Avaliação da capacidade antioxidante e antimicrobiana em algas da costa de Peniche (Portugal): identificação de compostos bioactivos com elevado potencial biotecnológico

Dissertação de Mestrado em Biotecnologia dos Recursos Marinhos apresentada à ESTM - Escola Superior de Turismo e Tecnologia do Mar do Instituto Politécnico de LeiriaNeste trabalho foi avaliado o potencial biotecnológico de dez algas da costa de Peniche relativamente à sua capacidade antioxidante e antimicrobiana. As algas analisadas foram Asparagopsis armata, Plocamium cartilagineum, Ceramium ciliatum, Sphaerococcus coronopifolius, Codium adhaerens, Ulva compressa; Fucus spiralis, Saccorhiza polyschides, Stypocaulon scoparium e Halopteris filicina. A identificação de compostos bioactivos das algas que revelaram o maior potencial biotecnológico também foi alvo de estudo. Os compostos bioactivos foram extraídos das algas com recurso a solventes, nomeadamente metanol, n-hexano e diclorometano. Estes extractos foram utilizados em todos os ensaios experimentais. A capacidade antioxidante foi determinada através da Quantificação Total de Polifenóis (QTP) pelo método de Folin-Ciocalteu e através da avaliação da capacidade de redução do radical 1, 1-Difenil-2-picrilhidrazil (DPPH). A capacidade antimicrobiana foi avaliada através do método de difusão em disco em Bacillus subtilis e Escherichia coli. Por outro lado, a actividade antifúngica foi avaliada pelo acompanhamento da curva de crescimento de Saccharomyces cerevisiae na ausência e na presença dos extractos das algas. A identificação de compostos bioactivos foi realizada por espectrometria de massa com ionização por “electrospray” (ESI-MS). A alga Fucus spiralis, na fracção metanólica, foi a alga que apresentou maior actividade antioxidante, com 90,01 ± 0,003 mg de equivalentes de ácido gálico/g extracto. As algas Saccorhiza polyschides e Fucus spiralis exibiram o efeito mais potente na capacidade de redução do DPPH, com IC50 de 0,049 e 0,099 mg de extracto metanólico/ml, respectivamente. Relativamente à capacidade antimicrobiana, não existiram resultados positivos contra E. coli. No entanto existiram vários resultados positivos contra Bacillus subtilis, tendo sido a alga Asparagopsis armata (fracção metanólica) a que apresentou o maior potencial com um halo de inibição de 10 mm para a menor concentração a que se obteve actividade (100 μg/disco). Quanto à actividade antifúngica, alga Sphaerococcus coronopifolius produziu um efeito inibitório muito potente no crescimento de Saccharomyces cerevisiae, com IC50 de 56,44; 78,9 e 40,2 μg/ml nas fracções metanólica, diclorometano e n-hexano, respectivamente. Não foi possível identificar as principais moléculas responsáveis pela actividade antimicrobiana e antioxidante, no entanto foram identificados dois ácidos gordos de cadeia longa, na fracção do diclorometano da alga Sphaerococcus coronopifolius, o ácido heptadecanóico e o ácido oleico. Pela realização deste trabalho podemos concluir que as algas castanhas Fucus spiralis e Saccorhiza polyschides foram as que apresentaram mais poder antioxidante. Por outro lado, as algas vermelhas Sphaerococcus coronopifolius e Asparagopsis armata destacaram-se pelo elevado potencial antimicrobiano. Das quatro algas referidas, as algas Fucus spiralis e Sphaerococcus coronopifolius, pela potência dos resultados obtidos na capacidade antioxidante e na capacidade antifúngica, respectivamente, são as algas que abrem maiores janelas para a identificação e obtenção de novas moléculas com potencial biotecnológico relevante

Neuroprotective effects of seaweeds against 6-hydroxidopamine-induced cell death on an in vitro human neuroblastoma model

Background: Parkinson’s disease (PD) is a progressive neurodegenerative disorder of the central nervous system. Although the causes of PD pathogenesis remain incomplete, some evidences has suggested that oxidative stress is an important mediator in its pathogenesis. The aim of this study was to evaluate the protective effects of seaweeds with high antioxidant activity on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in the human neuroblastoma cell line SH-SY5Y, as well as the associated intracellular signaling pathways. Methods: Cell viability studies were assessed by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium (MTT) bromide assay and the intracellular signaling pathways analyzed were: hydrogen peroxide (H2O2) production, changes in the mitochondrial membrane potential and Caspase-3 activity. Results: Exposure of SH-SY5Y cells to 6-OHDA (10–1000 μM) reduced cell’s viability in a concentration and timedependent manner. The data suggest that the cell death induced by 6-OHDA was mediated by an increase of H2O2 production, the depolarization of mitochondrial membrane potential and the increase of Caspase-3 activity. Extracts from S. polyshides, P. pavonica, S. muticum, C. tomentosum and U. compressa revealed to efficiently protect cell’s viability in the presence of 6-OHDA (100 μM; 24 h). These effects appear to be associated with the reduction of H2O2 cell’s production, the protection of mitochondrial membrane’s potential and the reduction of Caspase-3 activity. Conclusions: These results suggest that seaweeds can be a promising source of new compounds with neuroprotective potential.info:eu-repo/semantics/publishedVersio

Algae from Portuguese Coast Presented High Cytotoxicity and Antiproliferative Effects on an In vitro Model of Human Colorectal Cancer

Background: The marine environment has shown to be an interesting source of new antitumor agents, representing an important tool in cancer research. Objective: The aim of this study was to evaluate the antitumor activities of 12 algae from Peniche coast (Portugal) on an in vitro model of human colorectal cancer (Caco‑2 cells). Materials and Methods: The antitumor potential was accessed by evaluating Caco‑2 cell’s viability and proliferation through the 3‑[4, 5‑dimethylthiazol‑2‑yl]‑2, 5‑diphenyl tetrazolium bromide and calcein‑AM methods. Results: The dichloromethane extracts of Asparagopsis armata and Sphaerococcus coronopifolius induced the highest decrease on cell’s viability (1 mg/mL; 24 h), 98.96% ± 0.39% and 98.08% ± 0.89%, respectively, followed by the methanolic extracts of S. coronopifolius (96.47% ± 1.26%) and A. armata (92.68% ± 1.17%). Regarding cell proliferation, the highest decrease of Caco‑2 cell’s proliferation (1 mg/mL; 24 h) was induced by the dichloromethane extract of A. armata (100% ± 0.48%), S. coronopifolius (99.04 ± 0.51%), and Plocamium cartilagineum (95.05% ± 1.19%). The highest potency was shown by the dichloromethane extract of S. coronopifolius in both, cytotoxicity and antiproliferative tests, with an IC50 of 21.3 and 36.5 µg/mL, respectively. Conclusion: The extracts of A. armata and S. coronopifolius are promising sources of new bioactive molecules with application in cancer therapeutics.info:eu-repo/semantics/publishedVersio

Natural approaches for neurological disorders: The neuroprotective potential of Codium tomentosum

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, and is characterized by a progressive degeneration of the dopaminergic neurons in the substantia nigra. Although not completely understood, several abnormal cellular events are known to be related with PD progression, such as oxidative stress, mitochondrial dysfunction and apoptosis. Accordingly, the aim of this study was to evaluate the neuroprotective e ects of Codium tomentosum enriched fractions in a neurotoxicity model mediated by 6-hydroxydopamine (6-OHDA) on SH-SY5Y human cells, and the disclosure of their mechanisms of action. Additionally, a preliminary chemical screening of the most promising bioactive fractions of C. tomentosum was carried out by GC-MS analysis. Among the tested fractions, four samples exhibited the capacity to revert the neurotoxicity induced by 6-OHDA to values higher or similar to the vitamin E (90.11 +- 3.74% of viable cells). The neuroprotective e ects were mediated by the mitigation of reactive oxygen species (ROS) generation, mitochondrial dysfunctions and DNA damage, together with the reduction of Caspase-3 activity. Compounds belonging to different chemical classes, such as terpenes, alcohols, carboxylic acids, aldehydes, esters, ketones, saturated and unsaturated hydrocarbons were tentatively identified by GC-MS. The results show that C. tomentosum is a relevant source of neuroprotective agents, with particular interest for preventive therapeutics.info:eu-repo/semantics/publishedVersio

Sea cucumber Holothuria forskali, a new resource for aquaculture? Reproductive biology and nutraceutical approach

Sea cucumbers are highly marketable as a food product due to their nutritional value. Also, it has been suggested that sea cucumbers possess a wide range of bioactive compounds that can be used in the pharmaceutical industry. In this study, the reproductive biology of Holothuria forskali was performed by evaluating the gonadosomatic index (GI) and histological analyses of the gonadal tubules. The biotechnological potential was assessed through the evaluation of the antioxidant, antimicrobial and antitumor potential. Finally, the fatty acid profile was also evaluated. These three subjects were chosen to increase the interest and to focus the economic potential of this species rearing, predicting that it can be sold in Europe or export to Asia to be used for human consumption or for the pharmaceutical industry. The GI and the histological analysis of the gonadal tubules revealed that the range from February to March corresponds to the peak of gonads maturation. Furthermore, the methanolic fraction revealed the highest antimicrobial potential against Candida albicans with an IC50 of 233.2 mg mL -1. Also, this fraction presented the highest cytotoxic and anti-proliferative activities through the method for measuring cell proliferation method in both cell lines, with an IC50 of 238.2 and 396.0 mg mL -1 for MCF-7 cells respectively and 260.3 and 218.7 mg mL -1 for HepG-2 cells respectively. Regarding the fatty acid profile, the total fat content was 4.83% and the highest values were obtained for palmitic acid (9.96%), stearic acid (11.23%), eicosapentaenoic acid (10.49%) and arachidonic acid (20.36%).info:eu-repo/semantics/publishedVersio

Natural Approaches for Neurological Disorders—The Neuroprotective Potential of Codium tomentosum

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, and is characterized by a progressive degeneration of the dopaminergic neurons in the substantianigra. Although not completely understood, several abnormal cellular events are known to be related with PD progression, such as oxidative stress, mitochondrial dysfunction and apoptosis. Accordingly, the aim of this study was to evaluate the neuroprotective effects of Codium tomentosum enriched fractions in a neurotoxicity model mediated by 6-hydroxydopamine (6-OHDA) on SH-SY5Y human cells, and the disclosure of their mechanisms of action. Additionally, a preliminary chemical screening of the most promising bioactive fractions of C. tomentosum was carried out by GC-MS analysis. Among the tested fractions, four samples exhibited the capacity to revert the neurotoxicity induced by 6-OHDA to values higher or similar to the vitamin E (90.11 ± 3.74% of viable cells). The neuroprotective effects were mediated by the mitigation of reactive oxygen species (ROS) generation, mitochondrial dysfunctions and DNA damage, together with the reduction of Caspase-3 activity. Compounds belonging to different chemical classes, such as terpenes, alcohols, carboxylic acids, aldehydes, esters, ketones, saturated and unsaturated hydrocarbons were tentatively identified by GC-MS. The results show that C. tomentosum is a relevant source of neuroprotective agents, with particular interest for preventive therapeuticsThis work was supported by the Portuguese Foundation for Science and Technology (FCT) through the strategic project UID/MAR/04292/2020 to MARE—Marine and Environmental Sciences Centre and UID/Multi/04046/2020 and UIDB/04046/2020 granted to BioISI—BioSystems and Integrative Sciences Institute, through POINT4PAC project (Oncologia de Precisão: Terapias e Tecnologias Inovadoras, SAICTPAC/0019/2015-LISBOA-01-0145-FEDER-016405), through CROSS-ATLANTIC project (PTDC/BIA-OUT/29250/2017), co-financed by COMPETE (POCI-01-0145-FEDER-029250) and through Molecules for Health project (PTDC/BIA-BQM/28355/2017). This work was also funded by the Integrated Programme of SR&TD Smart Valorization of Endogenous Marine Biological Resources Under a Changing Climate (Centro-01-0145-FEDER-000018), co-funded by Centro 2020 Programme, Portugal 2020, European Union, through the European Regional Development FundS

The biotechnological and seafood potential of Stichopus regalis

Sea cucumbers are highly used not only for the production of the bêche-de-mer, but also recent studies have been showing the high potential of these marine invertebrates on the pharmaceutical industry, as they are rich in bioactive compounds with important functions. Stichopus regalis extracts (1 mg∙mL−1) were used in antioxidant, antimicrobial and antitumor assays. The lipid profile was also evaluated. No significant antioxidant activity was detected in both methanolic and dichloromethane extracts. The methanolic fraction showed the highest antimicrobial potential against Candida albicans with an IC50 of 475.4 μg∙mL−1. In the antitumor assays, the dichloromethane fraction showed a high potential for both cell lines, as revealed by the MTT method. The total fat content was 3.63% ± 0.11% and the fatty acid profile revealed the highest amount in C16:0 (9.43% ± 0.77%), C18:0 (12.43% ± 0.83%), C18:1 ω7 (5.63% ± 0.33%), EPA (12.49% ± 0.15%), DHA (7.35% ± 0.02%), ARA (19.29% ± 0.14%) and a ω3/ω6 ratio of 1.078. These findings led us to suggest the potential use of S. regalis as a new source of bioactive compounds with pharmacological potential and its nutritional benefits for human health.info:eu-repo/semantics/publishedVersio

From marine origin to therapeutics: the antitumor potential of marine algae-derived compounds

Marine environment has demonstrated to be an interesting source of compounds with uncommon and unique chemical features on which themolecularmodeling and chemical synthesis of new drugs can be based with greater efficacy and specificity for the therapeutics. Cancer is a growing public health threat, and despite the advances in biomedical research and technology, there is an urgent need for the development of new anticancer drugs. In this field, it is estimated that more than 60% of commercially available anticancer drugs are natural biomimetic inspired. Among the marine organisms, algae have revealed to be one of the major sources of new compounds of marine origin, including those exhibiting antitumor and cytotoxic potential. These compounds demonstrated ability to mediate specific inhibitory activities on a number of key cellular processes, including apoptosis pathways, angiogenesis, migration and invasion, in both in vitro and in vivo models, revealing their potential to be used as anticancer drugs. This review will focus on the bioactive molecules from algae with antitumor potential, from their origin to their potential uses, with special emphasis to the alga Sphaerococcus coronopifolius as a producer of cytotoxic compounds.info:eu-repo/semantics/publishedVersio

Sphaerococcus coronopifolius bromoterpenes as potential cancer stem celltargeting agents

Cancer is one of the major threats to human health and, due to distinct factors, it is expected that its incidence will increase in the next decades leading to an urgent need of new anticancer drugs development. Ongoing experimental and clinical observations propose that cancer cells with stem-like properties (CSCs) are involved on the development of lung cancer chemoresistance. As tumour growth and metastasis can be controlled by tumourassociated stromal cells, the main goal of this study was to access the antitumor potential of five bromoterpenes isolated from Sphaerococcus coronopifolius red alga to target CSCs originated in a co-culture system of fibroblast and lung malignant cells. Cytotoxicity of compounds (10–500 μM; 72 h) was evaluated on monocultures of several malignant and non-malignant cells lines (HBF, BEAS-2B, RenG2, SC-DRenG2) and the effects estimated by MTT assay. Co-cultures of non-malignant human bronchial fibroblasts (HBF) and malignant human bronchial epithelial cells (RenG2) were implemented and the compounds ability to selectively kill CSCs was evaluated by sphere forming assay. The interleucine-6 (IL-6) levels were also determined as cytokine is crucial for CSCs. Regarding the monocultures results bromosphaerol selectively eliminated the malignant cells. Both 12S-hydroxy-bromosphaerol and 12R-hydroxy-bromosphaerol steroisomers were cytotoxic towards non-malignant bronchial BEAS-2B cell line, IC50 of 4.29 and 4.30 μM respectively. However, none of the steroisomers induced damage in the HBFs. As to the co-cultures, 12R-hydroxy-bromosphaerol revealed the highest cytotoxicity and ability to abrogate the malignant stem cells; however its effects were IL-6 independent. The results presented here are the first evidence of the potential of these bromoterpenes to abrogate CSCs opening new research opportunities. The 12R-hydroxy-bromosphaerol revealed to be the most promising compound to be test in more complex living models.info:eu-repo/semantics/publishedVersio

Antioxidant and neuroprotective potential of the Brown Seaweed Bifurcaria bifurcata in an in vitro Parkinson’s disease model

Bifurcaria bifurcata is a marine brown seaweed mainly found on the Atlantic coast. Herein, we report the antioxidant and neuroprotective activities of seven fractions (F1–F7) obtained by normal phase chromatography from the B. bifurcata dichloromethane extract, as well as of its two major isolated diterpenes. Total phenolic content of fractions was determined by the Folin–Ciocalteu method, while antioxidant activity was evaluated by the DPPH, ORAC, and FRAP assays. Neuroprotective effects were evaluated in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y), while the mechanisms associated to neuroprotection were investigated by the determination of mitochondrial membrane potential, H2O2 production, Caspase-3 activity, and by observation of DNA fragmentation. Fractions F4 and F5 exhibited the best neuroprotective and antioxidant activities, respectively. F4 fraction prevented changes in mitochondrial potential, and induced a reduction of H2O2 levels production and an increase in cell viability, suggesting that it may contain multi-target compounds acting on different pathways. Hence, this fraction was subjected to purification steps, affording the known diterpenes eleganolone and eleganonal. Both compounds exhibited antioxidant potential, being interesting candidates for further neuroprotective studiesThis work was supported by the Portuguese Foundation for Science and Technology (FCT) through Strategic Projects UID/MAR/04292/2013 and UID/Multi/04046/2019 granted to MARE—Marine and Environmental Sciences Centre, and BioISI—BioSystems and Integrative Sciences Institute, respectively, through Red2Discovery Project (PTDC/MAR-BIO/6149/2014), co-financed by COMPETE (POCI-01-0145-FEDER-016791), through Oncologia de Precisão: Terapias e Tecnologias Inovadoras project (POINT4PAC) (SAICTPAC/0019/2015-LISBOA-01-0145-FEDER-016405) and through CrossAtlantic Project (PTDC/BIA-OUT/29250/2017), co-financed by COMPETE (POCI-01-0145-FEDER-029250). This work was also funded by the Integrated Programme of SR&TD Smart Valorization of Endogenous Marine Biological Resources Under a Changing Climate (reference Centro-01-0145-FEDER-000018), co-funded by Centro 2020 Programme, Portugal 2020, European Union, through the European Regional Development FundS