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Gestión De Calidad CONSEJO NACIONAL DE CIENCIA Y TECNOLOGIA –CONCYT- SECRETARIA NACIONAL DE CIENCIA Y TECNOLOGIA-SENACYT- FONDO NACIONAL DE CIENCIA Y TECNOLOGIA -FONACYT- FACULTAD DE CIENCIAS QUÍMICAS Y FARMACIA UNIVERSIDAD DE SAN CARLOS DE GUATEMALA INFORME FINAL “Evaluación química, nutricional y biológica de cinco especies de basidiomicetos para el diseño y formulación de un producto con aplicación medicinal, cosmética y alimenticia” PROYECTO FINDECYT/FODECYT 54-2017 Dra. Sully Margot Cruz Velásquez Investigador Principal Guatemala, mayo 2020. AGRADECIMIENTOS La realización de este trabajo, ha sido posible gracias al apoyo financiero dentro del Fondo Nacional de Ciencia y Tecnología -FONACYT-, otorgado por la Secretaría Nacional de Ciencia y Tecnología -SENACYT- y al Consejo Nacional de Ciencia y Tecnología –CONCYT-. i OTROS AGRADECIMIENTOS A la Universidad de San Carlos de Guatemala, Facultad de Ciencias Químicas y Farmacia por el aval institucional, la infraestructura brindada, materiales y equipo para la ejecución del proyecto. Al Laboratorio de Investigación de Productos Naturales (LIPRONAT), y Depto de Farmacognosia y Fitoquímica de la Escuela de Química Farmacéutica por brindar la infraestructura, materiales y apoyo del personal que colaboró para la ejecución del proyecto. Al Laboratorio de Productos Naturales Farmaya S.A. especialmente a Lic. Armando Cáceres por el apoyo, asesoría y colaboración brindada en el desarrollo de la investigación. Al equipo de investigación por su compromiso, entrega y dedicación especialmente a Dra. Dora Elena Chang, Lic. Roberto Cáceres, Licda. Nereida Marroquín, Br. Libny Pernillo, Akassia Stefania Hengstenberg, Jaqueline Lourdes Alvarado Cárdenas. ii ÍNDICE Página RESUMEN ABSTRACT 1 PARTE I I.1 INTRODUCCIÓN I.2 PLANTEAMIENTO DEL 1 PROBLEMA I.3 OBJETIVOS E HIPOTESIS 4 I.3.1 Objetivos I.3.1.1 General I.3.1.2 Específicos I.3.1.3 Hipótesis I.4 METODOLOGIA 8 I.4.1 Las Variables 1.4.1.1 Variables dependientes 1.4.1.2 Variables Independientes I.4.2 Indicadores I.4.3 Estrategia Metodológica I.4.3.1 Población y Muestra I.4.4 El Método I.4.5 La Técnica Estadística I.4.6 Los Instrumentos a utilizar I.4.7 Cronogramas PARTE II 20 MARCO TEÓRICO PARTE III 44 III. RESULTADOS III.1 Discusión de Resultados 65 PARTE IV IV.1 CONCLUSIONES 70 IV.2 RECOMENDACIONES 72 IV.3 REFERENCIAS 73 BIBLIOGRÁFICAS IV.4 ANEXOS 81 PARTE V V.1 INFORME FINANCIERO 99 iii Índice de Tablas y Cuadros Número Título de Tablas Página 1 Cronograma de actividades 25 2 Cronograma de desembolsos 26 Título de Cuadros 1 Información de colecta 51 2 Determinación de humedad 52 3 Cenizas totales y cenizas ácido insoluble 53 4 Determinación de mejor disolvente 54 5 Rendimiento de extracto 54 6 Identificación de cumarinas en material y extracto seco 55 7 Identificación de taninos en material fúngico y extracto 56 8 Identificacion de alcaloides en material fúngico y extracto 56 9 Cromatografía en capa fina de alcaloides en material fúngico 57 10 Identificación de saponinas en material fúngico 57 11 Cromatografía en capa fina de saponinas 58 12 Cromaografía en capa fina de flavonoides en extractos 59 13 Identificación de azúcares por CCF 60 14 Identificación de estándares de azúcares por HPLC 61 15 Detección de azúcares en extractos por HPLC 61 16 Cuantificación de azúcares en extractos por HPLC 62 17 Evaluación de la actividad antioxidante 63 18 Cuantificación de fenoles totales 64 19 Actividad antioxidante de base al poder reductor de hierro 64 (FRAP) 20 Evaluación de la actividad antiureasa por CCF en extractos 65 21 Porcentaje de rendimiento de aceite fijo 67 22 Composición nutricional de hongos 68 23 Evaluación de la actividad antibacteriana 69 24 Actividad citotóxica contra Artemia salina 69 25 Análisis de oligoelementos en muestras secas 70 26 Propuesta de productos en base a hongo fresco 71 27 Propuesta de productos en base a hongo seco y extracto 72 iv RESUMEN Los basidiomicetos también conocidos como macromicetos se consideran una fuente importante de productos naturales biológicamente activos. Por lo que la evaluación de la actividad biológica, composición química y nutricional de especies que crecen en zonas de alto endemismo o se cultivan en diferentes áreas rurales es importante para establecer su potencial para propiciar su conservación y su mejor aprovechamiento. Es por ello que se evaluó la antioxidante, antimicrobiana, enzimática y citotóxica, composición nutricional y química de cinco basidiomicetos y se desarrollaron productos a base de extractos y material fúngico con fines nutricionales. De acuerdo a los resultados se evidención el potencial antioxidante y valor nutricional de las especies. Se presentaron rendimientos de extracción muy promisorios hasta un 63.72% en extactos etanólicos y 28.18 % en extractos acuosos en Boletus edulis. Se determinó la presencia de alcaloides, saponinas, flavonoides, azúcares (trehalosa, glucose y fructosa) mediante ensayos macro y semimicro, cromatografía en capa fina y cromatografía líquida. Se demostró el valor nutricional por la presencia de fibra cruda (6.74-49.22%), proteína cruda (18.20-57.41%), carbohidratos (1.59-55.21%), minerales (7.08- 20.97%), y grasas (1.09-6.44%). Se demostró la actividad antioxidante por tres diferentes métodos, la mayor actividad se presentó en Agaricus (DPPH CI 50 1.99 mg/mL, ABTS 5.82 mg/mL, FRAP 4.33 g Fe+2/g extracto) y B. edulis (DPPH 2.46 mg/mL, ABTS 4.87 mg/mL, FRAP 3.53 g Fe+2/g extracto), dicha actividad mostró relación con la presencia de fenoles totales. Por lo que se demuestra el potencial de los hongos a nivel nutricional para el fomento de la salud. v ABSTRACT Basidiomycetes also known as macromycetes are considered an important source of biologically active natural products. Therefore, the evaluation of the biological activity, chemical and nutritional composition of species that grow in areas of high endemism or are cultivated in different rural areas is important to establish their potential to promote their conservation and better use. That is why the antioxidant, antimicrobial, enzymatic and cytotoxic, nutritional and chemical composition of five basidiomycetes was evaluated and products based on extracts and fungal material were developed for nutritional purposes. According to the results, the antioxidant potential and nutritional value of the species were evidenced. Very promising extraction yields were presented up to 63.72% in ethanolic extracts and 28.18% in aqueous extracts in Boletus edulis. The presence of alkaloids, saponins, flavonoids, sugars (trehalose, glucose and fructose) was determined by macro and semi-micro tests, thin layer chromatography and liquid chromatography. The nutritional value was demonstrated by the presence of crude fiber (6.74- 49.22%), crude protein (18.20-57.41%), carbohydrates (1.59-55.21%), minerals (7.08-20.97%), and fats (1.09-6.44%). Antioxidant activity was demonstrated by three different methods, the highest activity was found in Agaricus (DPPH CI 50 1.99 mg / mL, ABTS 5.82 mg / mL, FRAP 4.33 g Fe + 2 / g extract) and B. edulis (DPPH 2.46 mg / mL, ABTS 4.87 mg / mL, FRAP 3.53 g Fe + 2 / g extract), said activity was related to the presence of total phenols. So the potential of mushrooms at the nutritional level for the promotion of health is demonstrated. vi PARTE I I.1 INTRODUCCIÓN La extraordinaria biodiversidad fúngica de la región latinoamericana y especialmente la región mesoamericana, y la reducida información técnica y científica existente sobre su caracterización, actividad biológica o composición química, hace necesario realizar estudios sobre dichos recursos naturales poco explotados. Es conocido que ciertos componentes macromoleculares aislados de hongos superiores, tales como polisacáridos, glicoproteínas, ácidos nucleicos, etc., poseen actividad antitumoral, antimicrobiana y antioxidante relacionada con la activación que provocan dichas sustancias de la respuesta inmunológica. Los polisacáridos antitumorales producidos por basidiomicetos han ido recibiendo en los últimos años una creciente atención, apareciendo en la literatura reportes sobre la obtención de β-glucanos, polisacáridos antitumorales aislados a partir de hongos comestibles o medicinales tales como Grifola frondosa, Polyporus confluens, Hericium erinaceum, Lentinus edodes y Coriolus versicolor. Los basidiomicetos fueron usados como alimento, medicamento, y ritualmente por los mayas desde tiempos inmemorables, encontrándose evidencias arqueológicas de sus uso como los famosos hongos piedra. Desde mediados del siglo pasado se evidenció el consumo tradicional de basidiomicetos por la población, demostrándose en estudios posteriores un difundido uso en la población rural, por lo que se le llama una cultura micofílica, que aunado a una amplia diversidad de especies presentes, hace de este un tema prioritario para la investigación de nuestra diversidad y el planteamiento de su aprovechamiento racional, ya que son muy pocos los estudios químicos y farmacológicos que validen su uso popular y propiedades atribuidas. 1 I.2 PLANTEAMIENTO DEL PROBLEMA I.2.1 Antecedentes en Guatemala Los hongos comestibles se conocen desde tiempos inmemoriales. Se estima que cerca de 7,000 especies poseen varios grados de comestibilidad, y más de 3,000 especies de 31 géneros se consideran como las principales comestibles (Chang & Miles, 2004). Los análisis de la composición de los hongos cultivados han revelado que los hongos comestibles son ricos en proteínas y carbohidratos, moderados en fibra y cenizas, y bajos en grasas. Su valor energético es bajo, y son una
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