Evaluation of grape stems extracts from cultivar ‘Sousão’ as a source of compounds inhibitors of oxidative stress in vitro using keratinocytes
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2017
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A vinicultura é uma das mais importantes atividades sócio-económicas em Portugal, bem como em toda a bacia do Mediterrâneo. De todos os resíduos resultantes do processo de vinificação o engaço, que é descartado antes do início da produção do vinho, está entre os resíduos que aparecem em maiores quantidades.
O potencial antioxidante dos extratos de engaço de uva tem sido muito falado na ultima década, no entanto existe uma falta de informação sobre o potencial dos seus compostos fenólicos individuais e o estabelecimento de combinações desses compostos individuais com outros compostos antioxidantes. Neste trabalho, extratos polifenólicos de engaço de uvas da casta ‘Sousão’ (Vitis vinifera L.) foram analisados por HPLC-UV-Vis semi-preparativo obtendo-se compostos fenólicos isolados e purificados (isorhamnetina-3-O-(6-O-feruil)-glicosídeo, ácido caftárico, malvidina-3-O-glucosídeo, quercitina-3-O-rutinosídeo, quercitina-3-O-glucoronídeo, kaempferol-3-O-rutinosídeo, malvidina-3-O-(6-O-cafeoíl)-glucosídeo, malvidina-3-O-rutinosídeo e Σ-viniferina) que foram caracterizados por HPLC-PDA-ESi-MSn. Os compostos isolados e purificados foram testados para a sua capacidade anti-radicalar (DPPH e ABTS) para estabelecer o valor de metade da sua concentração máxima inibitória (IC50). De acordo com o potencial anti-radicalar destes compostos onde foi calculado o IC50 (malvidina-3-O-glucosídeo (0,41 μmol/L), quercetina-3-O-glucuronídeo (1,25 μmol/L), e malvidina-3-O-(6-O-cafeoíl)-glucosídeo (0,43 μmol/L)) foi testado o seu efeito na viabilidade celular, atividade anti-inflamatória, capacidade de modulação dos marcadores de stresse oxidativo e na supressão do mesmo e a capacidade de diminuir os processos apoptóticos usando os modelos celulares (in vitro) HaCaT (linha celular não tumoral de queratinócitos humanos) e RAW 264.7 (linha celular não tumoral de macrófagos murinos) sob condições basais e em ambiente oxidativo. Os resultados obtidos durante estas determinações permitiram identificar as combinações malvidina-3-O-glucosídeo + Vitamina E e quercetina-3-O-glucuronide + Vitamina C como as mais valiosas e efetivas no que diz respeito à atividade anti-inflamatória (inibiram cerca de 75 % da produção de NO nas células RAW 264.7 quando estimuladas por lipopolissacarídeo) e à atividade anti-radicalar (ex. mostrou os menores valores de ROS medidos pela técnica da citometria de fluxo). A quercitina-3-O-glucoronídeo e a malvidina-3-O-(6-O-cafeoil)-glucosídeo mostraram o maior potencial anti-apoptótico como comprovado pela diminuição em caspase-3 clivada que é um marcador pró-apoptótico. Estes resultados permitiram melhorar o conhecimento das atividades biológicas exercidas pelos extratos completos e pelos compostos individuais, indicando que estes exercem uma proteção celular benéfica contra o stresse oxidativo e que podem ser potenciais candidatos a ser usados no desenvolvimento de novos ingredientes funcionais para comidas, bebidas, cosméticos e também nutracêuticos.
Viticulture represents one of the most important socio-economic activities in Portugal, as well as in the whole area of the Mediterranean Basin. From all the residues resulting from the production of wine, grape stems, which are discarded at the beginning of the vinification process, are among the residues that appear in higher quantities. The antioxidant potential of phenolic extracts from grape stems have been widely reported in the last decade, whilst there is a lack of information on the potential of their individual compounds and the establishment of improved combinations with other antioxidant compounds. In this work, polyphenolic extract of grape stems (Vitis vinifera L.), from the cultivar ‘Sousão’ was processed resorting to semi-preparative HPLC-UV-Vis, allowing to obtain purified phenolics (isorhamnetin-3-O-(6-O-feruloyl)-glucoside, caftaric acid, malvidin-3-O-glucoside, quercetin-3-O-rutinoside, quercetin-3-O-glucuronide, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, malvidin-3-O-(6-O-caffeoyl)-glucoside, malvidin-3-O-rutinoside, and Σ-viniferin), which were fully characterized by HPLC-PDA-ESi-MSn. Isolated and purified compounds were featured on their radical scavenging capacity (DPPH and ABTS) in order to establish the half maximum inhibitory concentration (IC50). Those compounds on which could be established the IC50 according to their radical scavenging power (malvidin-3-O-glucoside (0.41 μmol/L), quercetin-3-O-glucuronide (1.25 μmol/L), and malvidin-3-O-(6-O-caffeoyl)-glucoside (0.43 μmol/L)) were further tested on cell viability, anti-inflammatory activity, capacity to modulate oxidative stress markers and overall oxidative stress and also on their capacity to decrease the apoptotic process in biological (in vitro) models HaCaT cells (human keratinocytes) and RAW 264.7 (murine macrophages from blood) under basal and under oxidative conditions. The results obtained through the plethora of determinations developed allowed to identify the combinations malvidin-3-O-glucoside + Vitamin E and quercetin-3-O-glucuronide + vitamin C as the most valuable and most effective regarding the anti-inflammatory (inhibited about 75 % the NO produced by RAW 264.7 cells, stimulated with lipopolysaccharide) and anti-radicalar activities (e.g. showed the lower values of ROS as measured by flow cytometry). Quercetin-3-O-glucoronide and malvidin-3-O-(6-O-caffeoyl)-glucoside showed the higher anti-apoptotic potential, as evaluated by the decrease in cleaved caspase-3 a pro-apoptotic marker. These results allowed to improve the knowledge on biological activities exerted by complete extracts, or their individual compounds, indicating beneficial cellular protection against oxidative stress and as potential candidates to be used in the development of new functional ingredients to foods, beverages, cosmetics and nutraceuticals.
Viticulture represents one of the most important socio-economic activities in Portugal, as well as in the whole area of the Mediterranean Basin. From all the residues resulting from the production of wine, grape stems, which are discarded at the beginning of the vinification process, are among the residues that appear in higher quantities. The antioxidant potential of phenolic extracts from grape stems have been widely reported in the last decade, whilst there is a lack of information on the potential of their individual compounds and the establishment of improved combinations with other antioxidant compounds. In this work, polyphenolic extract of grape stems (Vitis vinifera L.), from the cultivar ‘Sousão’ was processed resorting to semi-preparative HPLC-UV-Vis, allowing to obtain purified phenolics (isorhamnetin-3-O-(6-O-feruloyl)-glucoside, caftaric acid, malvidin-3-O-glucoside, quercetin-3-O-rutinoside, quercetin-3-O-glucuronide, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, malvidin-3-O-(6-O-caffeoyl)-glucoside, malvidin-3-O-rutinoside, and Σ-viniferin), which were fully characterized by HPLC-PDA-ESi-MSn. Isolated and purified compounds were featured on their radical scavenging capacity (DPPH and ABTS) in order to establish the half maximum inhibitory concentration (IC50). Those compounds on which could be established the IC50 according to their radical scavenging power (malvidin-3-O-glucoside (0.41 μmol/L), quercetin-3-O-glucuronide (1.25 μmol/L), and malvidin-3-O-(6-O-caffeoyl)-glucoside (0.43 μmol/L)) were further tested on cell viability, anti-inflammatory activity, capacity to modulate oxidative stress markers and overall oxidative stress and also on their capacity to decrease the apoptotic process in biological (in vitro) models HaCaT cells (human keratinocytes) and RAW 264.7 (murine macrophages from blood) under basal and under oxidative conditions. The results obtained through the plethora of determinations developed allowed to identify the combinations malvidin-3-O-glucoside + Vitamin E and quercetin-3-O-glucuronide + vitamin C as the most valuable and most effective regarding the anti-inflammatory (inhibited about 75 % the NO produced by RAW 264.7 cells, stimulated with lipopolysaccharide) and anti-radicalar activities (e.g. showed the lower values of ROS as measured by flow cytometry). Quercetin-3-O-glucoronide and malvidin-3-O-(6-O-caffeoyl)-glucoside showed the higher anti-apoptotic potential, as evaluated by the decrease in cleaved caspase-3 a pro-apoptotic marker. These results allowed to improve the knowledge on biological activities exerted by complete extracts, or their individual compounds, indicating beneficial cellular protection against oxidative stress and as potential candidates to be used in the development of new functional ingredients to foods, beverages, cosmetics and nutraceuticals.
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Dissertação de Mestrado em Bioquímica
Palavras-chave
Bioquímica , Vitis vinifera , Stresse oxidativo , Apoptose , Compostos fenólicos , Propriedades bioativas , Engaço