Correlation between chemical composition and bioactivity of Thymus carnosus Boiss. extracts
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2017
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O uso de plantas medicinais tem sido recorrente ao longo da História, mas atualmente o interesse em produtos naturais tem vindo a crescer como forma de reduzir a utilização de fármacos e de aumentar a ingestão de compostos bioativos. Embora muitas das espécies já estejam caracterizadas, os dados sobre a maioria dos fitofármacos que produzem, a sua bioatividade, nomeadamente vias metabólicas que afetam, são escassos. Diretamente implicadas em vários efeitos medicinais estão plantas da família Lamiaceae, como os tomilhos, mentas, rosmaninho, entre outros. Dentro do género Thymus encontramos uma espécie em perigo de extinção, o Thymus carnosus Boiss, devido à sua restrita distribuição geográfica, limitada ao sul e sudoeste da costa Portuguesa, e uma pequena porção da costa sul de Espanha. A literatura disponível restringe-se essencialmente ao estudo da composição dos óleos essenciais, onde borneol, canfeno, terpineno-4-ol e cis-sabineno hidratado são os principais componentes, e ao potencial bioativo desta planta como antioxidante, anti-inflamatório e antifúngico.
O objetivo deste estudo foca dois aspetos: aumentar o interesse por T. carnosus, tendo em vista a sua proteção enquanto planta endémica, e determinar a composição química de extratos aquosos e hidro-etanólicos, bem como a sua bioatividade, tentando desvendar algumas das vias metabólicas responsáveis pela sua ação.
Os extratos foram obtidos por decocção (água em ebulição) e agitação (hidro-etanólicos 20:80, v/v) a partir de material vegetal proveniente de duas colheitas. Os extratos foram caracterizados quanto ao teor em fenóis totais, orto-difenóis e flavonoides, que se verificou ser estatisticamente mais elevado nos extratos obtidos por agitação (não se verificaram diferenças significativas entre colheitas). A análise por HPLC-DAD e HPLC-ESI-MSn revelou presença dos ácidos: cafeico, rosmarínico, ursólico, salvianico A e salvianólico K, bem como dos flavonoides: luteolina, apigenina, eriodictiol e quercetina. O scavenging dos radicais ABTS•+, hidroxilo e óxido nítrico, para avaliar a capacidade antioxidante, indicou a decocção como tendo a melhor resposta. Nos extratos hidro-etanólicos foi recolhida uma nova fração na forma de pequenas partículas ou grânulos, onde se identificou totarol.
A bioatividade dos extratos foi avaliada em seis linhas celulares: HaCaT, Caco-2, HepG2, MCF-7, BT-474 e Raw 264.7; tendo as decocções induzido menor toxicidade (IC50 > 202,59 μg/mL) e os grânulos a maior toxicidade (IC50 < 74,90 μg/mL). As decocções (> 50 μg/mL) levam a perda de adesão celular, confirmado pela diminuição da expressão de E-caderina. Aparentemente a ação dos extratos não passa pelo dano no ADN, dado que não se
verificou indução de fragmentação (marcação com Hoescht) ou incremento na expressão do fator indutor de apoptose (AIF). Dados de citometria de fluxo, revelaram que na presença de peróxido de hidrogénio (H2O2), os extratos protegem as células, reduzindo o stresse oxidativo e a taxa de apoptose, em células HaCaT. Na ausência de H2O2, os extratos induzem oxidação, em pequena escala, e também podem induzir apoptose, dependente da concentração.
Todos os extratos revelaram bom potencial anti-inflamatório, com destaque para os extratos obtidos por decocção e para os ácidos rosmarínico e cafeico a assumirem-se como grandes contribuintes para o efeito. A elucidação completa das vias de sinalização subjacentes à ação bioativa destes extratos e seus componentes ainda requer estudos adicionais.
Medicinal plants have been used for a long time through history, but the interest in natural products has been growing to reduce the intake of medical drugs and to increase the presence of bioactive compounds in the diet. Although many species are characterized, data about the phytochemicals they produce, their bioactivity, as the metabolic pathways they affect, are far from being complete. Directly related to medicinal effects by plants is the Lamiaceae family, where we can find thyme, mint, rosemary and many others. Within the genus Thymus we find an endangered species, Thymus carnosus Boiss., due to its restricted location that is limited to south and southwest shore of Portugal and a small area in Spain south shore. The available knowledge about this species is mainly restricted to the characterization of essential oils, in which borneol, camphene, terpinene-4-ol and cis-sabinene hydrate appear as the main components. Concerning the bioactivity, the studies available in the literature show a potential application of the plant oils and extracts as antioxidant, anti-inflammatory and antifungal agents. The research in this dissertation is focused on two main topics: to increase the interest for T. carnosus, in order to protect its status as an important endemic plant of Portugal, by clarifying the chemical composition of aqueous and hydro-ethanolic extracts and their bioactivity, trying to unveil some of the mechanisms that may trigger biological effects. The extracts were obtained by decoction (boiling water) and agitation, with a water-ethanol solution (20:80, % v/v), using material obtained from two harvests. Decoction and agitation extracts were chemically characterized with the quantification of total phenols, ortho-diphenols and flavonoids content, where agitation had a significantly higher value between extraction methods in flavonoids and ortho-diphenols. HPLC-DAD and HPLC-ESI-MSn analysis showed the presence of the acids: caffeic, rosmarinic, salvianic A, salvianolic K, ursolic and the flavonoids: luteolin, apigenin, eriodictyol and quercetin. ABTS•+, hydroxyl, nitric oxide radicals scavenging was performed, with the extracts showing an antioxidant capacity, especially decoction. In the hydro-ethanolic extracts small isolated particles have been detected, which were named as granules. These have been analysed by GC-MS, revealing the presence of totarol. The bioactivity studies started with the cytotoxicity assessment for all the extracts in six cell lines: HaCaT, Caco-2, HepG2, MCF-7, BT-474 and Raw 264.7; with the decoctions inducing lower toxicity (IC50 > 202.59 μg/mL) and the granules higher toxicity (IC50 < 74.90 μg/mL). Decoction (> 50 μg/mL) caused loss of cell to cell adhesion while the decrease of E- cadherin expression was confirmed by immunocytochemistry. The lack of expression of AIF as well as low DNA fragmentation (assessed with Hoechst probe) indicated that DNA damage was not linked to the extracts potential mechanism of action. The extracts protected the HaCaT cells against the presence of H2O2, reducing the oxidative stress level and lowering the apoptosis rates. In the absence of H2O2 the extracts had a small contribution to oxidation events and potentially induced apoptosis, probably through intrinsic pathway. All the extracts showed significant anti-inflammatory activity, with decoction producing the best result, in which rosmarinic and caffeic acids have a major role. A full understanding of the signalling pathways still requires more research to correlate the extracts composition and their bioactivity.
Medicinal plants have been used for a long time through history, but the interest in natural products has been growing to reduce the intake of medical drugs and to increase the presence of bioactive compounds in the diet. Although many species are characterized, data about the phytochemicals they produce, their bioactivity, as the metabolic pathways they affect, are far from being complete. Directly related to medicinal effects by plants is the Lamiaceae family, where we can find thyme, mint, rosemary and many others. Within the genus Thymus we find an endangered species, Thymus carnosus Boiss., due to its restricted location that is limited to south and southwest shore of Portugal and a small area in Spain south shore. The available knowledge about this species is mainly restricted to the characterization of essential oils, in which borneol, camphene, terpinene-4-ol and cis-sabinene hydrate appear as the main components. Concerning the bioactivity, the studies available in the literature show a potential application of the plant oils and extracts as antioxidant, anti-inflammatory and antifungal agents. The research in this dissertation is focused on two main topics: to increase the interest for T. carnosus, in order to protect its status as an important endemic plant of Portugal, by clarifying the chemical composition of aqueous and hydro-ethanolic extracts and their bioactivity, trying to unveil some of the mechanisms that may trigger biological effects. The extracts were obtained by decoction (boiling water) and agitation, with a water-ethanol solution (20:80, % v/v), using material obtained from two harvests. Decoction and agitation extracts were chemically characterized with the quantification of total phenols, ortho-diphenols and flavonoids content, where agitation had a significantly higher value between extraction methods in flavonoids and ortho-diphenols. HPLC-DAD and HPLC-ESI-MSn analysis showed the presence of the acids: caffeic, rosmarinic, salvianic A, salvianolic K, ursolic and the flavonoids: luteolin, apigenin, eriodictyol and quercetin. ABTS•+, hydroxyl, nitric oxide radicals scavenging was performed, with the extracts showing an antioxidant capacity, especially decoction. In the hydro-ethanolic extracts small isolated particles have been detected, which were named as granules. These have been analysed by GC-MS, revealing the presence of totarol. The bioactivity studies started with the cytotoxicity assessment for all the extracts in six cell lines: HaCaT, Caco-2, HepG2, MCF-7, BT-474 and Raw 264.7; with the decoctions inducing lower toxicity (IC50 > 202.59 μg/mL) and the granules higher toxicity (IC50 < 74.90 μg/mL). Decoction (> 50 μg/mL) caused loss of cell to cell adhesion while the decrease of E- cadherin expression was confirmed by immunocytochemistry. The lack of expression of AIF as well as low DNA fragmentation (assessed with Hoechst probe) indicated that DNA damage was not linked to the extracts potential mechanism of action. The extracts protected the HaCaT cells against the presence of H2O2, reducing the oxidative stress level and lowering the apoptosis rates. In the absence of H2O2 the extracts had a small contribution to oxidation events and potentially induced apoptosis, probably through intrinsic pathway. All the extracts showed significant anti-inflammatory activity, with decoction producing the best result, in which rosmarinic and caffeic acids have a major role. A full understanding of the signalling pathways still requires more research to correlate the extracts composition and their bioactivity.
Descrição
Dissertação de Mestrado em Bioquímica
Palavras-chave
Bioquímica , Citotoxicidade , Citometria de fluxo , Thymus carnosus Boiss , Atividade antioxidante , Anti-inflamatório