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Facultad De Enfermería Maestría En Salud Pública METABOLITOS SECUNDARIOS, ACTIVIDAD CITOTÓXICA Y ANTIINFLAMATORIA DE SEIS E

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Facultad De Enfermería Maestría En Salud Pública METABOLITOS SECUNDARIOS, ACTIVIDAD CITOTÓXICA Y ANTIINFLAMATORIA DE SEIS E Facultad de Enfermería Maestría en Salud Pública METABOLITOS SECUNDARIOS, ACTIVIDAD CITOTÓXICA Y ANTIINFLAMATORIA DE SEIS ESPECIES VEGETALES PROVENIENTES DE LA AMAZONÍA PERUANA, IQUITOS - 2019 Tesis para optar el grado académico de Maestro en Salud Pública Presentado por: Mario Javier De la Cruz Flores Asesor(es): Dra. Dora Enith García de Sotero Dr. Víctor Erasmo Sotero Solís Iquitos - Perú 2019 Facultad de Enfermería Maestría en Salud Pública METABOLITOS SECUNDARIOS, ACTIVIDAD CITOTÓXICA Y ANTIINFLAMATORIA DE SEIS ESPECIES VEGETALES PROVENIENTES DE LA AMAZONÍA PERUANA, IQUITOS - 2019 Tesis para optar el grado académico de Maestro en Salud Pública Presentado por: Mario Javier De la Cruz Flores Asesor(es): Dra. Dora Enith García de Sotero Dr. Víctor Erasmo Sotero Solís Iquitos - Perú 2019 ii iii TESIS APROBADA EN SUSTENTACION PÚBLICA EL 27 DE MAYO DEL 2019 EN EL AUDITORIO DE LA ESCUELA DE POSTGRADO DE LA UNIVERSIDAD NACIONAL DE LA AMAZONÍA PERUANA, EN LA CIUDAD DE IQUITOS – PERÚ. iv DEDICATORIA A Dios todopoderoso que hizo los cielos y la tierra, y todo lo que en ello hay. Autor del amor, la vida y la ciencia; quien me mostró un propósito, el cual yo abrasé. A mis padres: Manuela de Jesús Flores Núñez y Sergio De la Cruz Peralta (quien descansa en paz), porque son el motor y apoyo incondicional para llegar a ser un profesional. A mis hijas Evelyn Gabriela De la Cruz Babilonia y Maricielo Samantha De la Cruz Babilonia, quienes son mi adoración, motivación e inspiración por seguir adelante. A mi hermano Serman De la Cruz Flores, quien me brinda el apoyo incondicional por salir adelante y seguir superándome día a día. v AGRADECIMIENTO A Dios por la vida, perseverancia y fuerza para ser mejor persona, por las pruebas que a diario tengo que soportar y esto me enseña a no rendirme ante los obstáculos que se me presenta en el camino; y gracias a EL estoy aquí de pie, con los deseos de cumplir con mis objetivos, para seguir siendo una persona y profesional de bien. A los todos los docentes de la maestría en Salud Pública de la Universidad Nacional de la Amazonía Peruana – UNAP, por haberme impartido conocimientos, educación y valores. A la Fundación para el Desarrollo Sostenible de la Amazonía Baja - FUNDESAB y CIENCIA ACTIVA, quienes hicieron posible el financiamiento total de este proyecto de investigación, apostando toda su confianza en mi persona, logrando todos los objetivos y metas durante la realización del trabajo. A la Dra. Dora Enith García de Sotero y al Dr. Víctor Erasmo Sotero Solís, quienes con su apoyo, guía, conocimientos científicos y paciencia; han permitido desarrollar y terminar de manera favorable ésta tesis. vi ÍNDICE DE CONTENIDO Pág. Carátula i Contracarátula ii Acta de sustentación iii Jurados iv Dedicatoria v Agradecimiento vi Índice de contenido vii Índice de tablas ix Índice de figuras x Resumen 1 Abstract 2 INTRODUCCIÓN 3 CAPITULO I: MARCO TEÓRICO 1.1 Antecedentes 5 1.2 Bases teóricas 8 1.3 Definición de términos básicos 23 CAPITULO II: VARIABLES E HIPÓTESIS 2.1 Variables y su operacionalización 24 2.2 Formulación de la hipótesis 28 CAPÍTULO III: METODOLOGÍA 3.1 Tipo y diseño de investigación 29 3.2 Población y muestra 29 3.3 Técnicas e instrumentos 30 3.4 Procedimiento de recolección de datos 30 3.5 Técnicas de procesamiento y análisis de datos 38 3.6 Aspectos éticos 38 CAPITULO IV: RESULTADOS Actividad citotóxica 40 Actividad antiinflamatoria in vitro 43 Actividad antiinflamatoria in vivo 44 Metabolitos secundarios 46 vii CAPITULO V: DISCUSIÓN DE LOS RESULTADOS 48 CAPITULO VI: PROPUESTA 50 CAPITULO VII: CONCLUSIONES 51 CAPITULO VIII: RECOMENDACIONES 52 CAPITULO IX: REFERENCIAS BIBLIOGRÁFICAS 53 ANEXOS 61 viii ÍNDICE DE TABLAS Pág. Tabla 1 Modelo experimental antiinflamatorio in vivo 36 Tabla 2 Cantidad de nauplios de Artemia franciscana 40 inyectados - citotoxicidad Tabla 3 Conteo de nauplios vivos y muertos de Artemia 41 franciscana en las disoluciones Tabla 4 Cálculo del porcentaje de efecto, valores probit 42 relacionados y CL50 Tabla 5 Porcentaje de inhibición antiinflamatoria – actividad 43 antiinflamatorio in vitro Tabla 6 Cálculo de IC50 a partir de la ecuación obtenida con su 43 respectivo R2 Tabla 7 Administración de extractos, según tratamiento y 44 tiempo – actividad antiinflamatorio in vivo Tabla 8 Porcentajes de inflamación de cada tratamiento 45 Tabla 9 Cuantificación de componentes químicos 46 Tabla 10 Cuantificación de componentes químicos alcaloides 47 Tabla 11 Cuantificación de componentes químicos saponinas 47 ix ÍNDICE DE FIGURAS Pág. Figura 1. Dilución del extracto para el experimento citotoxicidad 62 Figura 2. Configuración del experimento citotoxicidad 62 Figura 3. Flujograma de preparación de extractos hidroalcohólicos 63 (etanólico) Figura 4. Hoja de trabajo_ensayo de toxicidad por Artemia franciscana 64 Figura 5. Hoja de trabajo_conteo de larvas vivas/muertas de Artemia 65 franciscana Figura 6. Hoja de trabajo_preparación de extracto 66 Figura 7. Flujograma del ensayo de la actividad citotóxica 67 Figura 8. Flujograma del ensayo de la actividad antiinflamatoria in vitro 68 Figura 9. Flujograma del ensayo de la actividad antiinflamatoria in vitro 69 Figura 10. Constancia de certificación de especies vegetales 70 x RESUMEN El objetivo fue determinar metabolitos secundarios, actividad citotóxica y antiinflamatoria de seis especies vegetales: corteza V. pseudolea Rusby, G. macrophylla Mart., R. pedunculata (H. Karst.) Flueck, G. ulmifolia Lam, G. madruno (Kunth) Hammel y hojas C. cajan (L.) Huth. Muestras acondicionadas, secadas en deshumecedor a 35°C por seis días, posteriormente pulverizadas. La ACIT fue sobre nauplios de A. franciscana 1500, 1000, 500, 50, 10 ppm; obteniéndose CL50 vía Probit. La AAI in vitro 0,2 mL albúmina de huevo, 2,8 mL buffer fosfato y 2 mL de concentraciones de muestras, siendo concentraciones finales 31,25, 62,5, 125, 250, 500, 1000 ug/mL, absorbancia 660nm e IC50 por curva dosis-respuesta. La AAI in vivo por edema plantar en ratón inducido por formol 10%, extractos vía oral 125mg/kg y 250mg/kg vs Diclofenaco 10mg/kg. Los resultados obtenidos G. ulmifolia Lam CL50=9,12ppm; G. madruno (Kunth) Hammel CL50=2,14ppm, C. cajan (L.) Huth CL50=1,52ppm. La AAI in vitro G. macrophylla Mart. IC50=399,29 ug/mL; V. pseudolea Rusby IC50=321,21 ug/mL, R. pedunculata (H. Karst.) Flueck IC50=319,97 ug/mL. La AAI in vivo G. macrophylla Mart. presentó mejor AAI a 125 mg/kg con 29,99% inflamación vs Diclofenaco 32,00%; V. pseudolea Rusby 32.23% inflamación vs Diclofenaco 29,49%; R. pedunculata (H. Karst.) Flueck 43,30% inflamación vs Diclofenaco 26,81%. Se concluye que las especies en estudio presentan ACIT y AAI. Palabras claves: Actividad citotóxica, Actividad antiinflamatoria. Metabolitos secundarios 1 ABSTRACT The objective was to establish secondary metabolites, cytotoxic activity and anti-immflamatory of six plant species: barks from V. pseudolea Rusby, G. macrophylla Mart., R. pedunculata (H. Karst.) Flueck, G. ulmifolia Lam, G. madruno (Kunth) Hammel and leaves from C. cajan (L.) Huth. The samples were conditioned, dried in a dehumidifier at 35°C for six days, and subsequently in powder. The ACIT was about nauplii of A. franciscana 1500, 1000, 500, 50, 10 ppm; obtaining CL50 via Probit. The AAI in vitro 0.2mL of egg albumin, 2.8mL of phosphate-buffered-saline and 2mL of samples concentrations, being the final concentrations 31.25, 62.5, 125, 250, 500, 1000 ug/mL, absorbance 660nm and IC50 by dose-response curve. The AAI in vivo by plantar edema in mice induced for 10% formalin, were extracted orally 125mg/kg and 250mg/kg vs Diclofenac 10mg/kg. The obtained results G. ulmifolia Lam CL50=9.12ppm; G. madruno (Kunth) Hammel CL50=2.14ppm, C. cajan (L.) Huth CL50=1.52ppm. The AAI in vitro G. macrophylla Mart. IC50=399.29 ug/mL; V. pseudolea Rusby IC50=321.21 ug/mL, R. pedunculata (H. Karst.) Flueck IC50=319.97 ug/mL. The AAI in vivo G. macrophylla Mart. presented the best AAI to 125 mg/kg with 29.99% of immflamation vs Diclofenac 32.00%; V. pseudolea Rusby 32.23% of immflamation vs Diclofenac 29.49%; R. pedunculata (H. Karst.) Flueck 43.30% of immflamation vs Diclofenac 26.81%. It concludes that the species in the study present ACIT and AAI. Key words: Anti-immflamatory activity, cytotoxic activity, secondary metabolites 2 INTRODUCCIÓN En el Perú existen alrededor de 4440 especies vegetales potencialmente útiles según estudios etnobotánicos, los cuales indican que 1080 especies son utilizadas en la Amazonía para tratar diferentes enfermedades 1. Los productos naturales derivados de plantas medicinales, continúan en aumento y anualmente ingresan a los mercados nuevos productos, generando preocupación en torno a la seguridad de la salud; esto se debe a que muchos de estos remedios naturales con potenciales efectos beneficiosos, aún no han sido probados y su uso no está controlado. Esto hace que el conocimiento de sus potenciales efectos adversos sea limitado y la identificación de terapias seguras y eficaces, así como la promoción de su uso racional sea difícil 2. El estudio y la investigación de los metabolitos secundarios ayudan a comprender la bioquímica y fisiología de los organismos que los producen y su mejor aprovechamiento con fines farmacológicos 3. La importancia en el estudio de plantas medicinales de la Amazonía Peruana, radica en la forma en que éstas son utilizadas, qué parte es empleada y lo más importante cual es la finalidad de su uso; además del gran potencial para nuevos y mayores mercados nacionales y mundiales. En la actualidad, el poblador amazónico sigue dando crédito al uso de vegetales con propiedades bondadosas en el tratamiento de diversas enfermedades, muchas veces con resultados positivos, aunque con base científica muy deficiente. Por tal motivo nace el interés de generar nuevos conocimientos con base científica, acerca de la actividad citotóxica y antiinflamatoria que poseen algunos vegetales procedentes de la Amazonía y que son muy usadas por los pobladores, como lo son: Vitex pseudolea Rusby, Garcinia macrophylla Mart, Garcinia madruno (Kunth) Hammel, Cajanus cajan (L.) Remijia pedunculata (H.
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