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Abstract(s)
Desde la antigüedad se han utilizado las plantas y las setas como fuentes de nutrientes y 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 en alimentos funcionales, de ahí la importancia de su estudio químico y bioactivo.
Con el fin ampliar el conocimiento de estas fuentes naturales y por tanto de sus usos, en este trabajo se han estudiado dos especies de plantas medicinales, Calendula officinalis L. y Mentha cervina L., y una especie de seta comestible Macrolepiota procera (Scop.) Singer.
La caracterización química de C. officinalis y M. cervina 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-DAD, compuestos fenólicos por HPLC-DAD-MS y ácidos orgánicos por UFLC-DAD. También se evaluaron la actividad antioxidante de los extractos metanólicos e infusiones mediante ensayos in vitro: actividad captadora de radicales libres (DPPH), poder reductor, inhibición de la peroxidación lipídica TBARS e inhibición de la decoloración de β-caroteno; y la actividad antitumoral en cuatro líneas celulares tumorales humanas: MCF-7 (carcinoma de mama), NCI-H460 (carcinoma de pulmón), HCT-15 (carcinoma de colon), HeLa (carcinoma cervical) y HepG2 (carcinoma hepatocelular); y evaluación de la citotoxicidad en cultivos primarios de células de hígado de cerdo (PLP2) por el método de la sulforodamina B (SRB).
El valor nutricional de ambas plantas reveló una composición elevada en hidratos de carbono y baja en lípidos, con un contenido superior en proteínas para M. cervina, y valores energéticos muy similares. Sin embargo, presentaron perfiles fenólicos diferentes, C. officinalis presentó principalmente flavonoles glicosilados, siendo mayoritario el flavonoide isoramnetina-3-O-ramnosilrutinosa; mientras que M. cervina, con una composición total de compuestos fenólicos superior, presentó principalmente derivados de cafeoilo, siendo mayoritario el ácido rosmarínico. En cambio, C. officinalis mostró la mayor concentración total de azúcares, compuestos orgánicos y tocoferoles, siendo sus compuestos mayoritarios fructosa, ácido cítrico y α-tocoferol. Los perfiles de ácidos grasos también fueron diferentes, C. officinalis mostró una relación de ácidos grasos totales: SFA (76 %) > PUFA> MUFA, mayoritario el ácido palmítico; mientras que la relación de M. cervina fue de: PUFA (56 %) > SFA > MUFA, mayoritario el ácido α-linolénico. Los mejores resultados de actividad antioxidante y antitumoral se obtuvieron para el extracto hidrometanólico de M. cervina que presentó los valores más bajos de EC50 y que resultó tener actividad antitumoral frente a las cuatro líneas celulares tumorales evaluadas, siendo más activo frente al carcinoma cervical (HeLa). Las infusiones no mostraron actividad antitumoral y ningún extracto presentó hepatotoxicidad.
La caracterización química de M. procera 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 diferentes metodologías como extracciones ácido-base, extracción de alcaloides, reacciones de acetilación y metilación, cromatografías en columna y en capa fina preparativas y HPLC. Los diferentes extractos, fracciones y subfracciones se analizaron por diferentes técnicas como 1H-RMN, 13C-RMN, COSY, IR, UV, GC-MS y HRMS, y los resultados se compararon con patrones, bases de datos de espectros y con publicaciones, con el fin de identificar los compuestos presentes.
En total, fueron identificados 18 compuestos en los extractos de M. procera, principalmente productos del metabolismo primario: compuestos glucídicos o derivados, el polialcohol manitol y el disacárido trehalosa; compuestos lipídicos o derivados, los ácidos grasos (ácido pentadecílico, ácido palmítico y ácido linoleico), los ésteres metílicos de ácidos grasos (palmitato de metilo, oleato de metilo y linoleato de metilo), los acetilgliceroles (1,2 y 1,3-diacetilgliceroles y 1-monoacetilglicerol), glicerol, 1-octadeceno y dos derivados del ergosterol (α,α-epidioxiergosterol y β,β-epidioxiergosterol); compuestos de los ácidos nucleicos, la base pirimidínica uracilo y el nucleósido adenosina; y un ácido fenólico (ácido p-hidroxibenzoico).
De manera general, C. officinalis, M. cervina y M. procera poseen en su composición fitoquímicos de gran interés, lo que contribuye a ampliar su uso a nivel medicinal y su uso en la alimentación como ingredientes funcionales.
Since ancient times, plants and mushrooms have been used as sources of nutrients and medicinal compounds. Nowadays, the search for natural products that lead to new drugs, dietary supplements, nutraceuticals or functional foods, is still a hot topic, which justify the importance of their chemical characterization and evaluation of bioactive properties. In order to expand the knowledge on natural sources and therefore its use, in this work we have studied two species of medicinal plants, Calendula officinalis L. and Mentha cervina L., and an edible mushroom species, Macrolepiota procera (Scop.) Singer. The chemical characterization of C. officinalis and M. cervina included the determination of the nutritional and energy value by analysis of protein, fat, ash and carbohydrates, particularly sugars by HPLC-RI and fatty acids by GC-FID, and the determination of other phytochemicals as tocopherols by HPLC-DAD, phenolic compounds by HPLC-DAD-MS and organic acids by UFLC-DAD. The antioxidant activity of the methanol extracts and infusions was evaluated by in vitro assays: free radical scavenging activity (DPPH), reducing power, lipid peroxidation inhibition by TBARS and β-carotene bleaching inhibition. The cytotoxicity was also screened in four human tumor cell lines: MCF-7 (breast carcinoma), NCI-H460 (lung carcinoma), HCT-15 (colon carcinoma), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma), and in primary cultures of porcine liver cells (PLP2), by the method of the sulforhodamine B (SRB). The nutritional value of both plants revealed a high content of carbohydrates and low fat levels, with higher protein content for M. cervina, and very similar energy values. However, they presented different phenolic profiles; C. officinalis presented mainly glycosylated flavonols, with the flavonoid isorhamnetin-3-O-ramnosilrutinose as the most abundant; while M. cervina, with higher concentration in total phenolic compounds, presented mainly caffeoyl derivates, with rosmarinic acid as the most abundant. However, C. officinalis showed the highest concentration of total sugars, organic acids and tocopherols; the most abundant compounds were fructose, citric acid and α-tocopherol, respectively. The fatty acid profiles were also different; C. officinalis showed: SFA (76%)> PUFA> MUFA, mainly palmitic acid; while M. cervina gave: PUFA (56%)> SFA> MUFA, mainly α-linolenic acid. The best results of antioxidant and cytotoxic activities were obtained for the methanolic extract of M. cervina, which presented the lowest values of EC50 and exhibited cytotoxicity against the four tumor cell lines tested, being more active against cervical carcinoma (HeLa). Infusions showed no cytotoxicity for the tumor cell lines, and none the extracts showed toxicity against non-tumor cells. The chemical characterization of M. procera included the preparation of extracts and fractions, by sequential maceration, using different polarity solvents: hexane, dichloromethane, ethyl acetate, methanol and water. These extracts were fractionated by different methodologies as acid-base extractions, alkaloids extractions, acetylation and methylation reactions, column and thin layer preparative chromatographs and HPLC. The different extracts, fractions and subfractions were analyzed by different techniques such as 1H-NMR, 13C-NMR, COSY, IR, UV, GC-MS and HRMS, and the results compared with standards, spectral databases and publications, in order to identify the compounds present. In total, 18 compounds were identified in the extracts of M. procera, mainly products of primary metabolism: carbohydrates or compounds derivatives, the polyol mannitol and the disaccharide trehalose; lipids or compounds derivatives as fatty acids (pentadecylic acid, palmitic acid and linoleic acid), fatty acids methyl esters (methyl palmitate, methyl oleate and methyl linoleate), acetylated glycerols (1,2 and 1,3 diacetin and 1-monoacetin), glycerol, 1-octadecene and two derivatives of ergosterol (α,α-epidioxyergosterol and β,β-epidioxyergosterol); compounds of nucleic acids, the pyrimidine base uracil and the nucleoside adenosine; and a phenolic acid (p-hydroxybenzoic acid). Overall, C. officinalis, M. cervina and M. procera contain phytochemicals that are of great interest, which contribute to extend its use as functional ingredients, and in medical purposes.
Since ancient times, plants and mushrooms have been used as sources of nutrients and medicinal compounds. Nowadays, the search for natural products that lead to new drugs, dietary supplements, nutraceuticals or functional foods, is still a hot topic, which justify the importance of their chemical characterization and evaluation of bioactive properties. In order to expand the knowledge on natural sources and therefore its use, in this work we have studied two species of medicinal plants, Calendula officinalis L. and Mentha cervina L., and an edible mushroom species, Macrolepiota procera (Scop.) Singer. The chemical characterization of C. officinalis and M. cervina included the determination of the nutritional and energy value by analysis of protein, fat, ash and carbohydrates, particularly sugars by HPLC-RI and fatty acids by GC-FID, and the determination of other phytochemicals as tocopherols by HPLC-DAD, phenolic compounds by HPLC-DAD-MS and organic acids by UFLC-DAD. The antioxidant activity of the methanol extracts and infusions was evaluated by in vitro assays: free radical scavenging activity (DPPH), reducing power, lipid peroxidation inhibition by TBARS and β-carotene bleaching inhibition. The cytotoxicity was also screened in four human tumor cell lines: MCF-7 (breast carcinoma), NCI-H460 (lung carcinoma), HCT-15 (colon carcinoma), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma), and in primary cultures of porcine liver cells (PLP2), by the method of the sulforhodamine B (SRB). The nutritional value of both plants revealed a high content of carbohydrates and low fat levels, with higher protein content for M. cervina, and very similar energy values. However, they presented different phenolic profiles; C. officinalis presented mainly glycosylated flavonols, with the flavonoid isorhamnetin-3-O-ramnosilrutinose as the most abundant; while M. cervina, with higher concentration in total phenolic compounds, presented mainly caffeoyl derivates, with rosmarinic acid as the most abundant. However, C. officinalis showed the highest concentration of total sugars, organic acids and tocopherols; the most abundant compounds were fructose, citric acid and α-tocopherol, respectively. The fatty acid profiles were also different; C. officinalis showed: SFA (76%)> PUFA> MUFA, mainly palmitic acid; while M. cervina gave: PUFA (56%)> SFA> MUFA, mainly α-linolenic acid. The best results of antioxidant and cytotoxic activities were obtained for the methanolic extract of M. cervina, which presented the lowest values of EC50 and exhibited cytotoxicity against the four tumor cell lines tested, being more active against cervical carcinoma (HeLa). Infusions showed no cytotoxicity for the tumor cell lines, and none the extracts showed toxicity against non-tumor cells. The chemical characterization of M. procera included the preparation of extracts and fractions, by sequential maceration, using different polarity solvents: hexane, dichloromethane, ethyl acetate, methanol and water. These extracts were fractionated by different methodologies as acid-base extractions, alkaloids extractions, acetylation and methylation reactions, column and thin layer preparative chromatographs and HPLC. The different extracts, fractions and subfractions were analyzed by different techniques such as 1H-NMR, 13C-NMR, COSY, IR, UV, GC-MS and HRMS, and the results compared with standards, spectral databases and publications, in order to identify the compounds present. In total, 18 compounds were identified in the extracts of M. procera, mainly products of primary metabolism: carbohydrates or compounds derivatives, the polyol mannitol and the disaccharide trehalose; lipids or compounds derivatives as fatty acids (pentadecylic acid, palmitic acid and linoleic acid), fatty acids methyl esters (methyl palmitate, methyl oleate and methyl linoleate), acetylated glycerols (1,2 and 1,3 diacetin and 1-monoacetin), glycerol, 1-octadecene and two derivatives of ergosterol (α,α-epidioxyergosterol and β,β-epidioxyergosterol); compounds of nucleic acids, the pyrimidine base uracil and the nucleoside adenosine; and a phenolic acid (p-hydroxybenzoic acid). Overall, C. officinalis, M. cervina and M. procera contain phytochemicals that are of great interest, which contribute to extend its use as functional ingredients, and in medical purposes.
Description
Mestrado em cooperação com a Universidade de Salamanca