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Abstract(s)
Desde la antigüedad se han utilizado las plantas y las setas como fuentes de nutrientes y también por sus propiedades medicinales. En la actualidad, continua el interés en la búsqueda de productos naturales que den lugar a nuevos fármacos, complementos dietéticos o nutracéuticos, de ahí la importancia del estudio de sus constituyentes químicos y de sus actividades biológicas.
Con el fin de ampliar el conocimiento de estas fuentes naturales y por tanto de sus usos, en este trabajo se han estudiado tres especies de plantas medicinales, Kalanchoe pinnata (Lam.) Oken., Kalanchoe daigremontiana Raym.-Hamet & H.Perrier y Kalanchoe gastonis-bonnieri Raym.-Hamet & H.Perrier y una especie de hongo saprófito, Terana caerulea (Lam.) Kuntze.
El hongo Terana caerulea fue elegido mediante un estudio etnobotánico de la Selva de Irati, a través de entrevistas a 14 personas de 9 localidades diferentes, de un rango de edad de 27-94 años de los valles de Salazar y Aezkoa. Se recopilaron un total de 47 usos farmacológicos tradicionales pertenecientes a 25 plantas de 20 familias diferentes. También se recopilaron los usos de un liquen y dos hongos. Mediante este estudio etnobotánico se escogió el hongo Terana caerulea para su posterior estudio.
La caracterización química de este hongo consistió en la preparación de extractos y fracciones utilizando disolventes de diferentes polaridades: hexano, diclorometano, acetato de etilo, metanol y agua. Estos extractos fueron fraccionados mediante el uso de cromatografía en columna y cromatografías en capa fina.
Los diferentes extractos y fracciones se analizaron mediante espectrometría de masas (GC/MS y HRMS) y resonancia magnética nuclear (1H-RMN, 13C-RMN, COSY, HSQC, HMBC y ROESY), y los resultados se compararon con bases de datos de espectros y con la bibliografía, con el fin de identificar los compuestos presentes.
Se evaluó la actividad citotóxica de extractos, fracciones y compuestos aislados en cuatro líneas celulares tumorales humanas: MCF-7 (carcinoma de mama), NCI-H460 (carcinoma de pulmón), HeLa (carcinoma cervical) y HepG2 (carcinoma hepatocelular); y la evaluación de la citotoxicidad en cultivos primarios de células de hígado de cerdo (PLP2) por el método de la sulforodamina B (SRB).El extracto de AcOEt fue el que presentó los valores más bajos de GI50 en los ensayos de actividad citotóxica frente a las cuatro líneas celulares tumorales evaluadas, siendo más activo frente al carcinoma de mama (MCF-7). A partir de él, se aisló e identificó un compuesto no descrito anteriormente, un derivado del ácido telefórico con tres de los seis grupos hidroxilos metilados: 5',8,8'-trimetoxi-2,9':2',9-diepoxi-9,7'-ciclo-7,9'-neoligna-7(9'),8,7'-trieno-4,4',5-triol. Este presentó actividad citotóxica, pero en menor medida que los resultados del extracto total de AcOEt, con lo que se podría decir que no es el compuesto más citotóxico de toda la composición química de Terana caerulea o que en ese extracto existen sinergias entre diferentes compuestos para lograr una potencia que se pierde en el aislamiento.
La caracterización química de las tres Kalanchoes incluyó la determinación de su valor nutricional y energético mediante el análisis de proteínas, grasas, cenizas y carbohidratos, particularmente azúcares por HPLC-RI y ácidos grasos por GC-FID, y la determinación de otros fitoquímicos como tocoferoles por HPLC-Fluorescencia y ácidos orgánicos por UFLC-DAD. También se evaluó su actividad citotóxica frente a las mismas líneas celulares de los extractos de diferentes polaridades (hexano, DCM, AcOEt, MeOH y agua), infusiones y decocciones.
El valor nutricional de las tres plantas reveló una composición elevada en carbohidratos, baja en lípidos y valores energéticos bastante similares. Sin embargo, la composición de ácidos orgánicos varió mucho según la planta. Los tres Kalanchoes mostraron una relación de ácidos grasos SFA>PUFA>MUFA, siendo en todos los casos mayoritarios los ácidos grasos saturados. Respecto al contenido en azúcares, K. pinnata presentó una mayor cantidad. También contiene la mayor concentración en tocoferoles, siendo γ-tocoferol la isoforma mayoritaria. Todas las plantas presentaron actividad citotóxica, siendo el extracto de BuOH de K. daigremontiana el que mejores resultados mostró, y con lo cual, el extracto que se fraccionó para aislar e identificar los compuestos responsables de la actividad. En este caso se aisló e identificó el compuesto 3-O-β-D-xilopiranosil(1→2)α-L-ramnopiranósido de kaempferol mediante las mismas técnicas espectroscópicas, flavonoide no responsable de toda la bioactividad detectada en el extracto butanólico de K. daigremontiana.
Since Antiquity different plants and fungus have been used as a nutrient source and also for its medical properties. Nowadays, the interest of looking for diet complements or neurasthenic is due to the study of chemical constituents and biological activities. In order to know more about these natural sources and therefor their uses in this research, three plant species have been issued: Kalanchoe pinnata (Lam.) Oken., Kalanchoe daigremontiana Raym.-Hamet & H.Perrier and Kalanchoe gastonis-bonnieri Raym.-Hamet & H.Perrier, and a species of saprophytic fungus, Terana caerulea (Lam.) Kuntze. The fungus Terana caerulea was elected throughout an ethnobotanic study in Irati’s Forest, through interviewing 14 people of 9 different places, from an age-range between 27 to 94 in Salazar and Aezkoa valleys. A total of 47 pharmacological uses have been collected from 25 plants of 20 different families. In addition, the usage of a lichen and two fungi was recorded and through the ethnobotanical study the fungus Terana caerulea was chosen for investigation. Its chemical characteristics consisted on making extracts and fractions using solvents from different polarities: hexane, dichloromethane, ethyl acetate, methanol and water. These extracts were fractioned through column chromatography and using thin layer chromatography for checking the fractions. Different extracts and fractions were analyzed using mass spectrometry (GC/MS y HRMS) and nuclear magnetic resonance (1H-RMN, 13C-RMN, COSY, HSQC, HMBC y ROESY), and results were compared with spectrum data bases as well as bibliography to identify the compounds. Antitumoral activity of extracts, fractions and isolated compounds was assessed in four human tumor cell lines: MCF-7 (mama carcinoma), NCI-H460 (lung carcinoma), HeLa (cervical carcinoma) y HepG2 (hepatocellular carcinoma); and cytotoxicity in primary liver pig cells culture (PLP2) by sulphorodamine B method (SRB). EtOAc extract was presenting lowest GI50 in cytotoxic activity assay against the four tumor cell lines evaluated, being more active against mama carcinoma. (MCF-7). XIV Based on that, it was isolated and identified a compound not previously described, a derivate of telephoric acid: 5',8,8'-trimethoxy-2,9':2',9-diepoxy-9,7'-ciclo-7,9'-neoligna-7(9'),8,7'-trieno-4,4',5-triol. This showed cytotoxic activity, but to a lesser extent the results of the total extract of EtOAc, so that compound is not the most cytotoxic of all the chemical composition of Terana caerulea or that in this extract synergies between different compounds to reach a power which is lost in isolation. The chemical characterization of the three Kalanchoes included its nutritional and energetic value by an analysis of proteins, ashes and carbohydrates, particularly sugars by HPLC-RI, fatty acids by GC-FID and determination of other phytochemist like tocopherols by HPLC-Fluorescence and organic acids by UFLC-DAD. Their cytotoxic activities were evaluated against the same cell lines of the different polarity extracts (hexane, DCM, EtOAc, MeOH y water), infusions and decoction. Three plants´ nutritional values revealed a high composition in carbohydrates, low in lipids and energetic values quite similar. However, the composition of organic acids varied a lot depending on the plant. The three Kalanchoes showed a relation among fatty acids SFA>PUFA>MUFA, being mainly saturated fatty acids. Regarding to sugar content, K. pinnata, showed a higher quantity. It also presented the major concentration in tocopherols, being γ-tocopherol the major isoform. Every plant presented cytotoxic activity, being BuOH extract from K. daigremontiana which showed the best results and throughout the fractioned extract which isolated and identified the compounds responsible of activity. In this case the kaempferol 3-O-β-D-xylopyranosyl(1→2)α-L-rhamnopyranoside was isolated and identified with the same spectroscopic techniques. As before, this flavonoid is not the responsible of the whole bioactivity detected in K. daigremontiana butanolic extract.
Since Antiquity different plants and fungus have been used as a nutrient source and also for its medical properties. Nowadays, the interest of looking for diet complements or neurasthenic is due to the study of chemical constituents and biological activities. In order to know more about these natural sources and therefor their uses in this research, three plant species have been issued: Kalanchoe pinnata (Lam.) Oken., Kalanchoe daigremontiana Raym.-Hamet & H.Perrier and Kalanchoe gastonis-bonnieri Raym.-Hamet & H.Perrier, and a species of saprophytic fungus, Terana caerulea (Lam.) Kuntze. The fungus Terana caerulea was elected throughout an ethnobotanic study in Irati’s Forest, through interviewing 14 people of 9 different places, from an age-range between 27 to 94 in Salazar and Aezkoa valleys. A total of 47 pharmacological uses have been collected from 25 plants of 20 different families. In addition, the usage of a lichen and two fungi was recorded and through the ethnobotanical study the fungus Terana caerulea was chosen for investigation. Its chemical characteristics consisted on making extracts and fractions using solvents from different polarities: hexane, dichloromethane, ethyl acetate, methanol and water. These extracts were fractioned through column chromatography and using thin layer chromatography for checking the fractions. Different extracts and fractions were analyzed using mass spectrometry (GC/MS y HRMS) and nuclear magnetic resonance (1H-RMN, 13C-RMN, COSY, HSQC, HMBC y ROESY), and results were compared with spectrum data bases as well as bibliography to identify the compounds. Antitumoral activity of extracts, fractions and isolated compounds was assessed in four human tumor cell lines: MCF-7 (mama carcinoma), NCI-H460 (lung carcinoma), HeLa (cervical carcinoma) y HepG2 (hepatocellular carcinoma); and cytotoxicity in primary liver pig cells culture (PLP2) by sulphorodamine B method (SRB). EtOAc extract was presenting lowest GI50 in cytotoxic activity assay against the four tumor cell lines evaluated, being more active against mama carcinoma. (MCF-7). XIV Based on that, it was isolated and identified a compound not previously described, a derivate of telephoric acid: 5',8,8'-trimethoxy-2,9':2',9-diepoxy-9,7'-ciclo-7,9'-neoligna-7(9'),8,7'-trieno-4,4',5-triol. This showed cytotoxic activity, but to a lesser extent the results of the total extract of EtOAc, so that compound is not the most cytotoxic of all the chemical composition of Terana caerulea or that in this extract synergies between different compounds to reach a power which is lost in isolation. The chemical characterization of the three Kalanchoes included its nutritional and energetic value by an analysis of proteins, ashes and carbohydrates, particularly sugars by HPLC-RI, fatty acids by GC-FID and determination of other phytochemist like tocopherols by HPLC-Fluorescence and organic acids by UFLC-DAD. Their cytotoxic activities were evaluated against the same cell lines of the different polarity extracts (hexane, DCM, EtOAc, MeOH y water), infusions and decoction. Three plants´ nutritional values revealed a high composition in carbohydrates, low in lipids and energetic values quite similar. However, the composition of organic acids varied a lot depending on the plant. The three Kalanchoes showed a relation among fatty acids SFA>PUFA>MUFA, being mainly saturated fatty acids. Regarding to sugar content, K. pinnata, showed a higher quantity. It also presented the major concentration in tocopherols, being γ-tocopherol the major isoform. Every plant presented cytotoxic activity, being BuOH extract from K. daigremontiana which showed the best results and throughout the fractioned extract which isolated and identified the compounds responsible of activity. In this case the kaempferol 3-O-β-D-xylopyranosyl(1→2)α-L-rhamnopyranoside was isolated and identified with the same spectroscopic techniques. As before, this flavonoid is not the responsible of the whole bioactivity detected in K. daigremontiana butanolic extract.
Description
Mestrado em cooperação com a Universidade de Salamanca