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“Efectos Antidiabéticos En Extractos De Jarabes De Agave Y Evaluación De Su Contenido De Fenólicos Y Actividad Antioxidante”

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“Efectos Antidiabéticos En Extractos De Jarabes De Agave Y Evaluación De Su Contenido De Fenólicos Y Actividad Antioxidante” INSTITUTO TECNOLÓGICO Y DE ESTUDIOS SUPERIORES DE MONTERREY CAMPUS MONTERREY DIVISIÓN DE BIOTECNOLOGÍA Y ALIMENTOS “Evaluación del contenido de fenólicos, actividad antioxidante y efectos antidiabéticos de extractos de jarabes de agave (A. atrovirens Karw)” TESIS PRESENTADA COMO REQUISITO PARCIAL PARA OBTENER EL GRADO ACADÉMICO DE: MAESTRÍA EN CIENCIAS CON ESPECIALIDAD EN BIOTECNOLOGÍA POR: LIC. NUT. KARLA LETICIA CERDA DE LOS SANTOS MONTERREY, N. L. MAYO 2011 “Efectos antidiabéticos en extractos de jarabes de agave y evaluación de su contenido de fenólicos y actividad antioxidante” Resumen Agave L. pertenece a la familia Agavaceae y forma parte de las tradiciones religiosas, alimentarias y medicinales de México y Latinoamérica. Ancestralmente ha sido utilizado en el tratamiento de enfermedades crónico-degenerativas como la Diabetes Mellitus (DM). Dado que compuestos fenólicos de diversas matrices biológicas han reportado capacidad de secretar insulina y de inhibir la actividad de enzimas digestivas que hidrolizan hidratos de carbono, entre otros efectos, el objetivo de este proyecto fue evaluar la capacidad de los extractos de jarabes de agave (A. atrovirens Karw) para ejercer los mencionados efectos. Para profundizar nuestro conocimiento se caracterizaron dichos extractos obtenidos con 2 disolventes (acetona y metanol) a partir de 3 jarabes en función de la cantidad de compuestos fenólicos y su actividad antioxidante medida por el método ORAC. La capacidad secretora de insulina de los extractos se determinó por un método ELISA en células de insulinoma de páncreas y la inhibición de liberación de glucosa en un ensayo enzimático in vitro. Los valores más altos del contenido de fenólicos totales y de la actividad antioxidante fueron encontrados en los extractos obtenidos con acetona (19.26 ± 0.42 mg EAG/g extracto, 180.42 ± 2.62 µmol ET/g extracto), sin embargo su rendimiento de extracción (5- 6%) fue cinco veces menor que en los extractos obtenidos con metanol (20-27%). Los extractos metanólicos del jarabe Mimex y Nazario fueron los que presentaron actividad antidiabética más alta. El extracto metanólico del jarabe Mimex mostró un valor de insulina secretada (6.43 ± 0.76 ng/mL, concentración de 200µg/mL) similar al de la glibenclamida (6.99 ± 0.57 ng/mL, concentración de 200µM), un hipoglucemiante oral ampliamente utilizado en pacientes diabético. Mientras que el extracto metanólico del jarabe Nazario disminuyó el índice glucémico (28.27 ± 2.96 % inhibición en invertasa y 42.74 ± 3.06 % inhibición en amiloglucosidasa). Se determinó que el compuesto responsable de la capacidad secretora de insulina no pertenece al grupo de compuestos fenólicos. Mientras que la inhibición de la liberación de glucosa posiblemente se debe a la presencia de moléculas hidroxiflavononas que actúan en sinergia en el extracto. Finalmente se concluye que los extractos del jarabe de agave son alimentos nutracéuticos en la profilaxis y el tratamiento de DM. Sin embargo, se deben hacer análisis posteriores para determinar el o los compuestos presentes en los extractos metanólicos que ejercen las bioactividades antes mencionadas. i “Efectos antidiabéticos en extractos de jarabes de agave y evaluación de su contenido de fenólicos y actividad antioxidante” Agradecimientos Agradezco a Dios por la vida que me ha regalado, porque siempre está conmigo en cada momento sin soltarme de su mano. ¡Gracias niñito por tu ayuda con las células y enzimas! Gracias a mi familia por apoyarme en cada instante de mi vida. Mamá: la persona y profesional que soy hoy es gracias a ti y a tu ejemplo, gracias por siempre impulsarme y no dejarme rendir. Nana: gracias por hacerme de comer, aconsejarme y escucharme. Chiquirronga: gracias por tu empuje y tus consejos. Vale: gracias por tus sonrisas. Gracias Pris, Frida y Tanya: por su apoyo, comprensión y alegría. Gracias al ITESM y al Conacyt por brindarme el apoyo becario durante estos dos años. Gracias a mi asesora, la Dra. Janet por guiarme en el camino de la ciencia y la investigación, por su tiempo, sus ideas y sus conocimientos, pero sobre todo por su buena disposición y paciencia. Gracias al Dr. Serna por permitirme pertenecer a su cátedra de investigación y por su tiempo dedicado a este proyecto. Gracias al Dr. Silverio por su ayuda durante estos últimos meses, los cuales fueron cruciales para el desarrollo de este documento. Gracias a cada uno de mis maestros del posgrado, el compartir sus conocimientos contribuyó a mi formación profesional y personal. Especialmente al Dr. Welti, por iniciarme en el aprendizaje de la lectura y la redacción científica. Gracias a cada una de las personas que compartieron sus conocimientos prácticos en el laboratorio, en especial: Dra. Esther, M.C. Irasema, M.C. Cristina, M.C. Dulce, M.C. Liliana, M.C. Marisol, M.C. Daniel, Ing. Enrique y Felipe. Gracias al Lic. Albino Vargas, por la información brindada en todo momento sobre los jarabes de agave. Gracias a mis compañeros del posgrado y amigos de laboratorio: Marce, Dul, Kike, Kei, Lau, Uli, Vini, Fede, Victor, Marco, Betty, Chuy, Bertha, Alma, Edna. La experiencia no pudo ser más satisfactoria. Gracias al personal del Centro de Biotecnología: Don Francisco, Mary Carmen, Isela y Miriam. Por último, pero sin restarle importancia, quiero dedicar este proyecto a cada una de las personas que viven con Diabetes, recordando que es tiempo de permitir que los alimentos sean nuestro medicamento. ii “Efectos antidiabéticos en extractos de jarabes de agave y evaluación de su contenido de fenólicos y actividad antioxidante” Índice General Resumen i Agradecimientos ii Índice General iii Índice de Tablas vi Índice de Figuras viii Lista de Abreviaturas xi Capítulo I Introducción 1 Capítulo II Antecedentes 3 2.1 Agave 3 2.1.1 Fitoquímicos nutracéuticos del agave 5 2.1.2 Actividad biológica del agave 6 2.1.2.1 Estudios in vitro 7 2.1.2.2 Estudios in vivo 8 2.1.3 Composición del jarabe de agave 11 2.2 Diabetes Mellitus 13 2.2.1 Síntesis y secreción de insulina 14 2.2.2 Etiología 16 2.2.3 Estrés oxidativo en Diabetes Mellitus 19 2.2.4 Diagnóstico de Diabetes Mellitus 21 2.2.5 Tratamiento farmacológico de Diabetes Mellitus 23 2.2.6 Tratamiento nutracéutico de Diabetes Mellitus 24 2.3 Índice glucémico 26 2.3.1 Determinación del índice glucémico in vivo 27 2.3.2 Determinación del índice glucémico in vitro 28 2.3.3 Efecto de fitoquímicos sobre el índice glucémico 28 2.4 Justificación 30 2.5 Objetivos 30 iii “Efectos antidiabéticos en extractos de jarabes de agave y evaluación de su contenido de fenólicos y actividad antioxidante” CapítuloIII Materiales y Métodos 31 3.1 Fuentes de jarabes de agave 31 3.2 Extracción de compuestos 31 3.3 Caracterización de los extractos de jarabes de agave 32 3.3.1 Cuantificación de compuestos fenólicos totales 32 3.3.2 Identificación de ácidos fenólicos y flavonoides por HPLC 32 3.3.2.1 Preparación de muestras para análisis por HPLC 32 3.3.2.2 Detección de compuestos en HPLC 33 3.3.3 Determinación de la capacidad antioxidante (ORAC) 35 3.4 Bioensayo de secreción de insulina en la línea celular RIN-5F 35 3.4.1 Mantenimiento de la línea celular RIN-5F 36 3.4.2 Bioensayo de estimulación de secreción de insulina 36 3.4.3 Cuantificación del contenido de proteína total y porcentaje de viabilidad 37 3.4.4 Cuantificación de insulina 37 3.5 Determinación de la inhibición de glucosa liberada siguiendo la metodología de índice glucémico in vitro 38 3.5.1 Preparación de la muestra 38 3.5.2 Medición de glucosa libre 38 3.5.3 Medición de glucosa total 39 3.5.4 Cuantificación de azúcares en HPLC-ELSD 40 3.5.5 Evaluación del efecto de los extractos de jarabe de agave sobre la liberación de glucosa 41 3.5.5.1 Tratamiento enzimático con invertasa 41 3.5.5.2 Tratamiento enzimático con amiloglucosidasa 41 3.6 Análisis estadístico 41 CapítuloIV Resultados y Discusión 42 4.1 Rendimiento de extracción acetónica y metanólica de jarabes de agave 42 4.2 Contenido de fenólicos totales de los extractos de jarabes de agave 42 4.3 Identificación de ácidos fenólicos y flavonoides en los extractos de jarabes de agave 44 4.4 Capacidad antioxidante de los extractos de jarabes de agave 50 4.5 Capacidad de secreción de insulina en la línea celular RIN-5F de los extractos de jarabes de agave 51 iv “Efectos antidiabéticos en extractos de jarabes de agave y evaluación de su contenido de fenólicos y actividad antioxidante” 4.6 Efecto de los extractos de jarabe sobre la liberación de glucosa 54 CapítuloV Conclusiones y Recomendaciones 58 Referencias 60 Anexo A: Cromatogramas de los extractos 72 Anexo B: Espectros UV-visible de los compuestos encontrados en los extractos 76 Anexo C 79 v “Efectos antidiabéticos en extractos de jarabes de agave y evaluación de su contenido de fenólicos y actividad antioxidante” Índice de Tablas Tabla 2.1: Sapogeninas esteroidales en agave 5 Tabla 2.2: Efectos nutracéuticos de fitoquímicos asociados a la familia Agavacea con efectos positivos en la salud 10 Tabla 2.3: Características fisicoquímicas del aguamiel de tres variedades de maguey pulquero (Agave ssp) 12 Tabla 2.4: Prevalencia de Diabetes Mellitus e intolerancia a la glucosa en el mundo 13 Tabla 2.5: Factores y condiciones que incrementan la secreción de insulina 15 Tabla 2.6: Causas de resistencia a la insulina 18 Tabla 2.7: Criterios para el diagnóstico de prediabetes y Diabetes Mellitus (DM) 22 Tabla 2.8: Clasificación de alimentos según índice glucémico (IG) 27 Tabla 3.1: Jarabes de agave evaluados 31 Tabla 3.2: Estándares de compuestos fenólicos 34 Tabla 3.3: Gradiente utilizado
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