Uso de técnicas separativas acopladas a espectrometría de masas de alta resolución para estudios metabolómicos de nutracéuticos y matrices vegetales

  1. Borrás Linares, María Isabel
Dirigida por:
  1. Alberto Fernández Gutiérrez Director
  2. David Arráez Román Codirector
  3. Antonio Segura Carretero Codirector

Universidad de defensa: Universidad de Granada

Fecha de defensa: 10 de enero de 2014

Tribunal:
  1. Antonia Garrido Frenich Presidente/a
  2. Alberto Zafra Gómez Secretario
  3. Jaroslava Svarc Gajic Vocal
  4. Belén García-Villanova Ruiz Vocal
  5. Vicente Micol Molina Vocal
Departamento:
  1. QUÍMICA ANALÍTICA

Tipo: Tesis

Resumen

RESUMEN En esta memoria se reúnen los resultados obtenidos durante la realización de la tesis doctoral titulada ¿Uso de técnicas separativas acopladas a espectrometría de masas de alta resolución para estudios metabolómicos de nutraceúticos y matrices vegetales¿, donde se evalúa el potencial de las hojas de Rosmarinus officinalis y de los cálices de Hibiscus sabdariffa como fuentes de compuestos fenólicos bioactivos para el desarrollo de nutracéuticos, así como la bioactividad que presentan. La memoria se ha dividido en dos secciones: introducción y parte experimental. En la introducción se describen, en primer lugar, las principales características de los alimentos funcionales y los nutracéuticos, así como las diferencias que existen entre ellos. A continuación se detallan las principales características de las dos matrices vegetales bajo estudio, Rosmarinus officinalis e Hibiscus sabdariffa. Posteriormente, se describen los compuestos fenólicos y el lugar que ocupan dentro de los compuestos bioactivos, su estructura, clasificación y bioactividad, prestando especial atención a su papel protector frente a cáncer, infección microbiana y obesidad. También se describen algunos ensayos in vitro para la evaluación de la bioactividad de dichos compuestos y su absorción y metabolismo. Por último, se detallan las diferentes etapas del procedimiento analítico para la determinación de compuestos fenólicos que se han empleado en la parte experimental: tratamiento de muestra (tanto para muestras vegetales como biológicas), separación (en especial la técnica separativa empleada en la parte experimental, LC) y detección (absorción UV-Vis, TOF-MS, QTOF-MS, UHR-Qq-TOF-MS). La parte experimental se divide en dos secciones, metodología y resultados, ambas subdivididas en dos bloques cada una correspondientes a las matrices vegetales estudiadas, Rosmarinus officinalis e Hibiscus sabdariffa. En la metodología se describen para cada bloque los reactivos y las muestras utilizadas en cada uno, así como los métodos de extracción, tratamiento de muestra, cultivos celulares y ensayos para la evaluación de la bioactividad de los compuestos fenólicos de estas matrices usados en cada caso. También se detallan las técnicas analíticas utilizadas a lo largo de los diferentes capítulos y se describen con detalle los parámetros de los métodos utilizados en la determinación de estos compuestos. La sección resultados se divide en dos bloques, que a su vez constan de tres y cuatro capítulos respectivamente, correspondientes a las fuentes vegetales estudiadas, Rosmarinus officinalis e Hibiscus sabdariffa. En ambos bloques se abordan tanto la caracterización de ambas matrices, como estudios de absorción, metabolismo y bioactividad in vitro e in vivo de los compuestos fenólicos de dichas fuentes vegetales. El bloque I se ha centrado en la caracterización exhaustiva de diferentes extractos de hojas de Rosmarinus officinalis de distinta procedencia obtenidos con diferentes sistemas de extracción, con el objetivo de encontrar extractos de variada composición ricos en compuestos bioactivos. Posteriormente se ha evaluado la actividad anticancerígena de uno de los extractos obtenidos anteriormente así como de algunos de sus compuestos aislados en líneas celulares de adenocarcinoma de colon. En el capítulo 1, llevado a cabo en colaboración con el Instituto de Investigación en Ciencias de la Alimentación del Consejo Superior de Investigaciones Científicas (CSIC) de Madrid, se emplea HPLC-DAD-ESI-TOF-MS para estudiar la composición de cinco extractos de Rosmarinus officinalis obtenidos mediante SFE y PLE utilizando diferentes condiciones de extracción. El uso de una columna de pequeño tamaño de partícula que proporciona gran resolución, posibilita la separación de varios isómeros. La información complementaria proporcionada por los distintos detectores, familia del compuesto indicada por los máximos de absorción, masas exactas y distribución isotópica proporcionada por el analizador TOF-MS, permiten la identificación tentativa de compuestos fenólicos de romero para los que no se dispone de patrones comerciales. Como continuación en la búsqueda de fuentes de compuestos bioactivos, en el capítulo 2, realizado en colaboración con la Facultad de Tecnología de Novi Sad (Serbia), se estudia la composición cualitativa y cuantitativa de quince muestras de Rosmarinus officinalis recolectadas en diferentes zonas geográficas de Serbia. La extracción se realizó empleando MAE, y el análisis mediante UPLC-ESI-QTOF-MS. La elevada exactitud de masas y distribución isotópica tanto de los espectros de masas como de masas/masas proporcionada por el analizador QTOF-MS permite la identificación de un gran número de compuestos fenólicos en los diferentes extractos, la mayoría de ellos flavonoides, diterpenos fenólicos y triterpenos tipo abietano. En el capítulo 3, realizado en colaboración con el Instituto de Biología Molecular y Celular (IBMC) de la Universidad Miguel Hernández (Elche), se estudia el potencial bioactivo en tres líneas celulares de adenocarcinoma de colon (HT-29, SW 480 y HGUE-C-1) de un extracto de romero obtenido mediante SFE en el capítulo 1, así como de diferentes compuestos aislados del mismo mediante cromatografía semi-preparativa, en un intento de establecer qué compuestos fenólicos son los responsables de la actividad citotóxica mostrada por el extracto. El bloque II se ha centrado en el estudio de la composición de distintos extractos de Hibiscus sabdariffa, y en la evaluación tanto de su actividad como agente antimicrobiano, como su permeabilidad intestinal y absorción en adipocitos de algunos compuestos presentes en dicho extracto. Asimismo se han realizado estudios de biodisponibilidad y excreción urinaria en humanos de un extracto de Hibiscus sabdariffa enriquecido en polifenoles, para los que se han puesto a punto diferentes procedimientos analíticos para la determinación de estos compuestos en muestras de suero, plasma y orina. En el capítulo 4, realizado en colaboración con el laboratorio de Biología Molecular del Instituto Tecnológico de Tlajomulco (Méjico) se estudia la composición de 25 muestras de diferentes variedades de Hibiscus sabdariffa recolectadas en las principales zonas productoras de Méjico mediante HPLC-ESI-TOF-MS, determinándose el contenido total de compuestos fenólicos, flavonoides y antocianinas, así como los contenidos individuales de cianidina y delfinidina. También se evalúan la actividad antioxidante y la actividad antimicrobiana de los extractos frente a bacterias Gram positivas y Gram negativas, presentando algunos de ellos una capacidad antibacteriana similar a la mostrada por los antibióticos de amplio espectro utilizados como controles positivos. En el capítulo 5, realizado en colaboración con el Instituto de Biología Molecular y Celular (IBMC) de la Universidad Miguel Hernández (Elche), se estudia la permeabilidad en un modelo celular que simula la barrera intestinal de un extracto de Hibiscus sabdariffa enriquecido en polifenoles (PEHS), de su formulación liposomada y de algunos de los compuestos presentes en su composición. El análisis del extracto PEHS mediante UPLC-UHR-Qq-TOF revela la presencia de 7 nuevos compuestos no detectados previamente en dicho extracto. En el capítulo 6, también realizado en colaboración con el Instituto de Biología Molecular y Celular (IBMC) de la Universidad Miguel Hernández (Elche), se estudia la absorción de quercetina y quercetina-3-glucurónido, dos compuestos relacionados con la composición y metabolismo del extracto PEHS, en células pre-adipocíticas 3T3-L1, en las cuales dicho extracto había demostrado una potente actividad antiadipogénica disminuyendo la acumulación de triglicéridos. Para ello se analiza el contenido citoplasmático de estos compuestos a diferentes tiempos de incubación (0, 3, 6, 12, 18 horas) mediante HPLC-ESI-TOF, cuantificándose su contenido y el de los metabolitos encontrados. Por último, en el capítulo 7, realizado en colaboración con el Hospital Universitario Sant Joan de Reus, se evalúan diversos procedimientos de tratamiento de muestra y extracción para el análisis de los compuestos fenólicos del extracto PEHS en muestras de suero, plasma y orina, incluyendo la precipitación de proteínas con distintos disolventes, LLE y SPE utilizando diferentes fases sólidas. Los procedimientos desarrollados se aplican al análisis de muestras de suero, plasma y orina recolectados a diferentes tiempos de un voluntario tras la administración oral de 500 mg de PEHS. Parte de los trabajos experimentales realizados en los capítulos 2, 5 y 7 se desarrollaron en diferentes estancias realizadas en el la Facultad de Tecnología de Novi Sad en Serbia (capítulo 2), y en la sede de la empresa Bruker Daltonik en Bremen, Alemania (capítulos 5 y 7). SUMMARY This work summarizes all the results presented in the PhD thesis entitled: ¿Uso de técnicas separativas acopladas a espectrometría de masas de alta resolución para estudios metabolómicos de nutraceúticos y matrices vegetales¿ (Use of separative techniques coupled to high-resolution mass spectrometry for metabolomic studies of nutraceuticals and vegetable matrices), which assesses the potential of leaves from Rosmarinus officinalis and Hibiscus sabdariffa calyces as a source of bioactive phenolic compounds for developing nutraceuticals. The current report is divided into the Introduction and the Experimental Section. The Introduction presents noteworthy information concerning the main features of nutraceuticals and the differences between these and functional foods. Next, both plant sources under study, Rosmarinus officinalis and Hibiscus sabdariffa are described. Afterwards, phenolic compounds and their role as bioactive compounds are described (structure, classification, and healthy effects, focusing on anticancer, antimicrobial and anti-adipogenic activities) as well as their absorption, metabolism, and some in vitro bioactivity assays. Finally, the different stages of an analytical procedure for determining phenolic compounds used in the experimental section are described: sample treatment (for plant and biological samples), separation (especially the LC separation technique), and detection (UV-Vis spectroscopy, TOF-MS, QTOF-MS and UHR-Qq-TOF-MS). The Experimental Part is divided in two sections, Methodology and Results, with two subsections each one corresponding to the plant matrices under study, i.e. rosemary and roselle. In Methodology, all the reagents, samples, extraction methods, biological samples treatments, cell cultures and bioactivity assays used or applied in the subsequent studies are described for each subsection. The analytical techniques, the detection systems and the parameters of the chromatographic separation and detection systems are thoroughly described as well. The Results Section is composed by 7 chapters, three comprising Subsection I, which is focused on the study of Rosmarinus officinalis, and the other four make up Subsection II, which is related to the study of Hibiscus sabdariffa. In these sections, the composition of these plant matrices is studied, and different studies are conducted on the absorption, metabolism and bioactivity of the phenolic compounds present in these plants. Section I deals with the comprehensive characterization of the bioactive phenolic compounds from different extracts of Rosmarinus officinalis leaves obtained by various extraction systems. The aim of applying different extraction systems to the same plant matrix was to obtain separate extracts with varied compositions of bioactive compounds, and presumably different bioactivity. Then, the anticancer activity of one of these extracts and different compounds isolated by semi-preparative chromatography was assayed in different colon-cancer cell lines. In Chapter 1, HPLC-DAD-ESI-TOF-MS is used to study the composition of five extracts of Rosmarinus officinalis obtained by PLE and SFE under different extraction conditions. The use of a small-particle-size column provides high resolution, making it possible to separate several isomers. The complementary information provided by the different detectors, type of phenolic compound delimited by UV-visible spectroscopy, accurate mass measurements, and true isotopic pattern provided by TOF-MS, enabled the tentative identification of phenolic compounds from rosemary when commercial standards were not available. This work was conducted in collaboration with the Institute of Food Science Research from the Superior Council of Scientific Research (CIAL-CSIC). As a continuation of the search of bioactive phenolic compounds plant sources, in Chapter 2, the qualitative and quantitative composition of fifteen Rosmarinus officinalis samples obtained by MAE was studied by UPLC-ESI-QTOF-MS. The rosemary leaves were collected from different geographical zones of Serbia. The high mass accuracy and true isotopic pattern in both MS and MS/MS spectra provided by a QTOF-MS analyzer enable the identification of a wide range of phenolic compounds in the extracts, including flavonoids, phenolic diterpenes and abietane triterpenoids, among others. This work was conducted in collaboration with the Faculty of Technology from the University of Novi Sad (Serbia). In Chapter 3, the anticancer activities of a rosemary extract obtained by SFE and different compounds isolated from this extract by semi-preparative chromatography are assayed. These studies were conducted in order to determine which phenolic compounds are responsible for the cytotoxic activity showed by the extract, in three different cell lines of colon cancer (HT-29, SW 480 and HGUE-C-1). This work was performed in collaboration with the Institute of Molecular and Cellular Biology (IBMC) of Miguel Hernández University (Alicante, Spain). Section II is focused on the study of the composition of different Hibiscus sabdariffa samples, and their evaluation as an antimicrobial agent. The permeability of Hibiscus sabdariffa compounds and the uptake of some of them in adipocytes were studied. The different analytical procedures for the determination of phenolic compounds of this plant in biological matrices were also assessed. In Chapter 4, the aim was the characterization by HPLC-ESI-TOF-MS of different extracts obtained from 25 varieties of Hibiscus sabdariffa collected at different sites in Mexico. The total content in phenolics, flavonoids, anthocyanins, as well as the individual contents of delphinidin and cyanidin were subsequently determined. Furthermore, the antioxidant capacity and the antibacterial activity of the Hibuscus sabdariffa extracts were evaluated against Gram-negative bacteria (E. coli, S enteritidis) and Gram-positive ones (S. aureus, M. luteus), demonstrating that the extracts were effective against all the bacterial strains tested, as these showed similar antibacterial capacity to broad-spectrum antibiotic, such as gentamycin. This work was performed in collaboration with the Laboratory of Molecular Biology from the Technological Institute of Tlajomulco (Mexico). Chapter 5 evaluates the in vitro permeability of a phenolic-enriched Hibiscus sabdariffa extract (PEHS), a liposomed PEHS extract, and the individual compounds related to this matrix. The analysis of PEHS with the powerful analytical methodology UPLC-UHR-Qq-TOF-MS reveals the present of seven new compounds which had not previously been detected in the extract. Chapter 6 studies the in vitro uptake of quercetin and quercetina-3-glucuronide in adipocytes 3T3-L1 at different incubation times (0, 3, 6, 12, 18 h) by HPLC-ESI-TOF-MS. These compounds are related to the composition and metabolism of PEHS, which showed an anti-adipogenic activity, lowering the levels of triglycerides in these cells. The studies made in Chapter 5 and 6 were performed in collaboration with the Institute of Molecular and Cellular Biology (IBMC) of Miguel Hernández University (Alicante, Spain). Finally, Chapter 7 assesses different sample treatments and extraction procedures for the analysis of phenolic compounds from Hibiscus sabdariffa in serum, plasma and urine samples. The procedures tested include protein precipitation by using different solvents, LLE and SPE using several solid phases. 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