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Authors
Abstract(s)
Las plantas han sido fuente de curación desde la antigüedad. En la actualidad continua ese
interés en la búsqueda de productos naturales que den lugar a nuevos fármacos.
En este trabajo se ha estudiado la citotoxicidad y la composición química de las hojas y flores
macho de Castanea sativa Miller con el fin de purificar y aislar compuestos citotóxicos y así
poder ampliar más los conocimientos sobre estos residuos naturales como posible fuente de
curación.
Se obtuvieron distintos extractos progresivos en polaridad: n-hexano, diclorometano, Acetato
de etilo (AcOEt), en etanol:agua (70:30, v/v) que a su vez fue fraccionado en dos fases
inmiscibles (n-butanol y agua), y finalmente en agua caliente (decocción). Se evaluó la
actividad citotóxica de los extractos mediante el método de la sulforodamina B frente a líneas
celulares humanas: MCF7 (adenocarcinoma de pecho), NCI-H460 (carcinoma de pulmón),
HeLa (carcinoma cervical) y HepG2 (carcinoma hepatocelular). Para evaluar la toxicidad se
utilizaron líneas celulares PLP2 (cultivos primarios de células no tumorales de hígado de
cerdo).
Los extractos más activos fueron: diclorometano flores (DF), diclorometano hojas (DH) y
hexano hojas (HH). Los extractos HH y DH se fraccionaron mediante la técnica VLC
(cromatografía líquida a vacío). Las fracciones obtenidas se volvieron a ensayar frente a las
líneas celulares antes mencionadas, y los resultados mostraron que las que mayor actividad
tenían fueron: HD6, HD7, HD8, HD9, HH8 y HH9.
Se realizo la caracterización química de estas fracciones con el fin de identificar los
compuestos presentes mediante Resonancia Magnética Nuclear de 1H y de 13C y
Espectrometría de Masas (GC-MS y HRMS), comparando los resultados con bases de datos
de espectros y con la bibliografía.
Se identificaron 11 compuestos entre los que destacaban: lupeol (mayoritario en las fracciones
HH8 y HH9) y loliolida (en HD6, HD7 y HD8). Se consiguió el aislamiento de ambos
compuestos mediante una cromatografía de columna. Se consiguió purificar el Lupeol y la
Loliolida. Se evaluó la actividad citotóxica de los dos compuestos concluyendo que estos
compuestos no eran los responsables de toda la bioactividad observada, y en caso de serlo, su
actividad se vería potenciada por la presencia de otros compuestos presentes en las fracciones.
Since ancient times that plants have been considered a source of different therapeutic principles. Nowadays the search for natural products that could lead to new drugs, justifies the importance of their structure elucidation and bioactive properties evaluation. In order to increase the knowledge on natural products and their different uses, in particular on Castanea sativa Miller (chestnut), we have studied the cytotoxicity and chemical composition of its leaves and male flowers. The main objective was to isolate and purify the cytotoxic compounds present in these matrices. The extractions were performed by sequential maceration, using different polarity solvents: hexane, dichloromethane, ethyl acetate and ethanol: water (70:30, v/v); this extract was fractioned in two immiscible phases (n-butanol and water); and water (decoction). The evaluation of cytotoxicity was conducted by SRB (sulphorodamine B) assay in human tumor cell lines: MCF-7 (breast carcinoma), NCI-H460 (non-small lung carcinoma), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma) and in primary cultures of porcine liver cells (PLP2). The most actives extracts were: dichloromethane flowers (DF), dichloromethane leaves (DH) and hexane leaves (HH). The extracts HH and DH were fractioned by VLC (vacuum liquid chromatography) and the fractions obtained were also screened for their cytotoxicity. The results showed that the most active fraction were: HD6, HD7, HD8, HD9, HH8 y HH9. The chemical characterization was made with different spectral methods: 1H and 13C nuclear magnetic resonance and mass spectrometry (GC-MS and HRMS); the spectral dates were compared with standards, spectral databases and literature, in order to identify the compounds, present in the obtained fractions. Eleven compounds were identified and two of them were very interesting: Lupheol (mainly in fractions HH8 and HH9) and Loliolide (in HD6, HD7 and HD8). The isolation of both compounds was achieved by column chromatography and the purification was quite efficient. Both compounds were screened for their cytotoxicity and the results showed that they were not responsible for all the observed bioactivity, and if responsible, the bioactivity is enhanced by the presence of other compounds in the fractions.
Since ancient times that plants have been considered a source of different therapeutic principles. Nowadays the search for natural products that could lead to new drugs, justifies the importance of their structure elucidation and bioactive properties evaluation. In order to increase the knowledge on natural products and their different uses, in particular on Castanea sativa Miller (chestnut), we have studied the cytotoxicity and chemical composition of its leaves and male flowers. The main objective was to isolate and purify the cytotoxic compounds present in these matrices. The extractions were performed by sequential maceration, using different polarity solvents: hexane, dichloromethane, ethyl acetate and ethanol: water (70:30, v/v); this extract was fractioned in two immiscible phases (n-butanol and water); and water (decoction). The evaluation of cytotoxicity was conducted by SRB (sulphorodamine B) assay in human tumor cell lines: MCF-7 (breast carcinoma), NCI-H460 (non-small lung carcinoma), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma) and in primary cultures of porcine liver cells (PLP2). The most actives extracts were: dichloromethane flowers (DF), dichloromethane leaves (DH) and hexane leaves (HH). The extracts HH and DH were fractioned by VLC (vacuum liquid chromatography) and the fractions obtained were also screened for their cytotoxicity. The results showed that the most active fraction were: HD6, HD7, HD8, HD9, HH8 y HH9. The chemical characterization was made with different spectral methods: 1H and 13C nuclear magnetic resonance and mass spectrometry (GC-MS and HRMS); the spectral dates were compared with standards, spectral databases and literature, in order to identify the compounds, present in the obtained fractions. Eleven compounds were identified and two of them were very interesting: Lupheol (mainly in fractions HH8 and HH9) and Loliolide (in HD6, HD7 and HD8). The isolation of both compounds was achieved by column chromatography and the purification was quite efficient. Both compounds were screened for their cytotoxicity and the results showed that they were not responsible for all the observed bioactivity, and if responsible, the bioactivity is enhanced by the presence of other compounds in the fractions.
Description
Mestrado em cooperação com a Universidade de Salamanca