Publicação
Multifunctional health-promoting effects of Thymus plants as novel dietary products: modulation of redox, inflammatory and cell death pathways
| Resumo: | The higher incidence of intestinal tract diseases and the increased awareness about intestinal barrier homeostasis are partly responsible for current trends towards healthier diet choices, where functional foods and nutraceuticals assume a predominant role. The need for new functional foods is promoting the screening of previously unstudied plant species for their potential bioactivities. In this thesis, we studied the potential of Thymus carnosus and Thymus capitellatus as dietary antioxidant, anti-inflammatory and anti-tumoral agents. Hydroethanolic (HE) extracts of both species were shown to be rich in ursolic, oleanolic and rosmarinic acids, in addition to quercetin-(?)-O-hexoside. Aqueous decoction (AD) extracts did not present pentacyclic triterpenoids, but were shown to be rich in rosmarinic acid, salvianolic acids and flavonoid derivatives. In T. carnosus extracts was observed that interannual variability and geographic location induce significant changes in the phytochemical composition. However, despite the variability induced by the harvesting location and year, T. carnosus extracts’ antioxidant, neuroprotective and anti-diabetic potential was maintained, thus supporting its suitability for sustainable cultivation. Also, based on principal component analysis results, by selecting the location of harvest it should be possible to select specific chemotypes depending on the desired bioactivity. AD and HE extracts of T. carnosus and T. capitellatus induced cytotoxicity in two colorectal adenocarcinoma cell models (Caco-2 and HT-29) and a hepatocarcinoma cell model (HepG2), and the sensitivity to the extracts action increased as follows: HT-29 < Caco-2 < HepG2. HE extracts produced lower IC50 values than AD extracts. AD and HE extracts of T. carnosus induced mitochondrial membrane hyperpolarization in HepG2 cells, but induced mitochondrial membrane depolarization in Caco-2 cells. In HT-29 cells, mitochondrial membrane depolarization was only observed in cells exposed to AD extracts. AD and HE extracts of both species increased intracellular ROS levels in Caco-2 cells, but not in HT-29 cells. However, Annexin− /PI+ and Annexin+ /PI + staining was observed in the three cell lines, regardless of the extract used. Apoptosis induction in Caco-2 and HT-29 cells was confirmed by caspase-3 activation, and it was also confirmed that apoptosis is not the only cell death process involved in extracts-induced anti-proliferative activity, being hypothesized that necroptosis is also induced. Among the main phytochemicals, ursolic acid presented the highest correlation to HE extracts’ anti-proliferative activity, justifying the differences between extraction methods. AD and HE extracts of both species showed neuroprotective (anti-acetylcholinesterase and ant-tyrosinase activity) as well as anti-diabetic activity (anti-α-glucosidase activity). All extracts tested present free radical scavenging activity, evaluated as ABTS, hydroxyl, superoxide and nitric oxide radicals scavenging, and inhibition of β-carotene bleaching. At concentrations ≤ 50 μg/mL the extracts did not induce cytotoxicity (confirmed by Alamar Blue and Annexin/PI assays), oxidative stress, irritative or angiogenic effects, and also did not induce the production of nitric oxide (NO) and cytokines by Caco-2 cells, thus supporting their safety for dietary use. The oxidative damage induced by tert-butyl-hydroperoxide (t-BHP) in Caco-2 cells was effectively reduced in cells pre-exposed to T. carnosus and T. capitellatus AD and HE extracts. Nevertheless, cells pre-exposed to T. carnosus HE extract presented morphological changes and reduced cell viability when analysed 24 h after the oxidative stimulus, while cells exposed to AD extracts present normal morphology and no reduction in cell viability. AD extracts reduced t-BHP-induced increase in reactive oxygen species, lipid peroxidation, glutathione depletion and DNA damage. The effect of both AD extracts was shown to be dependent on increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression, and T. capitellatus AD extract also increased NAD(P)H quinone dehydrogenase 1 (NQO1) expression. Ursolic acid was correlated to the reduced potential of HE extracts, while salvianolic acid A presented an antioxidant effect similar to that observed for the extracts. The anti-inflammatory activity was evaluated in RAW 264.7 cells (mouse macrophages). Both AD and HE extracts of T. carnosus and T. capitellatus reduced lipopolysaccharides (LPS)-induced NO release, an effect mainly correlated to the presence of rosmarinic acid (in both extraction methods) and ursolic acid (HE extracts). In the absence of LPS, both AD extracts increased intracellular NO, and T. carnosus AD extract induced the production of IL(interleukin)-1β, IL-6 and TNF-α (tumour necrosis factor alpha), which may indicate a hormetic effect. In the presence of LPS, both extracts reduced IL-1β production. The modulation of NO production was not related to increased iNOS expression, as the extracts induced no/low effect in its expression. Overall, AD extracts present higher potential as antioxidant and anti-inflammatory dietary agents, being able to target various key signalling pathways at non-cytotoxic concentrations. HE extracts show higher potential as anti-proliferative agents and as sources of nutraceuticals. This work further unravels the potential of T. carnosus and T. capitellatus as new functional foods. |
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| Autores principais: | Gomes, Carlos Alexandre Martins |
| Assunto: | Thymus carnosus Thymus capitellatus Anti-proliferative activity Antioxidant activity Anti-inflammatory activity Functional foods |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Trás-os-Montes e Alto Douro |
| Idioma: | inglês |
| Origem: | Repositório da UTAD |
| Resumo: | The higher incidence of intestinal tract diseases and the increased awareness about intestinal barrier homeostasis are partly responsible for current trends towards healthier diet choices, where functional foods and nutraceuticals assume a predominant role. The need for new functional foods is promoting the screening of previously unstudied plant species for their potential bioactivities. In this thesis, we studied the potential of Thymus carnosus and Thymus capitellatus as dietary antioxidant, anti-inflammatory and anti-tumoral agents. Hydroethanolic (HE) extracts of both species were shown to be rich in ursolic, oleanolic and rosmarinic acids, in addition to quercetin-(?)-O-hexoside. Aqueous decoction (AD) extracts did not present pentacyclic triterpenoids, but were shown to be rich in rosmarinic acid, salvianolic acids and flavonoid derivatives. In T. carnosus extracts was observed that interannual variability and geographic location induce significant changes in the phytochemical composition. However, despite the variability induced by the harvesting location and year, T. carnosus extracts’ antioxidant, neuroprotective and anti-diabetic potential was maintained, thus supporting its suitability for sustainable cultivation. Also, based on principal component analysis results, by selecting the location of harvest it should be possible to select specific chemotypes depending on the desired bioactivity. AD and HE extracts of T. carnosus and T. capitellatus induced cytotoxicity in two colorectal adenocarcinoma cell models (Caco-2 and HT-29) and a hepatocarcinoma cell model (HepG2), and the sensitivity to the extracts action increased as follows: HT-29 < Caco-2 < HepG2. HE extracts produced lower IC50 values than AD extracts. AD and HE extracts of T. carnosus induced mitochondrial membrane hyperpolarization in HepG2 cells, but induced mitochondrial membrane depolarization in Caco-2 cells. In HT-29 cells, mitochondrial membrane depolarization was only observed in cells exposed to AD extracts. AD and HE extracts of both species increased intracellular ROS levels in Caco-2 cells, but not in HT-29 cells. However, Annexin− /PI+ and Annexin+ /PI + staining was observed in the three cell lines, regardless of the extract used. Apoptosis induction in Caco-2 and HT-29 cells was confirmed by caspase-3 activation, and it was also confirmed that apoptosis is not the only cell death process involved in extracts-induced anti-proliferative activity, being hypothesized that necroptosis is also induced. Among the main phytochemicals, ursolic acid presented the highest correlation to HE extracts’ anti-proliferative activity, justifying the differences between extraction methods. AD and HE extracts of both species showed neuroprotective (anti-acetylcholinesterase and ant-tyrosinase activity) as well as anti-diabetic activity (anti-α-glucosidase activity). All extracts tested present free radical scavenging activity, evaluated as ABTS, hydroxyl, superoxide and nitric oxide radicals scavenging, and inhibition of β-carotene bleaching. At concentrations ≤ 50 μg/mL the extracts did not induce cytotoxicity (confirmed by Alamar Blue and Annexin/PI assays), oxidative stress, irritative or angiogenic effects, and also did not induce the production of nitric oxide (NO) and cytokines by Caco-2 cells, thus supporting their safety for dietary use. The oxidative damage induced by tert-butyl-hydroperoxide (t-BHP) in Caco-2 cells was effectively reduced in cells pre-exposed to T. carnosus and T. capitellatus AD and HE extracts. Nevertheless, cells pre-exposed to T. carnosus HE extract presented morphological changes and reduced cell viability when analysed 24 h after the oxidative stimulus, while cells exposed to AD extracts present normal morphology and no reduction in cell viability. AD extracts reduced t-BHP-induced increase in reactive oxygen species, lipid peroxidation, glutathione depletion and DNA damage. The effect of both AD extracts was shown to be dependent on increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression, and T. capitellatus AD extract also increased NAD(P)H quinone dehydrogenase 1 (NQO1) expression. Ursolic acid was correlated to the reduced potential of HE extracts, while salvianolic acid A presented an antioxidant effect similar to that observed for the extracts. The anti-inflammatory activity was evaluated in RAW 264.7 cells (mouse macrophages). Both AD and HE extracts of T. carnosus and T. capitellatus reduced lipopolysaccharides (LPS)-induced NO release, an effect mainly correlated to the presence of rosmarinic acid (in both extraction methods) and ursolic acid (HE extracts). In the absence of LPS, both AD extracts increased intracellular NO, and T. carnosus AD extract induced the production of IL(interleukin)-1β, IL-6 and TNF-α (tumour necrosis factor alpha), which may indicate a hormetic effect. In the presence of LPS, both extracts reduced IL-1β production. The modulation of NO production was not related to increased iNOS expression, as the extracts induced no/low effect in its expression. Overall, AD extracts present higher potential as antioxidant and anti-inflammatory dietary agents, being able to target various key signalling pathways at non-cytotoxic concentrations. HE extracts show higher potential as anti-proliferative agents and as sources of nutraceuticals. This work further unravels the potential of T. carnosus and T. capitellatus as new functional foods. |
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