Avaliação do perfil químico de folhas de espécies de Eugenia (Myrtaceae) por CLAE, RMN e IES-EM com vistas ao desenvolvimento de cosméticos e alimentos funcionais
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Data
2017-06-02
Autores
Campideli, Mariana Bordin [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O gênero Eugenia pertence à família Myrtaceae e compreende cerca de 400 espécies de ocorrência em todo o território nacional. Algumas espécies são muito conhecidas pelas atividades antifúngica, antiviral e bactericida. Outras são descritas pelas propriedades apresentadas na redução dos níveis de triglicerídeos e colesterol. As espécies de Eugenia apresentam uma composição química diversificada, como relatos na literatura da presença de terpenos, derivados flavonoidicos (quercetina, metoxiquercetina, miricetina) e outros fenólicos como chalconas, taninos e flavonas. Mesmo tratando-se de espécies com frutos comestíveis, a literatura é bastante escassa quanto à composição química das folhas de E. handroana e E. pyriformis, e todo trabalho relatado até o presente trata-se especialmente dos óleos essenciais. Desta forma, este projeto tem como objetivo o estudo bioguiado dos extratos das folhas de três espécies do gênero: E. pyriformis Cambess, E. uniflora L. e E. handroana D. Legrand, visando identificar substâncias presentes nas frações bioativas nos ensaios antioxidante e de inibição da glicação com vistas ao desenvolvimento de cosméticos e alimentos funcionais. Extratos hidroetanólicos e suas frações foram analisados por CLAE-DAD e avaliados por ensaios biológicos (ensaio antioxidante com radical DPPH•, ação redutora sobre radicais peroxila e antiglicação), sendo a fração acetato de etila a mais ativa nas três espécies estudadas nos ensaios descritos. A fração acetato de etila de E. pyriformis e E. handroana foi submetida a cromatografia em coluna e as frações resultantes a ensaios biológicos. A fração C foi selecionada por apresentar resultados significativos nos ensaios antioxidantes e promissores no ensaio antiglicação. Esta fração foi submetida ao fracionamento por cromatografia líquida de alta eficiência (CLAE) em escala preparativa, levando a identificação de seis bandas na fração C de E. pyriformis e quatro bandas na fração C de E. handroana. A identificação estrutural das substâncias presentes nestas bandas foi feita por ressonância magnética nuclear (RMN) e espectrometria de massas com ionização por electrospray (IES-EM) por infusão direta, como também pela utilização de dados da literatura. Foram identificados os seguintes compostos: miricetina-3-O-α-L-ramnosídeo, quercetina-3-O-β-D-glicosídeo, quercetina-3-O-β-D-galactosídeo, quercetina-3-O-β-D-xilosídeo, quercetina-3-O-α-L-ramnosídeo, miricetina-3-O-(2″-O-galloil)-α-L-ramnosídeo, miricetina-3-O-(4″-O-galloil)-α-L-ramnosídeo, quercetina-3-O-(2″-O-galloil)-α-L-ramnosídeo e quercetina, que foram submetidos aos ensaios biológicos propostos nesse projeto. A fração acetato de etila de E. uniflora foi explorada por meio da técnica de desreplicação, devido ao seu perfil cromatográfico bastante semelhante ao das outras espécies em estudo. Assim a identificação tentativa das substâncias foi realizada através das técnicas de IES-EM, CLAE-DAD e dados de EM publicados na literatura, sendo provavelmente as mesmas substâncias observadas nas outras espécies em estudo.
The genus Eugenia belongs to the family Myrtaceae and comprises about 400 species of occurrence throughout the national territory. Some species are well known for antifungal, antiviral and bactericidal activities. Others are described by their properties in reducing triglyceride and cholesterol levels. The species of Eugenia have a diverse chemical composition, such as reports in the literature of the presence of terpenes, flavonoid derivatives (quercetin, methoxyquercetin, myricetin) and other phenolics such as chalcones, tannins and flavones. Even though it is species with edible fruits, the literature is rather scarce as to the chemical composition of the leaves of E. handroana and E. pyriformis, and all the literature reported to date is especially about the essential oils. In this way, this project aims at the bio-guided study with leaf extracts of three species of the genus: E. pyriformis Cambess, E. uniflora L. and E. handroana D. Legrand, aiming to identify substances present in the bioactive fractions, in the antioxidant and inhibition of glycation assays with a view to the development of cosmetics and functional foods. Hydroethanolic extracts and their respective fractions were analyzed by HPLC-DAD and accompanied by biological assays (antioxidant assay with DPPH• radical, reducing action on peroxyl radicals and antiglycation), with ethyl acetate fraction being the most active in the three species studied in the described assays. The ethyl acetate fraction of E. pyriformis and E. handroana was fractionated by column chromatography and the resulting fractions were submitted to biological assays. The fraction C was selected because for presented significant results in the antioxidant and promising results in the antiglycation assay. This fraction was then submitted to fractionation by preparative high performance liquid chromatography (HPLC), leading to the identification of six bands in the C fraction of E. pyriformis and four bands in the C fraction of E. handroana. The structural identification of the substances present in these bands were made through nuclear magnetic resonance (NMR) and electrospray ionisation mass spectrometry (ESI-MS) by direct infusion, as well as the use of data from the literature. The following compounds were identified: myricetin-3-O-α-L-rhamnoside, quercetin-3-O-β-D-glucoside, quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-xyloside, quercetin-3-O-α-L-rhamnoside, myricetin-3-O-(2"-O-galloyl)-α-L-rhamnoside, myricetin-3-O-(4"-O-galloyl)-α-L-ramnoside, quercetin-3-O-(2 "-O-galloyl) -α-L-rhamnoside and quercetin, which were submitted to the biological tests proposed in this project. The ethyl acetate fraction of E. uniflora was explored through the technique of dereplication because it presented a very similar chromatographic profile with that of the other species under study, so the tentative identification of the substances was performed through the ESI-MS, HPLC-DAD and MS data published in the literature, being probably the same substances observed in the other species under study.
The genus Eugenia belongs to the family Myrtaceae and comprises about 400 species of occurrence throughout the national territory. Some species are well known for antifungal, antiviral and bactericidal activities. Others are described by their properties in reducing triglyceride and cholesterol levels. The species of Eugenia have a diverse chemical composition, such as reports in the literature of the presence of terpenes, flavonoid derivatives (quercetin, methoxyquercetin, myricetin) and other phenolics such as chalcones, tannins and flavones. Even though it is species with edible fruits, the literature is rather scarce as to the chemical composition of the leaves of E. handroana and E. pyriformis, and all the literature reported to date is especially about the essential oils. In this way, this project aims at the bio-guided study with leaf extracts of three species of the genus: E. pyriformis Cambess, E. uniflora L. and E. handroana D. Legrand, aiming to identify substances present in the bioactive fractions, in the antioxidant and inhibition of glycation assays with a view to the development of cosmetics and functional foods. Hydroethanolic extracts and their respective fractions were analyzed by HPLC-DAD and accompanied by biological assays (antioxidant assay with DPPH• radical, reducing action on peroxyl radicals and antiglycation), with ethyl acetate fraction being the most active in the three species studied in the described assays. The ethyl acetate fraction of E. pyriformis and E. handroana was fractionated by column chromatography and the resulting fractions were submitted to biological assays. The fraction C was selected because for presented significant results in the antioxidant and promising results in the antiglycation assay. This fraction was then submitted to fractionation by preparative high performance liquid chromatography (HPLC), leading to the identification of six bands in the C fraction of E. pyriformis and four bands in the C fraction of E. handroana. The structural identification of the substances present in these bands were made through nuclear magnetic resonance (NMR) and electrospray ionisation mass spectrometry (ESI-MS) by direct infusion, as well as the use of data from the literature. The following compounds were identified: myricetin-3-O-α-L-rhamnoside, quercetin-3-O-β-D-glucoside, quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-xyloside, quercetin-3-O-α-L-rhamnoside, myricetin-3-O-(2"-O-galloyl)-α-L-rhamnoside, myricetin-3-O-(4"-O-galloyl)-α-L-ramnoside, quercetin-3-O-(2 "-O-galloyl) -α-L-rhamnoside and quercetin, which were submitted to the biological tests proposed in this project. The ethyl acetate fraction of E. uniflora was explored through the technique of dereplication because it presented a very similar chromatographic profile with that of the other species under study, so the tentative identification of the substances was performed through the ESI-MS, HPLC-DAD and MS data published in the literature, being probably the same substances observed in the other species under study.
Descrição
Palavras-chave
Eugenia, Antioxidantes, Antiglicação, Flavonoides, CLAE, RMN, IES-EM, Desreplicação