 Analytical Methods The effects of solvents on the phenolic contents and antioxidant activity of Stypocaulon scoparium algae extracts Aroa López, Milagros Rico ⇑ , Argimiro Rivero, Miguel Suárez de Tangil Departamento de Química, Universidad de Las Palmas de Gran Canaria, Campus de Tafira, 35017 Las Palmas de Gran Canaria, Canary Islands, Spain article info Article history: Received 20 May 2009 Received in revised form 15 March 2010 Accepted 28 September 2010 Keywords: Stypocaulon scoparium (S. scoparium) Total phenolic content (TPC) Polyphenols Reverse phase high performance liquid chromatography (RP-HPLC) Gallic acid (GA) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical scavenging activity (RSA), Dry alga powder abstract Water, water/methanol (1/1), methanol and ethanol crude extracts from a brown alga Stypocaulon scoparium were examined for total phenolic contents (TPC) using Folin–Ciocalteu method. DPPH scavenging assay was performed to measure the radical scavenging activities (RSA) of the extracts. Results showed a significant association between the antioxidant potency and the TPC. The aqueous extract showed both, the highest antioxidant activity and highest phenolic contents. The identification and quantification of phenolic antioxidants were carried out with a rapid and simple method of reverse phase high performance liquid chromatography (RP-HPLC). This method was developed for the simultaneous analysis of 14 polyphenols, namely gallic acid, catechin, epicatechin, rutin, p-coumaric acid, myricetin, quercetin and protocatechuic, vanillic, caffeic, ferulic, chlorogenic, syringic and gentisic acids. The chromatographic separation of 14 polyphenols was achieved in less than 40 min by RP-HPLC (Varian, Pursuit XRs C18 column, 5 l m, 250 mm 4.6 mm) using linear gradient elution of methanol and water (0.1% formic acid) with a flow rate of 1 ml/min. Gallic acid was by far the predominant polyphenol. Ó2010 Elsevier Ltd. All rights reserved. 1. Introduction Recently phytochemicals in herbal plants have attracted a great deal of attention mainly concentrated on their role in preventing diseases caused as a result of oxidative stress (Southon, 2000). Dietary antioxidants from plants are believed to help prevent ageing and many degenerative diseases such as cardiovascular diseases and cancers through radical scavenging activity (Dillard & German, 2000; Prior & Cao, 2000; Steinmetz & Potter, 1996; Virgili & Scaccini, 2003; Wargovich, 2000; Yang, Landau, Huang, & Newmark, 2001). There is considerable interest in preventive medicine in the development of natural antioxidants obtained from botanical sources (Kaur & Kapoor, 2002). Seaweeds are considered to be a rich source of antioxidants (Cahyana, Shuto, & Kinoshita, 1992; Lim, Cheung, Ooi, & Ang, 2002), and different types of antioxidants from various species of seaweeds have been reported (Yan, Chuda, Suzuki, & Nagata, 1999). The high level of solar radiation and the high temperature prevailing in the regions of Canary Islands (African Northwestern Coast) forces plants to develop defence mechanisms against ultraviolet radiation and excessive production of free radicals through the accumulation of antioxidant substances. This prompted us to evaluate total phenolic content of the crude extracts derived from Stypocaulon scoparium collected from Canary Islands. The effects of different extracting solvents have been tested for the extraction of polyphenols from plant material (Pinelo, Rubilar, Sineiro, & Nunez, 2004). Extraction yield is dependent on the solvent and the method of extraction (Hayouni, Abedrabba, Bouix, & Hamdi, 2007). Khokhar and Magnusdottír (2002) reported that aqueous ethanol was superior to methanol and acetone for extracting flavonoids from tea. Thus, the objective of this study was to investigate the effect of several extracting solvents on the total phenolic compounds of the crude extracts derived from S. scoparium. It is obvious that total phenolics measured by Folin– Ciocalteu procedure do not give a full picture of the quantity or quality of the phenolic constituents in the extracts. Several methods have been developed to identify polyphenols from plants, fruits and vegetables using RP-HPLC analysis (Andre et al., 2007; Keyhanian & Stahl-Biskup, 2007; Khokhar & Magnusdottír, 2002). We report here the rapid determination of 14 polyphenols in crude extracts derived from a brown alga S. scoparium, to compare the ability of each solvent to dissolve a selected group of polyphenols. In addition, we will investigate the correlation between TPC and RSA of the extracts. Several reports have convincingly shown a close relationship between antioxidant activity and 0308-8146/$ - see front matter Ó2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.09.101 ⇑ Corresponding author. Tel.: +34 928454418; fax: +34 9282922. E-mail address: [email protected] (M. Rico). Food Chemistry 125 (2011) 1104–1109 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem total phenolic content (Duan, Wu, & Jiang, 2007; Zhao, Yang, Wang, Li, & Jiang, 2006). However, other reports indicated that this correlation doesn’t exist and it was concluded that phenolic compounds are not responsible of the antioxidant activity (Kähkönen et al., 1999; Patthamakanokporn, Puwastien, Nitithamyong, & Sirichakwal, 2008; Rapisarda, Lo Bianco, Pannuzzo, & Timpanaro, 2008). Although valid in some cases, this interpretation does not consider factors such as differences in the phenolic profiles between samples (Jacobo-Velazquez & CisnerosZevallos, 2009). 2. Materials and methods 2.1. Chemicals Methanol (Panreac, Barcelona) and ethanol (Panreac, Barcelona) were of HPLC grade. Water was purified on a Milli-Q system from Millipore (Bedford, MA, USA). Formic acid provided by Merck (Darmstadt, Germany) was of analytical quality. Folin–Ciocalteu’s phenol reagent and sodium carbonate were from Sigma-Aldrich Chemie (Steinheim, Germany). Polyphenol standards were supplied as follows: gallic acid, protocatechuic acid, chlorogenic acid, () epicatechin, quercetin, myricetin, ferulic acid, p-coumaric acid, vanillic acid, syringic acid, (+) catechin, by Sigma–Aldrich Chemie (Steinheim, Germany); rutin and gentisic and caffeic acids by Merck (Darmstadt, Germany). 2.2. Plant material The brown alga S. scoparium used for this study was freshly collected from Canary Island, Spain, at 0–0.1 m depth between March and April 2008. The algae were rinsed carefully in fresh seawater and then frozen. The frozen samples were lyophilised, pulverised into powder by a blender (Moulinex, 600 W, France) and were kept in the dark at 20 °C under nitrogen. 2.3. Preparation of seaweed extracts for both, DPPH assays and TPC determinations Dried powders (2.0 g) were separately extracted for 2 h at room temperature in 30 ml of one of these solvents: absolute methanol, absolute ethanol, water and a mixture water/methanol (50%) by mixing using a magnetic stirrer. Each extract was filtered for removal of alga particles. After centrifugation at 2000gfor 20 min, the supernatant was collected and filtered through 0.45 l m filter paper and stored (10 ml) at 4 °C. Extraction solutions (20 ml) were dried by vacuum-evaporator. The dried residues were weighed and the yield for extractable substances was calculated based on the weight of dry alga powder. 2.4. Preparation of seaweed extracts prior to HPLC injection Approximately 50 mg of powdered freeze-dried material was mixed separately with 1.0 ml of each solvent (methanol, ethanol, water and methanol/water 50%). The mixture was homogenised using a vortex for 30 s and shaken for 60 min at room temperature in the darkness. After centrifugation at 2000gfor 20 min at 4 °C, the supernatant was collected. Supernatants were evaporated to dryness and residues were suspended in 500 l l of water and filtered through a 45 l m nylon syringe filter prior to injection. 2.5. Determination of total phenolics The amount of total phenolics in extracts was determined according to the Folin–Ciocalteu assay (Julkunen-Tiitto, 1985). Samples (100 l l) were introduced into test tubes containing 8.4 ml of water; 0.5 ml of Folin–Ciocalteu’s reagent and 1 ml of sodium carbonate (20%) were added. The tube were mixed and allowed to stand for 1 h in the darkness at room temperature. The absorbance was measured at 765 nm using a SHIMADZU 1700 UV–vis spectrophotometer. The estimation of phenolic compounds was carried out in triplicate, and the results were averaged. A calibration curve of gallic acid (ranging from 0.050 to 0.9 mg/ml) was prepared (in methanol), and the results, determined by the regression equation of the calibration curve (y= 0.00029x0.00025); correlation coefficient r= 0.9992), were expressed as gallic acid (GA) mg equivalents/100 g dry alga powder. 2.6. DPPH scavenging activity The free radical scavenging activity was measured using the method of Chu, Chang, and Hsu (2000) with some modification. One ml of 0.1 mM DPPH (1,1-diphenyl-2-pikryl-hydrazin) solution in methanol was added to 100 l l of the sample solution. The decline in absorbance was recorded at 515 nm against a methanol blank over a period of 20 min. The scavenging activity (%) (RSA) on DPPH radicals was calculated as follows: RSA (%) = (1 absorbance of sample/absorbance of control) 100. 2.7. Analytical data Chromatographic analysis was performed on a Varian ProStar 210 system, equipped with a vacuum degasser, a binary pump, a thermostatted column compartment and a diode array detector (DAD), connected to a ChemStation software. The separation was performed with a reverse phase Pursuit XRs C18 (250 mm 4.6 mm, 5 l m) column and a Pursuit XRs C18 (10 mm 4.6 mm, 5 l m) guard column (Varian, Barcelona). A gradient system was used involving two mobile phases. Eluent A was water with 0.1% formic acid and eluent B methanol. The flow rate was 1.0 ml/min, and the injection volume was 60 l l of crude extracts. The system operated at 27 °C. The elution conditions applied were: 0–5 min, 20% B isocratic; 5–30 min, linear gradient from 20% to 60% B; 30–35 min, 60% B isocratic; 35–40, linear gradient from 60% to 20% B and finally, washing and reconditioning of the column. Simultaneous monitoring was set at 270 nm (gallic acid, protocatechuic acid, catechin, vanillic acid, epicatechin and syringic acid), 324 nm (chlorogenic acid, gentisic acid, caffeic acid, coumaric acid and ferulic acid) and 373 nm (rutin, myricetin, and quercetin) for quantification. 2.7.1. Calibration curves Stock solution containing standards was prepared and diluted with methanol to appropriate concentration in the range of 1.0– 200 l g/ml for establishing calibration curves. For quantitative analysis, five different concentrations of fourteen analytes were injected in triplicate. The calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. 2.7.2. Selectivity The selectivity of the method was determined by analysis of standard compounds and samples. The peaks of polyphenols were identified by comparing their retention times and overlaying of UV spectra with those of standard compounds. 2.7.3. Linearity Previous experiments showed us the correct ranges for concentrations to construct calibration curves. Stock solution was prepared containing 200, 100, 50, 10, 2.0 l g/ml for gallic acid; 75, 40, 20, 10 and 5.0 l g/ml for catechin; 50, 25, 10, 5.0 and 1.0 l g/ ml for rutin; 25.0, 10.0, 5.0, 3.0 and 1.0 l g/ml for caffeic acid; 75, A. López et al. / Food Chemistry 125 (2011) 1104–1109 1105 50, 25, 10 and 2.0 l g/ml for epicatechin, myricetin, quercetin, protocatechuic acid, ferulic acid, coumaric acid, chlorogenic acid, gentisic acid, vanillic acid and syringic acid. The linearity was assessed by linear regression analysis, which was calculated by the least square method. Each point on the calibration plot was the mean from two area measurements. All correlation coefficients were not less than 0.9982 (Table 1). 2.7.4. Limit of detection and quantification Limits of detection (LOD) and limits of quantification (LOQ) were estimated from signal-to-noise ratio of the individual peaks, assuming a minimum detectable signal-to-noise level of 3 and 10, respectively (Taverniers, De Loose, & Van Bockstaele, 2004). The detection and the quantification limits are shown in Table 2. 2.7.5. Accuracy and precision Reproducibility, expressed as relative standard deviation (RSD), was obtained by analysing six replicate samples containing the following concentrations: 50 l g/ml for epicatechin, 35 l g/ml for catechin, 30 l g/ml for quercetin, 25 l g/ml for gallic and syringic acids; 20 l g/ml for rutin, protocatechuic acid, and gentisic, ferulic and coumaric acids; 15 l g/ml for myricetin and vanillic acid and 10 l g/ml for caffeic and chlorogenic acids. RSDs values ranged from 1.91% to 5.81%. The accuracy was expressed as the recovery of standard compounds added to the pre-analysed sample (Shabir, 2003). The recovery was found to be in the range of 87.97–115.79%. RSD values and recoveries are shown in Table 2. 3. Results and discussion 3.1. Extraction yields It is well known that the yield of chemical extraction depends on the type of solvents with varying polarities, pH, extraction time and temperature as well as on the chemical compositions of the sample. Under the same conditions of time and temperature, the solvent and the chemical properties of the sample are two most important factors. Earlier, solvents such as methanol, ethanol, butanol, acetone, chloroform and water have been commonly used for the extraction of phenolics from brown and red seaweeds (Chandini, Ganesan, & Bhaskar, 2008; Duan, Zhang, Li, & Wang, 2006; Ganesan, Kumar, & Bhaskar, 2008; Lim et al., 2002; Yuan & Walsh, 2006). Yields of different extracts of S. scoparium were examined and presented in Table 3. The highest yield for extractable substances was achieved by the polar solvents. The order of the yields from high to low was: water/methanol > water > methanol > ethanol. As compared to results of the present study, Chandini et al. (2008) observed lower yields of total methanol extracts of three brown seaweeds (12.31%, 5.76% and 5.45%). 3.2. Amounts of total phenolics The amount of total phenolics varied from 123.2 to 328.7 mg equivalent GA/100 g of dry alga powder (Table 3). TPC was strongly affected by the extracting solvent with the following order from high to low: water > water/methanol > methanol > ethanol. Similar findings were also reported by Kuda, Tsunekawa, Goto, and Araki (2005). No correlation was found between yield and TPC, which agrees with earlier reported results (Chandini et al., 2008). In spite of that water extract showed higher TPC than water/methanol extract, both extracts gave the same yield. By other way, water extraction gained only 2.7 times more total phenolics than ethanol extraction, although water gave 7.0 times higher yield than ethanol. That means that ethanol extract gave relatively high TPC, although yield was too low. 3.3. DPPH radical scavenging activity Antioxidant activities of the extracts are shown in Table 3. The highest activity (47.9%) was observed in the aqueous extract. Solvent polarity was significantly effective on the DPPH inhibition of crude extracts. Extracts with higher amounts of polyphenols gave also higher values of RSA. The lowest activity and phenolic content were observed when ethanol was used for maceration. This present finding corroborates well with earlier reports in other plants materials including brown/red seaweeds (Alothman, Bhat, & Karim, Table 1 Linearity data for calibration curves of fourteen polyphenols determined by RP-HPLC. Polyphenol Linearity range ( l g/ml) Regression equation Correlation coefficient (r) Gallic acid 2–200 y= 175,500x+ 41,647 0.9987 Protocatechuic 2–75 y= 179,055x6369 0.9995 Catechin 5–75 y= 40,283x+ 4319 0.9993 Vanillic acid 2–75 y= 165,648x+ 12,613 0.9994 Epicatechin 3–75 y= 82,583x+ 5895 0.9984 Syringic acid 2–75 y= 202,997x6454 0.9999 Chlorogenic acid 2–75 y= 127,234x+ 4450 0.9999 Gentisic acid 2–75 y= 98,060x4968 0.9992 Caffeic acid 1–25 y= 364,361x2983 0.9992 Coumaric acid 2–75 y= 424,019x2775 0.9997 Ferulic acid 2–75 y= 407,619x4259 0.9994 Rutin 1–50 y= 90,597x3719 0.9994 Myricetin 2–75 y= 274,627x4760 0.9982 Quercetin 2–75 y= 408,705x1420 0.9984 Table 2 Method validation data for the quantitative determination of fourteen polyphenols in extracts of S. scoparium alga by RP-HPLC. Compounds LOD a ( l g/ml) LOQ a ( l g/ml) Recovery b (%) RSD c (%) Gallic acid 0.0003 0.0008 99.92 ± 3.13 5.37 Protocatechuic 0.0015 0.0050 97.85 ± 3.04 4.88 Catechin 0.0069 0.0230 95.96 ± 0.64 2.93 Vanillic acid 0.0117 0.0390 97.28 ± 4.74 5.43 Epicatechin 0.0165 0.0550 102.7 ± 3.42 3.65 Syringic acid 0.1020 0.3400 99.85 ± 3.40 1.94 Chlorogenic acid 0.0032 0.0105 89.34 ± 1.52 3.60 Gentisic acid 0.0156 0.0520 95.45 ± 2.22 3.25 Caffeic acid 0.0267 0.0890 99.42 ± 3.56 5.81 Coumaric acid 0.0207 0.0690 102.9 ± 4.27 5.51 Ferulic acid 0.0315 0.1050 98.98 ± 3.87 3.95 Rutin 0.1230 0.4100 115.8 ± 4.18 3.17 Myricetin 0.0528 0.1760 91.28 ± 4.11 4.65 Quercetin 0.0410 0.0410 87.97 ± 3.48 1.91 a Detection limits are calculated as signal-to-noise ratio of six times. b Means ± standard deviation of three measurements. c n=6. Table 3 Comparative analysis of extraction yields, total phenolic contents and DPPH radical scavenging activities of S. scoparium extracts prepared using different solvents. Solvent Extraction yield a (%) TPC b RSA c Water 16.6 328.7 ± 2.87 47.9 ± 0.67 Water–methanol 16.8 292.3 ± 5.14 38.6 ± 0.95 Methanol 13.3 255.2 ± 1.59 34.8 ± 0.55 Ethanol 2.36 123.2 ± 3.36 17.0 ± 0.11 a EY: values expressed as % of dry algae powder. B TPC: values are expressed as mg GA equivalents/100 g dry alga powder (means ± standard deviation of three measurements). c RSA: values represented means of triplicate determinations ± standard deviation. 1106 A. López et al. / Food Chemistry 125 (2011) 1104–1109 2009; Kuda et al., 2005; Sun & Ho, 2005). However, some authors reported that no significant correlation could be found between the antioxidant activity and total phenolic content (Bozan & Karakaplan, 2007; Kähkönen et al., 1999). Ethanol extract gave also relatively high RSA as happened with TPC, although yield was too low. 3.4. HPLC analysis To achieve better resolution in a short period a mobile phase consisting of methanol/water with 0.1% formic acid as a gradient eluent was selected. Each standard was individually tested to determine its retention times (RT). Fourteen phenolics, namely gallic acid (RT: 5.3 min), protocatechuic (RT: 10.0 min), catechin (RT: 12.7 min), chlorogenic acid (RT: 14.9 min), gentisic acid (RT: 17.1 min), vanillic acid (RT: 17.7 min), epicatechin (RT: 17.9 min), caffeic acid (17.9 min), syringic acid (RT: 18.9 min), coumaric acid (RT: 23.4 min), rutin (RT: 28.1 min), ferulic acid (RT: 24.3 min), myricetin (RT: 30.6 min) and quercetin (RT: 34.6 min) were well resolved (Fig. 1). At 270 nm, epicatechin and caffeic acid were detected with a retention time of 17.9 min (peak 5). At 324 nm, only caffeic acid (peak 9) was detected whit this retention time. Epicatechin amount was determined subtracting the area of peak 9 from peak 5 area and using the corresponding calibration curve. The LODs were found to be in the range of 0.0003–0.1230 l g/ml and the LOQs were observed in the range of 0.0008–0.4100 l g/ml. This indicated that the proposed method gave a good sensitivity for the quantification of polyphenols. The presence of polyphenols in the extracts was confirmed by comparison of their retention times and overlying of UV spectra with those of standard compounds. Each extract showed the presence of all the fourteen polyphenols, among which quercetin, caffeic acid and coumaric acid were observed in lower amount than the other. In this analysis, gallic acid was by far the predominant polyphenol for each tested extract. However, an exception has been found, the ethanolic extract contain gallic acid as a minor constituent. In comparison to other polyphenols, significant amounts of catechin and epicatechin were also found in all the extracts. Few reports are available on the characterisation of polyphenols by HPLC techniques from algal material to compare results. Some of the first polyphenols found in algae were florotannins (Parys et al., 2007). As compared to results of the present study, Onofrejová et al. (2010) reported lower amounts of protocatechuic, p-coumaric, vanillic, caffeic and chlorogenic acids extracted from in vitro culture of two freshwater algae and from food products of marine macroalgae. As it can be observed in Table 4, changes on solvent polarity alter its ability to dissolve a selected group of antioxidant compounds. An increase in the polarity of the solvent gave an increase in the quantity of gallic acid in the tested extract. No significantly differences were found when the sums of the rest of identified polyphenols in Table 4 were calculated: 32.2 mg (water); 24.2 mg (water/methanol); 25.2 mg (methanol) and 23.3 mg (ethanol). It is difficult to describe the correlation between yield, total phenolic content and antioxidant activities, since each extract might have different classes of phenolics which have varying antioxidant potential. In this study we reported the extreme changes in the concentration of gallic acid when solvent Fig. 1. HPLC chromatograms of standard polyphenols: (A) 270 nm: peak 1 = gallic acid, peak 2 = protocatechuic acid, peak 3 = catechin, peak 4 = vanillic acid, peak 5 = epicatechin + caffeic acid, and peak 6 syringic acid; (B) 324 nm: peak 7 = chlorogenic acid, peak 8 = gentisic acid, peak 9 = caffeic acid, peak 10 = coumaric acid, and peak 11 = ferulic acid; and (C) 373 nm: peak 12 = rutin, peak 13 = myricetin, and peak 14 = quercetin. A. López et al. / Food Chemistry 125 (2011) 1104–1109 1107 polarity increases. However, solvent polarity does not change drastically the total amounts of a phenolics group, but the phenolic profile. 4. Conclusion The RP-HPLC method developed in this study was shown to be rapid, sensitive, and accurate in simultaneously detecting and quantifying of 14 polyphenols with good reproducibility. On the basis of this study, it can be concluded that the extracting solvent significantly affected total polyphenol content and antioxidant activity of several extracts from S. scoparium. The HPLC analysis showed that solubility of polyphenols changes with the polarity of the solvent but not always in the same way: with some of them it increases and with other ones it decreases. Thus, gallic acid was found to be increased in extracts prepared with polar solvents, while protocatechuic acid amount increases when polarity of solvent decreases. Ethanol extract gave relatively high RSA and TPC, although yield was too low. It seems that ethanol might dissolve more radical scavenging active polyphenols than other solvents, as happened with protocatechuic acid, and may be, a lower amount of no antioxidant active substances. Extracting solvents, phenolic content and antioxidant activity must be discussed when evaluating the antioxidant activity of extracts. In addition, the typical approach used to analyse correlations between total antioxidant activity and total phenolics do not reflect the characteristics of phenolics, which can act synergistically, additively or antagonistically (Jacobo-Velazquez & Cisneros-Zevallos, 2009). Differences in the phenolic profiles should be too considered. Our proposed analytical method can be used for the rapid analysis of bioactive phenols algae materials and their derived food products. Algae represent a source of interesting natural bioactive compounds for human nutrition. The result of this experiment may show S. scoparium as a natural source of well known antioxidant compounds such as gallic acid, catechin and epicatechin. Acknowledgments This research was supported partly by the Caja Insular de Ahorros de Canarias and the Consejería de Vivienda y Arquitectura, Agricultura, Ganadería y Pesca y Agua del Cabildo Insular de Gran Canaria. The authors would like to thank Begoña Rocha Sosa for providing algae. We also thank Maria Esther Torres Padron for suggestions in analysis. References Alothman, M., Bhat, R., & Karim, A. A. (2009). Antioxidant capacity and phenolic content of selected tropical fruits from Malaysia, extracted with different solvents. Food Chemistry, 115, 785–788. Andre, C. M., Oufir, M., Guignard, C., Hoffmann, L., Hausman, J.-F., Evers, D., et al. (2007). Antioxidant profiling of native Andean potato tubers (Solanum tuberosum L.) reveals cultivars with high levels of b-carotene, a -tocopherol, chlorogenic acid, and petanin. 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(2002). Total phenol, catechin and caffeine contents of teas commonly consumed in the United Kingdom. Journal of Agricultural and Food Chemistry, 50, 565–570. Kuda, T., Tsunekawa, M., Goto, H., & Araki, Y. (2005). Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan. Journal of Food Composition and Analysis, 18, 625–633. Lim, S. N., Cheung, P. C. K., Ooi, V. E. C., & Ang, P. O. (2002). Evaluation of antioxidative activity of extracts from a brown seaweed, Sargassum siliquastrum.Journal of Agricultural Food Chemistry, 50, 3862–3866. Table 4 Polyphenol amounts in extracts of S. Scoparium algae determined by RP-HPLC analysis. Compound S. scoparium (mg/100 g dry alga powder) a Ethanol Methanol Aqueous methanol Water Gallic acid 2.798 ± 0.081 34.42 ± 0.155 71.45 ± 2.455 90.62 ± 1.549 Protocatechuic 1.719 ± 0.104 1.153 ± 0.068 0.967 ± 0.052 0.904 ± 0.011 Catechin 4.995 ± 0.225 6.908 ± 0.001 5.906 ± 0.298 6.548 ± 0.223 Vanillic acid 1.271 ± 0.023 1.166 ± 0.081 1.467 ± 0.053 2.074 ± 0.037 Epicatechin 4.434 ± 0.126 5.069 ± 0.024 5.452 ± 0.045 7.649 ± 0.035 Syringic acid 1.276 ± 0.039 1.797 ± 0.080 1.439 ± 0.001 4.437 ± 0.102 Chlorogenic acid 1.767 ± 0.032 1.751 ± 0.018 1.841 ± 0.066 1.862 ± 0.008 Gentisic acid 1.953 ± 0.065 1.932 ± 0.005 2.157 ± 0.026 2.792 ± 0.146 Caffeic acid 0.659 ± 0.006 0.643 ± 0.016 0.659 ± 0.037 0.691 ± 0.014 Coumaric acid 0.747 ± 0.020 0.739 ± 0.046 0.676 ± 0.003 0.742 ± 0.019 Ferulic acid 1.188 ± 0.074 1.324 ± 0.087 0.668 ± 0.021 1.242 ± 0.003 Rutin 0.956 ± 0.025 0.804 ± 0.022 0.860 ± 0.002 1.176 ± 0.062 Myricetin 2.076 ± 0.017 1.851 ± 0.025 1.861 ± 0.057 1.838 ± 0.026 Quercetin 0.291 ± 0.023 0.079 ± 0.001 0.235 ± 0.007 0.211 ± 0.010 a Means ± standard deviation of three measurements. 1108 A. López et al. / Food Chemistry 125 (2011) 1104–1109 Capitulo 4: DISCUSIÓN Y RESULTADOS control) debido a la alta densidad celular en el cultivo (crecimiento celular de 471% respecto al control) que ayuda a reducir al mínimo el estrés sufrido por cada célula, disminuyendo el esfuerzo metabólico necesario para la asimilación de hierro (FIGURA 14). Aunque la cantidad de polifenoles exudados por cada célula disminuyó, la concentración de hierro en el agua de mar se mantiene prácticamente constante (24 nmol L-1 en el control y 28 nmol L-1 en el ensayo con hierro). Se encontraron diferencias significativas en el perfil fenólico de la diatomea Phaeodactylum tricornutum cultivada en agua de mar (control) y en agua de mar enriquecida con metales pesados (hierro y cobre) en función de las distintas necesidades de las células. Los extractos de las diatomeas expuestas a cobre presentaron mayores actividades antioxidantes que el resto, tanto mayor al incrementar la concentración de cobre en el agua de cultivo, probablemente, debido al requerimiento de la célula en esas condiciones de mayor estrés. Los perfiles fenólicos para estos extractos más activos fueron muy similares. Sin embargo, las algas expuestas a hierro mostraron diferente perfil fenólico que debe variar el potencial antioxidante. 98 Variability of the phenolic profile in the diatom Phaeodactylum tricornutum growing under copper and iron stress Milagros Rico, * Aroa Lo´pez, J. Magdalena Santana-Casiano, Aridane G. Gonza´lez, and Melchor Gonza´lez-Da´vila Departamento de Quı ´mica, Universidad de Las Palmas de Gran Canaria, Facultad de Ciencias del Mar, Campus de Tafira, Las Palmas de Gran Canaria, The Canary Islands, Spain Abstract Fourteen phenolic compounds were identified and quantified in extracts derived from in vitro culture of Phaeodactylum tricornutum diatom growing in natural seawater (control) and in natural seawater containing the following added copper (Cu) and iron (Fe) metals: Cu(II) (315 nmol L 21 and 790 nmol L 21 ) and Fe(III) (900 nmol L 21 ). The cell concentration was 471%for the last day of culture (expressed as a percentage of the control) for diatoms exposed to iron, while diatoms exposed to copper showed cell densities of 80%and 52.5%at concentrations of 315 nmol L 21 and 790 nmol L 21 , respectively. Each extract revealed the presence of 14 phenolic compounds, with the exceptions of gallic acid, which was detected only in the iron-enriched diatoms, and quercetin, which was not detected in the control diatom exudates. Significant differences in the phenolic profiles were found depending on the metal added to the culture media. These differences seem to be the response to the different needs of diatoms exposed to copper and iron concentrations. These results show that increasing levels of metal result in a general increase in levels of total phenolic contents in the cells of P. tricornutum. The great increase in phenolic compounds in the cells at the highest copper concentration was 2.4 times higher than in the control, which may reflect the involvement of these compounds in protection against copper toxicity. All the extracts revealed radical scavenging activity against the stable radical 1,1-diphenyl-2-picrylhydrazyl, with the most active extracts from the copper enrichments. Phenolic compounds are an important group of natural products involved in responses to different kinds of biotic and abiotic stresses (Treutter 2006). The main chemical process where polyphenols are involved is in the scavenging of reactive oxygen species (Neudorffer et al. 2006; Bentes et al. 2011), but phenolic compounds from plants can also act as antioxidants by chelating metal ions (Mira et al. 2002). Therefore, plants under metallic stress conditions accumulate elevated amounts of phenolic compounds for protection and recovery from heavy-metal injury. There is considerable evidence with respect to the implications of phenolic compounds in plants growing in conditions of metal deficiency and stress (Michalak 2006). Phenolic compounds are frequently reported as the main components of root exudates in response to iron deficiency (Wei et al. 2007). Stimulation of phenolic metabolism in response to the toxicity produced by several heavy metals has also been found in chamomile (Matricaria chamomilla L.), wheat (Triticum aestivum),andinmaize(Zea mays)(Dı ´az et al. 2001; WinkelShirley 2002; Kovacik and Backor 2007). Studies carried out in order to evaluate the effects of high soil concentrations of copper (Cu) and nickel on phenolics in Scots pine (Pinus sylvestris L.) showed that trees exposed to nickel had higher concentrations of condensed tannins than controls. However, concentrations of several other phenolic compounds decreased when seedlings were exposed to high levels of copper or to a combination of nickel and copper (Roitto et al. 2005). Loponen et al. (2001) reported both increased and decreased levels of individual phenolic compounds in mountain birch Betula pubescens ssp. czerepanovii leaves from forest sites polluted by copper and nickel. The important role of phenolic compounds has also been observed in mangroves (A. corniculatum) exposed to heavy metals where the total foliar phenolics were markedly enhanced (Guangqiu et al. 2007) and in waterlily (Nymphaeaceae), capable of accumulating heavy metals with no observed toxicity to the plant (Lavid et al. 2001). In addition, polyphenols and peroxidases seem to play a major role in heavy-metal accumulation and tolerance in Nymphaea plants, involving at least two mechanisms: direct chelation by polyphenols and binding and trapping of heavymetal precipitates during the process of polyphenol polymerization by peroxidases. In addition, planktonic algae are an important component of natural surface waters. They can regulate the speciation and bioavailability of trace metals through the production and release of organic ligands (Koukal et al. 2007). These organic ligands bind .99%of the total iron (Fe) and copper in natural waters (Gledhill and van den Berg 1994; Wu and Luther 1995). Wells et al. (2005) demonstrated that diatoms of the genus Pseudo-nitzschia have an unusual capacity for adapting to iron limitation in synthetic growth medium through the production of the strong iron-complexing organic ligand, domoic acid. Few reports have focused on the analysis of bioactive phenolic acids, flavonoids, or similar polyphenols and their implications in algae growing under metal stress (Onofrejova´ et al. 2010; Lo´pez et al. 2011). Recently, there have been reports of increases in the total phenolic content and flavonoids in submerged macrophyte Vallisneria natans exposed to lead (Pb) stress (Wang et al. 2011). The aim of this study was to determine the differences in the phenolic profile of the diatom Phaeodactylum tricornutum * Corresponding author: [email protected] Limnol. Oceanogr., 58(1), 2013, 144–152 E2013, by the Association for the Sciences of Limnology and Oceanography, Inc. doi:10.4319/lo.2013.58.1.0144 144 harvested in natural seawater (control) and in natural seawater enriched with heavy metals: Cu(II) (315 nmol L 21 and 790 nmol L 21 ) and Fe(III) (900 nmol L 21 ). These two metals were chosen because of their opposite effects. It is well known that iron acts as fertilizer in the ocean and in special in high-nutrient low-chlorophyll areas, whereas copper, a nutrient at low concentration, becomes toxic at high concentrations (Stumm and Morgan 1996; Franklin et al. 2001). Iron is needed by most phytoplankton (Ho et al. 2003) and, due to its redox properties, is a component of many enzymes and electron transferring proteins in photosynthesis and respiration (Raven et al. 1999). Copper, with its two oxidation states, is also a valuable cellular constituent, performing an important role in respiration and in the photosynthetic apparatus in some marine diatoms (Peers and Price 2006). Copper replaces iron in some metabolic functions and is also involved in the high-affinity iron transport system of some iron-limited diatoms (Maldonado and Price 2001). Thus, due to iron limitation, copper is important for the growth of oceanic phytoplankton. At physiological and molecular level, this interaction is, however, not well studied. The differences in the phenolic profile may have their roots in the response of the diatoms when exposed to different metal conditions. The effect of those metal concentrations on the growth of the diatoms (cell division) was also studied. A solid-phase extraction method for isolation of the phenolic compounds was developed. Reverse phase high-performance liquid chromatography (RP-HPLC) was applied for the analysis and quantification of 14 phenolic compounds. The antioxidant activity of diatom extracts in scavenging of the stable radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was also determined. Methods Chemicals—Methanol, hexane, and acetone were HPLC grade (Panreac). Milli-Q water (18MQ, Millipore) was used throughout the entire study. Formic and acetic acids (Merck) were analytical quality reagent. DPPH and polyphenol standard gallic acid, protocatechuic acid, chlorogenic acid, (2)-epicatechin, quercetin, myricetin, ferulic acid, p-coumaric acid, vanillic acid, syringic acid, (+)-catechin, sinapic acid, quercitrin, kaempferol, and apigenin were purchased from Sigma-Aldrich Chemie (Steinheim); rutin and gentisic and caffeic acids were supplied by Merck. The SPE cartridges used were Chromabon with polar modified polystyrene divinylbenzene (500 mg) from Macherey-Nagel. The stock solutions of nutrients were prepared using sodium nitrate (Sigma) (0.88 mol L 21 ), potassium hydrogen phosphate (Sigma) (0.03 mol L 21 ), and sodium silicate (Sigma) (0.14 mol L 21 ). Both the copper and iron were used direct from the stock solution for atomic absorption spectroscopy (Fluka). Algae cultivation and preparation—Axenic cultures of P. tricornutum were given support by the Spanish Bank of Algae at Taliarte (Gran Canaria, Spain). The main culture of Fig. 1. Growth rates (cell division) of diatoms exposed to iron and copper. Phaeodactylum tricornutum phenols 145 diatom was kept in a clean culture chamber (Friocell FC111) using f/2 media (Guillard 1975) at constant temperature (24uC) with permanent illumination at 8000 lux. Experimental cultures were carried out in seawater with f/2 nutrients (Guillard 1975). Seawater was ultraviolet treated (off the coast of Gran Canaria) and filtered by 0.45 mm. The f/2 nutrients ([NO{ 3]5883 mmol L 21 , [HPO2{ 4]529.3 mmol L 21 , [SiO2{ 3]5142 mmol L 21 )were added to the seawater. Under these conditions, P. tricornutum, with an initial cell density of 2 310 7 cells L 21 , reached the stationary phase after 8 d of growth (Fig. 1). This time of culture was used throughout to collect the samples in the same conditions under different conditions of metal concentrations. The experimental cultures were produced under four different conditions: (1) at the reference culture, in seawater with f/2 nutrients. The effect of copper was tested (2) with 315 nmol L 21 of Cu(II) and (3) 790 nmol L 21 of Cu(II). The effect of iron (4) was studied with 900 nmol L 21 of Fe(III). All of the experiments were triplicated. The cell concentration was counted daily using a light microscope (Microbiotest) with a hemocytometer and measuring the absorbance (640 nm) with a spectrophotometer (USB4000). Once the culture reached the stationary phase, the cell was collected by filtration in two steps: gravity (1.2 mm) and vacuum (0.1 mm). This treatment avoids rupturing cells. Both cells and enriched seawater were used in this study for the analysis of the phenolic compounds. Preparation of P. tricornutum diatom extracts for isolation and quantification of the phenolic compounds— The algae were freeze-dried and 1 g of the dry material was extracted by stirring with methanol (25 mL) for 1 h. After centrifugation at 3500 revolutions per minute (rpm) for 30 min, the supernatant was collected and evaporated. The dry residue (100 mg) and 4 mL of acetone : hexane (1 : 4) were mixed and stirred for 10 min. The supernatant was discarded and the residue was mixed and homogenized with 5 mL of methanol using a vortex (10 min). After centrifugation at 3000 rpm, the supernatant was separated and the residue was extracted twice with 5 mL of methanol. All the methanolic fractions were collected and evaporated in a rotary vacuum evaporator (Eppendorf, Concentrator plus) at 30uC (6800 rpm). The residues were resolved in 5 mL of HCl (1 mol L 21 ) and the samples were hydrolyzed at room temperature, 3800 rpm for 30 min. The hydrolysates were purified using solid-phase extraction (SPE). Solid-phase extraction—The cartridge Chromabon Easy (Macherey-Nagel, 500 mg, particle size 93 mm) was preconditioned by successive elution with 3 mL of methanol and 3 mL of deionized water. The hydrolysates (5 mL) were passed through the cartridges at a flow of 2.5 mL min 21 .The cartridge was then rinsed using 2%acetic acid in a 5% methanol solution (Onofrejova´ et al. 2010). The retained analytes were subsequently eluted using aqueous methanol solutions (5%,10%,and20%methanol, 2 mL for each). Prior to the analyses, the fractions were evaporated to dryness in a rotary vacuum evaporator at 25uC, 3500 rpm, resolved in 500 mL of methanol, filtered through a 45 mm nylon syringe filter, and injected directly into the HPLC system. Seawater enriched with diatom exudates (2.5 L) were passed through the conditioned cartridges (as described above) at a flow of 2.5 mL min 21 (,800 mL per cartridge, using three cartridges), following the same elution procedure described above for each cartridge. The fractions from the three cartridges were collected, evaporated as above, and the residue resolved in 500 mL of methanol, and then filtered to be injected into the HPLC system. Quantification of the phenolic compounds by RP-HPLC— Chromatographic analysis was performed on a Varian ProStar 210 system, equipped with a vacuum degasser, a binary pump, a thermostatted column compartment, and a diode array detector, connected to a ChemStation software. A reverse phase Pursuit XRs C18 (250 mm 34.6 mm, 5 mm) column and a Pursuit XRs C18 (10 mm 34.6 mm, 5 mm) guard column (Varian) with a gradient system involving twomobilephaseswereused.Theflowratewas 1.0 mL min 21 and the injection volume was 60 mL of crude extracts. The system operated at 27uC. The phenolic compounds gallic acid, protocatechuic acid, catechin, vanillic acid, epicatechin and syringic acid, chlorogenic acid, gentisic acid, caffeic acid, p-coumaric acid and ferulic acid, rutin, myricetin, and quercetin were quantified in line with previously reported methods (Lo´pez et al. 2011). In brief, the eluent A was Milli-Q water with 0.1%formic acid and eluent B was methanol. The elution conditions applied were 0–5 min, 20%B isocratic; 5–30 min, linear gradient from 20%to 60%B; 30–35 min, 60%B isocratic; 35–40 min, linear gradient from 60%to 20%B; and, finally, washing and reconditioning of the column. Each standard was individually tested to determine its retention times (RT) as follows: gallic acid (RT: 5.3 min), protocatechuic (RT: 10.0 min), catechin (RT: 12.7 min), chlorogenic acid (RT: 14.9 min), gentisic acid (RT: 17.1 min), vanillic acid (RT: 17.7 min), epicatechin (RT: 17.9 min), caffeic acid (17.9 min), syringic acid (RT: 18.9 min), coumaric acid (RT: 23.4 min), rutin (RT: 28.1 min), ferulic acid (RT: 24.3 min), myricetin (RT: 30.6 min), and quercetin (RT: 34.6 min) were well resolved. Simultaneous monitoring was set at 270 nm (gallic acid, protocatechuic acid, (+)-catechin, vanillic acid, (2)-epicatechin, and syringic acid), 324 nm (chlorogenic acid, gentisic acid, caffeic acid, p-coumaric acid, and ferulic acid), and 373 nm (rutin, myricetin, and quercetin) for quantification. Limits of detection (LODs) and limits of quantification (LOQs) were estimated from signal-to-noise ratio of the individual peaks, assuming a minimum detectable signal-tonoise level of 3 and 10, respectively. The LODs were found to be in the range of 1.59 310 23 nmol mL 21 to 0.52 nmol mL 21 and the LOQs were observed in the range of 4.25 310 23 nmol mL 21 to 0.17 nmol mL 21 . SPE accuracy and precision—The presence of polyphenols in the extracts was confirmed by comparison of the retention times and overlapping ultraviolet spectra with those of standard compounds. The precision of the SPE-RPHPLC method was estimated by measuring the repeatability and the relative standard deviations of 10 replicate samples 146 Rico et al. containing the following concentrations: 146.9 mmol L 21 for (+)-catechin; 117.6 mmol L 21 for gallic acid, 129.8 mmol L 21 for protocatechuic acid, 118.9 mmol L 21 for vanillic acid, 100.9 mmol L 21 for syringic acid, 129.8 mmol L 21 for gentisic acid, 32.7 mmol L 21 for rutin, and 73.4 mmol L 21 for (2)-epicatechin; 41.3 mmol L 21 for chlorogenic acid, 91.4 mmol L 21 for p-coumaric acid, 77.2 mmol L 21 for ferulic acid, 47.1 mmol L 21 for myricetin, 55.5 mmol L 21 for caffeic acid, and 33.1 mmol L 21 for quercetin. Relative standard deviation (RSD) values ranged from 2.1%to 6.4%. The accuracy was expressed as the recovery of the standards and was found to be in the range of 87.3–107.5%. RSD values and recoveries are shown in Table 1. Preparation of P. tricornutum diatom extracts for DPPH assay—The algae were freeze-dried and 1 g of the dry material was extracted by stirring with methanol (25 mL) for 1 h. After centrifugation at 3500 rpm for 30 min, the supernatant was collected and evaporated. The dry residue (100 mg) was extracted for 1 h at room temperature with 3 mL of methanol by mixing with a magnetic stirrer. Each extract was centrifuged at 7500 rpm for 20 min, and the supernatant collected and filtered through 0.45 mm filter paper. Free radical scavenging activity on DPPH—The reducing ability of antioxidants on DPPH radical was evaluated by measuring the loss of DPPH color at 515 nm after reaction with the test extracts (Bondet et al. 1997). The sample solution (100 mL) was rapidly mixed with 1 mL of a solution of 0.1 mmol L 21 DPPH. After 25 min incubation time in the dark at ambient temperature (23uC), the decline in absorbance (Abs) was measured against a methanol blank. The inhibition percentage values, expressed in terms of radical scavenging activity (RSA), were calculated by the equation: RSA~100|1{½Abs in the preence of sampleðÞ =Abs in the absence of sampleðÞ ð1Þ Results Effects of copper and iron on the growth of P. tricornutum—The cultures of P. tricornutum were produced at three metal concentrations in order to stimulate the production of exudates. To this effect, the diatom was harvested in seawater with f/2 nutrients (control seawater) as the reference growth (Fig. 1) in close concordance with recent studies (Vasconcelos and Leal 2008; Gonza´lez et al. 2012). At these conditions, the cell density increased from 2 310 7 cells L 21 to 5.9 310 8 cells L 21 after 8 d of culture. The slope for the exponential phase was 8.9 310 7 cells L 21 d 21 . The growth rate during the 8 d of culture was 7.6 310 7 cells L 21 d 21 . The growth of P. tricornutum was also carried out in the presence of 315 nmol L 21 of Cu(II) (Fig. 1). The cell density increased to 4.7 310 8 cells L 21 , with a growth rate of 2.6 310 7 cells L 21 d 21 over the 8 d of culture, meaning that the P. tricornutum cell density decreased 20%with respect to the reference culture. In addition, during the initial 5 d of culture, the growth curve showed a linear relationship with time, where the slope was 2.26 310 7 cells L 21 d 21 . Between the sixth and eighth days of culture, the cell density reached a slope of 10.4 310 7 cells L 21 d 21 , comparable to the reference culture in the absence of copper. The third culture of P. tricornutum was carried out under conditions of 790 nmol L 21 of Cu(II) (Fig. 1). This concentration allows for the determination of the polyphenolic profile under high copper stress. The growth curve showed that the cell density increased to 3.1 310 8 cells L 21 . The cell concentration decreased 47.5%with respect to the reference culture. In this experiment, a similar growth pattern was found as in the study at 315 nmol L 21 of Cu(II). The growth slope was only 0.53 310 7 cells L 21 d 21 through to the fifth day of culture and increased to 8.01 3 10 7 cells L 21 d 21 over the following days. The iron effect was studied at 900 nmol L 21 of Fe(III) added to the culture of P. tricornutum. Under these conditions, the cell density reached a maximum concentration of 2.70 310 9 cells L 21 , one order of magnitude higher than the reference culture. The growth slope in the exponential phase was 46.9 310 7 cells L 21 d 21 , and the growth rate was 5.4 310 8 cells L 21 d 21 for the 8 d of culture (the cell density increased 471%with respect to the reference culture). Polyphenolic profiles—The polyphenolic profile of P. tricornutum was determined both in the extracts of cells collected from the cultures and in seawater enriched with exudates. Tables 2 and 3 show all the polyphenols quantified in this work. The proposed polyphenols were identified in the extracts of cells, except for gallic acid, which was only detected in the iron enrichment experiment. In addition, in the seawater enriched with exudates, only quercetin was not detected. The phenolic compound content in cell extracts of P. tricornutum exposed to copper was strongly affected by the metal concentration (Table 2). As compared to the control, Table 1. Solid-phase extraction recoveries and relative standard deviations (RSD) (n510). Compound Recovery* (%) RSD (%) Gallic acid 9461 4.2 Protocatechuic acid 95.160.7 2.6 Catechin 9461 2.6 Vanillic acid 10861.0 3.9 Epicatechin 105.360.8 3.2 Syringic acid 96.860.8 3.1 Chlorogenic acid 93.660.5 2.9 Gentisic acid 91.160.5 2.1 Caffeic acid 106.560.8 6.4 Coumaric acid 9761.0 5.6 Ferulic acid 93.460.8 3.9 Rutin 11261 3.4 Myricetin 89.460.6 3.0 Quercetin 87.360.6 4.5 * Means 6standard deviation of 10 measurements. Phaeodactylum tricornutum phenols 147 the amount of most of the identified phenolic compounds increased slightly when the copper concentration was 315 nmol L 21 (growth rate 80%). Significantly, the greatest amount (around double with respect to the control) of all the identified phenolic compounds was found when the diatom P. tricornutum was exposed to copper concentration of 790 nmol L 21 (Table 2). Similar findings were given in the seawater samples enriched with exudates, with double the sum of all the identified phenolic compounds exuded per cell in the culture enriched with a copper concentration of 790 nmol L 21 as opposed to those in the control (Table 3). The concentration of polyphenols increased in the culture seawater enriched with copper at concentration 790 nmol L 21 to 39 nmol L 21 . In the iron enrichment experiments, the amount of the quantified phenolic compounds per cell increased under these conditions of high cell growth (471%). The sum of all the identified compounds was 1208 nmol of polyphenols, 1.3 times higher than the control values of 897 nmol of polyphenols (Table 2). In these experiments, myricetin and (+)-catechin were the predominant phenolic compounds, and gallic acid was detected only in the diatoms exposed to iron. The contents of polyphenols such as rutin, myricetin, and quercetin increased by 365%,273%, and 579%, respectively, whereas in the copper enrichment experiment ([Cu(II)] 5790 nmol L 21 ) the increases were inferior (249%, 142%, and 79%, respectively). However, gentisic acid decreased in the presence of iron and increased when Table 3. Phenolic compounds contents in seawater enriched with exudates of diatom P. tricornutum under high iron and copper concentrations. Phenolic compound Control [Fe(III)] 900 nmol L 21 [Cu(II)] 315 nmol L 21 [Cu(II)] 790 nmol L 21 Gallic acid* 2.360.1 10.360.5 0.5860.07 4.860.5 Protocatechuic acid* 23.260.6 5.960.5 19.760.9 4362 Catechin* 125611 11.660.1 11264 283615 Vanillic acid* 1961 4.360.3 18.360.9 3662 Epicatechin* 55.960.8 11.760.0 6667 10763 Syringic acid* 32.060.1 7.860.4 24625862 Chlorogenic acid* 18.260.7 3.560.0 2462 35.260.8 Gentisic acid* 48.360.7 6.860.2 61659367 Caffeic acid* 11.460.1 2.460.2 16.460.8 21.460.6 Coumaric acid* 14.860.3 2.960.2 19.660.7 27.360.6 Ferulic acid* 21.060.4 4.160.2 27634164 Rutin* 9.060.3 6.560.6 11.460.6 16.860.6 Miricetin* 32.460.9 216259636062 Quercetin* nd{5.560.2 2.260.2 1.960.1 Sum* 413617 10465 461630 828640 Sum{2461286230623962 * nmol of phenolic compound per cell 310 10 6standard deviation of two measurements. {nd, not detected. {nmol L 21 6standard deviation of two measurements. Table 2. PhenoliccompoundscontentsinextractsofdiatomPhaeodactylum tricornutum exposed to high iron and copper concentrations. Phenolic compound Control [Fe(III)] 900 nmol L 21 [Cu(II)] 315 nmol L 21 [Cu(II)] 790 nmol L 21 Gallic acid* nd{4364nd nd Protocatechuic acid* 46.960.4 65645963 144610 Catechin* 23663 27761 20864 670642 Vanillic acid* 53.860.8 596483669562 Epicatechin* 12365 12966 174613 234615 Syringic acid* 656476636762 11863 Chlorogenic acid* 506247626264 10662 Gentisic acid* 138647465 17468 278610 Caffeic acid* 3561 35.160.5 50627561 Coumaric acid* 38.260.2 38.160.6 60628162 Ferulic acid* 37.060.9 60657969 122610 Rutin* 12.960.3 606320624564 Myricetin* 56.160.5 20969 11969 13666 Quercetin* 5.360.3 3662 3.160.4 9.560.6 Sum{897622 1208649 1158664 21146108 * nmol of phenolic compound per cell 310 10 6standard deviation of two measurements. {nd, not detected. {nmol of total phenolic compounds per cell 310 10 6standard deviation of two measurements. 148 Rico et al. the diatom was exposed to copper, whereas the rest of the polyphenols showed major increases when the diatoms were exposed to a copper concentration of 790 nmol L 21 . Quantification of phenolic compounds in the seawater samples enriched with exudates from the iron enrichment experiments showed that the amount of single phenolic compounds exuded per cell declined dramatically as compared to the control, with the sum of all the identified compounds exuded per cell 4 times less than those in the control (Table 3). However, the concentration of the polyphenols in nmol L 21 increased in the culture seawater enriched with iron from 24 nmol L 21 (control) to 28 nmol L 21 due to the large number of cells. Antioxidant activities of the diatom extracts—Figure 2 shows the relative antioxidant efficiency of the diatom extracts against the DPPH radical. Antioxidants suppressed the absorbance at 515 nm on a time scale dependent on the antioxidant activity of the extracts. As may be observed, the extracts derived from the diatom exposed to copper showed the highest radical scavenging activities (16.2%and 11.9%) followed by the extract derived from cells under iron enrichment conditions (8.1%) and the control (7.6%). Discussion Copper and iron stress affect growth and the phenolic contents of the marine diatom P. tricornutum, but not in the same way. The behavior of P. tricornutum under copper additions suggested that the diatoms metabolically produced exudates capable of complexing the copper present in the solution, thereby decreasing the toxic effect. Once the conditions were optimal, the diatom growth began. The effect of copper on diatom growth concords well with results published in previous reports with respect to the effect of copper toxicity on microalgae growth. Kagalou et al. (2002) reported that Cu(II) concentrations of 15.7 mmol L 21 had significant effects on the growth rate of the microalgae Isochrysis galbana,whereaslower concentrations (157 and 1570 nmol L 21 ) produced less decline in algal growth. Franklin et al. (2002) concluded that the copper concentrations required to inhibit growth rate by 50%increased from 72.4 to 252 nmol L 21 for the tropical freshwater alga Chlorella sp. and from 104 to 268 nmol L 21 for the temperate species Selenastrum capricornutum as the initial cell density increased from 10 5 to 10 8 cells L 21 . The initial cell density is a very important parameter in toxicity tests carried out using microalgae. Moreno-Garrido et al. (2000) reported copper growth inhibition tests on four marine microalgal species including P. tricornutum. Diatoms were cultivated in artificial seawater enriched with a modification of f/2 medium, lacking ethylenediaminetetraacetic acid, which decreases the toxicity of heavy metals. In these conditions, MorenoGarrido et al. (2000) observed EC 50 values (representing the concentration of copper that caused a 50%reduction in the cell division rate compared with controls and calculated by semilogarithmic plotting of average inhibition data fits to straight line) for P. tricornutum of 154, 542, and 551 nmol L 21 for the initial cellular densities 10 6 ,53 10 6 ,and10 7 cells L 21 , respectively (after 72 h exposure to copper). Franklin et al. (2001) reported that copper had an inhibitory effect on the cell division rate of several algal species after 48 h and 72 h exposure to copper (initial cell density 2 310 7 to 4 310 7 cells L 21 and using filtersterilized seawater supplemented with [NO{ 3]5242 mmol L 21 and [PO3{ 4]515.8 mmol L 21 as a culture medium to maintain exponential growth over 72 h). In said study, the 48 h and 72 h EC 50 values were 142 647 and 158 6 63 nmol L 21 , respectively, and complete growth inhibition was observed at 11.8 mmol L 21 . The cell light scatter properties of P. tricornutum depended on the cell size and intracellular granularity. These properties were found to be Fig. 2. Relative activity of the extracts derived from diatom Phaeodactylum tricornutum to scavenge the stable radical DPPH under different conditions: control seawater, [Fe(III)] 5900 nmol L 21 , Cu(a) was [Cu(II)] 5315 nmol L 21 , and Cu(b) was [Cu(II)] 5 790 nmol L 21 . RSD values represented means of triplicate determinations 6standard deviation. Phaeodactylum tricornutum phenols 149 a useful indicator of chronic copper toxicity to the marine alga P. tricornutum. Franklin et al. (2001) found that copper caused an increase in cell size after 24 h exposure, with 50% or more of the cells larger than the controls at 3.15 mmol L 21 , whereas similar increases in cell size were observed after 48 h and 72 h at 157.5 nmol L 21 , with an EC 50 value of 126 6 47 nmol L 21 . Similar changes in cell size and granularity were also reflected in changes in side-angle light scatter with an EC 50 value of 189 nmol L 21 (after 48 h and 72 h exposure to copper). The diatoms P. tricornutum exposed to lower doses of copper in said study than those used by the present researchers at the same initial cell density, showed changes in cell size, granularity, and growth rate, considered to be indicators of copper toxicity. P. tricornutum showed the highest growth rate at iron concentration 900 nmol L 21 . Iron is well known as a fertilizer and as an essential micronutrient participating in essential metabolism processes such as photosynthesis, respiration, and nitrogen fixation (Stumm and Morgan 1996). Morel et al. (2008) have demonstrated that unchelated Fe is highly available for uptake but that chelated Fe is necessary in order to explain the total uptake system. There is even evidence of direct internalization of siderophores by microorganisms using reductive mechanisms with varying degrees of efficiency (Hopkinson and Morel 2009). Our results align well with Kudo et al. (2000), who reported the combined effect of iron nutrition and temperature on growth (cell division) of the diatom P. tricornutum, concluding that the growth rate of cells cultivated with Fe(III) 2.0 mmol L 21 was twice as fast as the growth rate of diatoms cultivated with lower Fe(III) concentration (2.0 nmol L 21 )at20uC. In this study, increased phenolic compound content has been reported in P. tricornutum exposed to increasing doses of Cu(II). At the highest copper concentration (790 nmol L 21 ), the phenolic content per cell was 2.4 times higher than in the control. In addition, the concentration of polyphenols increased in the culture seawater enriched with copper at concentration 790 nmol L 21 to 39 nmol L 21 (Table 3), despite the number of cells being much lower (52.5%), indicating that cells exposed to copper must excrete a larger amount of polyphenols. The diatom must make an extra metabolic effort in order to live in these levels of copper concentrations and must produce relevant amounts of polyphenols to slow down the toxicity of the copper in the solution, acting as a protective mechanism. This role has also been described for soil bacteria (Pseudomonas aureofaciens) (Gonza´lez et al. 2010) and plants that present mechanisms of detoxification, such as the exudation of phenols that act as chelate towards heavy metals to protect cells from induced oxidative damage (Jung et al. 2003). Increasing phenolic compounds may also contribute to enhancing the ability to cope with osmotic stress by binding metal ion to wallassociated polyphenols, countering metal toxicity at the cell surface (Suresh and Subramanyam 1998; Jung et al. 2003). Plant intracellular detoxification mechanisms may involve specific scavenging of reactive oxygen species through the action of enzymes and a wide array of metabolites, including phenolic components or direct chelate formation by polyphenols (Quideau et al. 2011). Wells et al. (2005) reported a synergistic link between the release of the ironcomplexing ligand domoic acid and the limitation by iron and copper in toxigenic diatoms Pseudo-nitzschia spp. In said study, copper was found to be a key factor facilitating the success of these diatoms in iron-deficient waters, by inducing a high-affinity iron uptake. Iron limitation is the primary trigger for domoic acid production, which doubled under iron deficiency and increased by one order of magnitude under copper-limiting conditions. However, similar increases in domoic acid production were detected under conditions of toxic copper concentration, presumed to reduce copper toxicity. The findings here evidenced increased phenolic compounds per cell under iron-rich media conditions ([Fe(III)] 5900 nmol L 21 ) as compared to the control. However, the amounts of phenolic compounds exuded per cell decreased drastically, resulting four times lower than the control. This decline in excreted polyphenol concentrations per cell may have its explanation in the high cell densities in the culture that help to minimize the stress suffered per cell, making the metabolic effort needed to uptake iron to decrease. P. tricornutum produced and exuded phenolic compounds mixtures with different phenolic profiles depending on the culture conditions. The extracts derived from the diatom exposed to copper showed the highest radical scavenging activities (16.2%and 11.9%). The phenolic profiles for these more active extracts were very similar. However, the diatoms exposed to iron showed different phenolic profiles that may vary the antioxidant potential. The antioxidant activity is not only dependent on the concentration of antioxidants, but also on the structure (Wright et al. 2001). The antioxidant activity is also dependent on the interaction among the antioxidants (phenolic compounds may act synergistically or antagonistically) (Jacobo-Velazquez and Cisneros-Zevallos 2009). Amoros et al. (1992) demonstrated the synergistic effect of binary flavone–flavonol combinations against herpes simplex virus type 1 in cell culture. Gao et al. (2011) reported that four phenolic compounds (vanillic acid, protocatechuic acid, ferulic acid, and caffeic acid) exerted additive and synergistic inhibition effects on the growth of Microcystis aeruginosa depending on the mix ratios. Tafesh et al. (2011) reported that hydroxytyrosol at 400 mgmL 21 caused growth inhibition of the four bacterial isolate gram-positive (Streptococcus pyogenes and Staphylococcus aureus) together with the gram-negative (Escherichia coli and Klebsiella pneumoniae), whereas gallic acid at 200 and 400 mgmL 21 inhibited the growth of S. aureus and S. pyogenes strains, respectively (no growth inhibition was observed for the gram-negative bacteria). However, the combination of both compound gallic acid and hydroxytyrosol at lower concentrations (100 and 200 mgmL 21 , respectively) caused a complete inhibition of the four bacterial strains. In the present study, significant differences in the phenolic profiles were found in P. tricornutum diatoms growing under iron fertilization conditions and under copper stress. These differences seem to be the response to the different needs of the diatoms when grown under different stress conditions: as compared to the control, the amount of gentisic acid decreased when the culture seawater was enriched with iron 150 Rico et al. and increased when the seawater was enriched with copper; cells under iron enrichment conditions revealed a higher content of quercetin, myricetin, and rutin than the other diatom extracts and gave no change for (2)-epicatechin or chlorogenic acid, which increased in diatoms exposed to copper (790 nmol L 21 ) (Table 2). As Gao et al. (2011) suggested, the profile of the phenolic compounds in a mixture and their mix ratios determine the joint action of the compounds, with either synergistic or additive effects. In addition, various different kinds of activities of the phenolic mixture may also depend on the mix ratios, where this mixture may be more active in complexing or reducing trace metals in solution. Therefore, further research is required to study the joint action of the phenolic compounds identified in cells exposed to multiple metal stresses and the influence of the mix ratio of said phenolic compounds on the kind and intensity of the mix activity. Polyphenols have attracted a great deal of attention recently, mainly focused on their role in preventing diseases produced as a result of oxidative stress (Dillard and German 2000; Dai and Mumper 2010). Dietary antioxidants from plants are believed to help prevent aging and many degenerative diseases, such as cardiovascular complaints and cancers, as a result of their radical scavenging activity (Virgili et al. 2003; Quideau et al. 2011). Therefore, there is considerable interest in the field of preventive medicine in the development of natural antioxidants obtained from botanical sources. Seaweeds are considered to be a rich source of antioxidants, and recent studies have suggested using microalgae as ‘‘health’’ foods (Chaco´n-Lee and Gonza´lezMarin˜o 2010). The results of this study confirm that P. tricornutum is a natural source of well-known antioxidant compounds (Silva et al. 2002) and afford more than sufficient arguments for researching the viability of the use of P. tricornutum diatom in the health and food industries in general, as well as in the pharmaceutical industry. Acknowledgments This study received financial support from the Project CTM2010-19517-MAR financed by the Ministerio de Economı ´a y Competitividad in Spain. Our thanks to the Spanish Bank of Algae (BEA) in Gran Canaria for providing algal strains. 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Complexation of iron (III) by natural organic ligands in the Northwest Atlantic Ocean by a competitive ligand equilibration method and a kinetic approach. Mar. Chem. 50: 159–177, doi:10.1016/0304-4203(95)00033-N Associate editor: Robert R. Bidigare Received: 14 June 2012 Accepted: 26 September 2012 Amended: 01 October 2012 152 Rico et al. enterobactin or desferrioxamine which form hexadentated complexes (Elhabiri et al., 2007). Catechol is a frequent functional group found in the catecholate siderophores and in polyphenols excreted by the phytoplankton (Rico et al., 2013). The catechol forms complexes with Fe(III) and subsequently decomposes to form Fe(II) in a two step redox process in seawater that is favored at pH values lower than 8 (Santana-Casiano et al., 2010). Therefore, the role of ligands that can simultaneously act as chelating agents for Fe(III) and iron reducing agents through intermolecular redox reactions should be considered. Iron in the marine environment exists in two different oxidation states, Fe(II) and Fe(III). In seawater, Fe(II) oxidizes to Fe(III), the thermodynamic stable form, through Eqs. (1)–(4) (Millero and Izaguirre, 1989). Fe(III) is less available for uptake of phytoplankton than Fe(II) because of its formation of ferric hydroxides and oxides and high stable ferric chelates, which are generally not directly bioavailable. In general, Fe(II) is much less strongly bound by organic ligands and does not form insoluble precipitates, and thus it is more bioavailable (Morel et al., 2008; Shaked et al., 2005; Shaked and Lis, 2012). However, Fe(III) can be reduced to Fe(II) by some specific organic compounds, depending on physical–chemical conditions, particularly pH, light and biological activity (Santana-Casiano et al., 2000, 2010; Strlic et al., 2002; Rose and Waite, 2003;Steigenberger et al., 2010; Shaked and Lis, 2012), and then oxidized back through Fenton type reactions (Eqs. (1)–(4)). In the presence of organic ligands (L), Eqs. ((5)–(9)) may take place and the generation of OH · from Eq. (3) may be strongly affected (Miller et al., 2013). Fe IIðÞþO2→Fe IIIðÞþO− 2ð1Þ Fe IIðÞþOÄ n− 2→Fe IIIðÞþH2O2ð2Þ ·- Fe IIðÞþH2O2→Fe IIIðÞþOHÄ nþOH−ð3Þ · Fe IIðÞþOHÄ n→Fe IIIðÞþOH− ð4Þ · Fe IIðÞþL¼Fe IIðÞLð5Þ Fe IIðÞLþO2→Fe IIIðÞLð6Þ Fe IIIðÞþL¼Fe IIIðÞLð7Þ Fe IIIðÞþL→Fe IIðÞþL0 ð8Þ Fe IIIðÞL→Fe IIðÞþL0 :ð9Þ In surface seawater, a small fraction of iron occurs as thermodynamically unstable Fe(II) and the reason is largely unknown (Roy et al., 2008) although it has been observed that the presence of organic ligands can regulate the Fe(II) oxidation (Roy and Wells, 2011). Moreover, the superoxide radical plays an important role in the biologically mediated Fe(III) reduction pathway (Rose et al., 2008). The aim of this study is to characterize the phenol compounds excreted by the diatom Phaeodactylum tricornutum (P. tricornutum) and their effect on the Fe(II) persistence in seawater. Two of the most abundant phenol exudates excreted by the diatom have been selected to study the possible reduction of Fe(III) to Fe(II), namely catechin and sinapic acid. P. tricornutum has been selected as a representative diatom species because it occupies a variety of habitats and it is one of the most abundant photosynthetic organismsin the marine environment. The diatoms are responsible for close to 40% of marine primary productivity (Falkowski et al., 1998). The P. tricornutum cells present adaptations to low-iron environments which include strategies to lower the cellular iron requirements and to enhance iron uptake (Lommer et al., 2012). Information about the organic compounds liberated by these algal species is very limited and a stronger effort must be made in order to characterize the composition of organic compounds that affect the speciation and behavior of iron in the ocean. 2. Experimental 2.1. Chemicals The stock solution of Fe(II) (4 × 10 −4 M) was prepared using ammonium iron(II) sulfate hexahydrate (Sigma), acidified at a pH of 2 with SuprapurHCl (Sigma) in NaCl(0.7 M).All solutionswereprepared with Milli-Q water and filtered by 0.1 μm. All chemicals used for the Fe(II) determination were trace analytical grade. Methanol was high performance liquid chromatography (HPLC) grade (Panreac). Formic and acetic acids (Merck) were the analytical quality reagents. Polyphenol standard gallic acid, protocatechuic acid, chlorogenic acid, (−) epicatechin, quercetin, myricetin, ferulic acid, p-coumaric acid, vanillic acid, syringic acid, (+) catechin, sinapic acid, quercitrin, kaempferol, and apigenin were purchased from Sigma-Aldrich Chemie (Steinheim, Germany); rutin and gentisic and caffeic acids were supplied by Merck (Darmstadt, Germany). The solid phase extraction (SPE) cartridges used were Chromabon with polar modified polystyrene divinylbenzene (500 mg) from Macherey-Nagel. 2.2. Culture of algae P. tricornutum was the phytoplankton species selected in order to study the exudates generated by the algae. This diatom was supplied by the Spanish Bank of Algae (BEA) of Taliarte (Gran Canaria). In order to obtain the exudates, the algae were cultured in seawater (filtered at 0.45 μm) by adding a modified f/2 nutrient media (Guillard, 1975). The seawater used in this study was collected off the coast of Gran Canaria. The seawater was enriched with nitrate, phosphate and silicate as for the f/2 media, but without addedchelators, metals or vitamins to minimize any change in the natural speciation (Re et al., 1999). The stock solutions of nutrients were sodium nitrate (Sigma) (0.88 M), potassium hydrogen phosphate (Sigma) (0.03 M) and sodium silicate (Sigma) (0.14 M).The concentration of each nutrient waskept constant at 8.82 × 10 −4 M, 2.93 × 10 −5 M and 1.42 × 10 −4 MforNO 3 − ,HPO 4 2− and SiO 3 2− , respectively. Cells were counted daily using a light microscope (Microbiotest, Inc.) with a hemacytometer and by measuring the absorbance (640 nm) with a spectrophotometer (USB2000). The initial cell concentration was 10 7 cell/L. The temperature was kept constant at 24 °C under permanent illumination (24 h) in a culture chamber (Friocell, FC111). The cultures reached the stationary phase with 4.98 × 10 8 cell/L after 8 days. Before each experiment, the culture was filtered by gravity with a 1.2 μmfilter in order to remove the cells and avoid cell rupture. The remaining solution, containing the exudates, was filtered by vacuum using a 0.1 μmfilter. All the filters used in this study were previously washed with HCl solution (10%) and kept in Milli-Q water for two hours before use. The control seawater was seawater enriched with the same f/2 nutrient concentrations and filtered at 0.1 μm. 2.3. Preparation of exudate extract from seawater Seawater and seawater enriched with diatom exudates (2.5 L) were passed through the pre-conditioned Chromabon Easy cartridges (successive elution with 3 mL of methanol and 3 mL deionized water) at a flow of 2.5 mL min −1 (800 mL per cartridge approximately, using 3 cartridges). In the elution procedure for each cartridge 2% acetic acid in a 5% methanol solution was used. The retained analytes were eluted using aqueous methanol solutions (5%, 10%, and 20% methanol, 2 mL 11J.M. Santana-Casiano et al. / Marine Chemistry 158 (2014) 10–16 for each). Prior tothe analyses, thefractions were evaporated to dryness in a rotary vacuum evaporator at 25 °C, 3500 rpm, dissolved in 500 μL of methanol, filtered through a 0.45 μm nylon syringe filter and injected directly into the HPLC system. 2.4. Determination of phenolic compounds Chromatographic analysis was performed using a Varian ProStar 210 system, equipped with a vacuum degasser, a binary pump, a thermostatted column compartment and a diode array detector connected to a ChemStation software. A reverse phase Pursuit XRs C18 (250 mm × 4.6 mm, 5 μm) column and a Pursuit XRs C18 (10 mm × 4.6 mm, 5 μm) guard column (Varian) were used for the separation processes. A gradient system was used involving two mobile phases. Eluent A was water with 0.1% formic acid, and eluent B was methanol. The flow rate was 1.0 ml/min. 60 μl of exudates extract were injected (rheodyne injector). The temperature was maintained at 27 °C. The peaks of polyphenols were identified by comparing their retention times (RT) and by overlaying UV spectra with those of standard compounds. To quantify the compounds sinapic acid, quercitrin, kaempferol and apigenin in the extracts the wavelength was fixed at 254 nm (López et al., 2013). The relative standard deviations (RSD) of ten replicate samples ranged from 7.40% to 10.33%. The accuracy, expressed astherecovery of the standard compounds, was in the range of 79.2%–102.6%. The limits of detection were found to be in the range of 8.12 × 10 −2 nmol mL −1 to 0.47 nmol mL −1 and the limits of quantification were observed in the range of 0.27 nmol mL −1 to 1.58 nmol mL −1 . The phenolic compounds gallic acid, protocatechuic acid, catechin, vanillic acid, epicatechin, syringic acid, chlorogenic acid, gentisic acid, caffeic acid, p-coumaric acid and ferulic acid, rutin, myricetin, and quercetin were quantified using previously reported methods (López et al., 2011; Rico et al., 2013). The RSD values ranged from 2.1% to 6.4%. The recovery was found to be in the range of 87.3%–108%. The limits of detection were found to be in the range of 1.59 × 10 −3 nmol mL −1 to 0.52 nmol mL −1 and the limits of quantification were observed in the range of 4.25 × 10 −3 nmol mL −1 to 0.17 nmol mL −1 . 2.5. Fe(II) oxidation experiments The Fe(II) oxidation rate was studied in the control seawater (SW n ) and in the seawater enriched with exudates (SW nE ) in air saturated conditions, by bubbling the solution with pure air for 1 h, prior to and during the experiments. The samples were stirred at 120 rpm with a teflon-coated magnetic stirrer. The pH (free scale and at 25 °C) was adjusted to the desired value and in order to keep the pH constant during the experiment to ±0.01, small additions of suprapur HCl 0.1 M were done using an automatic titration system (Titrino 719S, Metrohm). The studies were carried out in darkness in a glass thermostat vessel (250 mL) at a constant temperature of 25 ± 0.02 °C using an AG-2 ™ bath. The studies were done in triplicate and the average results are shown. The addition of the Fe(II) (25 nM) to the sample corresponds to the zero time of the reaction. The Fe(II) concentration was determined by UV–vis spectroscopy using the ferrozine method (Violler et al., 2000). The ferrozine and Fe(II) formed a peak at 562 nm. Every 30 s, 10 mL of the sample was added to a 25 mL glass flask containing ferrozine (50 μLof0.01M), acetate buffer (2 mL, pH 5.5) and NaF (50 μL of 7.1 × 10 −4 M) (González-Dávila et al., 2005). A 5 m long waveguide capillary flow cell (World Precision Instruments ™ ) was connected to the UV–vis detector USB2000 (Ocean Optics ™ ) and used to measure Fe(II) at nanomolar concentrations. The light used was a halogen light source (HL-2000-FHSA from Mikropack). The capillary flow cell and the detector were connected using optical fiber. The spectra were recorded using the OOIBase32 software by Ocean Optics. The sample was introduced into the column using a peristaltic pump (EXPETEC Perimax 12) with a flux of 1 mL/min. The Fe(II) oxidation rate equation in the presence of oxygen is defined by the equation (Santana-Casiano et al., 2005) dFeIIðÞ½ dt ¼−kapp Fe IIðÞ½O2 ½:ð10Þ Under airsaturated conditions, theplot of ln[Fe(II)]versustimegave a straight line and from its slope, the pseudo-first order rate constant is obtained according to the equation: dFeIIðÞ½ dt ¼−k0Fe IIðÞ½ ð11Þ where k′=k app [O 2 ]. 2.6. Fe(II) regeneration studies A 200 nM concentration of Fe was used in 100 ml of solution (NaCl or seawater) in a 200 ml thermostatted vessel controlled to 25 ± 0.02 °C. The experiments were done in a cover cell in order to avoid light effects and photooxidation reactions. The solution was initially aerated with air for two hours under oxygen saturation conditions in order to fully oxidize the added Fe(II) solution. After that time, 10 −3 mol L −1 ferrozine (FZ) was added. Fe(II) was not detected in the absence of the phenolic compounds. A blank was done for the solution containing the formed Fe(III) and the FZ. In order to keep a similar proportion between iron (Boye et al., 2006) and phenol compounds to that in seawater (Table 1), 1 μmol L −1 of each of the organic compounds (catechin or sinapic acid) was added to the solution and this was considered the time zero for the reaction. Some experiments were also done by adding Fe(III) directly instead of Fe(II) and no differences were observed. When both Fe(III) and catechin or sinapic acid were present in the solution, the Fe(II)–Ferrrozine complex was followed by UV–VIS spectroscopy at 562 nm. The measured sample was discharged after the analyses. The FZ method for the Fe(II) analysis was tested in the presence of both catechin and sinapic acid at pH 8.0 and 7.5 in NaCl and seawater. When the catechin was added, the spectrum showed a slight change with a maximum peak of absorbance at 450 nm, but did not interfere in the Fe(II)–FZ complex signal. In the presence of sinapic acid no interference in the Fe(II)–FZ complex signal was observed. Studies for Table 1 Concentrationsoftheeighteenindividualphenoliccompounds determinedfromseawater with nutrients at f/2 concentrations (SW n ) and with the presence of exudates from Phaeodactylum tricornutum after 8 days of culture (SW nE ). Compound SW n nM/L SW nE nM/L SW nE –SW n nM/L Gallic acid 0 0.13 0.13 Protocatechuic acid 0.81 1.37 0.56 Catechin 1.34 6.91 5.57 Vanillic acid 0.22 1.14 0.92 Epicatechin 0 3.08 3.08 Syringic acid 0 1.88 1.88 Chlorogenic acid 0 1.07 1.07 Gentisic acid 0 2.84 2.84 Caffeic acid 0 0.67 0.67 Coumaric acid 0.39 0.87 0.48 Ferulic acid 0.46 1.24 0.78 Rutin 0.04 0.49 0.45 Myricetin 1.03 1.90 0.87 Quercetin 0 0 0 Sinapic acid 2.76 7.30 4.54 Quercitrin 0 3.58 3.58 Kaempferol 1.07 4.25 3.18 Apigenin 0 4.30 4.3 TOTAL 8.12 43.02 34.9 12 J.M. Santana-Casiano et al. / Marine Chemistry 158 (2014) 10–16 sinapic acid were also done at pH 7.5 in artificial seawater (Millero, 2006) and there was no interference. 3. Results 3.1. Oxidation of Fe(II) in the presence of phytoplankton exudates The oxidation kinetic of 25 nM Fe(II) was studied in both seawater (SW n ) and seawater enriched with exudates (SW nE ), under oxygen saturated conditions, at two different pH values, 8.0 and 7.5. When the exudates were present in the solution, the oxidation rates of Fe(II) decreased at the two studied pH values, as is observed in Fig. 1,indicating that the presence of organic compounds excreted bythe diatomsaffected the rate of oxidation of Fe(II) in the solution. At pH = 8.0, in the reference seawater, the apparent rate constant (M −1 s −1 ) was log k app (SW n ) = 1.24 ± 0.00 while in the presence of exudates log k app (SW nE ) was 1.09 ± 0.02. The Fe(II) oxidation rate constant decreased by 29%. At pH = 7.5 the same behavior was observed, changing from log k app (SW n )=0.70±0.00 to log k app (SW nE ) = 0.34 ± 0.02. At pH 7.5, the Fe(II) oxidation rate constant decreased by 56%. The decrease observed in the rate of oxidation of Fe(II) in the presence of exudates is due to the interaction of these organic compounds with iron. These exudates result from an undetermined and uncharacterized mixture of saccharides, amino acids, phenolic compounds and other organic compounds that may react with iron changing its speciation and affecting the redox chemistry. 3.2. Phenol identification and quantification In order to elucidate the effects of the organic compounds present in the medium on the kinetics of oxidation of Fe(II), we focused this study on the identification, characterization and quantification of selected phenols exudated by the diatom. Eighteen polyphenols were considered and were resolved and identified in both extracts of SW n and SW nE .The total concentration of the polyphenols in each extract was quantified. In the seawater extract (SW n ) a total polyphenol concentration of 8 nM/L was determined, and 9 of the 18 analyzed polyphenols were found (Table 1). The vanillic acid, coumaric acid, ferulic acid, and rutin polyphenols were at concentrations below 0.5 nM/L. The most abundant polyphenols were the sinapic acid (2.8 nM/L), catechin (1.3 nM/L), myricetin (1.0 nM/L), kaempherol (1.0 nM/L) and protocatechuic acid (0.8 nM/L). The proportion of those individual compounds and dissolved Fe(II) in an oceanic seawater sample (0.1–0.2 nM, Boye et al., 2006) would range from 1:1 to 10:1. In the extract of seawater with exudates (SW nE ), a total concentration of 43 nM/L was obtained. In this extract 17 of the 18 polyphenols analyzed were found, the most important being the sinapic acid (7.3 nM/L), catechin (6.9 nM/L), kaempherol (4.3 nM/L), quercitrin (3.6 nM/L) and epicatechin (3.1 nM/L). Quercentin was not present in either SW n or SW nE . However, the quercitrin that was not present in SW n was found at a relatively high concentration in the SW nE , as was the case for the apigenin and epicatechin (Table 1). In the seawater enriched with exudates, the proportion of each individual phenolic compound and dissolved Fe(II) increased to reach ratios between 1:1 and 40:1. Table 1 shows the concentration of the polyphenols released by P. tricornutum after 8 days of culture. Taking into account the ability of some phenolic compounds to reduce Fe(III) (Mira et al., 2002; Santana-Casiano et al., 2010), the two major phenols released by the P. tricornutum with different characteristics, catechin and sinapic acid (Fig. 2) were selected for further experimentation. 3.3. Catechin Catechin is a flavonoid type compound with a catechol group in the B-ring, a resorcinol group in the A-ring and a hydroxyl group at position 3in theC-ring(Fig. 2). The reduction of Fe(III) to Fe(II) in the presence of catechin (initial Fe(III):catechin ratio, 1:5) in 0.7 M NaCl-2 mM NaHCO 3 wasfollowedbyaddingFZtotheFe(III)-catechin solutionatthreedifferent pH values 8.0, 7.5 and 6.0. The presence of the Fe(II) chelator FZ in the media acted as a sink for the released Fe(II), thereby preventing any back reactions and allowing the determination of the direct reduction process. Fig. 3 showsthatFe(II)wasefficiently formed and this process was pH dependent. The reduction of Fe(III) followed a pseudo-first order kinetic with the rate constant (k′in s −1 ) increasing as pH decreases from log k′=−5.35 ± 0.07 at pH = 8.00 to log k′=−3.68 ± 0.13 at pH 6.0 (Table 2). In seawater, log k′changed from −6.15 ± 0.02 at pH = 8.00 to −3.79 ± 0.03 at pH 6.0. In seawater the Fe(III) reduction was lower than in NaCl–NaHCO 3 solution. In Table 2,valuesforlogk′in NaCl–NaHCO 3 solutions with 0.05 M of Mg 2+ and 0.01 M of Ca 2+ are also included. The addition of seawater levels of Mg 2+ and Ca 2+ contributed to decrease the Fe(III) reduction rates. 3.4. Sinapic acid Sinapic acid is a phenylpropanoid member, a cinnamic acid derivative, which possesses 3, 5-dimethoxyl and 4-hydroxyl substitutions in the phenyl group of the cinnamic acid (Fig. 2). The regeneration of Fe(II) from the reduction of Fe(III) in the presence of sinapic acid (initial Fe(III):sinapic acid ratio, 1:5) was studied at three different pH values, 8.0, 7.5 and 6.0 in 0.7 M NaCl–2mMNaHCO 3 and in seawater. This process was also pH dependent (Fig. 3). Studies with both artificial seawater and NaCl solution with added Mg 2+ were also done at pH 7.5 in order to elucidate the effect caused by this major ion in seawater. In NaCl–NaHCO 3 solution the reduction of Fe(III) followed a pseudo-first order kinetic with rate constants increasing as pH decreased from log k′=−5.86 ± 0.08 at pH = 8.0 to log k′= −3.83 ± 0.02 at pH 6.0 (Table 3). In seawater, log k′changed from −6.57 ± 0.04 at pH 8.0 to −3.90 ± 0.10 at pH 6.0. In seawater, theregenerationofFe(II)wasalsolowerthaninNaCl–NaHCO 3 solution due to the presence of the major ions. In Table 3,valuesforlogk′ in NaCl–NaHCO 3 solutions with 0.05 M Mg 2+ added and artificial seawater are also included for pH 7.5. At this pH, the rate of Fe(III) reduction changed from log k′=−4.62 ± 0.07 in NaCl–NaHCO 3 solution to log k′=−6.10 ± 0.01 in NaCl–NaHCO 3 with added Mg 2+ ,and to log k′=−6.28 ± 0.08 in the artificial seawater. These values were quite similarto those found in seawater, log k′=−6.16 ± 0.00. No effects were observed when Ca 2+ ,K + ,F − were added to the solution. Fig. 1. Pseudo-first order plot for the oxidation kinetics of Fe(II) in reference seawater (SW n ) and in seawater enriched with exudates (SW nE ) at pH = 8.00 and pH = 7.50 at 25 °C. 13J.M. Santana-Casiano et al. / Marine Chemistry 158 (2014) 10–16 4. Discussion The presence of organic compounds in aqueous solutions is known to modify the oxidation rates of Fe(II) (Theis and Singer, 1974; Santana-Casiano et al., 2000; 2004; Rose and Waite, 2003). This study showed a decrease in the oxidation rate of Fe(II) due to the presence of the ligands exuded from the diatom P. tricornutum. This decrease could be explained by the formation of an Fe(II)–ligand complex that has a lower oxidation rate than the inorganic Fe(II), Eq. (6). Alternatively, the exudates could complex the Fe(III) formed from the oxidation of Fe(II), and facilitate the reduction of Fe(III) to Fe(II). The first mechanism is unlikely as Fe(II) only forms weak complexes with oxygen containing ligand groups such as OH − and carboxyl groups, which are the ligand groups in the extracellular compounds studied here (Fig. 2). The second mechanism, the regeneration of Fe(II) from the reduction of Fe(III) in the presence of organic compounds excreted by the P. tricornutum, should be considered as the most effective process. The studyofthebehaviorof Fe(III) in thepresence of two model ligands,catechin and sinapic acid, able to reduce Fe(III) to Fe(II), was the focus of this investigation.For this purpose, the identification and quantification of the phenols excreted by the phytoplankton cells were considered. The phenols identified in the exudates of the diatom P. tricornutum were simple phenols (gallic acid, protocatechuic acid, vanillic acid, syringic acid, gentisic acid), phenylpropanoids (caffeic acid, coumaric acid, ferulic acid, sinapic acid) and flavonoids.The structure and characteristic groups of these compounds have been fully described in the literature (Mira et al., 2002; Onofrejová et al., 2010) and it is known that they could playan important role in thechemistry of iron in seawater (Rico et al., 2013). The phenols that present a catechol group in the B-ring have the capacity to reduce Fe(III) to Fe(II). The capacity can be increased in some flavonoids with the simultaneous presence of both the catechol groups in the B-ring and the 3-hydroxyl group in the C-ring. The presence of the 2,3-double bond in conjugation with the4-oxo group in the C-ringis also particularly important for Fe(III) reducing activity, as it has been demonstrated by the comparison of quercetrin and catechin (Mira et al., 2002). It has been shown that the presence of catechin reduced Fe(III) to Fe(II) at the three selected pH values. The process was more efficient at low pH values in both NaCl–NaHCO 3 solution and seawater showing higher Fe(II) concentrations with increasing [H + ], which suggests that Fe(OH) 2 + is the most reactive species. However, in seawater the Fe(III) reduction rate was lower than in NaCl–NaHCO 3 solution. This is first, due to a change in the speciation of both iron and the added organic ligands and second, to the interaction of the major ions Mg 2+ and, to alesserextent,Ca 2+ (Table 4) with the catechin and with the benzoquinone radical, as was demonstrated by Santana-Casiano et al. (2010) for the studies done with catechol. The catechin forms a Fe(III)–catechin complex (Mira et al., 2002), that is reduced by intermolecular redox reactions in which the Fe(III) is reduced to Fe(II). The phenol complex is oxidized first to the semiquinone, and then to the corresponding quinone after reacting with another Fe(III) species. In the presence of Mg 2+ , this ion forms a complex with catechin that limits the free catechin concentration to form the Fe(III)–catechin complex, as can be seen in Table 4, where the Fe(II) formed in NaCl media with added Mg 2+ is less than10% of that formed in absence of Mg 2+ . Moreover, in the presence of Mg 2+ and Ca 2+ the semiquinone intermediate forms complexes with these major ions in weakly alkaline aqueous solutions, blocking any further oxidation to the quinone (Nikolićet al., 1988). This is a pH dependent process, and the amount of Fe(II) formed from the Fe(III)–catechin complex would decrease to half of that formed in the absence of any competing ion. This study has also demonstrated that sinapic acid was able to regenerate Fe(II) in the experimental conditions considered, especially at low pH values. The sinapic acid does not form complexes with Fe(III) and the reduction of Fe(III) to Fe(II) takes place by a single reaction step with an electron transfer reaction Fig. 2. Catechin and sinapic acid chemical structure. Fig. 3. Fe(II) regeneration from 200 nM Fe(III) in the presence of 1 μM catechin (continuous lines) and sinapic acid (dashed lines) as a function of pH. Table 2 Pseudo-first order rate constant for Fe(III) reduction in the presence of catechin (1 μM) at constant temperature (25 °C) in different media and pH values. Media pH k′(s −1 )logk′ NaCl 0.7 M; NaHCO 3 2mM 8.00 4.46·10 −6 −5.35 ± 0.07 7.51 1.17·10 −5 −4.93 ± 0.01 6.00 2.10·10 −4 −3.68 ± 0.13 Seawater 8.00 7.02·10 −7 −6.15 ± 0.02 7.50 2.86·10 −6 −5.54 ± 0.01 6.00 1.61·10 −4 −3.79 ± 0.03 NaCl 0.7 M; NaHCO 3 2 mM and Mg 2+ 0.05 M 8.00 Nd Nd 7.51 9.45·10 −7 −6.02 ± 0.04 NaCl 0.7 M; NaHCO 3 2 mM and Ca 2+ 0.01 M 8.00 2.25·10 −6 −5.65 ± 0.07 7.51 3.42·10 −6 −5.47 ± 0.02 14 J.M. Santana-Casiano et al. / Marine Chemistry 158 (2014) 10–16 between the sinapic acid and iron(III), resulting in the oxidation of sinapic acid (Hynes and O'Coinceanainn, 2004). The effect observed when Mg 2+ is added to the NaCl solution, and in seawater solutions, is explained by the complexation of sinapic acid with Mg 2+ that reduces the free sinapic concentration to be reduced in the presence of Fe(III) in a solution. Tables 4 and 5 compare the percentage of Fe(II) formed from the reduction of Fe(III) after 50 and 103 min in the presence of catechin and sinapic acid under the different media conditions. The major Fe(III) reduction is produced at low pH and in NaCl–NaHCO 3 solutions for both catechin and sinapic acid. For thesame initial concentration of ligands, 1 μM, the Fe(II) regeneration ishigher in thepresence of catechin than sinapic acid. The reduction of Fe(III) to Fe(II) decreased in seawater, due the presence of the major divalent ions that interacted with the phenolic compounds. At pH 8.0 in seawater with 200 nM of iron and after 103 min, Fe(II) regeneration was 0.4% in the presence of catechin and 0.1% for sinapic acid. When pH decreases to 7.5, Fe(II) regeneration was 1.7% in the presence of catechin and 0.5% for sinapic acid. At pH 6 Fe(II) regeneration increased to 60.4% in the presence of catechin and 46.3% for sinapic acid. The presence of phenols modified the behavior of iron in the medium and, when they are present, the reduction of Fe(III) to Fe(II) is favored in conditions where the pH (free scale and at 25 °C) is below 8.0 as observed at the depth of the chlorophyll maximum. According to the results presented in this work, in coastal upwelling regions and in a future scenario of ocean acidification, where pH is below 8.0 and the diatoms are expected to be the predominant species, the presence of this type of compounds can affect the iron redox chemistry. 5. Conclusions Phenolic compounds such as catechin andsinapic acid arepart of the organic compounds exudated by P. tricornutum that play an important role in both the decrease of the Fe(II) oxidation rate and the reduction of Fe(III) to Fe(II). Although the regeneration of Fe(II) in seawater is low compared to that in NaCl solution, the amount obtained is significant asthe pH of the seawaterdecreases. This study shows how theability of some organic compounds to favor the reduction of Fe(III) to Fe(II) is reduced due to the competitive effect of major ions such as Ca 2+ and Mg 2+ for the specific sites of complexation. A decrease in pH contributes to an increase in the amount of regenerated Fe(II), showing the acidification may contribute to an increase in the level of reduced iron in the environment. Acknowledgments This study was financed by the Project CTM2010-19517-MAR from the Ministerio de Economía y Competitividad in Spain. Our thanks to the Spanish Bank of Algae (BEA) in Gran Canaria for providing the algal strains. References Andjelković, M., Van Camp, J., De Meulenaer, B., Depaemelaere, G., Socaciu, C., Verloo, M., Verhe, R., 2006. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chem. 98, 23–31. Benner, R., 2011. Loose ligands and available iron in the ocean. Proc. Natl. Acad. Sci. 108, 893–894. 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González-Dávila, M., Santana-Casiano, J.M., Millero, F.J., 2005. Oxidation of nanomolar levels of iron(II) with H 2 O 2 in seawater. Geochim. Cosmochim. Acta 69, 83–93. Granger, J., Price, N.M., 1999. The importance of siderophores in iron nutrition of heterotrophic marine bacteria. Limnol. Oceanogr. 44, 541–555. Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum Publ Corp, New York, pp. 29–60. Table 3 Pseudo-first order rate constant for Fe(III) reduction in the presence of sinapic acid (1 μM) at constant temperature (25 °C) in different media and pH values. Media pH k′(s −1 )logk′ NaCl 0.7 M; NaHCO 3 2 mM 8.00 1.39·10 −6 −5.86 ± 0.087 7.51 2.39·10 −5 −4.62 ± 0.07 6.00 1.46·10 −4 −3.83 ± 0.02 Seawater 8.00 2.67·10 −7 −6.57 ± 0.04 7.50 7.00·10 −7 −6.16 ± 0.00 6.00 1.25·10 −4 −3.90 ± 0.01 NaCl 0.7 M; NaHCO 3 2 mM and Mg 2+ 0.05 M 8.00 Nd Nd 7.51 7.91·10 −7 −6.10 ± 0.01 Artificial seawater 7.50 5.21∙10 −7 −6.28 ± 0.08 Table 4 Fe(II) regenerated (%) from an initial Fe(III) concentration of 200 nM in the presence of 1μMcatechin. Catechin 1 μM Media pH Time (min) % Fe(II) regenerated NaCl 0.7 M and 2 mM NaHCO 3 6.003 50 46.8 6.003 103 84.6 7.508 50 3.4 7.508 103 6.8 7.997 50 1.3 7.997 103 2.2 Seawater 6.000 50 37.2 6.000 103 60.4 7.504 50 0.9 7.504 103 1.7 7.995 50 0.2 7.995 103 0.4 NaCl 0.7 M, 2 mM NaHCO 3 and Mg 2+ 0.05 M 7.508 50 0.3 7.508 103 0.6 NaCl 0.7 M, 2 mM NaHCO 3 and Ca 2+ 0.01 M 7.508 50 1.1 7.508 103 2.3 7.997 50 0.7 7.997 103 1.2 Table 5 Fe(II) regenerated (%) from an initial Fe(III) concentration of 200 nM in the presence of 1μM sinapic acid. Sinapic acid 1 μM Media pH Time (min) % Fe(II) regenerated NaCl 0.7 M and 2 mM NaHCO 3 6.003 50 38.0 6.003 103 56.5 7.508 50 6.9 7.508 103 11.4 7.997 50 0.5 7.997 103 0.7 Seawater 6.000 50 34.0 6.000 103 46.3 7.504 50 0.2 7.504 103 0.4 7.995 50 0.0 7.995 103 0.1 NaCl 0.7 M, 2 mM NaHCO 3 and Mg 2+ 0.05 M 7.508 50 0.2 7.508 103 0.4 ASW 7.504 53 0.2 7.504 103 0.3 15J.M. 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ThistableshowstherankingofthisjournalinitssubjectcategoriesbasedonImpactFactor. CategoryName TotalJournals inCategory JournalRank inCategory Quartile inCategory CHEMISTRY,MULTIDISCIPLINARY 157 47 Q2 OCEANOGRAPHY 61 9 Q1 CategoryBoxP lot  For2014,thejournalMARINECHEMISTRYhasanImpactFactorof2.735. Thisisaboxplotofthesubjectcategoryorcategoriestowhichthejournalhasbeenassigned.Itprovidesinformation aboutthedistributionofjournalsbasedonImpactFactorvalues.Itshowsmedian,25thand75thpercentiles,andthe extremevaluesofthedistribution. Key  A-CHEMISTRY, MULTIDISCIPLINARY B- OCEANOGRAPHY AcceptableUsePolicy Copyright©2015ThomsonReuters. Capitulo 4: DISCUSIÓN Y RESULTADOS 4.3. Dunaliella tertiolecta. Perfil fenólico de la microalga verde Dunaliella tertiolecta cultivada en presencia de altas concentraciones de hierro y cobre. El presente estudio se centra en determinar el perfil fenólico de exudados y de extractos de microalgas Dunaliella tertiolecta cultivadas en agua del mar en ausencia (control) y en presencia de Cu(II) (315 y 790 nmol L−1) y Fe (III) (900 nmol L−1) para identificar y cuantificar los compuestos fenólicos producidos bajo estas condiciones de estrés metálico. Se comprobó que la presencia de iones metálicos modifica el crecimiento celular, siendo la densidad celular 322% (respecto al control) en presencia de hierro, y 80% y 66% en presencia de 315 y 790 nmol L-1 de cobre, respectivamente. La concentración y el tipo de polifenoles detectados en los extractos preparados con las células y en los exudados están directamente relacionados con el metal y su concentración en los cultivos. El ácido gentísico, (+)-catequina y (−)-epicatequina fueron los compuestos fenólicos más abundantes detectados en los extractos de algas y mostraron elevada actividad antioxidante en la inhibición del radical DPPH. La microalga excreta polifenoles para adaptarse a las condiciones ambientales. La cantidad total de los compuestos polifenólicos exudados por cada célula aumentó ligeramente con la adición de cobre al agua de cultivo, llegando a ser 66 amol cell−1 a la máxima concentración de cobre estudiada (1,4 veces mayor que la cantidad excretada por cada célula en el 123 containing the following concentrations: 146.9 mmol L 21 for (+)-catechin; 117.6 mmol L 21 for gallic acid, 129.8 mmol L 21 for protocatechuic acid, 118.9 mmol L 21 for vanillic acid, 100.9 mmol L 21 for syringic acid, 129.8 mmol L 21 for gentisic acid, 32.7 mmol L 21 for rutin, and 73.4 mmol L 21 for (2)-epicatechin; 41.3 mmol L 21 for chlorogenic acid, 91.4 mmol L 21 for p-coumaric acid, 77.2 mmol L 21 for ferulic acid, 47.1 mmol L 21 for myricetin, 55.5 mmol L 21 for caffeic acid, and 33.1 mmol L 21 for quercetin. Relative standard deviation (RSD) values ranged from 2.1%to 6.4%. The accuracy was expressed as the recovery of the standards and was found to be in the range of 87.3–107.5%. RSD values and recoveries are shown in Table 1. Preparation of P. tricornutum diatom extracts for DPPH assay—The algae were freeze-dried and 1 g of the dry material was extracted by stirring with methanol (25 mL) for 1 h. After centrifugation at 3500 rpm for 30 min, the supernatant was collected and evaporated. The dry residue (100 mg) was extracted for 1 h at room temperature with 3 mL of methanol by mixing with a magnetic stirrer. Each extract was centrifuged at 7500 rpm for 20 min, and the supernatant collected and filtered through 0.45 mm filter paper. Free radical scavenging activity on DPPH—The reducing ability of antioxidants on DPPH radical was evaluated by measuring the loss of DPPH color at 515 nm after reaction with the test extracts (Bondet et al. 1997). The sample solution (100 mL) was rapidly mixed with 1 mL of a solution of 0.1 mmol L 21 DPPH. After 25 min incubation time in the dark at ambient temperature (23uC), the decline in absorbance (Abs) was measured against a methanol blank. The inhibition percentage values, expressed in terms of radical scavenging activity (RSA), were calculated by the equation: RSA~100|1{½Abs in the preence of sampleðÞ =Abs in the absence of sampleðÞ ð1Þ Results Effects of copper and iron on the growth of P. tricornutum—The cultures of P. tricornutum were produced at three metal concentrations in order to stimulate the production of exudates. To this effect, the diatom was harvested in seawater with f/2 nutrients (control seawater) as the reference growth (Fig. 1) in close concordance with recent studies (Vasconcelos and Leal 2008; Gonza´lez et al. 2012). At these conditions, the cell density increased from 2 310 7 cells L 21 to 5.9 310 8 cells L 21 after 8 d of culture. The slope for the exponential phase was 8.9 310 7 cells L 21 d 21 . The growth rate during the 8 d of culture was 7.6 310 7 cells L 21 d 21 . The growth of P. tricornutum was also carried out in the presence of 315 nmol L 21 of Cu(II) (Fig. 1). The cell density increased to 4.7 310 8 cells L 21 , with a growth rate of 2.6 310 7 cells L 21 d 21 over the 8 d of culture, meaning that the P. tricornutum cell density decreased 20%with respect to the reference culture. In addition, during the initial 5 d of culture, the growth curve showed a linear relationship with time, where the slope was 2.26 310 7 cells L 21 d 21 . Between the sixth and eighth days of culture, the cell density reached a slope of 10.4 310 7 cells L 21 d 21 , comparable to the reference culture in the absence of copper. The third culture of P. tricornutum was carried out under conditions of 790 nmol L 21 of Cu(II) (Fig. 1). This concentration allows for the determination of the polyphenolic profile under high copper stress. The growth curve showed that the cell density increased to 3.1 310 8 cells L 21 . The cell concentration decreased 47.5%with respect to the reference culture. In this experiment, a similar growth pattern was found as in the study at 315 nmol L 21 of Cu(II). The growth slope was only 0.53 310 7 cells L 21 d 21 through to the fifth day of culture and increased to 8.01 3 10 7 cells L 21 d 21 over the following days. The iron effect was studied at 900 nmol L 21 of Fe(III) added to the culture of P. tricornutum. Under these conditions, the cell density reached a maximum concentration of 2.70 310 9 cells L 21 , one order of magnitude higher than the reference culture. The growth slope in the exponential phase was 46.9 310 7 cells L 21 d 21 , and the growth rate was 5.4 310 8 cells L 21 d 21 for the 8 d of culture (the cell density increased 471%with respect to the reference culture). Polyphenolic profiles—The polyphenolic profile of P. tricornutum was determined both in the extracts of cells collected from the cultures and in seawater enriched with exudates. Tables 2 and 3 show all the polyphenols quantified in this work. The proposed polyphenols were identified in the extracts of cells, except for gallic acid, which was only detected in the iron enrichment experiment. In addition, in the seawater enriched with exudates, only quercetin was not detected. The phenolic compound content in cell extracts of P. tricornutum exposed to copper was strongly affected by the metal concentration (Table 2). As compared to the control, Table 1. Solid-phase extraction recoveries and relative standard deviations (RSD) (n510). Compound Recovery* (%) RSD (%) Gallic acid 9461 4.2 Protocatechuic acid 95.160.7 2.6 Catechin 9461 2.6 Vanillic acid 10861.0 3.9 Epicatechin 105.360.8 3.2 Syringic acid 96.860.8 3.1 Chlorogenic acid 93.660.5 2.9 Gentisic acid 91.160.5 2.1 Caffeic acid 106.560.8 6.4 Coumaric acid 9761.0 5.6 Ferulic acid 93.460.8 3.9 Rutin 11261 3.4 Myricetin 89.460.6 3.0 Quercetin 87.360.6 4.5 * Means 6standard deviation of 10 measurements. Phaeodactylum tricornutum phenols 147 the amount of most of the identified phenolic compounds increased slightly when the copper concentration was 315 nmol L 21 (growth rate 80%). Significantly, the greatest amount (around double with respect to the control) of all the identified phenolic compounds was found when the diatom P. tricornutum was exposed to copper concentration of 790 nmol L 21 (Table 2). Similar findings were given in the seawater samples enriched with exudates, with double the sum of all the identified phenolic compounds exuded per cell in the culture enriched with a copper concentration of 790 nmol L 21 as opposed to those in the control (Table 3). The concentration of polyphenols increased in the culture seawater enriched with copper at concentration 790 nmol L 21 to 39 nmol L 21 . In the iron enrichment experiments, the amount of the quantified phenolic compounds per cell increased under these conditions of high cell growth (471%). The sum of all the identified compounds was 1208 nmol of polyphenols, 1.3 times higher than the control values of 897 nmol of polyphenols (Table 2). In these experiments, myricetin and (+)-catechin were the predominant phenolic compounds, and gallic acid was detected only in the diatoms exposed to iron. The contents of polyphenols such as rutin, myricetin, and quercetin increased by 365%,273%, and 579%, respectively, whereas in the copper enrichment experiment ([Cu(II)] 5790 nmol L 21 ) the increases were inferior (249%, 142%, and 79%, respectively). However, gentisic acid decreased in the presence of iron and increased when Table 3. Phenolic compounds contents in seawater enriched with exudates of diatom P. tricornutum under high iron and copper concentrations. Phenolic compound Control [Fe(III)] 900 nmol L 21 [Cu(II)] 315 nmol L 21 [Cu(II)] 790 nmol L 21 Gallic acid* 2.360.1 10.360.5 0.5860.07 4.860.5 Protocatechuic acid* 23.260.6 5.960.5 19.760.9 4362 Catechin* 125611 11.660.1 11264 283615 Vanillic acid* 1961 4.360.3 18.360.9 3662 Epicatechin* 55.960.8 11.760.0 6667 10763 Syringic acid* 32.060.1 7.860.4 24625862 Chlorogenic acid* 18.260.7 3.560.0 2462 35.260.8 Gentisic acid* 48.360.7 6.860.2 61659367 Caffeic acid* 11.460.1 2.460.2 16.460.8 21.460.6 Coumaric acid* 14.860.3 2.960.2 19.660.7 27.360.6 Ferulic acid* 21.060.4 4.160.2 27634164 Rutin* 9.060.3 6.560.6 11.460.6 16.860.6 Miricetin* 32.460.9 216259636062 Quercetin* nd{5.560.2 2.260.2 1.960.1 Sum* 413617 10465 461630 828640 Sum{2461286230623962 * nmol of phenolic compound per cell 310 10 6standard deviation of two measurements. {nd, not detected. {nmol L 21 6standard deviation of two measurements. Table 2. PhenoliccompoundscontentsinextractsofdiatomPhaeodactylum tricornutum exposed to high iron and copper concentrations. Phenolic compound Control [Fe(III)] 900 nmol L 21 [Cu(II)] 315 nmol L 21 [Cu(II)] 790 nmol L 21 Gallic acid* nd{4364nd nd Protocatechuic acid* 46.960.4 65645963 144610 Catechin* 23663 27761 20864 670642 Vanillic acid* 53.860.8 596483669562 Epicatechin* 12365 12966 174613 234615 Syringic acid* 656476636762 11863 Chlorogenic acid* 506247626264 10662 Gentisic acid* 138647465 17468 278610 Caffeic acid* 3561 35.160.5 50627561 Coumaric acid* 38.260.2 38.160.6 60628162 Ferulic acid* 37.060.9 60657969 122610 Rutin* 12.960.3 606320624564 Myricetin* 56.160.5 20969 11969 13666 Quercetin* 5.360.3 3662 3.160.4 9.560.6 Sum{897622 1208649 1158664 21146108 * nmol of phenolic compound per cell 310 10 6standard deviation of two measurements. {nd, not detected. {nmol of total phenolic compounds per cell 310 10 6standard deviation of two measurements. 148 Rico et al. the diatom was exposed to copper, whereas the rest of the polyphenols showed major increases when the diatoms were exposed to a copper concentration of 790 nmol L 21 . Quantification of phenolic compounds in the seawater samples enriched with exudates from the iron enrichment experiments showed that the amount of single phenolic compounds exuded per cell declined dramatically as compared to the control, with the sum of all the identified compounds exuded per cell 4 times less than those in the control (Table 3). However, the concentration of the polyphenols in nmol L 21 increased in the culture seawater enriched with iron from 24 nmol L 21 (control) to 28 nmol L 21 due to the large number of cells. Antioxidant activities of the diatom extracts—Figure 2 shows the relative antioxidant efficiency of the diatom extracts against the DPPH radical. Antioxidants suppressed the absorbance at 515 nm on a time scale dependent on the antioxidant activity of the extracts. As may be observed, the extracts derived from the diatom exposed to copper showed the highest radical scavenging activities (16.2%and 11.9%) followed by the extract derived from cells under iron enrichment conditions (8.1%) and the control (7.6%). Discussion Copper and iron stress affect growth and the phenolic contents of the marine diatom P. tricornutum, but not in the same way. The behavior of P. tricornutum under copper additions suggested that the diatoms metabolically produced exudates capable of complexing the copper present in the solution, thereby decreasing the toxic effect. Once the conditions were optimal, the diatom growth began. The effect of copper on diatom growth concords well with results published in previous reports with respect to the effect of copper toxicity on microalgae growth. Kagalou et al. (2002) reported that Cu(II) concentrations of 15.7 mmol L 21 had significant effects on the growth rate of the microalgae Isochrysis galbana,whereaslower concentrations (157 and 1570 nmol L 21 ) produced less decline in algal growth. Franklin et al. (2002) concluded that the copper concentrations required to inhibit growth rate by 50%increased from 72.4 to 252 nmol L 21 for the tropical freshwater alga Chlorella sp. and from 104 to 268 nmol L 21 for the temperate species Selenastrum capricornutum as the initial cell density increased from 10 5 to 10 8 cells L 21 . The initial cell density is a very important parameter in toxicity tests carried out using microalgae. Moreno-Garrido et al. (2000) reported copper growth inhibition tests on four marine microalgal species including P. tricornutum. Diatoms were cultivated in artificial seawater enriched with a modification of f/2 medium, lacking ethylenediaminetetraacetic acid, which decreases the toxicity of heavy metals. In these conditions, MorenoGarrido et al. (2000) observed EC 50 values (representing the concentration of copper that caused a 50%reduction in the cell division rate compared with controls and calculated by semilogarithmic plotting of average inhibition data fits to straight line) for P. tricornutum of 154, 542, and 551 nmol L 21 for the initial cellular densities 10 6 ,53 10 6 ,and10 7 cells L 21 , respectively (after 72 h exposure to copper). Franklin et al. (2001) reported that copper had an inhibitory effect on the cell division rate of several algal species after 48 h and 72 h exposure to copper (initial cell density 2 310 7 to 4 310 7 cells L 21 and using filtersterilized seawater supplemented with [NO{ 3]5242 mmol L 21 and [PO3{ 4]515.8 mmol L 21 as a culture medium to maintain exponential growth over 72 h). In said study, the 48 h and 72 h EC 50 values were 142 647 and 158 6 63 nmol L 21 , respectively, and complete growth inhibition was observed at 11.8 mmol L 21 . The cell light scatter properties of P. tricornutum depended on the cell size and intracellular granularity. These properties were found to be Fig. 2. Relative activity of the extracts derived from diatom Phaeodactylum tricornutum to scavenge the stable radical DPPH under different conditions: control seawater, [Fe(III)] 5900 nmol L 21 , Cu(a) was [Cu(II)] 5315 nmol L 21 , and Cu(b) was [Cu(II)] 5 790 nmol L 21 . RSD values represented means of triplicate determinations 6standard deviation. Phaeodactylum tricornutum phenols 149 a useful indicator of chronic copper toxicity to the marine alga P. tricornutum. Franklin et al. (2001) found that copper caused an increase in cell size after 24 h exposure, with 50% or more of the cells larger than the controls at 3.15 mmol L 21 , whereas similar increases in cell size were observed after 48 h and 72 h at 157.5 nmol L 21 , with an EC 50 value of 126 6 47 nmol L 21 . Similar changes in cell size and granularity were also reflected in changes in side-angle light scatter with an EC 50 value of 189 nmol L 21 (after 48 h and 72 h exposure to copper). The diatoms P. tricornutum exposed to lower doses of copper in said study than those used by the present researchers at the same initial cell density, showed changes in cell size, granularity, and growth rate, considered to be indicators of copper toxicity. P. tricornutum showed the highest growth rate at iron concentration 900 nmol L 21 . Iron is well known as a fertilizer and as an essential micronutrient participating in essential metabolism processes such as photosynthesis, respiration, and nitrogen fixation (Stumm and Morgan 1996). Morel et al. (2008) have demonstrated that unchelated Fe is highly available for uptake but that chelated Fe is necessary in order to explain the total uptake system. There is even evidence of direct internalization of siderophores by microorganisms using reductive mechanisms with varying degrees of efficiency (Hopkinson and Morel 2009). Our results align well with Kudo et al. (2000), who reported the combined effect of iron nutrition and temperature on growth (cell division) of the diatom P. tricornutum, concluding that the growth rate of cells cultivated with Fe(III) 2.0 mmol L 21 was twice as fast as the growth rate of diatoms cultivated with lower Fe(III) concentration (2.0 nmol L 21 )at20uC. In this study, increased phenolic compound content has been reported in P. tricornutum exposed to increasing doses of Cu(II). At the highest copper concentration (790 nmol L 21 ), the phenolic content per cell was 2.4 times higher than in the control. In addition, the concentration of polyphenols increased in the culture seawater enriched with copper at concentration 790 nmol L 21 to 39 nmol L 21 (Table 3), despite the number of cells being much lower (52.5%), indicating that cells exposed to copper must excrete a larger amount of polyphenols. The diatom must make an extra metabolic effort in order to live in these levels of copper concentrations and must produce relevant amounts of polyphenols to slow down the toxicity of the copper in the solution, acting as a protective mechanism. This role has also been described for soil bacteria (Pseudomonas aureofaciens) (Gonza´lez et al. 2010) and plants that present mechanisms of detoxification, such as the exudation of phenols that act as chelate towards heavy metals to protect cells from induced oxidative damage (Jung et al. 2003). Increasing phenolic compounds may also contribute to enhancing the ability to cope with osmotic stress by binding metal ion to wallassociated polyphenols, countering metal toxicity at the cell surface (Suresh and Subramanyam 1998; Jung et al. 2003). Plant intracellular detoxification mechanisms may involve specific scavenging of reactive oxygen species through the action of enzymes and a wide array of metabolites, including phenolic components or direct chelate formation by polyphenols (Quideau et al. 2011). Wells et al. (2005) reported a synergistic link between the release of the ironcomplexing ligand domoic acid and the limitation by iron and copper in toxigenic diatoms Pseudo-nitzschia spp. In said study, copper was found to be a key factor facilitating the success of these diatoms in iron-deficient waters, by inducing a high-affinity iron uptake. Iron limitation is the primary trigger for domoic acid production, which doubled under iron deficiency and increased by one order of magnitude under copper-limiting conditions. However, similar increases in domoic acid production were detected under conditions of toxic copper concentration, presumed to reduce copper toxicity. The findings here evidenced increased phenolic compounds per cell under iron-rich media conditions ([Fe(III)] 5900 nmol L 21 ) as compared to the control. However, the amounts of phenolic compounds exuded per cell decreased drastically, resulting four times lower than the control. This decline in excreted polyphenol concentrations per cell may have its explanation in the high cell densities in the culture that help to minimize the stress suffered per cell, making the metabolic effort needed to uptake iron to decrease. P. tricornutum produced and exuded phenolic compounds mixtures with different phenolic profiles depending on the culture conditions. The extracts derived from the diatom exposed to copper showed the highest radical scavenging activities (16.2%and 11.9%). The phenolic profiles for these more active extracts were very similar. However, the diatoms exposed to iron showed different phenolic profiles that may vary the antioxidant potential. The antioxidant activity is not only dependent on the concentration of antioxidants, but also on the structure (Wright et al. 2001). The antioxidant activity is also dependent on the interaction among the antioxidants (phenolic compounds may act synergistically or antagonistically) (Jacobo-Velazquez and Cisneros-Zevallos 2009). Amoros et al. (1992) demonstrated the synergistic effect of binary flavone–flavonol combinations against herpes simplex virus type 1 in cell culture. Gao et al. (2011) reported that four phenolic compounds (vanillic acid, protocatechuic acid, ferulic acid, and caffeic acid) exerted additive and synergistic inhibition effects on the growth of Microcystis aeruginosa depending on the mix ratios. Tafesh et al. (2011) reported that hydroxytyrosol at 400 mgmL 21 caused growth inhibition of the four bacterial isolate gram-positive (Streptococcus pyogenes and Staphylococcus aureus) together with the gram-negative (Escherichia coli and Klebsiella pneumoniae), whereas gallic acid at 200 and 400 mgmL 21 inhibited the growth of S. aureus and S. pyogenes strains, respectively (no growth inhibition was observed for the gram-negative bacteria). However, the combination of both compound gallic acid and hydroxytyrosol at lower concentrations (100 and 200 mgmL 21 , respectively) caused a complete inhibition of the four bacterial strains. In the present study, significant differences in the phenolic profiles were found in P. tricornutum diatoms growing under iron fertilization conditions and under copper stress. These differences seem to be the response to the different needs of the diatoms when grown under different stress conditions: as compared to the control, the amount of gentisic acid decreased when the culture seawater was enriched with iron 150 Rico et al. and increased when the seawater was enriched with copper; cells under iron enrichment conditions revealed a higher content of quercetin, myricetin, and rutin than the other diatom extracts and gave no change for (2)-epicatechin or chlorogenic acid, which increased in diatoms exposed to copper (790 nmol L 21 ) (Table 2). As Gao et al. (2011) suggested, the profile of the phenolic compounds in a mixture and their mix ratios determine the joint action of the compounds, with either synergistic or additive effects. In addition, various different kinds of activities of the phenolic mixture may also depend on the mix ratios, where this mixture may be more active in complexing or reducing trace metals in solution. Therefore, further research is required to study the joint action of the phenolic compounds identified in cells exposed to multiple metal stresses and the influence of the mix ratio of said phenolic compounds on the kind and intensity of the mix activity. Polyphenols have attracted a great deal of attention recently, mainly focused on their role in preventing diseases produced as a result of oxidative stress (Dillard and German 2000; Dai and Mumper 2010). Dietary antioxidants from plants are believed to help prevent aging and many degenerative diseases, such as cardiovascular complaints and cancers, as a result of their radical scavenging activity (Virgili et al. 2003; Quideau et al. 2011). Therefore, there is considerable interest in the field of preventive medicine in the development of natural antioxidants obtained from botanical sources. Seaweeds are considered to be a rich source of antioxidants, and recent studies have suggested using microalgae as ‘‘health’’ foods (Chaco´n-Lee and Gonza´lezMarin˜o 2010). The results of this study confirm that P. tricornutum is a natural source of well-known antioxidant compounds (Silva et al. 2002) and afford more than sufficient arguments for researching the viability of the use of P. tricornutum diatom in the health and food industries in general, as well as in the pharmaceutical industry. Acknowledgments This study received financial support from the Project CTM2010-19517-MAR financed by the Ministerio de Economı ´a y Competitividad in Spain. Our thanks to the Spanish Bank of Algae (BEA) in Gran Canaria for providing algal strains. 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Complexation of iron (III) by natural organic ligands in the Northwest Atlantic Ocean by a competitive ligand equilibration method and a kinetic approach. Mar. Chem. 50: 159–177, doi:10.1016/0304-4203(95)00033-N Associate editor: Robert R. Bidigare Received: 14 June 2012 Accepted: 26 September 2012 Amended: 01 October 2012 152 Rico et al. 29/9/2015 JCR-Web4.5ImpactFactorBoxplot http://admin-apps.webofknowledge.com/JCR/JCR?RQ=IF_CAT_BOXPLOT&rank=1&journal=LIMNOL+OCEANOGR 1/1    2013JCRScienceEdition RankinCategory:LIMNOLOGYANDOCEANOGRAPHY JournalRanking For2013,thejournalLIMNOLOGYANDOCEANOGRAPHYhasanImpactFactorof3.615. ThistableshowstherankingofthisjournalinitssubjectcategoriesbasedonImpactFactor. CategoryName TotalJournals inCategory JournalRank inCategory Quartile inCategory LIMNOLOGY 20 2 Q1 OCEANOGRAPHY 59 5 Q1 CategoryBoxP lot  For2013,thejournalLIMNOLOGYANDOCEANOGRAPHYhasanImpactFactorof3.615. Thisisaboxplotofthesubjectcategoryorcategoriestowhichthejournalhasbeenassigned.Itprovides informationaboutthedistributionofjournalsbasedonImpactFactorvalues.Itshowsmedian,25thand75th percentiles,andtheextremevaluesofthedistribution. Key  A- LIMNOLOGY B- OCEANOGRAPHY AcceptableUsePolicy Copyright©2015ThomsonReuters. Capitulo 4: DISCUSIÓN Y RESULTADOS 4.4. Diferencias entre las dos especies de microalgas En la FIGURA 18 se muestran las densidades celulares alcanzadas por cada microalga en las diferentes condiciones de cultivo. Como se puede observar, la diatomea es menos tolerante a la presencia de cobre y, prácticamente, se llega a alcanzar la inhibición del crecimiento celular. FIGURA 18.- Densidades celulares de los cultivos de microalgas en % con respecto al control Las diferencias encontradas entre las dos especies de microalgas están de acuerdo con los resultados de los estudios de tolerancia llevados a cabo por Levy y colaboradores (2008), que han observado la adsorción e internalización de cobre de tres 139 Molecules 2013, 18 4943 1. Introduction The botanical name of Aloe vera is Aloe barbadensis Miller. It belongs to the Asphodelaceae (Liliaceae) family, and is a shrubby or arborescent, perennial, xerophytic, succulent pea-green colored plant [1]. The origins of these plants are the dry regions of Africa, Asia, and Southern Europe, especially in the Mediterranean regions. In the Canary Islands, Aloe vera plants naturally grow anywhere and everywhere and there is a considerable generally shared belief in the beneficial action of the gel among the population, estimated to be one of the few botanical medications in widespread domestic use. In folk medicine, the brown juice has been used traditionally for its purgative effects, and the fresh leaf gel in cosmetics and nutraceutical formulations [2]. Among the purported benefits of Aloe vera not supported by experimental or clinical data are the following: treatment of acne, haemorrhoids, psoriasis, anemia, glaucoma, petit ulcer, tuberculosis, blindness, seborrhoeic dermatitis, and fungal infections [2]. Several reports have demonstrated the antioxidant, antinociceptive and anti-inflammatory activities of the aloe species [3,4]. In addition, recent studies have shown the anti-cancer effect of aloe-emodin, an anthraquinone compound present in the leaves of Aloe vera [5]. Studies of the in vitro antimicrobial properties of the ethanolic extract of Aloe vera leaf gel revealed that it was active against most of the studied pathogenic bacteria and fungi, even at very low doses [6]. The list of different illnesses and conditions aided by the use of Aloe vera is indeed impressive, covering everything from burns and slight infections to extremely serious medical conditions. Several reviews have focused on the main scientific discoveries on Aloe vera reported over the last three decades [2,7–12]. These reviews deal with the botany, the chemical properties, the gel stabilization technique, the biological functions, and the current uses and applications of Aloe vera (mainly focusing on the exudate and gel of the Aloe vera leaves). Plant polyphenols have been implicated in diverse functional roles, including plant resistance against microbial pathogens and animal herbivores such as insects (antibiotic and antifeeding actions), protection against solar radiation, besides reproduction, nutrition, and growth [13]. Phenolic compounds have also been reported to prevent diseases resulting from oxidative stress [14–16]. Screening of the phytochemical (qualitative and quantitative) analysis of the Aloe vera leaf (leaf skin and gel) showed that almost all of the chemical constituents are present: tannin, phlobatannins, saponin, flavonoids, steroids, terpenoids, and cardiac glycosides anthroquinones, which are used for medicinal purposes [17]. Phenolic compounds are the second major substances found in Aloe vera. The main active constituent of the Aloe vera plant extract is aloine, an anthraquinone heteroside [18]. Several papers have also been published that focus on the identification of the main phenolic compounds from the gel and leaf exudate of Aloe. Okamura et al. developed a procedure for determination of aloesin, 2'-O-feruloylaloesin, aloeresin A, barbaloin, isobarbaloin, aloenin, aloe-emodin, 8-C-glucosyl-7-O-methyl-(S)-aloesol, isoaloeresin D and aloeresin E, which are phenolic constituents of aloe [19]. Thirteen phenolic compounds from Aloe barbadensis (syn. A. vera) and A. arborescens were identified and quantified: aloesin, 8-C-glucosyl-7-O-methyl-(S)-aloesol, neoaloesin A, 8-O-methyl-7-hydroxyaloin A and B, 10-hydroxyaloin A, isoaloeresin D, aloin A and B, aloeresin E and aloe-emodin from A. barbadensis; and aloenin, aloenin B, 10-hydroxyaloin A, aloin A and B, and aloe-emodin from A. arborescens [20]. A mixture of phenolic compounds, mainly anthrones (aloenin, aloenin B, isobarbaloin, barbaloin and other aloin derivatives from Aloe Molecules 2013, 18 4944 secundiflora (Aloeaceae) has been determined from the leaf exudate [21]. So far, little attention has been paid to the flowers or the leaf skin of the Aloe. Previous studies have suggested using Aloe vera flowers for phytotherapeutical purposes due to the presence of some polyphenols [22]. The aim of this study was to determine the differences in the phenolic profile of the methanol extracts derived from the leaf skin and flowers of Aloe vera (L.) Burm. f. (syn. A. barbadensis Mill.) from the Canary Islands to investigate the potential of the flowers and the leaf skin for uses in the health food industry, as well as in pharmaceuticals. As a result eighteen phenolic components were identified and quantified by reverse phase-high performance liquid chromatography (RP-HPLC). The antioxidant activities of the extracts were studied, as well as the preliminary in vitro susceptibility of some mycoplasma strains. Mollicutes, trivially known as mycoplasmas, are phylogenetically related to the Gram-positive branch of the eubacteria and can be divided into five phylogenetic groupings, including the anaeroplasma, asteroleplasma, spiroplasma, pneumoniae, and hominis groups [23]. Mycoplasmas are commensals or parasites on vertebrate, insect, and plant hosts, representing many significant pathogens in human and veterinary medicine. They are bacteria characterized by their lack of a cell wall and for their small genomes and highly structural and functional simplicity. Besides, they do not synthesize nucleotides or amino acids, express an unusual form of RNA polymerase, and certain species produce atypical ribosomes. All these characteristics make them naturally resistant to many antibiotics, reducing treatment options to tetracyclines, macrolides, and fluoroquinolones. Therefore, they represent magnificent targets for anti-microbe testing [24]. 2. Results and Discussion 2.1. Determination of the Phenolic Profile by HPLC The presence of polyphenols in the extracts was confirmed by comparing retention times (RT) and overlapping UV spectra with those of standard compounds. The phenolic compounds sinapic acid (RT: 6.9 min), quercitrin (RT: 7.6 min), kaempferol (RT: 10.8 min) and apigenin (RT: 11.3 min) were well resolved. Limits of detection (LOD) and limits of quantification (LOQ) were estimated from signal-to noise-ratio of the individual peaks, assuming a minimum detectable signal-to-noise level of 3 and 10 respectively [25]. The LODs were found to be in the range of 0.032–0.127 μg·mL−1 and the LOQs were observed in the range of 0.106–0.355 μg·mL−1 (Table 1). This indicated that the proposed method offers adequate sensitivity for the quantification of polyphenols. Reproducibility, expressed as the relative standard deviation (RSD), was obtained by analysing six replicate samples containing 20 μg·mL−1 of each of the four compounds. The accuracy was expressed as the recovery of standard compounds added to the pre-analysed sample [26]. The results are summarized in Table 1. The phenolic compounds gallic acid, protocatechuic acid, catechin, vanillic acid, epicatechin and syringic acid, chlorogenic acid, gentisic acid, caffeic acid, p-coumaric acid and ferulic acid, rutin, myricetin, and quercetin were quantified according to a previously reported method [27]. Molecules 2013, 18 4945 Table 1. Method validation data for the quantitative determination of four phenolic compounds using RP-HPLC. Compounds Regression equation (r) LOD a μg·mL−1LOQ a μg·mL−1Recovery b (%) RSD c (%) Sinapic acid y = 34493x − 25074 (0.9988) 0.10661 0.3554 118 ± 3 2.57 Quercitrin y = 88302x − 20416 (0.9976) 0.03640 0.1235 116 ± 4 3.69 Kaempferol y = 15436x − 28177 (0.9998) 0.09775 0.3258 105 ± 8 7.61 Apigenin y = 20306x − 67494 (0.9993) 0.03196 0.1065 99 ± 3 2.66 a Detection limits are calculated as signal to noise ratio of ten times; b Means ± standard deviation of three measurements; c Reproducibility was obtained by analyzing six replicate samples containing 20 μg·mL−1 for every standard. The proposed polyphenols were identified in the extracts, except for gallic acid, which was only detected in the flower extract. In addition, quercetin was only detected in the leaf skin extract. The results here evidenced that catechin, sinapic acid, gentisic acid and epicatechin were the most abundant compounds of those under study and their mix ratio changed depending on the source of the extract (aloe leaf skin or flowers). Catechin and sinapic acid were most abundant in the leaf skin extract and gentisic acid and epicatechin were predominant in the flower extract (Table 2). Table 2. The polyphenol contents in aloe extracts presented as average values ± standard deviation of two measurements. Phenolic compound Leaf skin a Flowers a Sinapic acid 54 ± 3 15.0 ± 0.6 Quercitrin 23 ± 1 31.9 ± 0.5 Kaempferol 4.03 ± 0.03 2.86 ± 0.01 Apigenin 3.3 ± 0.4 3.03 ± 0.00 Gallic acid nd b 12.6 ± 0.2 Protocatechuic 1.1 ± 0.0 0.57 ± 0.02 Catechin 95 ± 3 7.6 ± 0.2 Vanillic acid 2.30 ± 0.04 0.8 ± 0.1 Epicatechin 16.2 ± 0.7 58.0 ± 0.1 Syringic acid 4.9 ± 0.5 5.0 ± 0.3 Chlorogenic acid 7.8 ± 0.2 2.8 ± 0.2 Gentisic acid 6.0 ± 0.3 101 ± 2 Caffeic acid 4.9 ± 0.1 9.3 ± 0.1 Coumaric acid 0.8 ± 0.0 7.6 ± 0.4 Ferulic acid 7.9 ± 0.4 3.1 ± 0.1 Rutin 22.3 ± 2 11.6 ± 0.2 Miricetin 19.6 ± 0.7 1.76 ± 0.02 Quercetin 34.4 ± 2 nd b Sum 307.5 274.5 a mg per 100 gram of freeze-dried aloe material; b nd indicates not detected. Molecules 2013, 18 4946 The chemistry of the aloe plant has been studied for many years from a number of viewpoints [3–12]. Interest has centered on the parenchyma gel and its well-known therapeutic properties. Previous studies regarding the content of polyphenols in the leaf skin were not found. However, various phenolic compounds such as chlorogenic, caffeic, p-coumaric and ferulic acids were detected in the Aloe flowers [22]. In the present study, we detected and quantified eighteen phenolic compounds (Table 2), confirming that the Aloe leaf skin and flowers are natural sources of well-known antioxidant compounds [28]. 2.2. Radical Scavenging Activity (RSA) on DPPH and Ferric Reducing Antioxidant Power (FRAP) The reducing ability of antioxidants on the DPPH radical was evaluated by measuring the loss of DPPH color at 515 nm after reaction with the test samples. The leaf skin extract was more active to DPPH than the flower extract (Table 3). The FRAP assay was used to study the ability of the antioxidants in these extracts to reduce the ferric iron to a ferrous form. The redox reaction is carried out at pH 3.6. At more acidic conditions than the physiological pH, the reducing capacity may be suppressed due to protonation on the phenolics. However, the same behaviour as was observed in the DPPH assay was observed instead (the extract of the leaf skin was more active than the flower extract) (Table 3). Table 3. Antioxidant and antimycoplasmic activities of the aloe extracts. Extract RSA a,b FRAP a,c Antimycoplasmal activity a,d M. mycoides capri M. agalactiae Acholeplasma laidlawii M. gallisepticum Leaf skin 58.8 ± 0.4 2.4 ± 0.1 239 ± 51 b CCM 2253 ± 123 1466 ± 213 Flowers 53 ± 2 1.7 ± 0.0 - - - - a Values represented mean ± standard deviation of three measurements; b % inhibition; c mmol of Fe (III) reduced to Fe(II); CCM: changes in the colony morphology around the disc - indicates no growth inhibition; d in micrometers. In general, the literature reports that there is a relation between the content of phenolic compounds and the antioxidant properties [29]. Our results confirm the correlation between the phenolic content (calculated as the sum of the identified polyphenols) and the antioxidant activity (the extract with higher phenolic content gave the highest activities), allowing us to conclude that phenolic constituents are mainly responsible for the observed antioxidant activity in the extracts. 2.3. Antimycoplasmic Activity The four mycoplasmas were selected as representatives of four of the five mycoplasma phylogenetic groups [23]. The group without representation is the anaeroplasma group, composed of anaerobic commensals found in ovine and bovine rumen. Also the three mycoplasmas included in the genus Mycoplasma are ruminant and poultry pathogens, while the mycoplasma from the genus Acholeplasma is a frequent contaminant of eukaryotic cell cultures. The results of the in vitro susceptibility test are given in Table 3. The concentration of the extracts is slightly higher than the ones used by Al-Momani et al. [30]. Antimycoplasmic activity was only found in the leaf skin extract (from simple changes in the colony morphology (CCM) as occurred in the case of Mycoplasma agalactiae (M. agalactiae) or in greater inhibition zones (Acholeplasma laidlawii and Mycoplasma Molecules 2013, 18 4947 gallisepticum)). Further studies have to be undertaken to validate and extend these observations over a wider selection of bacteria and fungi in order to determine minimum inhibitory concentrations (MIC) of these extracts. The extract from the flowers did not show antimycoplasmic activity against the bacterial strains tested in the present study, but might express strong properties against other test organism or other kind of properties depending on its composition in phenolic compounds. Rodríguez Vaquero et al. reported that bacterial species exhibited different sensitivities towards the different concentrations of pure phenolic compounds [31]. Escherichia coli was the most sensitive bacterium and Flavobacterium sp. was resistant against all phenolic compounds tested. The activity of the extracts is not only dependent on the concentration of the phenolic compounds but also on the structure and nature of the compounds [32]. Several reports have suggested that the biological activity of the extracts is also dependent on the interaction among the phenolic compounds [33,34]. Tafesh et al. [35] reported that hydroxytyrosol at 400 µg·mL−1 caused growth inhibition of the four bacterial isolate gram-positive (Streptococcus pyogenes and Staphylococcus aureus) together with the gram-negative (Escherichia coli and Klebsiella pneumoniae), while gallic acid at 200 and 400 µg mL−1 inhibited the growth of the S. aureus and S. pyogenes strains, respectively (no growth inhibition was observed for the gram-negative bacteria). However, the combination of both compound gallic acid and hydroxytyrosol at lower concentrations (100 and 200 μg·mL−1, respectively) caused a complete inhibition of the four bacterial strains. Gao et al. [36] reported that four phenolic compounds of those under study (vanillic acid, protocatechuic acid, ferulic acid and caffeic acid) exerted additive and synergistic inhibition effects on the growth of Microcystis aeruginosa depending on the mix ratios. In said study, the authors concluded that the profile of the phenolic compounds in a mixture and their mix ratios determine the joint action of the compounds, with either synergistic, antagonistic and/or additive effects. The efficacy of a combination of different phenolic compounds structures might be greater than that of other combinations on a kind of activity. Therefore, various different kinds of activities of a phenolic mixture may also depend on the mix ratios, where the mixture may be more active in contributing to UV-B protection or warding off microbial infection or protecting the plants from herbivores. The differences in the phenolic profile in the present work may be the result of the involvement of these compounds in different functional roles in the flowers from in the leaf skin. Further research is required to study the joint action of the phenolic compounds identified in the extracts and the influence of the mix ratio of said phenolic compounds on the kind and intensity of the mix activity. 3. Experimental 3.1. Chemicals Methanol (of HPLC grade), ferric chloride (FeCl3·6H2O), ferrous sulphate (FeSO4·7H2O) and glacial acetic acid were obtained from Panreac (Barcelona, Spain) with formic acid and sodium acetate provided by Merck (Darmstadt, Germany) of analytical quality. The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) were from Sigma-Aldrich Chemie (Steinheim, Germany). Molecules 2013, 18 4948 The antimicrobial activity of the plant extracts was tested using susceptibility test disks (Oxoid, CT0998B, Ø = 5 mm). Polyphenol standards of gallic acid, protocatechuic acid, chlorogenic acid, (−) epicatechin, quercetin, myricetin, ferulic acid, p-coumaric acid, vanillic acid, syringic acid, (+) catechin, sinapic acid, quercitrin, kaempferol and apigenin were purchased from Sigma-Aldrich Chemie; rutin and gentisic and caffeic acids were supplied by Merck (Hohenbrunn, Germany). 3.2. Mycoplasmas Four types of strains of mollicutes: Mycoplasma mycoides subsp. Capri (Y-goat) (M. mycoides Capri), Mycoplasma agalactiae (PG2) (M. agalactiae), Acholeplasma (A.) laidlawii (PG8) (A. laidlawii) and Mycoplasma gallisepticum (PG31) (M. gallisepticum) were used in this study with the former two cultivated in PH medium [37] and the latter two in SP4-II medium [38]. The mycoplasmas were cultured under aerobic conditions at 37 °C. 3.3. Plant Material The Aloe vera leaves and flowers were collected fresh in February 2010. The plant was identified in the Herbarium of the Viera y Clavijo Botanical Gardens in Gran Canaria where a voucher specimen was deposited (LPA: 27058-27060). A voucher specimen was deposited in the Herbarium of the Viera y Clavijo Botanical Gardens in Gran Canaria (LPA: 27058-27060). Soon after collection, the skin of the leaves and the flowers were separated, shaken and frozen. The leaf skin was separated and cleaned with a knife. The frozen samples were then freeze-dried and pulverized into powder using a blender (Moulinex, 600 W, Ecully Cedex, France) and were subsequently kept in the dark at −20 °C under nitrogen. 3.4. Preparation of Aloe Extracts Freeze-dried plant material (10.0 g of leaf skin and 10.0 g of flowers separately) was extracted with solvent (175 mL) in a Soxhlet extractor for 3 h. The extraction was repeated four times, using the same plant material, but different solvents: hexane, acetone, ethanol and methanol were used consecutively. Several studies have reported that high levels of polyphenols may be associated with the use of polar solvents in the extraction [27,39]. Therefore, hexane, acetone and ethanol extracts were discarded. After extraction, 10 mL of methanol extract was reserved for the antioxidant activity assays. To prepare the samples for the antimycoplasmic activity determination, the methanol extract was evaporated under reduced pressure to give semi-solid residues. In order to avoid problems related to the use of plant extracts, their solubility and the use of solubilising agents [40], the only product used for solubilising the residues from methanol extracts was ultra-pure sterile water. Working concentrations were 111 mg·mL−1 for flower residues and 112 mg·mL−1 for the leaf skin residues. 3.5. Free Radical Scavenging Activity on DPPH The reducing ability of the antioxidants on the DPPH radical was evaluated by measuring the loss of 1,1-diphenyl-2-picrylhydrazyl (DPPH) colour at 515 nm after reaction with test extracts [41]. The sample solution (100 μL of extracts) was rapidly mixed with 1 mL of a solution of 0.2 mM DPPH. After 30 min incubation in darkness at ambient temperature (23 °C), the reduction of the DPPH radical Molecules 2013, 18 4949 was measured by monitoring the decline in absorbance (Abs) against a methanol blank at 515 nm using a Shimadzu 1700 UV-Vis spectrophotometer. The percentage inhibition was calculated by application of the equation: RSA = 100 (1 − Abs in the presence of sample/Abs in the absence of sample). 3.6. Ferric Reducing Antioxidant Power Assay (FRAP Assay) Reducing power was determined according to [42]. This method is based on the reduction of Fe3+ to Fe2+, which is recorded by measuring the formation of a blue coloured Fe2+-tripyridyltriazine compound from the colourless oxidized Fe3+ form via the action of electron-donating antioxidants. The FRAP reagent consists of 300 mM acetate buffer (3.1 g sodium acetate + 16 mL glacial acetic acid, made up to 1 litre with distilled water; pH = 3.6), 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3·6H2O in a ratio of 10:1:1. The extract (50 μL) was added to 1.5 mL freshly prepared and pre-warmed (37 °C) FRAP reagent. The mixture was incubated at 37 °C for 10 min and the absorbance was measured against a reagent blank (1.5 mL FRAP reagent + 50 μL distilled water) at 593 nm. A standard curve of Fe(II) was constructed over the concentration range of 0.1 mM to 2.0 mM. The results were determined by the regression equation of the curve (Abs = 0.00221[Fe(II)] + 0.02464, r = 0.9998) and expressed as µmol ferric ions reduced to ferrous form. 3.7. Determination of the Phenolic Profile by RP-HPLC To prepare the samples for the HPLC quantification, the freeze-dried plant material (50 mg) and 2 mL of methanol were mixed and homogenized using a vortex for 30 s. The mixture was stirred in a rotator (SB 3, Stuart, Staffordshire, UK) for 60 min at room temperature in darkness. After centrifugation at 7,000 ×g for 20 min at 4 °C, the supernatant was collected and evaporated. The dry residue and 0.5 mL of water were mixed and filtered through a 45 μm nylon syringe filter prior to injection. Chromatographic analysis was performed on a Varian ProStar 210 system, equipped with a vacuum degasser, a binary pump, a thermostat column compartment and a diode array detector (DAD), connected to ChemStation software. The separation was performed with a reverse-phase Pursuit XRs C18 (250 mm × 4.6 mm, 5 micrometers (μm)) column and a Pursuit XRs C18 (10 mm × 4.6 mm, 5 μm) guard column (Varian, Barcelona, Spain). A gradient system, involving two mobile phases, was used. Eluent A was water with 0.1% formic acid and eluent B, methanol. The flow rate was 1.0 mL/min, and the injection volume was 60 μL of crude extracts (rheodyne injector). The system operated at 27 °C. The elution conditions applied were: 0–4 min, linear gradient from 20% to 30% B; 4–10 min, 30% B isocratic; 10–13 min, linear gradient from 30% to 50% B; 13–15 min, linear gradient from 50% to 80% B and finally, washing and re-conditioning of the column. Monitoring was set at 254 nm for quantification. Method 1: to quantify the compounds sinapic acid, quercitrin, kaempferol and apigenin in the extracts, five different concentrations of the analytes were injected in triplicate. The calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The linearity was assessed by linear regression analysis, which was calculated by the least squares method. Each point on the calibration plot was the mean of two area measurements. All correlation coefficients were over 0.9976 (Table 2). The wavelength was fixed at 254 nm for quantification. The selectivity of the method was determined by analysis of standard compounds and samples. The peaks of polyphenols Molecules 2013, 18 4950 were identified by comparing their retention times (RT) and overlaying of UV spectra with those of standard compounds. Method 2: the phenolic compounds gallic acid, protocatechuic acid, catechin, vanillic acid, epicatechin and syringic acid, chlorogenic acid, gentisic acid, caffeic acid, p-coumaric acid and ferulic acid, rutin, myricetin, and quercetin were quantified in line with a previously reported method [27]. Briefly, eluent A was Milli-Q water with 0.1% formic acid and eluent B was methanol. The elution conditions applied were: 0–5 min, 20% B isocratic; 5–30 min, linear gradient from 20% to 60% B; 30–35 min, 60% B isocratic; 35–40, linear gradient from 60% to 20% B and finally, washing and reconditioning of the column. Each standard was individually tested to determine its retention times (RT) as follows: gallic acid (RT: 5.3 min), protocatechuic (RT: 10.0 min), catechin (RT: 12.7 min), chlorogenic acid (RT: 14.9 min), gentisic acid (RT: 17.1 min), vanillic acid (RT: 17.7 min), epicatechin (RT: 17.9 min), caffeic acid (17.9 min), syringic acid (RT: 18.9 min), coumaric acid (RT: 23.4 min), rutin (RT: 28.1 min), ferulic acid (RT: 24.3 min), myricetin (RT: 30.6 min), and quercetin (RT: 34.6 min) were well resolved. Simultaneous monitoring was set at 270 nm (gallic acid, protocatechuic acid, (+) catechin, vanillic acid, (−) epicatechin and syringic acid), 324 nm (chlorogenic acid, gentisic acid, caffeic acid, p-coumaric acid and ferulic acid), and 373 nm (rutin, myricetin, and quercetin) for quantification. The limits of detection (LOD) and limits of quantification (LOQ) were estimated from the signal-to noise-ratio of the individual peaks, assuming a minimum detectable signal-to-noise level of 3 and 10, respectively. The LODs were found to be in the range of 0.0003–0.1230 μg·mL−1 and the LOQs were observed in the range of 0.0008–0.4100 μg·mL−1. This indicated that the proposed method was suitably sensitive for the quantification of polyphenols. The linearity was assessed by linear regression analysis, which was calculated by the least square method. Each point on the calibration plot was the mean from two area measurements. All the correlation coefficients were no less than 0.9982. Reproducibility, expressed as the relative standard deviation (RSD), was obtained by analyzing six replicate sample RSDs values ranging from 1.91% to 5.81%. The accuracy was expressed as the recovery of standard compounds added to the pre-analyzed sample. The recovery was found to be in the range of 87.97%–115.79%. 3.8. Evaluation of the Antimycoplasmic Activity The antimycoplasmic activity of all plant extracts was determined using a modified disc diffusion method as described for growth inhibition tests elsewhere [43]. A total of 25 μL of each extract was tested in the disc diffusion assay against four strains of mycoplasmas. The plates were subsequently incubated and examined daily for colonies (1–2 days) under the conditions described above. Organisms were considered resistant when their growth was not inhibited by the 25 µL extract-impregnated wafers (5-mm sterilized filter paper discs). The presence of a zone of inhibition, as well as any changes in the colony morphology or in the colony concentration, was considered to be indicative of antimycoplasmic activity. The inhibition zones were measured in µm using an optical microscope Olympus CKX41 (Olympus, Hamburg, Germany), with a digital camera ProgRes C12 plus (Jenoptik, Jena, Germany) inserted, and using ProgRes® Image Capture Software for the measurements. Each antimycoplasmal assay was performed at least in triplicate and inhibition zones were measured at least three times per well, at perpendicular angles. Mean values and standard Molecules 2013, 18 4951 deviations (SD) were registered and calculated as mean ± SD to the effects of this study. Filter discs impregnated with 25 μL of Tilmicosin (0.4 μg/mL) were used as positive control for antimicrobial activity and impregnated with 25 μL of distilled sterilised water were used as negative controls. 4. Conclusions Phenolic compounds are active principles of medicinal plants and exhibit pharmacological effects that contribute towards human health. 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