Marcelo Dias Catarino Florotaninos Da Alga Fucus Vesiculosus: Extração, Caracterização Estrutural E Efeitos Ao Longo Do Trato Gastrointestinal

Universidade de Aveiro Departamento de Química 2021

Marcelo Dias Catarino Florotaninos da alga Fucus vesiculosus: Extração, caracterização estrutural e efeitos ao longo do trato gastrointestinal

Fucus vesiculosus phlorotannins: Extraction, structural characterization and effects throughout the gastrointestinal tract

Universidade de Aveiro Departamento de Química Ano 2021

Marcelo Dias Catarino Florotaninos da alga Fucus Vesiculosus: Extração, caracterização estrutural e efeitos ao longo do trato gastrointestinal

Fucus vesiculosus phlorotannins: Extraction, structural characterization and effects throughout the gastrointestinal tract Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Química Sustentável, realizada sob a orientação científica da Doutora Susana Maria Almeida Cardoso, Investigadora Doutorada do Departamento de Química da Universidade de Aveiro, Professor Doutor Artur Manuel Soares da Silva, Professor Catedrático do Departamento de Química da Universidade de Aveiro e do Professor Doutor Nuno Filipe da Cruz Baptista Mateus, Professor Associado do Departamento de Química e Bioquímica da Faculdade de Ciências da Universidade do Porto

Estudo efetuado com apoio financeiro do projeto PTDC/BAA-AGR/31015/2017, suportado pelos orçamentos do Programa Operacional de Competitividade e Internacionalização— POCI, na sua componente FEDER, e da Fundação para a Ciência e a Tecnologia, na sua componente de Orçamento de Estado. A Universidade de Aveiro e a Fundação para a Ciência e para a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES) financiaram o Laboratório Associado para a Química Verde (LAQV) da Rede de Química e Tecnologia (REQUIMTE) (UIDB/50006/2020), através de fundos nacionais e, quando aplicável, co-financiado pela FEDER, no âmbito do Portugal 2020. A FCT financiou a bolsa de PhD de Marcelo D. Catarino (PD/BD/114577/2016).

Dedico este trabalho à minha família e amigos, pela presença constante.

“A person who never made a mistake never tried anything new.” Albert Einstein

o júri presidente Prof. Doutor João Carlos de Oliveira Matias Professor Catedrático da Universidade de Aveiro

vogais Doutor Victor Armando Pereira Freitas Professor Catedrático da Universidade do Porto

Maria Manuela Estevez Pintado Professora Associada da Universidade Católica Portuguesa do Porto

Maria Teresa Cruz Rosete Professora Auxiliar da Universidade de Coimbra

Diana Cláudia Gouveia Alves Pinto Professora Auxiliar da Universidade de Aveiro

Doutora Susana Maria de Almeida Cardoso (Orientadora) Investigadora Doutorada (nível 3) da Universidade de Aveiro

agradecimentos Tendo chegado ao final deste longo e intrincado percurso, não posso deixar de dar uma palavra de agradecimento às pessoas que dele fizeram parte.

Em primeiro lugar à Doutora Susana Cardoso, orientadora deste trabalho, pela confiança, motivação, apoio e ensinamentos transmitidos.

Ao Professor Doutor Artur Silva e Professor Doutor Nuno Mateus, pela disponibilidade e apoio à realização deste trabalho.

Agradeço à antiga Unidade de Química Orgânica, Produtos Naturais e Agro- Alimentares (QOPNA) e ao recente Laboratório Associado para a Química Verde da Rede de Química e Tecnologia (LAQV-REQUIMTE) do Departamento de Química da Universidade de Aveiro, por ter disponibilizado todas as condições possíveis para a realização deste trabalho.

Agradeço à Fundação para a Ciência e a Tecnologia (FCT) pelo financiamento através de uma bolsa de doutoramento (PD/BD/114577/2016).

Aos meus colegas da FCUP, Iva Fernandes e Hélder Oliveira, pela ajuda preciosa que foram na realização de algumas tarefas.

À Professora Doutora Manuela Pintado e restantes membros da equipa, Teresa Bonifácio, Débora Campos e Manuela Machado, pelo acompanhamento e ensinamentos, e por me acolherem na ESB e disponibilizar condições.

Também à Professora Doutora Maria Teresa Cruz e restantes membros da equipa, Ana Silva, Cátia Sousa, Myléne Carrascal, Isabel Ferreira, por me terem acolhido no CNC, disponibilizando também condições e tempo para me acompanhar e transmitir ensinamentos. Ao Daniel Ferreira e Adriana Tavares, pela amizade e momentos de boa disposição.

Aos atuais e ex-colegas do meu grupo e grupos vizinhos Ana Rita Circuncisão, Andreia Silva, Cátia Oliveira, Élia Maricato, Letícia Costa, Mariana Mesquita, Mariana Vallejo, Pedro Fernandes, Ricardo Ferreira, Rita Bastos, Sofia Queirós, Sónia Ferreira e Soraia Silva pelo convívio e camaradagem. Um especial agradecimento à Catarina Marçal, pelo apoio e colaboração.

Aos meus bons amigos Daniel Oliveira, Diogo Carreiras, Gilberto Alkimin, Joana Martins, Luis Santos, Maria Gonçalves, Mariana Pires, Sónia Russo, Patrícia Vasconcelos, Renata Vicente, Sara Vallejo, Telmo Alves e Thomas Martins pelos momentos de convívio e boa disposição, força e motivação.

Ao Rui Soares pelo ânimo que me tem dado nesta etapa final.

À minha família, por serem a minha pedra basilar.

Obrigado a todos!

palavras-chave Fucus vesiculosus, florotaninos, extração, propriedades bioativas, antioxidante, anti-inflamatório, antitumoral, prébiotico.

resumo Desde a antiguidade que as macroalgas marinhas têm sido tradicionalmente usadas pelos povos de extremo oriente, quer para consumo direto, como fonte de nutrientes, como para fins medicinais. Nos últimos anos estes organismos marinhos têm vindo a ganhar uma crescente popularidade entre as populações ocidentais que começam a estar cada vez mais conscientes do potencial que estas possuem como uma possível fonte de compostos bioativos de grande interesse. De entre os seus compostos destacam-se os florotaninos, um grupo de compostos fenólicos derivados do floroglucinol, reivindicados pelos seus benefícios para a saúde, e cuja ocorrência é exclusiva das macroalgas castanhas. Apesar de várias propriedades bioativas terem já sido demonstradas para estes compostos, existe ainda um longo percurso a percorrer até à compreensão dos mecanismos por detrás destas, sendo isto particularmente verdade no que diz respeito às propriedades anti-inflamatórias e antitumorais dos florotaninos provenientes da macroalga Fucus vesiculosus. Além disso, pouco se sabe acerca do destino destes compostos durante a sua passagem pelo trato gastrointestinal. Neste contexto, neste trabalho pretendeu-se otimizar a extração de florotaninos a partir desta macroalga e elucidar a composição destes extratos através da técnica de UHPLC-DAD-ESI-MSn, bem como clarificar as potencialidades bioativas de extrato bruto ou frações do ponto de vista de atuação ao longo to trato gastrointestinal, nomeadamente os efeitos antioxidante, anti-inflamatório, antitumoral e prébiotico. Recorrendo à técnica de superfície-resposta, as condições determinadas para o máximo de recuperação de florotaninos da alga F. vesiculosus foram: acetona a 67% (v/v) numa proporção de 70 mL/g de farinha de alga a 25 ºC. Após um passo de purificação intermédio, a fração rica em florotaninos (EtOAc) revelou um complexo perfil cromatográfico composto por vários fucóis, fucofloretóis, fualóis e outros derivados de florotaninos. Foram ainda detetados três potenciais novos derivados de florotaninos, nomeadamente o fucofurodifloretol, fucofurotrifloretol e o fucofuropentafloretol. Tanto o extrato bruto como a EtOAc revelaram boa atividade antioxidante, em particular contra o radical de óxido nítrico, um importante interveniente na cascata sinalizadora da inflamação. De facto, uma atividade anti-inflamatória pronunciada foi confirmada em células RAW 264.7 estimuladas com lipopolissacarídeo (LPS). A partir da EtOAc foram ainda obtidas 9 sub-frações de florotaninos com diferentes pesos moleculares que, após testadas na mesma linha celular, revelaram algumas diferenças ao nível da atividade anti- inflamatória que possivelmente estarão relacionados com a sua complexidade e peso molecular. Um efeito anti-inflamatório bastante marcado foi ainda observado para uma sub-fração em particular, a F2, caracterizada pela presença de um composto derivado de florotaninos com peso molecular de 508 g/mol.

Esta fração, a 200 µg/mL, foi capaz de inibir a fosforilação e degradação da proteína IκBα, consequentemente bloqueando a cascata sinalizadora da inflamação ao nível transcricional. A fração EtOAc bem como algumas das posteriores sub-frações, nomeadamente F1 e F5, demostraram ainda propriedades antitumorias promissoras, provocando um efeito citotóxico seletivo apenas contra células tumorais de cancro gástrico (MKN-28) e cancro do colon (Caco-2 e HT-29), não afetando células normais. Das três amostras, a F5, caracterizada pela presença de fucóis, fucofloretóis, um fucofurodifloretol e eckstolonol, foi a que revelou o efeito mais acentuado em todas as linhas celulares tumorais, demonstrando valores de IC50 de 56.3 ± 14.7, 97.4 ± 11.6 e 118.8 ± 19.7 µg/mL para MKN-28, Caco-2 e HT-29, respetivamente. Curiosamente, enquanto a EtOAc exerceu este efeito através do bloqueio do ciclo celular e ativação de mecanismos de morte celular, a sub-fração F1 apenas promoveu o bloqueio do ciclo celular enquanto a sub-fração F5 apenas ativou a morte celular via ativação de mecanismos de apoptose/necrose. Por fim, verificou-se uma boa capacidade inibidora das enzimas digestivas por parte destes compostos, em particular da enzima α-glucosidase cujo IC50 foi 45 a 250 vezes inferior ao da acarbose, revelando potencial terapêutico contra a diabetes do tipo-II. No entanto, à semelhança dos polifenóis das plantas, os florotaninos revelaram-se suscetíveis a degradação e perda de atividade antioxidante durante a sua passagem ao pelo trato gastrointestinal, com menos de 15% dos florotaninos totais da amostra inicial ficando bioacessíveis e disponíveis para absorção. Em contrapartida, apesar de a fração não bioacessível das amostras de F. vesiculosus terem apenas contribuído com um efeito modestamente positivo na modulação da microbiota humana, estas demonstraram uma capacidade interessante de estimular a produção de propionato e butirato, dois ácidos gordos de cadeia curta com propriedades benéficas para a saúde do hospedeiro bem estabelecidas. Em conclusão, este estudo não só permitiu compreender melhor a composição dos florotaninos presentes na alga F. vesiculosus, como também demonstrou que os seus extratos e/ou frações purificadas podem ter um impacto relevante na manutenção de uma boa saúde gastrointestinal atuando em diferentes níveis. Em última instância, este trabalho contribui para a valorização da espécie F. vesiculosus como uma possível fonte de compostos naturais com grande potencial biológico e de aplicabilidade.

keywords Fucus vesiculosus, phlorotannins, extraction, bioactive properties, antioxidant, anti-inflammatory, antitumor, prebiotic.

abstract Marine macroalgae have a long tradition of usage and applications among the far Eastern populations, either for direct consumption and nutrition or for medicinal purposes. During the recent years, these marine organisms have received increasing popularity among the Western populations which are becoming aware of their potential to be exploited as a source of valuable bioactive compounds. Among these compounds are the phlorotannins, which are a group of phloroglucinol-derived phenolic compounds occurring exclusively on brown seaweeds and claimed for their promising health-promoting effects. Although several bioactive properties have been demonstrated for these compounds, there is still a long way to go before understanding the mechanisms behind them, and this is particularly true for the anti-inflammatory and antitumor properties of phlorotannins from Fucus vesiculosus. Moreover, little is known about the fate of these compounds when crossing the gastrointestinal tract. In this context, the aim of this work was to maximize the extraction of phlorotannins from this species for further characterization through UHPLC- DAD-ESI-MSn analysis and evaluation of the bioactive properties that could be relevant from a performance point of view throughout the gastrointestinal tract, namely antioxidant, anti-inflammatory, antitumor and prebiotic effects. Through the response-surface methodology, the conditions determined for maximum recovery of F. vesiculosus phlorotannins were acetone 67% (v/v) in a proportion of 70 mL/g of seaweed powder at 25 °C. After an intermediate purification step, the phlorotannin-rich fraction (EtOAc) revealed a complex chromatographic profile composed of several fucols, fucophlorethols, fuhalols and several other phlorotannin derivatives. Additionally, three potential new phlorotannin derivatives, namely fucofurodiphlorethol, fucofurotriphlorethol and fucofuropentaphlorethol have been tentatively identified in this extract. Both the crude extract and EtOAc revealed good antioxidant activity, particularly against the nitric oxide radical, which is as very important player in the inflammatory signalling cascade. Indeed, strong anti-inflammatory activity was confirmed in a cellular system of inflammation using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. From the EtOAc further subfractions with phlorotannins of increasing molecular weights were obtained and tested in the same cell line, revealing some differences between their anti-inflammatory activity which could be related to their complexity and molecular weight. A remarkably strong anti-inflammatory activity was observed for a particular subfraction, F2, characterized by the presence of a major phlorotannin derivative with molecular weight of 508 g/mol.

At 200 µg/mL this subfraction was capable of inhibiting IκBα phosphorylation and degradation, and consequently blocking the pro-inflammatory signalling cascade at the transcriptional level. The EtOAc and some of its subfractions, namely F1 and F5, also demonstrated promising antitumor properties exerting a selective cytotoxic effect against gastric (MKN-28) and colon cancer cells (Caco- 2 and HT-29) but not on normal cells. From these samples, F5, characterized for the presence of fucols, fucophlorethols, a fucofurodiphlorethol and eckstolonol, revealed the strongest effect in all the three tumor cell lines, exhibiting IC50 values of 56.3 ± 14.7, 97.4 ± 11.6 and 118.8 ± 19.7 µg/mL for MKN-28, Caco-2 and HT- 29, respectively. Interestingly, while the EtOAc displayed this activity via induction of cell cycle arrest and activation of cell death mechanisms, the subfraction F1 only caused cell cycle arrest and the subfraction F5 only triggered cell death via activation of the apoptotic/necrotic mechanisms. Finally, these compounds were found to exert promising inhibitory capacity against some digestive enzymes, particularly α-glucosidase, in which the IC50 values were 45 a 250 times lower compared to acarbose, thus revealing a therapeutical potential for the treatment of type-II diabetes. However, similar to plant polyphenols, phlorotannins were found to be susceptible to degradation and loss of antioxidant activity during their passage through the gastrointestinal tract, with less than 15% of the initial total phlorotannins becoming bioaccessible and available for absorption. In turn, although the non-bioaccessible fraction of F. vesiculosus samples only contributed with a modest positive effect for the modulation of human microbiota, they could stimulate the production of propionate and butyrate,’pç which are two short-chain fatty acids with well- established health-promoting properties for the host. In conclusion, this study not only allowed better understanding of the phlorotannin composition of F. vesiculosus, but also demonstrates that its extracts and/or purified fractions may have a relevant impact in the maintenance of a positive gastrointestinal health acting at different levels. Ultimately, this work contributes to the valorization of this species as a possible supply of natural compounds with great bioactive potential and applicability.

Table of Contents

Chapter 1 . 1 Introduction 3 1.1 The value of the ocean 3 1.2 Seaweeds 4 1.2.1 Brown Seaweeds 6 1.3 Bioactive compounds from brown seaweeds 7 1.3.1 Phlorotannins 9 1.3.1.1 Biosynthesis and chemistry of phlorotannins 16 1.3.1.2 Extraction, purification and quantification of phlorotannins 17 1.4 Oxidative stress, inflammation and cancer 18 1.4.1 Involvement of phlorotannins in oxidative stress, inflammation and cancer 21 1.5 Gut microbiota, human health and prebiotics 30 1.5.1 Prebiotic effects of phlorotannins 31 Motivation and Aims 33

Chapter 2 . 35 2.1 Optimization of phlorotannins extraction from F. vesiculosus 37 2.2 Materials and methods 37 2.2.1 Chemicals 37 2.2.2 Single-factor experiments 37 2.2.3 Experimental design for optimization of phlorotannins extraction 38 2.2.4 Preparation and purification of F. vesiculosus optimal extract 39 2.2.5 Determination of total phlorotannins contente (TPhC) 40 2.2.6 UHPLC-DAD-ESI/MS analysis 40 2.2.7 Statistical analysis 40 2.3 Results and discussion 41 2.3.1 Single-factor experiments 41 2.3.1.1 Effect of the acetone concentration on TPhC 41 2.3.1.2 Effect of the solvent-solid ratio on TPhC 42 2.3.1.3 Effect of the temperature on TPhC 43 2.3.1.4 Effect of time on TPhC 43 2.3.2 Analysis of the response surface methodology 44 2.3.2.1 Fitting the model 44 2.3.2.2 Effect of the independent variables on the TPhC 45 2.3.2.3 Optimization and validation of the models 46 2.3.3 Total phlorotannin content of the F. vesiculosus extract and respective fractions 47 2.3.4 Characterization of phlorotannins-rich fraction 47 2.4 Conclusions 55

Chapter 3 . 57 3.1 Antioxidant and anti-inflammatory properties of F. vesiculosus phlorotannins 59 3.2 Materials and methods 59 3.2.1 Chemicals 59 3.2.2 Extraction, purification and UHPLC-DAD-ESI/MS analysis of phlorotannins from F. vesiculosus 60 3.2.3 Antioxidant assays 61 3.2.3.1 Oxygen radical absorbance capacity (ORAC) 61 ●– 3.2.3.2 Superoxide anion (O2 ) scavenging assay 61 3.2.3.3 Xanthine oxidase (XO) assay 61 3.2.3.4 Nitric oxide (NO●) scavenging assay 61 3.2.4 Anti-inflammatory experiments 62 3.2.4.1 Cell culture 62 3.2.4.2 Assessment of cell viability 62 3.2.4.3 Inhibition of LPS-stimulated NO● 62 3.2.4.4 Preparation of total protein extracts and western blotting 63 3.2.5 Statistical analysis 63 3.3 Results and discussion 64 3.3.1 Antioxidant properties of F. vesiculosus crude extract and phlorotannin-rich fraction 64 3.3.2 Anti-inflammatory properties of F. vesiculosus in Raw 264.7 cells 66 3.3.2.1 Effects on cell viability and LPS-induced NO● production 66 3.3.2.2 Effects on the expression of iNOS, COX-2 and IL-1β 68 3.3.2.3 Effects on the NF-κB signaling pathway 71 3.3.3 Characterization of F2 73 3.4 Conclusions 75

Chapter 4 . 77 4.1 Antitumor activity of F. vesiculosus phlorotannins through activation of apoptotic signals 79 4.2 Materials and methods 79 4.2.1 Chemicals 79 4.2.2 Extraction, purification and UHPLC-DAD-ESI/MS analysis of phlorotannins from F. vesiculosus 80 4.2.3 Cell culture 80 4.2.4 Assessment of cell viability 80 4.2.5 Flow cytometric analysis of the cell cycle and apoptosis via annexin V-PI double staining 81 4.2.6 Statistical analysis 81 4.3 Results and discussion 81 4.3.1 Effect of CRD, EtOAc and purified fractions on tumor cells viability 81 4.3.2 Effects on cell cycle after exposure to F. vesiculosus samples 83 4.3.3 Apoptosis/necrosis detection on cells treated with F. vesiculosus samples 84 4.3.4 Characterization of F5 86 4.4 Conclusions 89

Chapter 5 . 91 5.1 Impact of phlorotannin extracts from F. vesiculosus on human digestive enzymes and gut microbiota 93 5.2 Material and methods 93 5.2.1 Chemicals 93 5.2.2 Extraction procedure 94 5.2.3 Effects on the metabolic enzymes 94 5.2.3.1 α-Amylase inhibition assay 94 5.2.3.2 α-Glucosidase inhibition assay 95 5.2.3.3 Pancreatic lipase inhibition assay 95 5.2.4 Gastrointestinal digestion simulation 95 5.2.5 Determination of the phlorotannin content and antioxidant activities 96 5.2.6 Prebiotic potential 96 5.2.7 In vitro fermentation assays 97 5.2.8 Gut microbiota evaluation 97 5.2.8.1 DNA extraction 97 5.2.8.2 Real-time PCR for microbial analysis at stool 97 5.2.8.3 Determination of organic acids 99 5.2.9 Statistical analysis 99 5.3 Results and discussion 99 5.3.1 Effects on the metabolic enzymes 99

5.3.2 Stability, bioaccessibility and antioxidant activity of F. vesiculosus extracts throughout the simulated GIT 101 5.3.3 Prebiotic effect 103 5.3.4 Evolution of the gut microbiota profile groups 105 5.3.5 Organic acids profile and pH variation 109 5.4 Conclusions 113

Chapter 6 115 Conclusions and future perspectives 117

References 119

Publications and communications The data collected along this work and presented in this thesis led to the publication of several papers in distinct international scientific peer-reviewed journals, as well as several communications (either oral or poster) in national and international meetings.

Publications in peer-reviewed journals Catarino, Marcelo D.; Silva, Artur, M. S.; Cardoso, Susana M. “Fucaceae: A Source of Bioactive Phlorotannins”, International Journal of Molecular Sciences, 2017, 18, 1327

Catarino, Marcelo D.; Silva, Artur, M. S.; Cardoso, Susana M. “Phycochemical Constituents and Biological Activities of Fucus spp.”, Marine Drugs, 2018, 16, 249

Catarino, Marcelo D.; Silva, Artur, M. S.; Mateus, Nuno; Cardoso, Susana M. “Optimization of Phlorotannins Extraction from Fucus vesiculosus and Evaluation of Their Potential to Prevent Metabolic Disorders”, Marine Drugs, 2019, 16, 162

Catarino, Marcelo D.; Silva, Ana; Cruz, Maria T.; Mateus, Nuno; Silva, Artur, M. S.; Cardoso, Susana M. “Phlorotannins from Fucus vesiculosus: Modulation of inflammatory response by blocking NF-κB signaling pathway”, International Journal of Molecular Sciences, 2020, 21, 6897

Catarino, M.D.; Fernandes, I.; Oliveira, H.; Carrascal, M.; Cruz, M.T.; Mateus, N.; Silva, A.M.S.; Cardoso, S.M. Antitumor activity of Fucus vesiculosus phlorotannins through activation of apoptotic signals. In preparation

Catarino, M.D.; Marçal, C.; Bonifácio, T.; Campos, D.; Ferreira, R.; Pintado, M.; Mateus, N.; Silva, A.M.S.; Cardoso, S.M. Impact of phlorotannin extracts from Fucus vesiculosus on human gut microbiota. In preparation.

Oral Communications Catarino, Marcelo D.; Fernandes, Iva; Oliveira, Hélder; Silva, Ana; Mateus, Nuno; Silva, Artur, M. S.; Cruz, Teresa; Cardoso, Susana M. “Fucus vesiculosus phlorotannins as potential anti-inflammatory and antitumor agents”, Iberphenol, Coimbra, Portugal, 2020, 5-6 November

Catarino, Marcelo D.; Cruz, Maria T.; Silva, Artur, M. S.; Cardoso, Susana M. “Phlorotannins from Fucus vesiculosus and their potential to prevent diseases of the modern civilization”, XXV Encontro Luso Galego de Química, Santiago de Compostela, Spain, 2019, 20-22 November

Catarino, Marcelo D.; Silva, Artur, M. S.; Cardoso, Susana M., “Phlorotannins from Fucus vesiculosus: potential for prevention of diabetes and obesity”, XXIV Encontro Luso Galego de Química, Porto, Portugal, 2018, 21-23 November Poster Communications Catarino, Marcelo D.; Fernandes, Iva; Mateus, Nuno; Silva, Artur, M. S.; Cardoso, Susana M. “Phlorotannins from Fucus vesiculosus and their potential benefits towards the gastrointestinal tract” XVI International Symposium on Marine Natural Products and XV European Congress on Marine Natural Products joint meeting, Peniche, Portugal, 2019, 1-5 September

Catarino, Marcelo D.; Mateus, Nuno; Silva, Artur, M. S.; Cardoso, Susana M. “Optimization of phlorotannins extraction from Fucus vesiculosus and their potential to prevent metabolic disorders” 2nd International Conference on Food Bioactives & Health, Lisbon, Portugal, 2018, 26-28 September

List of Figures

Figure 1.1. Number of publications in the period between 1980 – 2020 by simple search in Scopus using the keyword “phlorotannins” (A) and number of patents retrieved between 2011 – 2020 by searching for “phlorotannins” in the patent database of World Intellectual Property Organization (B)...... 15 Figure 1.2. Schematic representation of the phloroglucinol biosynthetic pathway (image retrieved from [56]) ...... 16 Figure 1.3. Representation of the different classes of phlorotannins (image retrieved from [56]) ...... 17 Figure 1.4. Schematic overview of the signaling cascades that mediate the oxidative stress and inflammatory conditions leading to neoplastic transformation...... 19 Figure 1.5. Number of publications in the period between 2000 – 2020 by simple search in Scopus using the keyword “polyphenols” AND “prebiotics”...... 32 Figure 2.1. Effect of (A) acetone concentration, (B) solvent-solid ratio, (C) temperature and (D) extracting time on the TPhC of F. vesiculosus extracts in the single-factor experiments. Initial extraction conditions consisted of 70% acetone, in a proportion of 1:20 (m:v) at room temperature during 24 h. Before moving to the next experiment, the previous condition was fixed at the point that showed the best TPhC. Data represent the mean ± SD of at least 3 independent assays and the results are expressed in mg of phloroglucinol equivalents/g of dried seaweed. Different letters represent statistical significance (one-way ANOVA followed by Tukey’s post hoc test; p ≤ 0.05)...... 42 Figure 2.2. Response surface plots for the total phlorotannin content (TPhC in mg PGE/g DW) from F. vesiculosus extracts with respect to acetone concentration (%, X1) and solvent-solid ratio (mL/g, X2). The variable temperature was kept at its zero level...... 45 Figure 2.3. Total ion chromatogram (TIC) of the EtOAc fraction. Peaks marked with numbers correspond to the tentatively identified compounds represented in Table 2.7...... 48 Figure 2.4. Structure of phlorotannin compounds tentatively identified in F. vesiculosus EtOAc fraction and proposed fragmentation patterns: (A) phloroglucinol ([M − H]− at m/z 125), (B) trifucol ([M − H]− at m/z 373), (C) tetrafucol ([M − H]− at m/z 497), (D) hexafucol ([M − H]− at m/z 745), (E) trifucophlorethol ([M − H]− at m/z 621), (F) trifucotriphlorethol ([M − H]− at m/z 869), (G) difucotetraphlorethol ([M − H]− at m/z 869), (H) trifucotetraphlorethol ([M − H]− at m/z 993), (I) dibenzodioxine-1,3,6,8-tetraol ([M − H]− at m/z 247), (J) fucofurodiphlorethol ([M − H]− at m/z 479), (K) fucofurotriphlorethol ([M − H]− at m/z 603). Fragmentations with simultaneous loss of water are only representative. Cleavage of the OH group may occur at different sites...... 49 Figure 3.1. Flowchart for extraction and fractionation of phlorotannins from F. vesiculosus. F1 to F9 represent nine subfractions obtained from the Sephadex LH-20 column chromatography with a solvent system of decreasing polarity. S/L—solid/liquid ratio (g/mL), RT—room temperature, MeOH— methanol, Act—acetone...... 60 Figure 3.2. Effects of F. vesiculosus crude extract (CRD), ethyl acetate fraction (EtOAc) and subsequent subfractions (F1–F9) on the NO● production (grey bars) and viability (◼) of LPS-stimulated Raw 264.7 cells. Data represent the mean ± SEM from at least 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, indicate that NO● production is significantly different from the positive control (with LPS) and # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 indicate that cells viability are statistically different from the negative control (CTRL, without LPS), as determined by one- way ANOVA followed by Dunnet’s post hoc test...... 67 Figure 3.3. The effects of F. vesiculosus extract and partitioned fractions on the expression of pro IL-1β, iNOS and COX-2 in LPS-stimulated Raw 264.7 cells. The immunoblots presented are representative of three independent blots; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicate significant differences from the positive control (LPS), as determined by one-way ANOVA, followed by Dunnett’s post hoc test. CRD—crude extract, EtOAc—ethyl acetate fraction...... 69 Figure 3.4. Effects of crude extract, F2 and F3, on the activation of the NF-κB signaling in LPS-stimulated Raw 264.7 cells after 15 min (pIκBα) and 25 min (IκBα) of incubation. The immunoblots presented are representative of 3 independent blots. * p < 0.05, ** p < 0.01 and **** p < 0.0001 indicate significantly differences from the positive control (with LPS) for pIκBα and negative control (without LPS) for IκBα, as determined by one-way ANOVA followed by Dunnett’s post hoc test. CRD—Crude extract ...... 72 Figure 3.5. Chromatographic profile at 280 nm (A) and total ion chromatogram (B) of F2. Peaks marked with numbers correspond to the tentatively identified compounds represented in Table 3.2...... 74 Figure 4.1. Effect of F. vesiculosus samples on the cell viability of Caco-2, HT-29, MKN-28 and HFF-1 cells after 48 h. Data are expressed as percentage of survival compared to the negative control and are given as the means ± SEM of at least three independent experiments...... 82 Figure 4.2. Effect of F. vesiculosus samples on the cell cycle distribution of Caco-2 and MKN-28 cells after 48 h. Data are expressed as percentage of PI+ cells and are given as the means ± SEM of at least three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 when compared to control; the symbols *, # and × were used for G0/G1, S, G2/M, respectively)...... 84 Figure 4.3. Detection of apoptosis/necrosis after the 48 h treatment with F. vesiculosus samples via annexin V-FITC/PI labelling. The populations of early apoptotic cells, late apoptotic cells, necrotic cells, and viable cells, were evaluated as a percentage of total cells and are given as the means ± SEM of at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 when compared to control; the symbols *, # and × were used for viable, apoptotic and necrotic cells, respectively)...... 85 Figure 4.4. UV chromatograms of recorded at 280 nm and extracted ion chromatograms of the major compounds detected in F5. Peaks marked with numbers correspond to the tentatively identified compounds listed in Table 4.1...... 87 Figure 5.1. Growth curves of L. casei (L01), L. acidophilus (LA-5), B. animalis (BB0) and B. animalis spp. lactis (BB12) in presence of different concentrations of digested crude (CRD) and ethyl acetate fraction (EtOAc)...... 105 Figure 5.2. Evolution of the gut microbiota groups (relative differences to negative control in %) and Firmicutes:Bacteroidetes (F:B) ratio along the fermentation...... 108

List of Tables

Table 1.1. Nutrient composition of selected edible brown macroalgae species in % of dry weight (adapted from [23]) ...... 8 Table 1.2. Overview of the phenolic content of brown algae from different locations, harvesting periods and extracted with different conditions (adapted from [51,52]) ...... 11 Table 2.1. Independent variables and their coded levels used in the BBD ...... 38 Table 2.2. Box-Behnken experimental design matrix and the experimental and predicted values observed for TPhC ...... 39 Table 2.3. Regression coefficients and results of ANOVA analysis of the model...... 45 Table 2.4. Predicted and experimental values obtained for TPhC according to the predicted optimum conditions...... 46 Table 2.5. Extraction yield (as w/w of algal powder for crude extract and w/w of crude extract for the fractions) and total phlorotannin content of F. vesiculosus crude extract and subsequent fractions. ... 47 Table 2.7. Tentative assignment of the compounds present in the EtOAC of F. vesiculosus extract, analyzed by LC-ESI-MS/MS...... 53 Table 3.1. Antioxidant activities of F. vesiculosus CRD, EtOAC and the respective reference compounds. ... 65 Table 3.2. Tentative assignment of the compounds detected in the F2 analyzed by UHPLC-ESI-MS/MS...... 75 Table 4.1. Tentative assignment of the compounds detected in the F5 analyzed by UHPLC-ESI-MS/MS...... 89 Table 5.1. Primer sequences and real-time PCR conditions used for gut microbiota analysis ...... 98 Table 5.2. Inhibition of α-glucosidase, α-amylase and pancreatic lipase by F. vesiculosus crd, EtOAC and the respective reference compounds...... 100 Table 5.3. Total phlorotannin content and antioxidant activity of F. vesiculosus CRD and EtOAC through the different stages of the gastrointestinal digestion...... 102 Table 5.4. Fecal microbiota composition of volunteer participants...... 106 Table 5.5. Concentration of organic acids (succinic, lactic, acetic, propionic and butyric) throughout fermentation of digested FOS, CRD and EtOAC with human microbiota (mg/mL) ...... 112 List of Abbreviations

[M − H]− CRD Deprotonated molecular ion Crude extract 1H-NMR Ctrl Proton nuclear magnetic resonance Control AA DAD Arachidonic acid Diode array detector AAPH DCM 2,2’-Azobis(2-amidinopropane)di- Dicloromethane hydrochloride DE ABTS+ Dry extract 2,2′-Azinobis(3-ethylbenzothiazoline-6- DMBA sulphonic acid Dimethoxybenzaldehyde Akt DMEM Protein kinase B Dulbecco’s modified Eagle’s medium AP-1 DMSO Activator protein 1 Dimethyl sulfoxide AR DNS Aqueous residue Dinitrosalicylic acid ATF3 DPPH● Activating transcription factor 3 1,1-Diphenyl-1,2-picrylhydrazyl radical Aβ25-35 DW Amyloid β-protein fragment 25-35 Dry weight Bak EEZ Bcl-2 homologous antagonist/killer Exclusive economic zone Bax EGFR Bcl-2-associated X protein Epidermal growth factor receptor BBD EIC Box-Behnken design Extracted ion chromatogram Bcl-2 ERK B-cell lymphoma 2 Extracellular signal-regulated protein kinase Bcl-xL ESI B-cell lymphoma extra large Electrospray ionization BHT EtOAc Butylated hydroxytoluene Ethyl acetate fraction Bid EtOH BH3 interacting-domain death agonist Ethanol Bim EU Bcl-2-like protein 11 European union CAT FAO Catalase Food and agriculture organization Chl FBS Chloroformm Fetal bovine serum CID FLIP Collision induced dissociation FLICE-like inhibitory protein COX-2 FOS Cyclooxygenase-2 Fructo-oligosaccharides CRC GIT Colorectal cancer Gastrotintestinal tract

GOS MAE Galacto-oligosaccharides Microwave-assisted extraction GR MAPK Glutathione redutase Mitogen-activated protein kinase GSH MCP1 Glutathione Monocyte chemoattractant protein 1 GSH-px MeOH Glutathione peroxidase Methanol GST MIP-1α - macrophage inflammatory protein Glutathione-S-transferase 1α Hex MMP Hexane Metalloproteinase HMGB-1 MPO High-mobility group box-1 Myeloperoxidase HO-1 MRS Heme oxygenase 1 Man-Rogosa-Sharp HPLC MS High-pressure liquid chromatography Mass spectrometry IBD mTOR Inflammatory bowel disease Mechanistic target of rapamycin IBS MTT - Irritable bowel syndrome 3-(4,5-Dimethylthiazol-2-yl)-2,5- IC50 diphenyltetrazolium bromide Half inhibitory concentration NADH ICAM-1 Nicotinamine adenine dinucleotide Intercellular cell adhesion molecule 1 NBT ICE Nitroblue tetrazolium Interleukin-1 converting enzyme NF-κB IDF Nuclear factor-κB Insoluble dietary fibre NIK IFNγ NF-κB inducing kinase Interferon γ NMR IGF-1R Nuclear magnetic ressonance Insulin-like growth factor-1 receptor NO• IκBα Nitric oxide radical Inhibitor of nuclear factor kappa B NOX IKK - NADPH oxidase IκB kinase NQO1 IL NAD(P)H:quinine oxidoreductase 1 Interleukin Nrf2 IMTA Nuclear factor erythroid 2-related factor 2 ●– Integrated multi-trophic aquaculture O2 iNOS Superoxide radical Inducible nitric oxide synthase OD ISAPP Optical density International Scientific Association of OH● Probiotics and Prebiotics Hydroxyl radical JNK ORAC c-Jun-amino-terminal kinase Oxygen radical absorbance capacity LOX OXA Lipoxygenase Oxazolone LPS p38 Lipopolysaccharide p38 Mitogen-activated protein kinase

p53 SEM Tumor protein 53 Standard error of the mean PBS SFE Phosphate buffer saline Supercritical fluid extraction PCR SLE Polymerase chain reaction Solid liquid extraction PGE SNP Phloroglucinol equivalents Sodium nitroprusside PGE2 SOD Prostaglandin E2 Superoxide dismutase PGU STZ Phloroglucinol unit Streptozotocin PI TAK1 Propidium iodide Transforming growth factor-β-activated PI3K kinase 1 Phosphoinositide 3-kinase TBS-T pIκBα Tris-buffered saline with tween 20 Phosphorylated inhibitor of nuclear factor TE κB Trolox equivalents PLE TIC Pressurized liquid extraction Total ion chromatogram PMA TIMP Phorbol myristate acetate Tissue inhibitor of MMPs PMS TLR Phenazine methasulfate Toll-like receptor pNPB TNF-α 4-Nitrophenil butyrate Tumor necrosis factor α pNPG TPA 4-Nitrophenyl α-D-glucopyranoside 12-O-Tetradecanoylphorbol-13-acetate PPARγ TPhC Peroxisome proliferator-activated receptor γ Total phlorotannins content PVDF TSB Polyvinylidene fluoride Trypticase soya broth Ras UAE Rat sarcoma protein [1]Ultrasound-assisted extraction RCOO● UHPLC Peroxyl radical Ultra-high-pressure liquid chromatography RNS UV Reactive nitrogen species Ultraviolet ROS VCAM-1 Reactive oxygen species Vascular cell adhesion molecule 1 RPMI VEGF Roswell Park Memorial Institute medium Vascular endothelial growth factor RSM WHO Response surface methodology World Health Organization RT XIAP Room temperature X-linked inhibitor of apoptosis S/L XO Solid/liquid ratio Xanthine oxidase SCFA XYL Short-chain fatty acid Xylene SD Standard deviation

Chapter 1 .

This chapter includes data from the papers:

Catarino, M.D.; Silva, A.M.S.; Cardoso, S.M. Fucaceae: A Source of Bioactive Phlorotannins. International Journal of Molecular Sciences 2017, 18, 1327. and

Catarino, M.D.; Silva, A.M.S.; Cardoso, S.M. Phycochemical Constituents and Biological Activities of Fucus spp. Marine Drugs 2018, 16, 249

CHAPTER 1

Introduction

1.1 The value of the ocean

With an economic zone covering 25 million km2, the European Union (EU) owns the largest maritime territory of the world. According to the EU blue economy report from 2019 [2], in 2017 the economic activities related to oceans, seas and coastal areas in the EU employed over 4 million people, generating a gross profit of €74.3 billion. In Portugal, the member with third largest economic zone of the EU and 20th of the world (eventually 10th if the application for the extension of the continental shelf zone is validated), the blue economy employed approximately 180 thousand people and generated almost €4.1 billion, according to the same report. Indeed, from the mined minerals and drilled crude oil to the fisheries and recreational activities, the ocean represents one of the most valuable natural resources not just for the EU, but for the entire world. However, the biodiversity in the seas is far from being fully explored, and as Tibor Navracsics, Commissioner for Education, Culture, Youth and Sport; responsible for the joint research center said:

« Though our oceans cover more than 70% of the earth’s surface, we know less about what lies beneath the waves than we do about faraway planets» [3]

Great efforts have however been taken during the last decades towards the exploration of the ocean secrets, and the search for novel natural products with bioactive properties is one of the topics that has drawn the attention of many researchers globally. Since marine organisms are exposed to such complex habitats and extreme conditions, they have developed unique adaptation mechanisms that greatly influenced the biochemical nature of their primary and/or secondary metabolites which have demonstrated significant biological activities and utility for application in several sectors, such as agriculture, food, textile and pharmaceutical industries. Therefore, marine organisms are viewed as a great reservoir of a wide variety of specific and potent active substances that cannot be found elsewhere [4]. Seaweeds are a good example of marine organisms that provide valuable bioactive compounds with great economic impact. Their polysaccharides (alginate, carrageenan and agar-agar) are perhaps among the most significant marine-derived compounds with very important industrial applications in numerous fields including textile, material, cosmetic, biomedical, pharmaceutical, but above all, in the food industry where they are mostly used as thickeners, gels, emulsifiers and stabilizers for improving the textural quality of numerous products [4]. In addition to polysaccharides, seaweeds can provide a myriad of other compounds with bioactive and commercial interest including fatty acids, steroids, carotenoids, lectins, acids, halogenated compounds, polyphenols and several others.

INTRODUCTION

Overall, more than 15000 novel compounds were reported in seaweeds during the last years due to the great intensification of algal research driven by the recent trends in the search for drugs from natural sources [5]. As a result from this increasing demand for seaweed products, algae farming and controlled algae cultivation in a large scale has grown at a steady pace. According to FAO, the world’s farmed seaweed production increased from 13.5 to 30.1 million tons between 2005 and 2016. The Asian countries dominate the sector with 99.4% of the global production in 2016. Of these, China remains the major producer (47.9%) followed by Indonesia (38.7%), adding up to 86.6% of the worldwide farmed seaweed production [6]. In turn, European seaweed industries keep relying on harvesting natural resources while seaweed farming is still a recent phenomenon, although it has been gaining ground during the recent years. According to FAO, Norway is the European country with the largest wild seaweed output and the third globally [7], whereas France was the major producer of farmed seaweeds in 2014 [8]. In Portugal, there is only one company dedicated to the production of seaweed and seaweed-based products in controlled environment, which was the first in Europe to adopt the integrated multi-trophic aquaculture (IMTA) concept, using nutrient rich effluent water that flows from a semi-intensive organic aquaculture of seabream (1/3) and seabass (2/3) to the seaweed tanks, after which it is discharged into a settling basin connected to the coastal lagoon, with a significantly lower load of nutrients [9]. This means that there is still much raw potential to consider in this seaside country in respect to seaweed exploitation.

1.2 Seaweeds

Seaweeds, a colloquial term for macroscopic, multicellular benthic marine algae, are aquatic photosynthetic organisms different from the higher plants as they do not possess true roots, stems, leaves or vascular tissues (xylem and phloem), making them thalloid in nature [10]. Typically, seaweeds occupy the rocky shores of coastal ecosystems, including the intertidal and the shallow subtidal zones, mostly until 40 m deep, although in very clear waters they can grow down to 200 m in depth [11]. Based on their pigmentary composition, nature of their cell walls, and reserve polysaccharides, three major groups of macroalgae can be distinguished: o Green algae (phylum: Chlorophyta), considered the ancestors of terrestrial plants, are characterized by the presence of chlorophyll a and b, β-carotene, lutein and several other xanthophylls in a proportion resembling terrestrial plants. They accumulate starch as storage product and the structure of their cell walls is mainly composed of cellulose, although xylans and mannans can appear in some species. From the 6000 documented species, only 10% are reported from the oceans,

CHAPTER 1

distributed from the Artic to Antarctic regions, typically growing in the upper shallow zones due to their greater need for light [12]. o Red algae (phylum: Rhodophyta), considered the oldest eukaryotic algae, are characterized by the presence chlorophyll a, phycobiliproteins (red phycoerythrin, red phycocyanin) and carotenoids, mostly α- and β-carotene, lutein and zeaxanthin. These seaweeds usually grow at the greatest depths since phycobiliproteins allow them to make use of blue light, which penetrates deeper in the water column. Their cell walls are made of pectic and cellulosic molecules in combination with agar and carrageenan, and the main storage product is floridean starch, although mannitol, sorbitol, and dulcitol also occur. Over 7000 species of red algae have been documented, the majority of them growing in marine waters from temperate to tropical regions [12]. o Brown algae (phylum: Ochrophyta; class: Phaeophyaceae), are the largest group in terms of dimensions and biomass production and are characterized by the presence chlorophyll a and c, β-carotene, violaxanthin, diatoxanthin and large amounts of fucoxanthin, which is the pigment responsible for their brown-yellowish color. Instead of starch, these organisms accumulate either laminarans or mannitol as storage products, and the structure of their cell walls is composed of cellulose, alginic acid, fucoidan and phlorotannins. From the approximately 2000 species documented of brown seaweeds, over 95% grow in marine waters and because they tolerate less light than green algae but not as much as red algae, they usually occupy relatively deep waters preferentially from the sub-polar to equatorial regions [12]. As primary producers, seaweeds constitute one of the largest biomass producers in the marine environment playing a fundamental role in the food chain of all aquatic ecosystems. Moreover, these organisms have an important structural role in many marine habitats, providing shelter for a rich and varied fauna of epiphytic and epibenthic communities, promoting sediment stabilization, and supplying atmospheric oxygen [13]. To mankind, seaweeds also represent a great deal of importance, particularly for coastal communities that have traditionally used these marine resources in both non-consumptive (as medicine, inputs into industrial processes, fertilizers and animal feed, and for other domestic purposes such as building materials) and consumptive forms (in raw for preparation of salads, soups and main dishes or processed for flavorings, chips and snacks) [14]. For the East Asian countries, seaweeds are of extreme importance being deeply rooted to their culture over centuries. It is estimated that seaweeds are served in approximately 21% of Japanese meals, with more than 20 species of red, green, and brown algae being used in everyday cookery, making this country the largest and most

INTRODUCTION

sophisticated consumer of seaweeds [15]. In turn, on the Western countries, their exploitation has been rather confined to extraction of phycocolloids and, to a lesser extent, certain fine biochemicals [16]. However, this panorama is currently shifting as seaweeds are becoming more and more popular among consumers driven by the raising awareness of seaweeds’ wealthy nutritional value. Indeed, seaweeds are low in fat, rich in complex polysaccharides and fibers, minerals, proteins, vitamins and several other phycochemicals capable of acting on a wide spectrum of disorders and/or diseases [17]. Notably, the regular consumption of seaweeds has even been pointed out as one of the reasons why the Japanese have the world’s longest life expectancy [16]. Based on this evidence, marine macroalgae have earned the status of “superfoods” and are presently pointed out as the plant-based food of the future, with their consumption among Western population becoming an upward trend [18].

1.2.1 Brown Seaweeds

While green and red seaweeds belong to the Kingdom Plantae, brown seaweeds are placed in the Kingdom Chromista, as, unlike plants, they do not use glucose or starch for energy storage and contain modified chlorophyll and pigments that are not found in plants. Although not all brown algae are structurally complex, they generally possess a root-like structure called holdfast that serves to anchor them on the substrate, a stem-like structure called stipe, and leaves-like structures that are termed as blades, laminas or fronds. Some species also exhibit pneumocysts, which are gas-filled bladders that can only be seen in brown algae and serve to keep their photosynthetic parts floating near the surface of the water [12]. These seaweeds present a wide morphological diversity, varying from groups of threadlike cells with few centimeters (Ectocarpus) to giant kelps such as Macrocystis, which are the largest seaweeds known, capable of growing for more than 100 m long, and are responsible for the formation of dense underwater communities known as kelp forests [19]. Others such as Sargassum natans and Sargassum fluitans are pelagic species, i.e., free-floating species, that form a massive floating ecosystem known as the Sargasso Sea [20]. Brown seaweeds are endowed of a great economic value mainly in the hydrocolloid industry as an important source of alginate, but also in the food, pharmaceutical, medical, cosmetic and several other industries. Currently, Saccharina and Undaria are the two main genus of brown seaweeds produced in aquaculture, representing 29 and 9% of the world macroalgae production, respectively [9]. The former is mostly exploited for the alginate, iodine, and mannitol production, while the latter is most commonly used as a food crop and as a source of medical nutrients (e.g., high levels of essential minerals, eicosapentaenoic acid and fucoxanthin) [21]. In Portugal, the harvesting and fertilization of agricultural fields with “sargaço”, a seaweed mixture composed mainly of brown seaweeds from Saccorhiza, Laminaria and Fucus and some red seaweeds from Codium, Palmaria,

CHAPTER 1

Chondrus and Gelidium, used to be a socially and economically important activity in the region between Minho and Douro River until the mid-twentieth century. Nowadays this practice has fallen into disuse, although it still remains in some settlements like Póvoa de Varzim and Viana do Castelo [22]. In the Archipelago of the Azores, swollen receptacles of Fucus spiralis are a much-appreciated delicacy known as sea lupines and have long been consumed by the local people. More recently, to revive the age-old tradition of foraging and consumption of seaweeds, the project Designing Food Cultures brought together the local communities and professional schools in order to document the traditional knowledge of edible seaweed in the Azores and revitalize their foraging and consumption through the co-development of recipes for the creation of a cookbook [23].

1.3 Bioactive compounds from brown seaweeds

In order to adapt to such a highly competitive environment, seaweeds have developed defense strategies that resulted in an enormous diversity of compounds that make their chemical composition particularly interesting not only for nutritional purposes but also for medicinal and industrial applications. Their composition is, however, highly susceptible to variability, depending on multiple factors such as geographical origin or area of cultivation, seasonality, environmental conditions, physiological variations, water temperature and salinity, harvesting/processing methods and several others [24]. Nevertheless, according to Pereira et al. [25], brown seaweeds’ content in carbohydrates, proteins, lipids and ashes generally vary between 33 – 66%, 3 – 26%, 0.1 – 4.5% and 10 – 40% of dry weight (DW), respectively (Table 1.1.). Compared to the others, brown seaweeds typically have the highest ash contents, with exceptional levels of iodine, and the lowest protein contents, although they usually contain all the essential amino acids [26]. They also contain several vitamins including vitamin A, vitamins from B complex, being particularly abundant in B3 (niacin) [27], vitamin C and vitamin E, the latter being amply present in the genus Fucus [18]. Brown macroalgae are known to produce different types of polysaccharides from which alginates, fucoidans and laminarans correspond to the most representative ones. Alginates and fucoidans are generally present in the cell walls serving different structural roles such as contributing for algae flexibility or preventing desiccation, while laminarans are storage saccharides used by brown seaweeds as carbon source [28]. Alginates are the most abundant polysaccharides in all brown seaweeds and are undoubtedly the most widely exploited. These are polyuronic saccharides consisting essentially of β-D-mannuronic and α-L-glucuronic acid units and due to their excellent gel properties, as well as stabilizing and water-holding capacities, they have become an essential industrial product in several fields, including textile, material, cosmetic and medical/pharmaceutical, but above all, in the food industry, where they are mostly used as thickeners, gels, emulsifiers and stabilizers for improving the textural quality of several products such as salad dressings, ice-creams,

INTRODUCTION

beers, jellies, lactic drinks, and many others [29]. In turn, fucoidans are polysaccharides typically

extracted from Fucus spp. composed mainly of L-fucose and sulphate, although other monosaccharides (mannose, galactose, glucose, xylose, etc.), uronic acids or even acetyl groups and proteins may be present [30]. Over the last years, promising preclinical results have revealed extensive biological activities of fucoidans (e.g., antitumor, antioxidant, immunoregulatory, antiviral, anti-inflammatory among others) [31]. However, it is as anticoagulant and antithrombotic agents that these polysaccharides have shown the most promising results, being even compared to heparin, a commercial animal-derived sulphated polysaccharide currently used as an anticoagulant drug [32]. As for laminarans, these are small linear glucans of 5 kDa that are usually predominant in Laminariales. Since these polysaccharides do not form viscous solutions nor gels, their applications in an industrial scale are still rather limited. Nevertheless, commercial interest of laminarans (or derivatives) is emerging due to their interesting bioactivities, such as antioxidant, antitumor, antimicrobial, immunomodulation and anticoagulant properties, that have recently been reported in literature, thus indicating that these polysaccharides might have potential to be used with medical/pharmaceutical purposes [28].

Table 1.1. Nutrient composition of selected edible brown macroalgae species in % of dry weight (adapted from [25])

Species Carbohydrates Protein Lipids Ash Alaria esculenta 46-51 9-20 1.0-2.0 - Eisinia bicyclis 61 8 0.1 10 Fucus spiralis - 11 - - Fucus vesiculosus 47 3-14 1.9 14-30 Himanthalia elongata 44-61 5-15 0.5-1.1 27-36 Laminaria digitata 48 8-15 1.0 38 Laminaria ochroleuca - 7 0.9 29 Laminaria japonica 52 7-8 1.0-1.9 27-33 Saccharina latissima 52-61 6-26 0.5-1.1 35 Sargassum fusiforme 31 12 1.4 20 Undaria pinatifida 45-51 12-23 1.1-4.5 26-40

The main sugar produced by brown seaweeds is mannitol, which is a sugar alcohol that also has a wide range of applications mainly in food, pharmaceutical, medical and chemical industries. It is commonly used as a sugar substitute for production of “low‐calorie” and “diabetic-friendly” products [33] or as a powerful osmotic diuretic mostly used for preventing or treating acute renal failure, reducing elevated eye pressure, as in glaucoma, and brain edema or elevated intra cranial pressure [34]. Due to its effectiveness and safety, mannitol has been included in the WHO Model List of Essential Medicines, a list that contains the medications considered to be most effective and safe, and should meet the most important needs in any health system [35].

CHAPTER 1

Although they are low in fat, the fat-soluble fraction of seaweeds is still rich in some interesting bioactive compounds. In this field, brown seaweeds are particularly rich in long chain unsaturated fatty acids, which are considered the most advantageous for human health. In fact, of the three algal groups, brown seaweeds are the most abundant in both mono- and polyunsaturated fatty acids, accumulating high levels of omega-3 fatty acids, which are well known for their preventive role in cardiovascular diseases [24]. Moreover, contributing for 83 to 97% of the total sterol content in certain brown seaweed species, fucosterol appears as the major sterol of this seaweeds group. Like phytosterols, fucosterol acts as a competitor during the absorption of cholesterol contributing for the decrease of the cholesterol blood levels, which is also an important aspect for the maintenance of a good cardiovascular health [36]. Additionally, this algal sterol has been regarded with a multitude of other bioactive properties including anti-cancer, anti-diabetic, antioxidant, hepatoprotective, anti- photodamaging, anti-osteoporotic and several others [37]. Another important compound present in the fat-soluble fraction of brown seaweeds is undoubtedly fucoxanthin. This carotenoid is an essential constituent of the photosynthetic machinery of brown seaweeds, responsible for the light harvesting during photosynthesis. Although this xanthophyll, is endowed with many physiological functions and biological properties, it has earned particular attention in recent years mainly as a result of its promising anti-obesity effects, as it is capable of interfering with the lipid metabolism at different levels, from the inhibition of the expression of several factors that regulate the cellular lipid uptake, to the stimulation of the metabolic thermogenesis by promoting fatty acid β-oxidation activity [38]. In virtue of these properties, numerous fucoxanthin supplements and nutraceuticals, mostly targeting weight loss, are currently available in the market. Moreover, the use of this compound as a pharmaceutical drug for treating metabolic syndrome is currently in a phase 2 clinical trials [39]. Other important bioactive secondary metabolites from brown seaweeds include terpenoids, small acetogenins and various halogenated compounds (mainly brominated and chlorinated) that comprise a vast and heterogeneous group of primary and secondary metabolites, all presenting different bioactive properties with pharmacological interest [24,40]. One of the most characteristic groups of these secondary metabolites is, undoubtedly, the group of phlorotannins, which are the focus of this dissertation and will be discussed with more detail further in below.

1.3.1 Phlorotannins

Phlorotannins are essentially phenolic compounds biosynthesized exclusively by brown macroalgae that serve vital roles in their physiology including structural functions and cell wall rigidification, response to biotic stress (working as herbivore deterrents, algicidals and bactericidals), and abiotic stress (as photoprotective agents against ultraviolet radiation or as heavy metals

INTRODUCTION

chelators). They also participate in the regulation of cell functions and survivability, acting as antioxidants, and in the algal embryogenesis contributing for the zygote’s cell walls formation and avoiding multiple fertilizations by inhibiting spermatozoid movement [41,42]. Unbound phlorotannins are known to accumulate in physodes, i.e., specialized membrane-bound vesicles of the cell cytoplasm, reaching up to concentrations of 25% of seaweed’s DW [43]. The content and profile of phlorotannins in seaweeds are, however, highly susceptible to variability. For instance, some authors demonstrated that seaweeds growing at shallower depths tend to accumulate higher phlorotannin contents than those growing at lower depths [44–46], while others have reported that the peak of phlorotannins accumulation in seaweeds usually occur during the spring/summer season [46–48]. In both scenarios the higher concentrations of phlorotannins seems to occur in seaweeds under higher solar exposure, which is in agreement with other works that reported an increase of phlorotannins accumulation in seaweeds after UV irradiation or in seaweed tissues with more light exposure [49–51]. Water salinity also seems to influence phlorotannins accumulation since higher concentrations of these compounds were reported on seaweeds growing on habitats with higher salinities rather than lower salinities waters [52]. Several other factors such as genetic differences, environmental conditions, nutrient availability, geographical origin, harvesting/post- harvesting/extraction conditions and others have great influence on phlorotannins accumulation, and therefore their concentrations in seaweeds usually fluctuates greatly even within the same species. Concentration of phlorotannins in some brown seaweed species are given in Table 1.2.

Table 1.2. Overview of the phenolic content of brown algae from different locations, harvesting periods and extracted with different conditions (adapted from [53,54])

Extraction Methods Phlorotannin Species Location Harvesting period Solvent S/L ratio Mode content (% DW) Alaria esculenta France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 2.03

H2O 13.8* Iceland May 2007 1:20 SLE, 24 h, RT 70% ACE 15.9*

H2O 0.02 UAE, 35.61 W cm-2, 15 min 0.1 M HCl 0.01 May 2014 1:20 H2O 0.02 SLE, 150 min, 70 ºC 0.1 M HCl 0.01 Ascophyllum nodosum H2O 1:20 2.24 Ireland 80% EtOH SLE, 24 h, RT 1.38 1:10 70% ACE 2.10 March 2010 H2O PLE, 120 ºC, 1500 psi 2.02

80% EtOH - 0.75 PLE, 100 ºC, 1000 psi 70% ACE 1.08

Asperococcus bullosus France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 1.11

Bifurcaria bifurcata France September 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 0.96 50% MeOH → 70% 1:40 SLE, 24 h, RT 8.6 Carpophyllum flexuosum New Zealand July 2014 ACE H2O 1:30 MAE 11.4 50% MeOH → 70% Carpophyllum pumosum New Zealand July 2014 1:40 SLE, 24 h, RT 7.5 ACE Cladostephus spongiosus Tunisia July 2015 50% EtOH 1:5 SLE, 30 min, 50 ºC 0.01

50% MeOH → 70% Cystophora subfarcinata Australia November 2014 1:40 SLE, 24 h, RT 2.2 ACE Cystoseira compressa Spain - 70% MeOH - - 4.83

Cystoseira foeniculacea Spain - 70% MeOH - - 2.16

Cystoseira sedoides Tunisia July 2015 50% EtOH 1:5 SLE, 30 min, 50 ºC 0.09

Cystoseira tamariscifolia France April - June 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 10.91

Desmarestia ligulata France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 1.22

Dictyopteris polypodioides Spain - 70% MeOH - - 0.09

Dictyota ciliolata Spain - 70% MeOH - - 0.08

France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 1.88 Dictyota dichotoma Malaysia - MeOH 1:10 SLE, 72 h, RT 1.42

H2O Boiling 20.7 Ecklonia cava Korea - - 30% EtOH SLE, 2 h, 50 ºC 45.3

H2O 9.7 Ecklonia kurome Japan March 2006 1:20 SLE, 20 min, 75 ºC 80% EtOH 6.2

H2O 7.4 Ecklonia stolonifera Japan March 2006 1:20 SLE, 20 min, 75 ºC 80% EtOH 7.3

MeOH 0.78 EtOH 0.19

ACE 0.41 Eisenia bicyclis Japan - 1:10 SLE, 24 h, dark, 75 ºC Chl 1.44 EtOAc 0.91 Hex 1.68

Fucus ceranoides France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 5.47

France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 2.82

Fucus serratus H2O 16.9* Iceland May 2007 1:20 SLE, 24 h, RT 70% ACE 24.0*

Spain - 70% MeOH - - 2.17

H2O 1:20 2.78

80% EtOH SLE, 24 h, RT 2.94 1:10 Fucus spiralis 70% ACE 2.08 Ireland May 2010 H2O PLE, 120 ºC, 1500 psi 3.60

80% EtOH - 2.92 PLE, 100 ºC, 1000 psi 70% ACE 3.56

H2O 1.76* Iceland May 2007 1:20 SLE, 24 h, RT 70% ACE 2.42* Fucus vesiculosus 30-35% EtOH SLE, 4 h, RT 27.7* France - 1:10 50-75% EtOH SLE, 2 h, RT 16.3*

Halidrys siliquosa France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 1.60

Halopteris scoparia Spain - 70% MeOH - - 0.16

H2O 0.04 UAE, 35.61 W cm-2, 15 min 0.1 M HCl 0.03 Laminaria hyperborea - May 2014 1:20 H2O 0.04 SLE 150 min, 70 ºC 0.1 M HCl 0.04

Lobophora variegate Spain - 70% MeOH - - 1.20

Padina sp. Malaysia - MeOH 1:10 SLE, 72 h, RT 3.31

Spain - 70% MeOH - - 0.69 Padina pavonica Tunisia July 2015 50% EtOH 1:5 SLE, 30 min, 50 ºC 0.01

H2O 1:20 1.09

80% EtOH SLE, 24 h, RT 0.69 1:10 70% ACE 0.73 Pelvetia canaliculata Ireland May 2010 H2O PLE, 120 ºC, 1500 psi 1.65

80% EtOH - 0.98 PLE, 100 ºC, 1000 psi 70% ACE 1.17

Saccorhiza polyschides France May 2007 DCM:MeOH (1:1) - PLE, 75º, 1500 psi 1.66 50% MeOH → 70% Sargassum aquifolium Australia November 2014 1:40 SLE, 24 h, RT 0.2 ACE Sargassum desfontainesii Spain - 70% MeOH - - 1.68 50% MeOH → 70% Sargassum flavicans Australia August 2014 1:40 SLE, 24 h, RT 1.5 ACE Sargassum furcatum Spain - 70% MeOH - - 2.97

Sargassum polycystum Malaysia - MeOH 1:10 SLE, 72 h, RT 0.18 50% MeOH → 70% Sirophysalis trinodis Australia August 2014 1:40 SLE, 24 h, RT 2.5 ACE Stypopodium zonale Spain - 70% MeOH - - 1.22

Zonaria tournefortii Spain - 70% MeOH - - 1.06 *Phlorotannin content expressed in g/100g extract. ACE - acetone, EtOH - ethanol, MeOH - methanol, DCM – dichloromethane, Chl - chloroform, EtOAc – ethyl acetate, Hex - hexane, SLE – solid-liquid extraction, S/L – solid/liquid ratio, PLE – pressurized liquid extraction, MAE – microwave assisted extraction, UAE – ultrasound assisted extraction, RT – room temperature.

CHAPTER 1

These compounds are acknowledged for their vast array of bioactive properties and benefits to human health showing promising effects against oxidative stress, photodamage, cancer, allergy, diabetes, obesity, inflammation, viral, fungal and microbial infections, and several others [55]. For this reason, many researchers have raised their interest in the algae phenolics, triggering an exponential increase of the number of phlorotannins-related publications in the last 20 years (Figure 1.1. A). Moreover, over the last 5 years between 40 to 70 new patents for products containing phlorotannins or phlorotannins-rich extracts have been filled yearly (Figure 1.1. B), and there are already some dietary supplements currently available in the market (for example, BioPure® PC Ecklonia Cava, with phlorotannins-rich extract from E. cava or InSea2®, with an extract mixture of A. nodosum and F. vesiculosus).

INTRODUCTION

1.3.1.1 Biosynthesis and chemistry of phlorotannins

Although the exact biosynthetic pathway by which phloroglucinol gives origin to phlorotannins is not yet fully understood, it is known that this compound is biosynthesized by the acetate-malonate pathway, also known as polyketide pathway, in a process involving polyketide synthases-type enzyme complexes (Figure 1.2). It starts with conversion of an acetyl-CoA molecule into malonyl- CoA by the addition of a carbon dioxide molecule, changing the acetyl group into a highly reactive methylene that will allow the polymerization process to occur with low energy expenditure. The polymerization gives rise to a polyketic chain that transforms into a hexacyclic ring (triketide) via intramolecular cyclization with the concomitant loss of a water molecule. Because the triketide structure is not stable, the molecule tautomerizes into its aromatic form, which is thermodynamically more stable, giving phloroglucinol (1,3,5-trihydroxybenzene) [56]. Phloroglucinol then undergoes through oxidative coupling reactions with other phloroglucinol moieties forming polymeric structures that may range from simple molecules of 126 Da (1 phloroglucinol unit) to very large and complex polymers [57].

Figure 1.2. Schematic representation of the phloroglucinol biosynthetic pathway (image retrieved from [58])

According to the nature of the structural linkages between phloroglucinol units, they can be characterized into different subclasses namely phlorethols (containing only ether linkages), fucols (containing only C-C linkages), fucophlorethols (containing both ether and C-C linkages) and eckols (containing dibenzodioxin linkages). Within the subclass of phlorethols there are also the fuhalols which are ether-linked phlorotannins that possess at least one additional hydroxyl group. Likewise, eckols with additional hydroxyl groups are known as carmalols (Figure 1.3) [59].

CHAPTER 1

Figure 1.3. Representation of the different classes of phlorotannins (image retrieved from [58])

1.3.1.2 Extraction, purification and quantification of phlorotannins

The extraction of phlorotannins is a demanding task due to their chemical structure complexity, susceptibility to oxidation and interaction with other matrix components. Many factors, such as solvent composition, solvent polarity, time of extraction, temperature, solvent-solid ratio and particle size, may significantly influence the solid–liquid extraction (SLE) of phenolic compounds [60]. The protocols most commonly used for the extraction of phlorotannins are typically based on aqueous mixtures of acetone, ethanol or methanol (see Table 1.2.), usually performed in acidic conditions, under nitrogen atmosphere or with solvents containing antioxidants such as K2S2O5 or ascorbic acid to prevent oxidative degradation [61]. Alternative methods such as pressurized liquid extraction (PLE), microwave-assisted extraction (MAE) or ultrasound-assisted extraction (UAE) (see Table 1.2.) have also been tested, although their applicability to routine isolation of phlorotannins is still not very common, due to the necessity of complex apparatus with limited capacities and/or high costs. The purification of these compounds is usually carried out using solvent partitioning, solid-phase extraction (SPE) or column chromatography for polarity-based separation [62–64], or molecular size discrimination through dialysis or ultrafiltration steps [65–67]. Other methods have also been reported including cellulose adsorption [68], thin-layer chromatography [69] or even the combination of both polarity- and size-based approaches [66,70].

INTRODUCTION

Monitorization of the total phlorotannins content in the extracts is usually carried out with the Folin-Ciocalteu method. However, even though phlorotannins are the major phenolic compounds present in brown seaweeds, several authors have reported the presence of other non-phlorotannin phenolic compounds in brown seaweeds. Therefore, this is not the most suitable approach since these minor phenolics, together with other unspecific reactions inherent to this colorimetric assay (e.g. with reducing sugars or ascorbic acid) may result in the overestimation of the phlorotannins levels. The most appropriate methodology for the specific quantification of phlorotannins is the dimethoxybenzaldehyde (DMBA) assay. In this reaction, the 2,4-dimethoxybenzaldehyde reacts specifically with the OH groups positioned at the 1,3- and 1,3,5-substituted phenols, thus being much less sensitive to the interference of other compounds. It can, however, form chromophores with some other non-polar compounds, although this issue can be easily surpassed with a defatting step prior to the DMBA assay [71].

1.4 Oxidative stress, inflammation and cancer

Oxidative stress can be defined as an imbalance between production of free radicals and reactive metabolites, commonly known as reactive oxygen and nitrogen species (ROS and RNS), and their elimination by protective mechanisms, referred to as antioxidants. In biological systems, the most common source of reactive species is oxygen, mainly through mitochondria activity [72] which is

responsible for the consumption of ~90% of cellular O2 to produce several short-lived intermediates,

●– ● including O2 , H2O2 and OH [73]. Reactive species can also be produced via other means, namely cellular enzymes such as xanthine oxidase (XOX), nicotinamide adenine dinucleotide phosphate oxidase (NOX) and inducible nitric oxide synthase (iNOS) [74]. To prevent the destructive effect of undesired oxidation reactions, cells make use of a plethora of antioxidant mechanisms consisting of several enzymatic [superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase (CAT), and others] and non-enzymatic (vitamins A, C, E) elements, allowing them to keep ROS and RNS in low concentrations [75]. When the homeostasis between antioxidants and reactive species is disturbed, overproduction of ROS and RNS becomes toxic to cells and tissues, reacting uncontrollably with endogenous macromolecules including lipids, proteins and DNA [76]. This self-aggression together with exogenous aggressions (ultraviolet light, ionizing radiation, pollutants, traumatisms, pathogens, among others) will cause the cells to enter in an oxidative stress state [77]. Under a sustained oxidative stress environment, serious damage occurs in the cells’ structure and functions, which can trigger the initiation of other pathophysiological events such as chronic inflammation and cancer (Figure 1.4).

CHAPTER 1

Figure 1.4. Schematic overview of the signaling cascades that mediate the oxidative stress and inflammatory conditions leading to neoplastic transformation.

Inflammation is, in its essence, the host mechanism of defense against harmful stimuli. It usually begins in a restricted area, although it can quickly become systemic depending on the injury´s severity. The acute phase of inflammation starts immediately upon injury and rapidly turns severe, persisting only for a short period of time [78]. During this event, several mediators such as cytokines [e.g. tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6] and chemokines [e.g. monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1α (MIP-1α) and IL-8] are released by phagocytic and nonphagocytic cells stimulating the surrounding cells and recruiting others from the immune system such as neutrophils and macrophages [79]. Upon interaction with their specific receptors on the cell surface, these mediators will trigger a series of intracellular signaling cascades that will stimulate transcriptional activity of NF-κB. This transcription factor is perhaps one of the most crucial regulators of the inflammatory signaling cascade since it is

INTRODUCTION

responsible for the transcription of multiple genes encoding for several pro-inflammatory markers including cytokines, chemokines and enzymes (including cycloocygenase-2 (COX-2), lipoxygenase (LOX) and iNOS) [80]. Among the cytokines, the IL-1 family (IL-1α and IL-1β) are major mediators of immune response, inducing the gene expression of multiple pro-inflammatory genes including COX-2, iNOS, and IL- 1 themselves, serving as a positive-feedback circle that amplifies the IL-1 response in an autocrine or paracrine manner [81]. In turn, COX-2 is a pivotal player in the arachidonic acid pathway controlling the biosynthesis of prostaglandins, considered as potent mediators locally released at the inflammation site and responsible for the classical cardinal signs of inflammation: redness, swelling, heat, and pain [82]. Likewise, the up-regulation of iNOS, mainly in macrophages and endothelial cells, greatly increases the production and release of NO●, which together with other mediators, acts on the endothelial capillaries causing their dilatation and increase of the blood flow on the affected area. This results in an increased permeability that allows the blood proteins, neutrophils and macrophages to be recruited to the interstitial spaces of the inflamed area, increasing the uptake of oxygen and accumulation of ROS in an event known as respiratory burst which is the second major endogenous source of reactive species [83]. Until they are contained, all these events result in a self- sustaining cycle where ROS and RNS stimulate the pro-inflammatory chemical mediators that in turn stimulate the production of more reactive species, establishing a perfect environment for the development of a chronic inflammation and ultimately, cell transformation and cancer [83]. Cancer is a multistep process defined in three main stages: initiation, promotion, and progression. Oxidative stress participates in all the three stages, contributing for the introduction of gene mutations and structural alterations of the DNA during the initiation stage, abnormal gene expression, blockage of cell to cell communication, and modification of second messenger systems during the promotion stage, leading to an increased cell proliferation or a decreased apoptosis of the mutated cells, and finally contributing for the addition of further DNA alterations during the progression stage [84]. Moreover, it has been demonstrated that ROS are involved in the link between chronic inflammation and cancer, and that an important characteristic of tumor promoters is their ability to recruit inflammatory cells and to stimulate them to generate ROS [79]. One of the key characteristics of tumor cells is their increased survivability and capacity to resist apoptosis, which is a major barrier against tumorigenesis. Once triggered, the apoptotic signaling cascade unfolds in an orchestrated series of steps that will cause the disruption of cellular membranes, the breakdown of nuclear and cytoplasmic skeleton, extrusion of cytosol, degradation of the chromosomes and fragmentation of the nucleus. In this process, there are essentially two types of components: the regulators, which are responsible for monitoring the extracellular and intracellular environment for conditions of normality or abnormality that influence whether a cell should live or

CHAPTER 1 die, and the effectors, which are responsible for the execution phase in which the cell progressively disassembles and gets consumed either by its neighbors or by phagocytic cells [85]. Many of the stimuli that trigger apoptosis converge on the mitochondria, which responds with the release of cytochrome c, a potent catalyst of apoptosis. These signals are, however, controlled by counterbalancing pro- and antiapoptotic members of the B-cell lymphoma 2 (Bcl-2) family of regulatory proteins. The antiapoptotic proteins [such as Bcl-2, B-cell lymphoma extra-large (Bcl-xL), and others] act by binding and suppressing the proapoptotic proteins [Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak)] in the mitochondrial outer membrane. When this suppression is lifted, Bax and Bak will induce the permeabilization of the outer mitochondrial membrane causing the release of the cytochrome c, which in turn will activate the caspase signaling cascade, culminating in the activation of caspase-3, considered the most important of the executioner caspases, and other effector proteins that will execute the selective obliteration of subcellular structures, organelles and genome [86]. In order to survive, tumor cells develop a variety of strategies that allow them to escape or limit apoptosis. One of the most common is the loss of function of tumor suppressor protein p53, which has been described as “the guardian of the genome” since it is responsible for controlling the stability of the DNA and upregulate the expression of Bax upon sensing DNA damage, thus inducing apoptosis [77]. In fact, the inactivation of this protein is commonly observed in more than 50% of human cancers and emerging evidence suggests that the dysfunction of p53 also promotes inflammation and endorses tumor immune evasion, thereby serving as an immunological driver of tumorigenesis [87,88]. Additionally, tumors may evade apoptosis by upregulating the expression of antiapoptotic proteins such as Bcl-2 and Bcl-xL. Indeed, the overexpression of Bcl-2 has been linked to the cancer resistance to chemo- and immunotherapy [89]. Other tumor evasion strategies include overexpression of survival signals, downregulation of multiple proapoptotic factors or short- circuiting the extrinsic ligand-induced death pathway. The variety of apoptosis-evading mechanisms presumably reflects the different apoptosis-inducing stimuli that tumor cell populations face and overcome during their development and evolution to the malignant state [90].

1.4.1 Involvement of phlorotannins in oxidative stress, inflammation and cancer

As phenolic compounds, the most characteristic biological property of phlorotannins is their antioxidant activity, which has been extensively explored and is encompassed by a large number of studies in literature contemplating numerous phlorotannin-rich extracts or isolated phlorotannins from multiple algal species and geographic origins, obtained with different extraction procedures and evaluated by a multitude of antioxidant assays. Not only these compounds are capable of chelating metals and scavenging free radicals in the intracellular environment [91–93], they also inhibit pro-

INTRODUCTION

oxidant enzymes and redox-sensitive mediators [94], and increase auto-antioxidant defenses through positive regulation of phase II detoxifying enzymes [95–97]. The most studied parameter for measuring the antioxidant activity is the formation of ROS. This parameter is usually evaluated by the scavenging activities of 1,1-diphenyl-1,2-picrylhydrazyl (DPPH), 2,2′-azinobis(3-

+ ● ● ● ●- ethylbenzothiazoline-6-sulphonic acid (ABTS ), peroxyl (RCOO ), alkoxyl (RO ), OH , and O2 assays and a decrease of ROS in the presence of phlorotannins has been consensually observed [91,96,98–102]. In many cases, their effects are even stronger when compared to well-known antioxidant compounds such as ascorbic acid, α-tocopherol, butylated hydroxytoluene (BHT) and others. For instance, both soluble and membrane bound phlorotannin extracts from A. nodosum were

● shown to exert markedly higher DPPH scavenging activity (IC50 = 6.3 – 7.7 µg/mL) than BHT (IC50

= 51 µg/mL), and those from F. vesiculosus (IC50 = 3.8 – 4.7 µg/mL) were even stronger than

ascorbic acid (IC50 = 6.3 µg/mL) [103]. Likewise, a phlorotannin purified extract from F. vesiculosus,

● displayed a DPPH scavenging activity stronger than α-tocopherol (IC50 = 3.76 against 5.93 µg/mL)

and comparable to that of BHT (IC50 = 3.28 µg/mL) [65]. In a different study, the phlorotannin

●- fraction purified from Sargassum ringgoldianum showed approximately five times stronger O2

scavenging activity than chatechin (IC50 = 1.0 against 4.6 µg/mL) [104]. Some isolated compounds from Ecklonia stolonifera, namely eckstolonol, dieckol,

phlorofucofuroeckol A also revealed lower IC50 values (8.8, 6.2 and 4.7 µM, respectively) than ascorbic acid (10.3 µM) on DPPH● scavenging assay, thus evidencing strong antioxidant activity [102]. In turn, eckol, phlorofucofuroeckol A, dieckol and 8,8′-bieckol isolated from Eisenia bicyclis,

● Ecklonia cava and Ecklonia kurome, were twice more effective scavengers of DPPH (IC50 = 26, 12,

13 and 15 µM, respectively) than catechin, ascorbic acid and α-tocopherol (IC50 = 32, 30 and 52 µM,

●- respectively). The same tendency was observed for the O2 scavenging in which these compounds

exhibited considerably lower IC50 values (10.7, 8.4, 7.6 and 6.5 µM, respectively) than resveratrol,

ascorbic acid or α-tocopherol (IC50 = 21, 16 and 12 µM, respectively) [105]. Promising results have been observed in different cellular systems of oxidative stress as well, with several authors reporting a decrease of the intracellular ROS on cells treated either with phlorotannin- rich extracts or pure compounds [65,106–112]. Decrease of the DNA oxidative degradation in oxidative stress-induced cells has been described for phlorotannin extracts from species such as F. vesiculosus, F. serratus or A. nodosum [113–116], and pure compounds including phloroglucinol, eckol, dieckol and 2,7″-phloroglucinol-6,6′-bieckol isolated from E. cava [93,117,118]. Strong dose- dependent inhibitions of myeloperoxidase (MPO) activity, an enzyme that produces HOCl from

- H2O2 and Cl , were also observed in HL-60 cells treated with phlorofucofuroeckol A, diphlororethohydroxycarmalol, 7-phloroeckol and 6,6′-bieckol prior to a pro-oxidant stimulus [98,119]. In addition to the cellular models, in vivo experiments have shown that phlorotannin

CHAPTER 1 extracts from Lessonia vadosa and Macrocystis pyrifera were able to prevent UV-B-induced ROS and malformations in zebrafish [120]. Likewise, compounds such as dieckol, phlorofucofuroeckol A 6,6′-bieckol, phloroglucinol, eckol, eckstolonol and triphloroethol A, all isolated from E. cava, were reported as effective inhibitors of intracellular ROS, lipid peroxidation and DNA damage either in ethanol, 2,2’-azobis-2-methyl-propanimidamide dihydrochloride (AAPH), high glucose or UV-B- exposed zebrafish [91,99,121,122]. In a different model, the prolonged consumption of high molecular weight phlorotannins from Sargassum kjellmanianum by Kunming mice (5.0 g/kg body weight/day over 30 days) was shown to significantly protect the liver against the lipid peroxidation induced either by CCl4 or FeSO4-VC [123]. Notably, in a randomized controlled trial, modest improvements in the DNA damage were observed in obese people after eight weeks of consuming 400 mg/day of an A. nodosum phenolic-rich extract [124]. Apart from the direct antioxidant properties, the capacity of phlorotannins to boost the auto- antioxidant defenses is a subject that has also shown promising results. For instance, SOD and CAT, which are two important detoxifying enzymes that are usually downregulated in cells under oxidative stress, were shown several times to be increased on different cell lines treated either with phlorotannin-rich extracts (from F. vesiculosus, F. serratus, Pelvetia canaliculata and A. nodosum) [106,115,125] or isolated phlorotannin compounds including phloroglucinol, eckol, eckstolonol, dieckol and triphlorethol A [95,96,111,112,126–128]. Likewise, glutathione (GSH), GSH peroxidase (GPx), GSH reductase (GR) and GSH-S-transferase (GST), which are crucial players in the neutralization of ROS and intimately associated to the maintenance of the redox balance in living organisms, were reported in multiple studies to be upregulated in response to the treatment of A. nodosum, H. elongata, F. serratus, F. vesiculosus, Pelvetia canaliculata, E. cava and Eisenia bicyclis phlorotannin extracts [108,113,125,129] as well as phloroglucinol, triphlorethol A, eckol, phlorofucofuroeckol A, 7-phloroeckol and 6,6′-bieckol [95,98,128,130,131]. In fact, it is possible that these observations might be occurring due to the possible interference of the phlorotannins with the transcriptional activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) which is the major regulator of the phase II detoxifying enzymes [132]. Indeed, dieckol was demonstrated to enhance the levels of detoxifying enzymes heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase 1

(NQO1) and GST via upregulation of the Nrf2 transcriptional activity in H2O2-induced HepG2 cells [96]. Similar results have been described for eckol, which was equally capable of stimulating the expression of HO-1 in V79-4 cells [133] and H2O2-induced HepG2 cells [95], both through activation of the nuclear translocation and transcriptional activity of Nrf2. The phlorotannins capacity to upregulate the auto-antioxidant defenses has even been confirmed in vivo. According to Moreira et al. [134], the incorporation of H. elongata in restructured meat given to hypercholesterolemic rats over 35 days significantly contributed for the restoration of the levels

INTRODUCTION

of GR and Cu,Zn-SOD in their livers. In a different approach, the administration of 55 mg/kg/day of L. japonica extract to streptozotocin-induced diabetes in Sprague-Dawley rats over 5 days not only significantly reduced the expression of XO but also increased the levels of GSH, GPx and GR in the liver of the treated animals [135]. Similarly, the prolonged oral administration of Sargassum pallidum

to Wistar rats with CCl4-induced hepatic injury was shown to remarkably increase both GSH and SOD levels in the livers of the treated animals [136]. Upregulated SOD, GPx, GSH and CAT have also been reported in injured liver tissues of mice treated either with eckol [137] or dieckol [97,138]. Phloroglucinol was even demonstrated to ameliorate the midbrain lesions in rats with 6- hydroxydopamine-induced Parkinson’s disease, most likely through restoration of the reduced levels of CAT and GPx via stimulation of Nrf2 activity [139]. In addition to their antioxidant properties, phlorotannins are also closely related with numerous inflammatory events, being capable of inhibiting the expression of pro-inflammatory cytokines, regulate the expression and/or activity of important enzymes, and even interfere with the transcriptional regulation, giving them great potential as therapeutic agents for mitigating inflammation and inflammatory-related diseases including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), coeliac disease and cancer [140–142]. Due to their wide distribution and ecological importance, seaweeds from the genus Eisenia and Ecklonia are perhaps the most studied in this field. The majority of the phlorotannins obtained from these seaweeds were proven of being able to inhibit the expression of cellular adhesion molecules, like, ICAM-1 and VCAM-1, and cytokines, namely TNF-α, IL-1β and IL-6, in several cells of the

● immune system [117,143–146]. Likewise, the release of NO and PG2, as well as the expression of enzymes responsible for their synthesis, i.e., iNOS and COX-2, respectively, were found reduced in cells treated with E. cava, E. stolonifera and E. arborea extracts [147–151], or phlorotannin compounds from them isolated including phloroglucinol, dieckol, fucofuroeckol A, 6,6′-bieckol and phlorofucofuroeckol A [102,144–146,152,153]. Other seaweeds have proven their capability of inhibiting multiple pro-inflammatory markers as well. Barbosa et al. [68] recently demonstrated that phlorotannin-purified extracts from several species of Fucus, namely F. vesiculosus, F. guiryi, F. spiralis and F. serratus, dose-dependently inhibited the LPS-induced NO● secretion in Raw 264.7 macrophages, which is in line with previous studies from Zaragozá et al. [106], who also described a dose-dependent inhibition of NO● production on PMA-stimulated macrophages after being treated with a phlorotannin-rich F. vesiculosus ethanol extract. Likewise, phlorotannin extracts of several species of Sargassum including S. horneri, S. sagamianum and S. patens were shown to affect the

● expression of NO , PG2, IL-1β, IL-6 and TNF-α in LPS-stimulated Raw 264.7 macrophages [154– 156], whereas in HaCaT cells exposed to UV-B radiation, the addition of S. fulvellum ethanolic

● extract prevented the expression of NO , PG2, TNF-α, COX-2 and iNOS [157]. Notably, different

CHAPTER 1 purified fractions of a polyphenol-extract from Macrocystis pyrifera not only exerted inhibitory effects towards NO●, TNF-α and iNOS but also stimulated the expression of IL-10 which is an important anti-inflammatory cytokine responsible for the suppression of cytokines secretion [151]. Evidences have shown that the anti-inflammatory capacity of these compounds is much likely to be related with their modulatory effects on important transcription factors that mediate the inflammatory signaling cascade. In fact, it has been proven that the addition of E. cava ethanolic extract to LPS-stimulated Raw 264.7 macrophages dose-dependently diminished the levels of TNF-

● α, IL-1β, IL-6, iNOS, COX-2, NO and PG2 via inhibition of the nuclear NF-κB translocation [147]. Notably, the E. cava ethanolic extract ability to inhibit NF-κB nuclear translocation and binding to DNA in LPS-stimulated BV2 microglia cells was caused by the inhibition of both IκBα degradation and mitogen-activated protein kinase (MAPK) pathway, which also contributes for the regulation of pro-inflammatory cytokines biosynthesis [148]. Yayeh et al. [158] reported that the inactivation of LPS-induced NF-κB transcriptional activity in dieckol-treated Raw 264.7 macrophages occurs via inhibition of the phosphorylation of NF-κB p65 subunit and its upstream kinases namely PI3K, Akt, IKK-α/β and IκBα, which is an inhibitory route very similar to that described for phlorofucofuroeckol A in LPS-stimulated Raw 264.7 cells [159] and 6,6’-bieckol in LPS-stimulated BV2 cells [160]. In phloroglucinol-treated LPS-stimulated Raw 264.7 macrophages, the inactivation NF-κB occurred via a different path, through inhibition of the upstream kinases IKK and NF-κB inducing kinase (NIK) [117]. In a different study, Lee et al. [161] reported that in addition to the Akt/IκB-mediated NF-κB inactivation, dieckol also caused the decrease of p38 and ERK phosphorylation in three different human hepatic cell lines, which is partially in agreement with a previous work carried out on LPS- stimulated BV2 cells in which this compound only inhibited p38-MAPK but not ERK nor JNK [145].

However, in the case of Aβ25-35-induced PC12 cells, the treatment with dieckol not only suppressed the expression of p38 but also the phosphorylation of both ERK 1/2 and JNK [109], suggesting that its mechanism of action also depends on the pro-inflammatory stimulus. Again, similar observations were described for phlorofucofuroeckol A, which inhibited the phosphorylation of p38, ERK and JNK in LPS-stimulated Raw 264.7 cells [159], and for phloroglucinol, which blocked the phosphorylation of ERK in HT1090 cells [117], both resulting in the suppression of AP-1 activity, another transcription factor involved in the regulation of some pro-inflammatory mediators such as matrix metalloproteinases, a group of enzymes involved in the tissue remodeling during chronic inflammation and cell motility further in a tumor environment. The inhibitory effects of these and other isolated phlorotannins such as eckol, eckstolonol, 6,6’-bieckol, 8,8’-bieckol, fucofuroeckol-A over NF-κB and MAPKs have been reported in multiple cell lines elicited with different pro- inflammatory stimulus, including LPS-induced BV2 microglia cells, THP-1 or Raw 264.7

INTRODUCTION

macrophages, Aβ25-35-stimulated PC12 pheochromocytoma cells, PMA-induced MG-63 human osteosarcoma cells, high glucose-induced HUVEC endothelial cells, hypoxia-induced primary mouse hepatocytes and gamma-irradiated primary rat hepatocytes [109,131,145,146,152,162–167], suggesting that these compounds may act in a wide range of inflammatory conditions. Additional in vivo experiments verified that the topical administration of Myagropsis myagroides ethanolic extract in ICR mice ears (90 μg/ear) as well as 6,6’-bieckol (30 μg/ear) isolated from the same species prior to the induction of ear edema with PMA significantly suppressed the ear swelling in approximately 67% and 64%, respectively, which was very close to the effects observed for indomethacin, used as positive control (at 1 mg/ear) [168]. Similarly, attenuation of ear swelling was described for phlorotannin purified extracts of Cystoseira sedoides, Cladostephus spongiosis and Padina pavonica as well as pure compounds such as eckol, 6,6′-bieckol, 6,8′-bieckol, 8,8′-bieckol, phlorofucofuroeckol A and phlorofucofuroeckol B in mice ear edemas induced with several different sensitizers including arachidonic acid (AA), 12-O-tetradecanoylphorbol-13-acetate (TPA), oxazolone (OXA) and xylene (XYL) [169–172]. Paw edema induced by carrageenan injection was also attenuated on Wistar rats treated with intraperitoneal injection of a phlorotannin purified extract of C. sedoides [172]. In BALB/c mice, topical application of Sargassum fulvellum phlorotannin purified fraction prior to UV-B irradiation effectively suppressed the protein expression of TNF-α,

● COX-2 and iNOS, and their downstream products, PGE2 and NO , respectively [157], while in UV‐ B‐irradiated zebrafish embryos, the treatment with phloroglucinol, triphlorethol A or eckstolonol all prevented the rise of the levels of NO● [173]. Likewise, diminished NO● levels as well as iNOS and COX-2 expression were described on zebrafish exposed to high glucose levels and treated with dieckol [99]. Oral administration of dieckol to LPS-induced septic shock in C57BL/6 mice over 7

● days, was also found to significantly decrease the blood serum levels of NO , PGE2 and high- mobility group box-1 (HMGB-1), improving their survival rates in a dose-dependent manner [174], whereas the oral administration of eckol to Kunming mice for an equal period of time effectively

suppressed the expression of TNF-α, IL-1β and IL-6, and enhanced the levels of IL-10 on the CCl4- injured livers [137]. More recently, Sanjeewa et al. [175] reported that the expression of these pro- inflammatory cytokines as well as iNOS and COX-2 were inhibited at the mRNA level and the phosphorylation of MAPKs were suppressed in the lung tissues of particular matter and ovalbumin- exposed BALB/c mice treated with S. horneri ethanol extract by oral ingestion over 15 days. Notably, in an intervention study carried out on human volunteers, the oral administration of a capsule containing A. nodosum polyphenol extract seems to attenuate the ex vivo production of IL-6 in blood samples cultured with LPS, although no statistical significance was achieved [176]. Because oxidative stress and inflammation are conditions that are intimately correlated with carcinogenesis, the fact that phlorotannins can counteract both states indicate that indirectly they can

CHAPTER 1 display some potential to prevent and/or mitigate oxidative stress- or inflammation-based tumor initiation and progression. Notwithstanding, the role of phlorotannins as anti-neoplastic agents has been suggested to go much further, with several authors reporting that these compounds may act as anti-proliferative, antimetastatic and anti-angiogenic agents at different types of cancer. According to Nwosu et al. [177], both phlorotannin-purified extracts from A. nodosum and Alaria esculenta exhibited dose-dependent anti-proliferative effects on Caco-2 growth showing IC50 values of 33 μg/mL and 7 μg/mL, respectively. Similarly, the phenolic-rich extracts of three different Cystoseira species (C. sedoides, C. compressa and C. crinita) demonstrated promising growth inhibitory effects against three different tumor cell lines, namely A549 lung cell carcinoma cells, HCT15 colon cell carcinoma cells and MCF-7 breast adenocarcinoma cells, although their IC50 values (17.9 – 90.3 µg/mL) were much higher when compared with those of cisplatin (1.5 – 1.9 µg/mL), an important chemotherapeutic drug used for the treatment of several neoplasms. However, when tested on normal cells, i.e., non-tumor cells (Mardin–Darby canine kidney cells and rat fibroblasts), it was found that all the three Cystoseira extracts presented much lower toxicity than cisplatin, suggesting that, although their antitumor capacity may be weaker compared to the commercial drug, their specificity for tumor cells is much higher, and thus administration of higher doses could be possible for achieving the desired antitumor effect without compromising the viability of normal cells [178]. In fact, according to the work of Ahn et al. [179] carried out in xenograft mice models implanted with SKOV3 ovarian cancer cells, the oral administration of dieckol (300 mg/kg/week) was even more effective than cisplatin (9 mg/kg/week) at suppressing the tumor growth without demonstrating any liver or kidney toxicity, while the cisplatin-treated mice revealed increased blood urea nitrogen and serum creatinine which are indicative of kidney dysfunction. Many authors have reported a causality link between the phlorotannin content of the extracts and the extracts’ anti-proliferative effects. This is the case of the work reported by Imbs et al. [180], who found that among three different seaweeds, namely Fucus evanescens, Laminaria cichorioides and Costaria costata, the 60% ethanolic extract from the former, which was the highest in polyphenols (10.1% dry matter), displayed the strongest inhibitory effects on DLD-1 and HT-29 human colorectal adenocarcinoma cells growth (67 and 63%, respectively), while the extract from L. cichorioides harvested in May, which had the lowest polyphenol content (1.4% dry matter), exhibited the lowest inhibitory capacity (9 and 23% respectively). However, when looking at the extracts of the same Laminaria species collected from July and September, which had phenolic contents of 2.7 and 5.5% dry matter, respectively, the growth inhibitory effects increased substantially for 64 and 50% in DLD-1 cells, and 56 and 52% in HT-29 cells, respectively. The authors reported identical observations for C. costata, since extracts from samples collected in May (phenolic content of 2.0% dry matter) exhibited slightly lower inhibitory effects than the extract from samples collected in July

INTRODUCTION

(phenolic content of 2.7% dry matter). These results are in agreement with a previous study carried out on HeLa cervical adenocarcinoma cells in which Macrocystis integrifolia and Nereocystis leutkeana methanolic extracts, both containing a phenolic content of 3.9 µg gallic acid equivalents/g extract, showed stronger anti-proliferative effect than the extract of Laminaria setchellii which contained only 1.8 µg gallic acid equivalents/g extract [181]. More recently, it has been demonstrated that ethanol extracts from S. muticum collected along the North Atlantic coast during the same harvesting season exhibited different anti-proliferative activities towards HT-29 cells according to their total phlorotannin content, i.e., the extracts from samples collected in Norway which had the highest phlorotannin contents (approximately 5 mg/g extract) displayed growth inhibitions almost two times stronger than those observed for samples from Portugal which contained approximately 4 mg/g extract [182]. One of the most common mechanisms responsible for phlorotannins anti-proliferative activity is their capacity to interact with multiple mediators of the apoptotic signaling cascade and thus trigger cell death by inducing apoptosis [183–187]. In this context, a hydrophilic fraction obtained from F. vesiculosus acetone extract was shown to induce apoptosis in different pancreatic cell lines through cell cycle arrest, which is comparable to the effects of common chemotherapeutic drugs clinically used, such as gemcitabine [188]. Compounds such as phloroglucinol or dieckol have been reported for their capacity to stimulate release of cytochrome c from the mitochondria or the expression of several caspases including caspase-3, -7, -8 and -9 in different tumor cell lines [179,185– 187,189,190]. Additionally, dieckol was found to dose-dependently upregulate the expression of Bid and Bim pro-apoptotic proteins in Hep3B hepatocarcinoma cells [187] and downregulate the expression of the XIAP, FLIP, and Bcl-2 anti-apoptotic proteins in SKOV3 ovarian cancer cells [179], while in MCF-7 breast cancer cells, the treatment with eckstolonol caused a dose-dependent downregulation of the anti-apoptotic protein Bcl-2 along with the upregulation of the pro-apoptotic protein Bax and tumor suppressor p53 [189]. Likewise, simultaneous increase of the pro-apoptotic

proteins Bak and Bax, and decrease of the anti-apoptotic proteins Bcl-2 and Bcl-xL was described in

HT-29 cells treated with increasing concentrations of phloroglucinol (12.5 – 50 µg/mL) [186], although Kang et al. [191] reported that this compound may also exert pro-apoptotic effects on this cell line via an alternative mechanism involving the suppression of insulin-like growth factor-1 receptor (IGF-1R) expression and consequently blocking the PI3K/Akt/mTOR and Ras/ERK-MAPK signaling pathways, both playing key roles in the cell survival, growth and metabolism in colon cancer. Interestingly, phlorofucofuroeckol A seems act via another path, through stimulation of the expression of activating transcription factor 3 (ATF3), which was shown to consequently induce apoptosis in four different colorectal tumor cell lines namely HCT116, SW480, LoVo and HT-29 cells [192].

CHAPTER 1

In vivo antitumor effects of eckol in a sarcoma xenograft-bearing mice model, were demonstrated to be not only resultant from its capacity to interfere with the expression of caspase-3, caspase-9, Bcl-2 and Bax, but also from its capacity to inhibit the expression of epidermal growth factor receptor (EGFR), a transmembrane protein that is usually overexpressed in many cancer types, and to stimulate the mononuclear phagocytic system, recruiting and activating dendritic cells, promoting the tumor-specific Th1 responses, increasing the CD4+/CD8+ T lymphocyte ratio, and enhancing cytotoxic T lymphocyte responses. Therefore, in addition to its anti-proliferative properties, eckol also exerts antitumor effects via stimulation of the host immune response [185]. Using a different in vivo model, Sadeeshkumar et al. [193] observed that more than activating the pro-apoptotic mechanisms (modulating the Bcl-2 family proteins, cytochrome c release and caspases expression), the administration of dieckol (40 mg/kg bw over 15 weeks) to Wistar rats with N- nitrosodiethylamine-induced hepatocarcinogenesis significantly inhibited the expression of vascular endothelial growth factor (VEGF), involved in the tumor angiogenesis, and metalloproteinases-2 and -9, both participating in the extracellular matrix processing and intimately related with tumor invasion and migration [193]. Indeed, antimetastatic effect is another characteristic of phlorotannins that greatly contributes for their antitumor properties. One of the mechanisms by which phlorotannins exert antimetastatic effects is through inhibition of the expression of matrix metalloproteinases (MMPs), which are important enzymes involved in degradation of extracellular matrix proteins and tissue remodeling, and are associated with various physiological or pathological processes such as morphogenesis, angiogenesis and metastasis. Among these, MMP-2 and MMP-9 are of particular relevance since they are usually overexpressed in tumor cells and are capable of degrading type IV collagen of basement membrane, the first barrier for cancer invasion [194]. Inhibition of the expression of these two enzymes were described for E. cava phlorotannin-rich extracts on human fibroblasts, HT1080 fibrosarcoma cells [195] and A549 human lung cancer cells [196], while different solvent-partitioned fractions of Ishige okamurae were shown to not only inhibit MMP-2 and MMP-9 expressions, but also to enhance the levels of tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 in HT1080 cells, at both gene and protein levels [197]. In TPA-induced human hepatoma cells, dieckol suppression of MMP-9 occurred via inhibition of the activity of ERK 1/2 and JNK kinases [198], while in HT1080 cells treated with 6,6'-bieckol both MMP-2 and MMP-9 were suppressed via inhibition of the NF- κB signaling pathway [199], evidencing the potential role of phlorotannins in inflammation-based tumor development and invasion. Identical findings were reported recently in LPS-stimulated MDA- MB-231 breast cancer cells, in which the treatment either with dieckol or phlorofucofuroeckol A (at 50 and 20 μM, respectively) significantly inhibited the cell migration and invasion via suppression of TLR-4/NF-κB-mediated expression of MMP-9 [200]. In a different breast cancer cell line (MCF-

INTRODUCTION

7 cells), Kim et al. [201] demonstrated that in addition to the inhibition of MMP-9 gene expression, dieckol was also capable stimulate the expression of TIMP-1 and TIMP-2, and even block the expression of VEGF, indicating that this compound may also contribute for the inhibition of tumor vascularization. An alternative antimetastatic mechanism of dieckol was demonstrated in A549 lung cancer cells, in which migratory and invasive phenotype were remarkable abrogated via activation of tumor suppressor protein E‐cadherin, an adhesion molecule that plays a key role in the epithelial- mesenchymal transition process, i.e., the mechanism by which epithelial cells lose their cell-cell adhesion characteristics and gain migratory/invasive properties [190].

1.5 Gut microbiota, human health and prebiotics

The human intestinal tract harbours a complex community of microorganisms, collectively termed as intestinal or gut microbiota. The microbial colonization of the gastrointestinal tract starts right after birth and undergoes a symbiotic co-evolution along with their host, importantly contributing for the maintenance of intestinal homeostasis, development and integrity of mucosal barrier, production of various nutrients, protection against microbial pathogens, maturation of immune system and many other functions [202]. An adult gastrointestinal tract contains predominantly Firmicutes and Bacteroidetes, with lower numbers of Actinobacteria, Proteobacteria and Verrucomicrobia. Eukaryotes (mainly yeasts), methanogenic archaea (mainly Methanobrevibacter smithii), viruses (mainly bacteriophages) and protozoa are also present [203]. Throughout adulthood, the intestinal microbiota is regarded as relatively stable, although it may be affected by several extrinsic factors including dietary habits, medication (especially with antibiotics), environmental pollution and exposure to xenobiotics, physical activity, hygiene and others [204]. When these factors cause significant changes in the composition and/or function of the gut microbiota, the whole microbial ecosystem is perturbed to an extent that exceeds its resistance and resilience capabilities, leading to a condition known as dysbiosis. Consequently, dysbiosis has been associated to an increasingly list of diseases which include IBD, irritable bowel syndrome IBS, coeliac disease and colorectal cancer (CRC) [205]. The development of IBD (Chron’s disease and ulcerative colitis), characterized by chronic relapsing inflammation affecting the intestinal mucosa, is perhaps one of the best examples in which the gut microbiota and dysbiosis is deeply implicated. Overall, patients with IBD present a decrease in both microbial population and functional diversity characterized by the reduction of Firmicutes together with an increase in Bacteroidetes and facultative anaerobes such as Enterobacteriaceae [203]. Indeed, low levels of Faecalibacterium prausnitzii, one of the most abundant and important commensal bacteria of the human gut microbiota, was described in both Chron’s disease and

CHAPTER 1 ulcerative colitis, being also associated with IBS, coeliac disease, CRC, diabetes and obesity, although there is still some controversy about the later [206,207]. This bacteria is one of the main butyrate producers found in the intestine, which is a short-chain fatty acid of extreme importance in gut physiology and host wellbeing since this is the main energy source for the colonocytes and can reduce intestinal mucosa inflammation through inhibition of interferon γ (IFNγ) levels and NF-κB transcriptional activity, and upregulation of peroxisome proliferator-activated receptor γ (PPARγ) and IL-10 [208]. Intestinal dysbiosis has even been linked to extra-intestinal disorders like asthma [209], systemic lupus erythematosus [210] or cardiovascular disease [211]. Moreover, preclinical and clinical studies have shown the existence of a bidirectional communication between the central nervous system and gut microbiota known as gut–brain axis, and that the manifestation of many mental illnesses including autism, anxiety, depression and chronic pain or even neurodegenerative diseases such as Parkinson’s, Alzheimer’s or Huntington’s can be linked to a dysfunctional gut microbiota [212–214]. Based on these evidences, it is clear that the manipulation of gut microbiota could be regarded as a promising strategy to treat disease and improve health. In this context, prebiotics appear as important tools capable of manipulating and modifying the gut microbiota composition and promote the host’s health status. These were first described in 1995 [215] and are currently defined as “a substrate that is selectively utilized by host microorganisms conferring a health benefit” [216]. In other terms, prebiotics are non-digestible dietary components that act as substrates that selectively stimulate the growth and/or biological activity of health promoting bacteria residing in the host’s colon. Common prebiotics include several non-digestible polysaccharides such as resistant starch and pectin; and oligosaccharides like fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose and inulin which are found mainly in several land-vegetables, fruits and milk [217], although increasing evidences have shown that other compounds such as polyphenols and polyunsaturated fatty acids may also display modulatory effects on gut microbiota populations through selective prebiotic effects and antimicrobial activities against gut pathogenic bacteria [218,219]. In contrast, studies regarding prebiotic potential of seaweeds (particularly brown) are still rather scarce and essentially focused on the in vitro effects of their polysaccharides, although some in vivo studies have already been carried out over the last few years [220].

1.5.1 Prebiotic effects of phlorotannins

After the recent update of the prebiotic definition by the International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus, the concept of prebiotic was broadened to enable the inclusion of other non-carbohydrates substances such as polyphenols and polyunsaturated fatty acids.

INTRODUCTION

This triggered an upsurge of the number of works focusing on the prebiotic effects of polyphenols (Figure 1.5).

Figure 1.5. Number of publications in the period between 2000 – 2020 by simple search in Scopus using the keyword “polyphenols” AND “prebiotics”

The prebiotic effect of phlorotannins on the other hand remains a subject to which little attention has been given, although some interesting results are already emerging. According to Lee et al. [221], E. cava powder not only exhibited the capacity to stimulate the growth of different Lactobacillus strains in vitro (L. brevis, L. pentosus and L. plantarum) but also improved mortality and diminished the inflammatory state in vivo, on Edwardsiella tarda-infected zebrafish co-treated with E. cava and L. plantarum. E. cava powder is, however, a very complex matrix containing multiple compounds and therefore further experiments would be necessary to attest if phlorotannins could be contributing for the observed results. More recently, the administration of Lessonia turbeculata polyphenol-rich extract to STZ-induced diabetic rats was found to significantly restore the relative abundance of Bacteroidetes, Firmicutes and Proteobacteria, the Firmicutes/Bacteroidetes ratio and the overall bacterial diversity to levels similar to the negative control. Additionally, it was observed that the total levels of SCFAs in the extracts-fed animals’ stool were significantly improved when compared to the diabetic animals, mostly by increasing the levels of acetic acid and butyric acid which is also indicative of an alteration of the gut microbiota composition and/or function [222]. Notably, experiments conducted with human gut microbiota demonstrated that different E. radiata samples (raw powder, enzyme assisted extract, acidic water extract, free sugar fraction, polysaccharide fraction and extract residue), promoted the growth of beneficial bacteria such as Bifidobacterium, Lactobacillus and Clostridium coccoides, as well as the SCFAs with particular increase of butyrate [223]. Later, the same research group reported that the increased levels of these beneficial bacteria

CHAPTER 1 was associated with the phlorotannin-enriched fermentation of E. radiata, since higher numbers of Lactobacillus, F. prausnitzii, C. coccoides, Firmicutes and E. coli were observed in phlorotannin- supplemented fermentations rather than in inulin fermentations. In contrast, the number of Enterococcus in both fermentations decreased approximately ten-fold relative to the initial counts [224]. Despite these scientific evidences of the prebiotic activity of phlorotannins, there is currently an emergent need to conduct further studies and clinical trials in order to demonstrate and solidify the prebiotic health claim of these compounds.

Motivation and Aims Fucus vesiculosus, an abundant and widely distributed species of brown, perennial and edible seaweed has earned increasing attention throughout the last years. This species is characterized by a greenish brown trisected thallus, consisting of a holdfast, a small stipe and flattened dichotomously branched blades with terminal receptacles that swell during the reproductive season. Moreover, it possesses pneumocysts which presents an almost spherical shape and are symmetrically paired, one on each side of the central-thickened area called midrib [11]. Popularly it is known as bladder-wrack or “bodelha-do-mar” (in Portuguese) and occupies the cold-temperate waters from the littoral and sublittoral regions along the rocky shorelines of the northern hemisphere, extending from the White Sea to the south of the Canary Islands at the east, and from south Greenland to North Carolina at the west of the North Atlantic shores. Although in less extent, it also occurs along the Northeast-Pacific shores, extending from Alaska to California [225]. In Portugal this species grows profusely along the central to northern coast of the country although it also occurs in the south and islands [226]. Apart from its nourishment purposes, F. vesiculosus has long been recognized in the folk medicine for its therapeutic properties. In fact, this species became very popular due to its high content in iodine which was described by Moro and Basile [227] as the most important active principle of F. vesiculosus, due to its significance for the production of thyroid hormones, which in turn are responsible for the increase of the metabolism in most tissues and consequently raise the basal metabolic rate [228]. Therefore, the most relevant therapeutic uses of F. vesiculosus nowadays are for the treatment of goiter and thyroid-related complications as well as for weight management and obesity [229]. Notwithstanding, this species is also an excellent source of bioactive compounds which have been repeatedly shown to possess important therapeutic properties including for the treatment of cellulite, blood clot formations, rheumatoid arthritis, asthma, atherosclerosis, diabetes, psoriasis and skin diseases, cancer and other oxidative and inflammatory related conditions [18]. Among these compounds, phlorotannins have drawn much attention during the recent years due to their numerous bioactivities such as antioxidant, anti-inflammatory, antimicrobial, antidiabetic and several other which give them a wide spectrum of possible applications, although there is still a long

INTRODUCTION

way to go before understanding of the mechanisms behind them, particularly for the anti- inflammatory and antitumor properties of phlorotannins from F. vesiculosus. Moreover, little is known about the fate of these compounds when crossing the gastrointestinal tract. In this context, the first approach of this work was to optimize the extraction of phlorotannins from F. vesiculosus using a response surface methodology and characterize the extracts using UHPLC-DAD-ESI/MS (chapter 2). Evaluation of the anti-inflammatory potential of F. vesiculosus phlorotannins was also carried out since inflammation plays a major role in the pathogenicity of several diseases that occur in the digestive tract, particularly in inflammatory bowel disease (chapter 3). As there is an intimate relation between inflammation and cancer, the following approach was to evaluate the potential anti-tumor properties of these compounds in cellular models of colon and stomach cancer, focusing on the possible molecular mechanisms underlying cell death and apoptosis (chapter 4). Finally, and because there is still very little knowledge about the fate of phlorotannins during their passage through the gastrointestinal tract, evaluation of their inhibitory effects on selected digestive enzymes as well as a simulation of the gastrointestinal digestion followed by colon fermentation was carried out with in order to evaluate their stability, bioaccessibility and modulatory effects on gut microbiota growth and production of short chain fatty acids (chapter 5).

Chapter 2 .

This chapter includes data from the paper:

Catarino, M.; Silva, A.; Mateus, N.; Cardoso, S. Optimization of Phlorotannins Extraction from Fucus vesiculosus and Evaluation of Their Potential to Prevent Metabolic Disorders. Marine Drugs 2019, 17, 162.

CHAPTER 2

2.1 Optimization of phlorotannins extraction from F. vesiculosus

Due to their complex chemical structure, susceptibility to oxidation and interaction with other matrix components, the extraction of phlorotannins can be a demanding task. Many factors, such as solvent composition, solvent polarity, time of extraction, temperature, solvent-solid ratio and particle size, may significantly influence the solid–liquid extraction of phenolic compounds [60]. The most common protocols used for the extraction of phlorotannins are based on aqueous mixtures of acetone, ethanol or methanol [125,230,231]. Indeed, Koivikko et al. [43] studied the influence of different solvents namely ethyl acetate, ethanol, methanol, acetone and water on the amount of phlorotannins extracted from F. vesiculosus and found that a water:acetone (30:70) mixture was the most suitable solvent system for the extraction of phlorotannins from this species. However, a proper study to evaluate the effects of cross interactions between different factors on F. vesiculosus phlorotannins extraction has not been performed yet. In this context, the first approach of this thesis was to clarify the effects of different extraction parameters, namely solvent concentration, solvent-solid ratio, temperature and time, on the total phlorotannin content of F. vesiculosus collected on the northern Portuguese coast and to optimize the recovery yields of these compounds using a Box-Behnken design (BBD), i.e., one instrument of response surface methodology (RSM) which uses quantitative data from an appropriate experimental design to determine or simultaneously solve a multivariate equation. Finally, a comprehensive interpretation of the UHPLC-DAD-ESI-MSn data will allow new insights into the composition of phlorotannins from the genus Fucus.

2.2 Materials and methods

2.2.1 Chemicals

Grounded F. vesiculosus samples from July 2017 were purchased from Algaplus Lda (production site located at Ria de Aveiro coastal lagoon, Northern Portugal, 40º36’43” N, 8º40’43” W) an enterprise dedicated to the production of edible seaweeds in an integrated multi-trophic aquaculture (IMTA) system. HPLC grade acetone, ethanol, methanol, n-hexane, ethyl acetate, acetonitrile, hydrochloric acid, glacial acetic acid, were acquired from Fisher (Pittsburgh, PA, USA). Formic acid and 2,4-dimethoxybenzaldehyde (DMBA) were purchased from Sigma (St. Louis, MO, USA). All reagents were of analytical grade or of the highest available purity

2.2.2 Single-factor experiments

Extractions were performed using the conventional mechanical stirring solid-liquid method at atmospheric pressure. For each experiment, 1 g of dried F. vesiculosus powder was loaded into glass flasks covered with aluminum foil. Initial extraction conditions were set as described by Neto et al. [232], i.e., dispersing 1 g of seaweed powder in 20 mL of 70% acetone for 24 h at room temperature.

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

The single-factor experiments were then carried by varying one condition at a time, namely solvent concentration (10–90% v/v), temperature (25–50 ºC), solvent-solid ratio (10–110 mL/g) and extraction time (1–9 h). The flasks were all screw capped to control solvent evaporation and kept under constant agitation. Finally, the extracts were centrifuged at 6000 rpm at 4 ºC for 10 min and the supernatant was filtered and stored at –20 ºC until subsequent use.

2.2.3 Experimental design for optimization of phlorotannins extraction

A three level, three-variable Box–Behnken experimental design (BBD) was employed in this study for evaluating the effects of solvent concentration (% v/v, X1), solvent-solid ratio (mL/g, X2) and extraction time (h, X3) on the total phlorotannin content (TPhC, mg PGE/g dry seaweed) of F. vesiculosus. The levels of these three variables (Table 2.1.) were set according to the single-factor tests outlined above.

Table 2.1. Independent variables and their coded levels used in the BBD

Levels Symbols Independent Variables −1 0 1 X1 Solvent concentration (% v/v) 30 50 70 X2 Solvent-solid ratio (mL/g) 30 50 70 X3 Temperature (°C) 15 25 35

A total of 15 different experiments, including three replicates at central point (Table 2.2.), were conducted in a randomized order. Using the response surface methodology, the experimental design and analysis of variance (ANOVA) were carried out in the statistical software JMP, version 10.0.0 (Cary, NC, USA), to generate the following second-order polynomial equation that represents the total phlorotannin content as a function of the coded independent variables:

푘 푘 푘 2 푌 = 훽0 + ∑ 훽푖푋푖 + ∑ 훽푖푖푋푖 + ∑ 훽푖푗푋푖푋푗 푖=1 푖=1 푖≠푗=1 where Y is the predicted response; β0 is the constant coefficient; βi, βii and βij are the linear,

quadratic and interactive coefficients of the model, respectively; and Xi and Xj are the coded independent variables.

CHAPTER 2

Table 2.2. Box-Behnken experimental design matrix and the experimental and predicted values observed for TPhC

Extract Independent Variables TPhC (mg PGE/g DW) No. X1 X2 X3 Experimental Predicted 1 30 30 25 2.21 2.19 2 30 70 25 2.13 2.12 3 70 30 25 2.55 2.56 4 70 70 25 2.94 2.97 5 50 30 15 2.56 2.54 6 50 30 35 2.53 2.57 7 50 70 15 2.73 2.69 8 50 70 35 2.72 2.74 9 30 50 15 1.98 2.03 10 70 50 15 2.67 2.68 11 30 50 35 2.12 2.11 12 70 50 35 2.73 2.68 13 50 50 25 2.63 2.69 14 50 50 25 2.70 2.69 15 50 50 25 2.73 2.69

Model adequacy was evaluated using the coefficient of determination (R2) and the lack-of-fit test represented at 5% level of significance, accordingly. Three-dimensional response surface plots were used for visualization of the effects of independent variables and their mutual interactions in the response. To validate the accuracy of the model, triplicate experiments were carried out at the optimal conditions predicted for TPhC, and the obtained experimental data were compared to the values predicted by the regression model.

2.2.4 Preparation and purification of F. vesiculosus optimal extract

The extracts were prepared following the optimum conditions determined through the response surface method. For that, 30 g of dried algal powder were dispersed in 2100 mL of 70% acetone solution and incubated in for 3 h at room temperature under constant agitation. The mixture was filtered through cotton to remove the solid residues and then through a G4 glass filter. Afterwards the extract was concentrated in a rotary evaporator to about 250 mL. Concentrated extract was defatted using n-hexane (1:1, v/v) for several times (until a colorless nonpolar fraction was obtained, and the aqueous phase was further submitted to liquid-liquid extraction with ethyl acetate (1:1, v/v) for three times, to obtain a phlorotannin-purified fraction (EtOAc). Finally, the solvents from each fraction, including aqueous residue, were removed by rotary evaporation and subsequently stored at −20 ºC until further analysis.

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

2.2.5 Determination of total phlorotannins contente (TPhC)

Quantification of the TPhC was carried out according to the 2,4-dimethoxybenzaldehyde (DMBA) colorimetric method [233]. For that, equal volumes of the stock solutions of DMBA (2%, m/v) and HCl (6%, v/v), both prepared in glacial acetic acid, were mixed prior to use (work solution). Afterwards, 250 µL of this solution was added to 50 µL of each extract in a 96-wells plate and the reaction was incubated in the dark, at room temperature. After 60 min, the absorbance was read at 515 nm and the phlorotannin content was determined by using a regression equation of the phloroglucinol linear calibration curve (0.06–0.1 mg/mL). The results were expressed as mg phloroglucinol equivalents/g dry seaweed (mg PGE/g DW).

2.2.6 UHPLC-DAD-ESI/MS analysis

Chromatographic analysis of F. vesiculosus phlorotannin-rich ethyl acetate fraction was carried out in Ultimate 3000 (Dionex Co., San Jose, CA, USA) an apparatus consisting of an autosampler/injector, a binary pump, a column compartment and an ultimate 3000 Diode Array Detector (Dionex Co., San Jose, CA, USA), coupled to a Thermo LTQ XL (Thermo Scientific, San Jose, CA, USA) ion trap mass spectrometer equipped with an ESI source. The UHPLC separation was conducted using a method adapted from Ferreres et al. [234] with a Hypersil Gold (ThermoScientific, San Jose, CA, USA) C18 column (100 mm length; 2.1 mm i.d.; 1.9 µm particle diameter, end-capped) maintained at 30 ºC and a binary solvent system composed of (A) acetonitrile and (B) 0.1% of formic acid (v/v). The solvent gradient started with 5–40% of solvent (A) over 14.72 min, from 40–100% over 1.91 min, remaining at 100% for 2.19 more min before returning to the initial conditions. The flow rate was 0.2 mL/min and UV–Vis spectral data for all peaks were accumulated in the range of 200–700 nm while the chromatographic profiles were recorded at 280 nm. Control and data acquisition of MS were carried out with the Thermo Xcalibur Qual Browser data system (ThermoScientific, San Jose, CA, USA). Nitrogen above 99% purity was used, and the gas pressure was 520 kPa (75 psi). The instrument was operated in negative mode with the ESI needle voltage set at 5.00 kV and an ESI capillary temperature of 275 ºC. The full scan covered the mass range from m/z 100 to 2000. CID–MS/MS experiments were performed for precursor ions using helium as the collision gas with a collision energy of 25–35 arbitrary units. All solvents used were of HPLC-MS grade.

2.2.7 Statistical analysis

All data was expressed as mean ± standard deviation (SD) of three similar and independent experiments performed in triplicate. JMP, version 10.0.0 (Cary, NC, USA) and Minitab, version 17.3.1. (Paris, France) softwares were used to construct the BBD and to analyze the results. Data

CHAPTER 2 from single-factor experiments and BBD were analyzed using ANOVA (p < 0.05) followed by Tukey’s post hoc test.

2.3 Results and discussion

2.3.1 Single-factor experiments

Prior to implementing the BBD, preliminary single-factor experiments concerning the relevant variables that could affect the phlorotannin extraction were conducted to narrow the range of selected factors in the BBD experiment.

2.3.1.1 Effect of the acetone concentration on TPhC

Usually, phenolic compounds are easily soluble in solvents less polar than water, and therefore, the most common extractants used for these compounds are methanol, ethanol and acetone or aqueous mixtures of these. Particularly concerning phlorotannins, several studies have shown that the highest extraction yields were achieved with acetone [43,64,66,102,235]. These results were also in line with our preliminary experiments, which confirmed a superior recovery of TPhC with acetone compared with ethanol or methanol (data not shown). Therefore, different concentrations of this solvent were further tested in a range of 10 to 90% (v/v). As depicted in Figure 2.1. A, the recovery of TPhC from F. vesiculosus increased proportionally for acetone concentrations between 10 to 50% (0.77 ± 0.05 to 1.77 ± 0.06 mg PGE/g DW) while maximum values were obtained for acetone concentrations between 50 to 70% (1.77 ± 0.06 and 1.73 ± 0.07 mg PGE/g DW, respectively). The increment of acetone above 70% caused a downward tendency in the TPhC of the extracts, suggesting that, in such conditions, the polarity of the solvent is above the ideal for phlorotannin extraction. Other authors demonstrated that concentrations between 40 to 80% of acetone were more effective than pure acetone or water to produce brown algae extracts with high content in phlorotannins [43,236–238]. Interestingly, Leyton et al. [239] demonstrated that water was the best solvent for phlorotannin extraction in Macrocystis pyrifera. However, for higher water percentages, polysaccharides and proteins are solubilized easily, resulting in more complex and/or less pure extracts that consequently require additional purification steps. In the particular case of F. vesiculosus, after comparing water, acetone and an aqueous mixture of 70% acetone, Koivikko et al. [43] concluded that the latter was the most effective solvent for phlorotannin extraction, which was further confirmed by Wang et al. [240], who showed that 70% acetone produced the extracts with the highest phlorotannin content and simultaneously the strongest antioxidant effects. Consequently, for the BBD experiment, an acetone concentration range between 30–70% was selected.

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

Figure 2.1. Effect of (A) acetone concentration, (B) solvent-solid ratio, (C) temperature and (D) extracting time on the TPhC of F. vesiculosus extracts in the single-factor experiments. Initial extraction conditions consisted of 70% acetone, in a proportion of 1:20 (m:v) at room temperature during 24 h. Before moving to the next experiment, the previous condition was fixed at the point that showed the best TPhC. Data represent the mean ± SD of at least 3 independent assays and the results are expressed in mg of phloroglucinol equivalents/g of dried seaweed. Different letters represent statistical significance (one-way ANOVA followed by Tukey’s post hoc test; p ≤ 0.05).

2.3.1.2 Effect of the solvent-solid ratio on TPhC

Based on the mass transfer principle, the higher the volume of solvent used, the greater the concentration gradient will be, driving the transference of the solutes from the sample matrix to the external solvent [241]. In this study, the effect of different solvent-solid ratios ranging from 10 to 110 mL/g on the phlorotannin recovery from F. vesiculosus were examined (Figure 2.1. B). Increasing phlorotannin recoveries were observed when the ratios varied from 10 to 30 mL/g (1.63 ± 0.03 to 2.21 ± 0.03 mg PGE/g DW). In turn, TPhC remained constant for ratios between 30 to 50 mL/g and decreased for higher ratios. Similar findings have been previously reported by Boi et al. [242] who showed that the recovery of TPhC from Sargassum serratum gradually increased until the ratio reached 40 mL/g, after which it tends to decrease. Our results suggest that increasing volumes of solvent enhances the extraction of phlorotannins until reaching an equilibrium (between 30 to 50

CHAPTER 2 mL/g) after which other compounds start to be co-extracted. Considering the results obtained for this experiment, the solvent-solid ratio interval selected for the BBD experiment was set at 30 to 70 mL/g.

2.3.1.3 Effect of the temperature on TPhC

Higher extraction temperatures are often used to improve extraction yields since they increase molecular movement and decrease solvents’ viscosity, making them more prone to penetrate the sample matrix and dissolve target compounds easily. However, for thermolabile compounds such as phenolics, the use of temperature may trigger their degradation and consequently hinder their extraction [243]. Therefore, it is necessary to select a proper extraction temperature that ensures the maximum extraction yields without damaging the target compounds. For this reason, five different extractions were conducted at room temperature (approximately 17 °C), 25, 37.5 and 50 °C. As depicted in Figure 2.1. C, there was a significant increase in phlorotannin recovery when the extracting temperatures rise from room temperature to 25 °C (2.46 ± 0.05 to 2.78 ± 0.11 mg PGE/g DW), but higher temperatures caused a gradual decrease in TPhC from 2.78 ± 0.11 mg PGE/g DW (at 25 °C) to 2.03 ± 0.06 mg PGE/g DW (at 50 °C). Some authors have reported optimum temperatures between 50 to 60 °C for the extraction of phlorotannins from different brown seaweed species, including M. pyrifera [239] and Saccharina japonica [183]. However, it is possible that the phlorotannins present in the species herein studied, i.e., F. vesiculosus, are more thermolabile, which could explain why lower temperatures are more appropriate for their extraction. In fact, the curve observed in Figure 2.1. C is identical to that reported by Li et al. [63], who also observed a significant enhancement in phlorotannin extraction from S. fusiforme when the temperature was increased from 15 to 25 °C followed by a drastic decline for temperatures above. Based on these results, the temperature interval chosen for testing in the BBD experiment was 15 to 35 °C.

2.3.1.4 Effect of time on TPhC

The determination of an adequate extraction time is important not only for ensuring an efficient extraction of the target compounds, but also for minimizing energy and other associated costs [244]. Therefore, in this study the extraction time was monitored in periods of 2 h over 23 h, to evaluate its effect on the phlorotannin content and antioxidant activity of the extracts. Figure 2.1. D shows that the variation of the extraction time did not significantly influence the TPhC of the extracts, which remained constant at approximately 2.8 mg PGE/g DW from 1 to 23 h. It is possible that equilibrium for phlorotannin extraction is achieved for a time point below 60 min, which explains the inexistence of variations for the range of periods tested. A previous study carried out with F. vesiculosus showed that 1 h was enough to extract approximately 80% of the phlorotannins from this seaweed [43]. More recently, Kadam et al. [245] reported that the extraction of phenolic compounds from A. nodosum,

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

another brown algae species belonging to Fucaceae, was not affected at all by time variations between 5 to 25 min. It is common to find in literature numerous studies where the extraction procedures used for phlorotannin recovery and analysis take about 24 h, which is very time consuming [67,246,247]. The results herein presented show that it is possible to reduce the extraction time of F. vesiculosus for 3 h or less without compromising the recovery of phlorotannins from the matrix, and for this reason, the extraction time used for the BBD experiment was fixed at 3 h.

2.3.2 Analysis of the response surface methodology

2.3.2.1 Fitting the model

Fitting the models for TPhC values is important to assess how precisely this response surface method can predict the ideal variances and determine the correlations of the selected parameters to the corresponding response. The experimental values obtained for the extracts’ TPhC (see Section 3.2.2.) were fitted to a quadratic polynomial model (in bellow) and used to study the correlations between the independent variables and corresponding responses, as well as to determine the optimum conditions for maximization of phlorotannin extraction from F. vesiculosus. Through an analysis of variance (ANOVA), it was possible to determine the significance of the coefficients, which can be observed in Table 2.3. The results show that the independent variables with a higher impact on TPhC were the acetone concentration (p < 0.001) followed by the solvent-solid ratio (p < 0.01), whereas no effect on temperature was seen. The acetone concentration also demonstrated a significant quadratic effect on phlorotannin recoveries (p < 0.001) as well as the interaction between the acetone concentration and solvent-solid ratio (p < 0.001).

TPhC = 2.69 + 0.31X1 + 0.08X2 + 0.02X3 – 0.25X12 + 0.02X22 – 0.07X32 + 0.12X1X2 – 0.02X1X3 + 0.01X2X3

The ANOVA analysis also allowed us to further confirm the reliability of the model. With high F-values and low associated p-values, the model was shown to be remarkably significant. The elevated R2 value (0.99) indicated that 99% of the variations are explained by the fitted model, while

2 2 the adjusted determination coefficient (R Adj) of 0.96, which is very close to R , revealed a great correlation between the observed and predicted values. Furthermore, the lack-of-fit test showed p- values much higher than 0.05, which means that the models can adequately fit to the experimental data. All these results indicate that the fitted model reliably explains the relation between the response and the independent variables and is suitable for predicting the response.

CHAPTER 2

Table 2.3. Regression coefficients and results of ANOVA analysis of the model.

Parameter Regression Coefficient β0 2.69 *** X1 0.31 *** X2 0.08 ** X3 0.02 X1 X1 −0.25 *** X2 X2 0.02 X3 X3 −0.07 X1 X2 0.12 ** X1 X3 −0.02 X2 X3 0.01 R2 0.99 2 R Adj 0.96 Model F-value 39.24 Model p-value <0.001 Lack-of-fit p-value 0.46

β0 – constant coefficient; X1 – acetone concentration (%); X2 – solvent-solid ratio (mL/g); X3 – extraction temperature (°C). **, *** represent statistical significance with p < 0.01 and 0.001, respectively.

2.3.2.2 Effect of the independent variables on the TPhC

The interactive effects of the significant terms, i.e., acetone concentration and solvent-solid ratio, on TPhC can be visualized on the three-dimensional response surface plot shown in Figure 2.2. that demonstrates the effects of these two independent variables on phlorotannin yields.

. . . . .

Figure 2.2. Response surface plots for the total phlorotannin content (TPhC in mg PGE/g DW) from F. vesiculosus extracts with respect to acetone concentration (%, X1) and solvent-solid ratio (mL/g, X2). The variable temperature was kept at its zero level.

According to the graphical representation, it is unequivocal that acetone was the variable that most affected the TPhC values, with the maximum yield being achieved for a concentration of 67.2%. A slight decay of the phlorotannin content is observable when the acetone concentration increases up to 70%, particularly for low solvent-solid ratios, thus evidencing the quadratic effect that this

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

variable has on this response. This confirms that the presence of water is important to confer a moderate polar medium to acetone that facilitates the extraction of hydrophilic phenolic compounds, which is the case of phlorotannins [248]. The variable solvent-solid ratio exhibited a directly proportional effect on the extracts’ TPhC, i.e., the higher the ratio, the greater the phlorotannin extraction from F. vesiculosus, reaching their maximum at 70 mL/g. This observation is however not in agreement with the single-factor experiments, since the total phlorotannin content of F. vesiculosus extracts were constant for ratios between 30 and 50 mL/g and decreased for ratios above (Figure 2.1. C). This difference might be explained by the interactive effects that this variable establishes with the acetone concentration, which are very perceptible in the Figure 2.2., and were not considered in the single-factor experiments. According to this picture, for high acetone concentrations, the response undergoes a great impact from the solvent-solid ratio variations, whereas for low acetone concentrations, the effect of this variable on TPhC is very tenuous. Consistent with these findings, other authors demonstrated that the extraction of phlorotannins from different brown seaweeds is greatly influenced by the solvent concentration or solvent-solid ratio [239,249], but none have yet shown the importance of the interaction between these two variables. Interestingly, although the single-factor experiments revealed that temperature significantly affected phlorotannin extraction from F. vesiculosus, this was not observed in the experimental design, suggesting that the extraction of phlorotannins may be carried out at the most convenient temperature within the interval tested without significantly affecting the extraction yields.

2.3.2.3 Optimization and validation of the models

Using the predictive equation mentioned above, the optimum conditions for the extraction of phlorotannins from F. vesiculosus were predicted as follows: acetone concentration at 67% (v/v), solvent-solid ratio at 70 mL/g, and temperature at 25 °C. Under these conditions the model predicted that the maximum phlorotannin recovery would be 2.97 mg PGE/g DW (Table 2.4.).

Table 2.4. Predicted and experimental values obtained for TPhC according to the predicted optimum conditions. Optimum Conditions Results Response X1 X2 X3 Predicted Experimental TPhC (mg PGE/g DW) 67 70 25 2.97 2.92 ± 0.05

X1 – acetone concentration (%, v/v); X2 – solvent-solid ratio (mL/g); X3 – temperature (°C); TPhC – total phlorotannin content; PGE – phloroglucinol equivalents.

To validate the reliability of the model, verification experiments were carried out three times under the predicted optimum parameters, and the experimental value of 2.92 ± 0.05 mg PGE/g DW was obtained. The good correlation that was observed between the experimental and predicted value confirms that this model is reliable and accurate.

CHAPTER 2

2.3.3 Total phlorotannin content of the F. vesiculosus extract and respective fractions

The crude extract obtained under the established optimum conditions was sequentially separated into three fractions, namely n-hexane (Hex) and ethyl acetate soluble (EtOAc) fractions and the final aqueous residue (AR), by liquid−liquid partitioning. The differences in the extraction yields and total phlorotannin content between the crude extract and its subsequent fractions are depicted in Table 2.5.

Table 2.5. Extraction yield (as % w/w of algal powder for crude extract and % w/w of crude extract for the fractions) and total phlorotannin content of F. vesiculosus crude extract and subsequent fractions. Sample Yield (%) TPhC (mg PGE/g ext) CRD 28.2 ± 2.1 10.7 ± 1.5 b Hex 15.5 ± 1.2 b 4.0 ± 0. 9 c EtOAc 3.9 ± 0.6 c 17.1 ± 1.5 a AR 82.2 ± 2.3 a 3.7 ± 0.5 c PGE – phloroglucinol equivalents; ext – extract; CRD – crude extract; Hex - n-hexane fraction; EtOAc - ethyl acetate fraction; AR - aqueous residue. Data expressed as mean ± standard deviation. Different letters within a column mean significantly differences at p < 0.05 using student’s t test.

On the one hand, a 28.2 ± 2.1% yield was obtained for the crude extract of F. vesiculosus, a value that is slightly higher than that described by Wang et al. [65] (20.2% w/w), and almost twice the yield reported by Liu et al. [64] (14.7% w/w), using both the same species and solvent. On the other hand, the total TPhC of the extract herein prepared was 10.7 ± 1.5 mg PGE/g extract, which is approximately 35 and 4 times lower compared to the values reported by those authors, respectively. Among several factors, this variability might be related to the geographical origin and/or harvest season of the algal material, as well as the methodology used to quantify the total phlorotannin content, since in the work of Wang et al. [65] the Folin-Ciocalteu was used instead of 2,4- dimethoxybenzaldehyde DMBA and only the latter is selective for phlorotannins [71]. After solvent partitioning, about 15.5%, 3.9% and 82.2% of the extract was distributed in the n-hexane, EtOAc and AR fractions, respectively. The highest level of TPhC was found in the EtOAc with a value of 17.1 ± 1.5 mg PGE/g of dry residue, while Hex and AR fractions showed the lowest TPhC both presenting approximately 4 mg PGE/g of dry residue. The use of ethyl acetate to selectively extract phlorotannins from various algae extracts has been a common practice [250]. The results herein obtained indicate that this solvent can be used for concentrating/enriching phlorotannins from F. vesiculosus crude extract as well, which is in agreement with previous studies conducted in this species [64,65].

2.3.4 Characterization of phlorotannins-rich fraction

The presence of phlorotannins in brown seaweeds is widely acknowledged, although, due to their structural complexity and similarity, as well as the lack of commercial standards, the identification

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

and characterization of these compounds is usually a challenging task. Due to its higher TPhC, the UHPLC-MS analysis was carried out using the EtOAc fraction. The total ion chromatogram (Figure 2.3.) obtained was characterized by a region of reasonably well separated peaks (up to 10 min), corresponding to phlorotannin oligomers, and another region where higher polymeric phlorotannin structures are eluted together in a hump [230,251]. Overall, a total of 21 peaks were analyzed, all exhibiting a UV-max around 270 nm, which is in line with what has been reported in the existing literature on phlorotannins [252] and is very close to that of phloroglucinol (267 nm). Moreover, in almost every identified compound, the base peak at MS2 corresponded to the loss of one or two water molecules, which is also a common characteristic in these compounds. The tentative identification of these compounds was further carried out based on an MS2 fragmentation pattern as well as by comparison with data previously reported in the literature.

Figure 2.3. Total ion chromatogram (TIC) of the EtOAc fraction. Peaks marked with numbers correspond to the tentatively identified compounds represented in Table 2.6.

Several types of phlorotannins could be noted in this fraction, including fucols, fuhalols and fucophlorethols. Fucols consist of polymers of phloroglucinol (A in Figure 2.4.) linked together through C−C bonds, while phlorethols are polymers of the same compound linked through C−O−C linkages. This is a relevant detail when it comes to the identification of these compounds through MS, since C−C bonds usually require higher energy to break than C−O−C linkages, and therefore, although fucols, phlorethols and fucophlorethols with the same degree of polymerization will have the same molecular weight, they can produce different fragmentation patterns [253]. In turn, fuhalols are ether-linked phloroglucinol units that contain at least one additional hydroxyl group [63].

CHAPTER 2

A: m/z 125 -H OH -H B: m/z 373 -H C: m/z 497 m/z 207 m/z 165 OH m/z 111 HO OH -84 HO OH OH OH m/z 165 -28 HO OH -44 m/z 315 OH OH HO OH -14 OH HO OH HO OH m/z 331 OH HO OH OH HO D: m/z 745 -H E: m/z 621 -H G: m/z 869 -H m/z 331 m/z 437 m/z 429 m/z 495 m/z 411 HO OH HO OH HO OH m/z 479 OH m/zOH 703 OOHH OH OH m/z 605 HO OH HO O HO OH HO O HO O HO OH OH OH OH

OH OH OH HO OH HO OH HHOO OH HO HO OH O OH OH OH OH OH OHHO OH HO OH HO OH m/z 455 m/z 289 m/z 725 m/z 289 m/z 355 m/z 331 m/z 455 m/z 677

m/z 413 m/z 455 F: m/z 869 -H I: m/z 247 -H m/z 121 OH m/z 81 m/z 579 OH m/z 759 OH O OH HO OH HO OH O OH OH OH HO O O OH O OH m/z 155 OH OH OH m/z 725 OH HO OH HO OH m/z 371 m/z 285 m/z 743 m/z 537

m/z 389 J: m/z 479 OH -H G: m/z 869 -H m/z 371 m/z 331 m/z 353

OH OH m/z 339 m/z 703 m/z 605 HO OH OH O OH HO OH HO O HO O HO OH m/z 313 OH HO OH

HO O HO O O O OH OH OH OH m/z 315 m/z 263 HO OH m/z 725 OH m/z 331 m/z 355 m/z 271 m/z 207 m/z 677

K: m/z 603 m/z 413 m/z 455 -H -H H:F: m/z m/z 993 869 m/z 689 -H OH m/z 663 m/z 867/868 m/z 579m/z 579 m/zOH 373 m/z 759 OH OH HO OH HO OH m/z 851 OH OH m/z 604/605 OHO OH OH HO OH HO OH m/z 351 OH HO O m/z 313 HO OH O HO O HO OH O OH O OH OH O OH OH OH HO OH HO OH OH HO OH O HO m/z 725 O OH HO OH HO OH m/z 709 m/z 413 HO OHOH OH m/z 437 m/z 371 m/z 849 O m/z m/zm/z 285 787 m/z 479 m/z 743 827 m/z 493 m/z 395 O m/z 537 m/z 331 m/z 271 OH Figure 2.4. Structure of phlorotannin compounds tentatively identified in F. vesiculosus EtOAc fraction and proposed fragmentation patterns: (A) phloroglucinol ([M − H]− at m/z 125), (B) trifucol ([M − H]− at m/z 373), (C) tetrafucol ([M − H]− at m/z 497), (D) hexafucol ([M − H]− at m/z 745), (E) trifucophlorethol ([M − H]− at m/z 621), (F) trifucotriphlorethol ([M − H]− at m/z 869), (G) difucotetraphlorethol ([M − H]− at m/z 869), (H) trifucotetraphlorethol ([M − H]− at m/z 993), (I) dibenzodioxine-1,3,6,8-tetraol ([M − H]− at m/z 247), (J) fucofurodiphlorethol ([M − H]− at m/z 479), (K) fucofurotriphlorethol ([M − H]− at m/z 603). Fragmentations with simultaneous loss of water are only representative. Cleavage of the OH group may occur at different sites.

Based on MS/MS analysis as well as data reported in literature, three compounds were tentatively identified as belonging to the group of fucols and, despite the fact that their exact structural features were not possible to disclose, these consisted of trifucol ([M − H]− at m/z 373, peak 1), tetrafucol ([M − H]− at m/z 497, peak 2) and hexafucol ([M − H]− at m/z 745, peak 5). Overall, the MS2 spectrum of

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

these compounds exhibited common losses of 14, 44 and 84 Da (as depicted in structure A of Figure 2.4.), and/or their combinations with water (60 and 102 Da, respectively), phloroglucinol units (e.g., 166 and 208 Da, respectively) or water plus phloroglucinol units, which are indicative of cross-ring cleavages (Table 2.6., B–D in Figure 2.4.), while product ions resulting from the loss of phloroglucinol moieties were present in very low intensities or even completely absent. Note that to our knowledge, this type of fragment has rarely been described in previous studies on the interpretation of MS product ions aiming at phlorotannin identification. In fact, although losses of 44, 28 or 14 Da (and their derivatives) have been previously assigned in these phenolic compounds [65,253], other possible cross-ring cleavages are being herein elucidated for the first time. In contrast, the compounds with [M − H]− at m/z 621, eluting in peak 3, [M − H]− at m/z 869, eluting in peaks 6 and 7, and [M − H]− at m/z 993, eluting in peaks 9, 10, 11 and 12, were identified as fucophlorethols since their MS2 spectra presented product ions resultant from cross-ring cleavages, and simultaneously, several originated from the loss of one or more phloroglucinol moieties (−124/126 Da): O-phloroglucinol (−140/142) and its fuhalol derivatives and/or resorcinol (108/110), which are indicative of ether bond cleavages [252]. The identification of the correct structure of these compounds through HPLC-MS is, however, very difficult, particularly for those with high molecular weights, since linkage positions cannot be assigned. Therefore, note that structures in Figure 2.4. are representative of compounds that may occur in different isomeric forms. In this context, the compound with [M − H]− at m/z 621 exhibited two product ions at m/z 495 and 479 (corresponding to the loss of phloroglucinol and O-phloroglucinol moieties, respectively) in its MS2 spectrum, thus suggesting a fucophlorethol pentamer structure with at least one ether bond in its backbone, as represented in E from Figure 2.4. In turn, the compounds with [M − H]− at m/z 869 presented slightly different fragmentation patterns. As detailed in structure F from Figure 2.4., the MS2 spectrum of the one eluting in peak 6 exhibited product ions at m/z 743 (−126 Da), 725 (−126–18 Da), 759 (−110 Da) and 371 (−498 Da), corresponding to the loss of a phloroglucinol, a phloroglucinol plus water, a resorcinol and four phloroglucinol units, respectively, whereas the compound eluted in peak 7 (G in Figure 2.4.) showed the product ions at m/z 725 (loss of 1 PGU plus water), m/z 605 (loss of bifuhalol) and at m/z 355 (loss of tetrafuhalol). In these compounds, the losses of resorcinol or bifuhalol moieties are particularly relevant for unveiling additional information about their structural arrangement. The former indicates that the terminal phloroglucinol unit contains two free OH groups (see highlights in the compound F from Figure 2.4.), while the latter indicates that the terminal phloroglucinol unit contains three free OH groups (see highlights in the structure G from Figure 2.4.). Based on these MS data, it is possible to conclude these two compounds are both fucophlorethol heptamers, containing at least two and three C−O−C bonds in their backbones, respectively.

CHAPTER 2

The fragmentation patterns among the four compounds with [M − H]− at m/z 993 also revealed some differences that evidenced their structural diversity. The appearance of the product ions at m/z 867 (loss of phloroglucinol), at m/z 849 (loss of phloroglucinol plus water) and at m/z 709 (loss of bifuhalol plus water), clearly indicates that the nonamer eluting in peak 9 contains at least 3 C−O−C linkages, while based on the MS fragmentation pattern of the compound eluting in peak 10 it is only possible to confirm the presence of one ether linkage (i.e., only m/z at 867 was observed). In turn, the fragmentation pattern of the compound eluting in peak 11 showed the product ions at m/z 867, 849, 831 (loss of PGU, PGU plus water and PGU plus 2 waters, respectively), 604 (loss of trifuhalol + 1), 479 (loss of tetrafuhalol) and 373 (loss of 5 PGUs) as represented in H from Figure 2.4., thus suggesting the presence of at least four ether linkages in the backbone of this isomer. Interestingly, even though MS2 spectra from the compound eluting in peak 12 also indicate the presence of four ether linkages, this compound produced a different fragmentation pattern, yielding fragment ions at m/z 851 (loss of O-phloroglucinol), 605 (loss of trifuhalol), 493 (loss of 4 PGUs + 2) and 351 (loss of 5 PGUs plus water + 2), thus evidencing structural dissemblance compared with the previous one. Note that despite fucophloretols of variable DP have been already documented in F. vesiculosus, the examination of their structural features is not commonly approached. In fact, to our knowledge, a detailed scrutinization of structural features of F. vesiculosus phlorotannin compounds by HPLC- MS has only been carried out by Lopes et al. [253], for those with DP below 6 units. Other compounds with more than 8 phloroglucinol units have also been detected in this F. vesiculosus fraction, although due to their low intensities in the MS spectra and the impossibility to go on with further fragmentation in tandem MS, additional structural details were not obtained. Nevertheless, some deprotonated molecular ions at m/z 1117 and 1241, consistent with the molecular weight of phlorotannin nonamers and decamers, respectively [251], were found co-eluting in peaks 13, 14 and 15 (data not shown). Moreover, several minor compounds co-eluting in the peaks 11–17 showed deprotonated molecular ions at m/z 806, 868, 930, 992, 1116, 1054, 1178, 1240, 1302 and 1364 (data not shown) which are consistent with the doubly charged ions of phlorotannins with DP 13–22, respectively [254]. Notably, compounds with such polymerization degrees in Portuguese- sourced F. vesiculosus were not detected in the study of Lopes et al. [253], although Steevensz et al. [254] described the presence of phlorotannin polymers with up to 39 units in samples of the same species, collected in Nova Scotia, Canada. The group of identified fuhalols comprised a hydroxytetrafuhalol ([M − H]− at m/z 529) and two isomers of pentafuhalol ([M − H]− at m/z 637), which, in addition to the evident extra OH groups, showed MS/MS fragmentation patterns concordant to previous data reported in the literature [182]. Notably, although the former has been described in F. vesiculosus before, pentafuhalol has not been detected yet in the genus Fucus [255].

OPTIMIZATION OF PHLOROTANNINS EXTRACTION FROM F. VESICULOSUS

Apart from the fucols, fucophlorethols and fuhalols, other unusual phlorotannin compounds were identified in this F. vesiculosus fraction as well. This is the case of the phloroglucinol dimmer with m/z at 247 eluting in peak 3. The MS/MS of this compound produced the main product ion at m/z 203 ([M-H-44]−) and ions at m/z 121, 81 and 155, corresponding to the [M-H-PGU]−, [M-H- dihydroxybenzodioxin]- and a methoxy-phloroglucinol moiety, respectively, suggesting the presence of dibenzodioxine-1,3,6,8-tetraol, i.e., a precursor of an eckol type phlorotannin (I in Figure 2.4.). Interestingly, small phlorotannins, in particular those with DP 2, are not commonly described in this species. In fact, Fucus are usually described to be more abundant in phlorotannin oligomers of higher DPs (5–10 units) [251,254]. Moreover, to our knowledge, this compound has only been described once in A. nodosum extracts among Fucaceae [176]. Additionally, two uncommon molecular ions were found at m/z 479 (eluting in peaks 7 and 8) and 603 (eluting in peak 9). The MS spectrum of the former suggests two isomers of a dehydroxylated phlorotannin tetramer both further producing atypical fragments at m/z 271 (PGU-84 Da) as well as an atypical loss of 148 Da that were not visible in the other tetramers and indicate the presence of a furan ring (J in Figure 2.4.). Likewise, [M − H]− at m/z 603 resembles a pentamer lacking an OH group, and also presented fragments ([M − H]− at m/z 271 and 315) that suggest the presence of a furan ring as well (K in Figure 2.4.). Based on their MS spectra and the published literature [250], it is possible that the structures of these tetramers and pentamer resemble that of fucofuroeckol ([M − H]− at m/z 477) and phlorofucofuroeckol ([M − H]− at m/z 601), respectively, although with an ether linkage instead of a dioxin ring between the two inner phloroglucinol moieties, which would explain the 2 Da difference between their deprotonated ions. Previous works have already reported fucofuroeckol derivatives in the genus Fucus [253], however, such compounds have never been described before, thus a proper purification and isolation of these compounds would be necessary to allow further spectroscopic analysis, such as NMR, aiming the better elucidation of their structural features. Following the same logic, the [M − H]− at m/z 851 (eluting in peak 14) is likely to belong to the same group of compounds, containing two additional phloroglucinol units, although there is no previous literature describing resembling structures. Notably, even though their structural elucidation were not achieved, many other compounds were identified as phlorotannin derivatives (Table 2.6.) based on their MS/MS spectra which exhibited fragmentation patterns similar to those of phlorotannin compounds, either yielding product ions that are indicative of one or multiple phloroglucinol units (e.g., m/z 125, 247/249, 371, 495/497, 621), or fragments that are indicative of phloroglucinol or phloroglucinol derivative losses (e.g., −124/126, −140/142, −144, −166, −248/250, −266, −374), as well as the usual water losses or cross ring cleavages.

Table 2.6. Tentative assignment of the compounds present in the EtOAc of F. vesiculosus extract, analyzed by UHPLC-ESI-MS/MS.

RT [M − H]− Peak MS/MS Fragments (-loss) * Tentative Assignment (min) (m/z) 1 1.8 373 355 (−18), 329 (−44), 207 (−166), 165 (−PGU−84), 289 (−84), 111 (−2PGU−14), Trifucol 497 479 (−18), 331 (−166), 461 (−36), 453 (−44), 435 (−44−18), 395 (−84−18), 165 (−2PGU−84), 315 (−166−18), 413 (−84) Tetrafucol 2 1.9 529 511 (−18), 493 (−36), 467 (−44−18), 411 (−84−36, +2), 449 (−44−36), 485 (−44), 347 (−166−18, +2), 405 (−PGU), 377 (−PGU−28) Hydroxytetrafuhalol 689 605 (−84), 497 (−192), 621 (−68), 553 (−136), 671 (−18), 653 (−36), 537 (−PGU−28), 643 (−46), 575 (−114), 507 (−182), 345 (−2PGU−96) Phlorotannin derivative 603 (−18), 455 (−166), 585 (−36), 331 (−PGU−166), 577 (−44), 559 (−44−18), 519 (−84−18), 289 (−2PGU−84), 429 (−192), 537 (−84), 495 (−PGU), 621 Trifucophlorethol 3 2.8 479 (−O–PGU), 411 (−PGU-84) 247 202 (−45), 121 (−PGU), 81 (−166), 155 (−PGU−29) Dibenzodioxine-1,3,6,8-tetraol 537 (−18), 511 (−44), 519 (−36), 389 (−166), 331 (−224), 363 (−192), 393 (−PGU−36), 413 (−O–PGU), 430 (−PGU, −1), 305 (−2PGU), 247 (−308), 4 3.1 555 Phlorotannin derivative 223 (−2PGU−84), 165 (−trifuhalol) 5 4.2 745 727 (−18), 455 (−PGU−166), 709 (−36), 579 (−166), 289 (−3PGU−84), 701 (−44), 683 (−44−18), 643 (−84−18) 437 (−PGU−166−18) Hexafucol 623 495 (−110−18), 477 (−110−36), 605 (−18), 369 (−2PGU, −2), 249 (−3PGU) Phlorotannin derivative 6 5.2 851 (−18), 833 (−36), 743 (−PGU), 841 (−28), 725 (−PGU−18), 313 (−2PGU−166−18), 759 (−110), 413 (−2PGU−166), 579 (−PGU−166), 537 (−2PGU- 869 Trifucotriphlorethol 84), 285 (−4PGU−72−18), 825 (−44), 455 (−3PGU−84), 371 (−4PGU) 869 833 (−36), 851 (−18), 703 (−166), 677 (−192), 767 (−84−18), 785 (−84), 725 (−PGU−18), 605 (−PGU−140), 355 (−tetrafuhalol), 331 (−3PGU−166) Difucotetraphlorethol 7 5.8 479 461 (−18), 435 (−44), 433 (−28−18), 389 (−72−18), 313 (−166), 315 (−164), 271 (−PGU−84), 443 (−36), 339 (−140), 371 (−108), 451 (−28), 207 (−272) Fucofurodiphlorethol 8 6.2 479 461 (−18), 435 (−44), 433 (−28−18), 389 (−72−18), 315 (−164), 443 (−36), 371 (−108), 271 (−208), 331 (−148), 353 (−126), 451 (−28), 263 (−216) Fucofurodiphlorethol 9 7.5 993 975 (−18), 965 (−28), 827 (−166), 849 (−PGU-18), 868 (−PGU, +1), 957 (−36), 413 (−4PGU-84), 709 (−bifuhalol−18) Pentafucodiphlorethol 603 585 (−18), 559 (−44), 437 (−166), 395 (−PGU−84), 313 (−PGU−166), 271 (−2PGU−84), 331 (−272) Fucofurotriphlorethol 385 259 (−PGU), 367 (−18), 341 (−44), 245 (−140), 357(−28), 261 (−PGU), 313 (−72), 219 (−166) Phlorotannin derivative 10 8.2 993 975 (−18), 579 (−2PGU−166), 957 (−36), 827 (−166), 849 (−PGU−18), 909 (−84), 891 (−84−18), 867 (−PGU), 949 (−44), 413 (−4PGU−84) Hexafucophlorethol 623 605 (−18), 579 (−44), 495 (−110−18), 535 (−88), 561 (−44−18), 551 (−72), 357 (−bifuhalol), 437 (−PGU−44−18), 457 (−166) Phlorotannin derivative 363 319 (−44), 345 (−18), 222 (−O–PGU, −1), 331 (−32), 301 (−44−18), 178 (−185), 327 (−36), Phlorotannin derivative 975 (−18), 965 (−28), 867 (−PGU), 604 (−trifuhalol, +1), 579 (−2PGU−166), 849 (−PGU−18), 479 (−tetrafuhalol), 831 (−PGU-36), 787 (−PGU−84), 993 Tetrafucotetraphloretol 11 10.0 373 (−5PGU) 753 (−18), 727 (−44), 735 (−36), 496 (−274), 471 (−300), 477 (−294), 504 (−bifuhalol, +1), 615 (−156), 263 (−508), 587 (−184), 643 (−110−18), 613 771 Phlorotannin derivative (−158), 373 (−398) 361 317 (−44), 343 (−18), 178 (-183), 331 (−30), 273 (−88), 299 (−44−18), 289 (−72) Phlorotannin derivative 12 10.2 975 (−18), 965 (−28), 957 (−36), 851 (−O–PGU), 493 (−4PGU, −2), 663 (−4PGU−166), 689 (−2PGU−56), 351 (−5PGU-18), 457 (−3PGU−84), 605 993 Tetrafucotetraphloretol (−trifuhalol) 403 261 (−O–PGU), 385 (−18), 259 (−44), 217 (−186), 327 (−76), 371 (−32), 309 (−94), 341 (−44−18), 353 (−50), 193 (−PGU−84), 141 (−262), 125 (−278) Phlorotannin derivative 13 11.1 711 693 (−18), 623 (−88), 229 (−482), 563 (−148), 429 (−282), 579 (−132), 249 (−462) Phlorotannin derivative 637 619 (−18), 496 (−141), 511 (−126), 335 (−248−54), 593 (−44), 575 (−62) 436 (−182−17), 371 (−266), 601 (−36), 261 (−374−18), 245 (−266−126) Pentafuhalol 317 273 (−44), 176 (−141), 299 (−18), 255 (−44−18), 245 (−72), 229 (−88), 187 (−130), 124 (−193) Phlorotannin derivative 526 482 (−44), 438 (−88), 508 (−18), 494 (−32), 466 (−60), 406 (−120), 349 (−177), 275 (−251), 263 (−263), 249 (−277) Unidentified 14 11.5 833 (−18), 709 (−O–PGU), 817 (−34), 691 (−160), 587 (−bifuhalol, −2), 435 (−2PGU−166), 455 (−3PGU−18), 761 (−90), 601 (−2PGU), 297 (−554), 851 Fucofuropentaphlorethol 583 (−268) 619 (−18), 496 (−141), 601 (−36), 335 (−2PGU−54), 577 (−60), 436 (−201), 471 (−166), 525 (−112), 575 (−44−18), 593 (−44), 555 (−84, −2), 419 15 11.7 637 Pentafuhalol (−218), 247 (−390), 373 (−bifuhalol, −2), 385 (−2PGU) 610 566 (−44), 592 (−18), 449 (−161), 534 (−76), 462 (−148), 367 (−243), 229 (−381), 245 (−365), 309 (−301), 496 (−114) Unidentified 16 12.2 317 299 (−18), 274 (−43), 245 (−72), 259 (−58), 194 (−123), 125 (−192) Phlorotannin derivative

631 (−80), 693 (−18), 565 (−146), 639 (−72), 675 (−36), 395 (−316), 313 (−398), 469 (−242), 427 (−284), 371 (−340), 267 (−444), 229 (−482), 479 711 Phlorotannin derivative (−232), 513 (−198) 881 (−18), 863 (−36), 741 (−158), 755 (−PGU−18), 693 (−206), 759 (−140), 471 (−428), 453 (−446), 371 (−528), 263 (−636), 507 (−3PGU−18), 565 899 Phlorotannin derivative (−334), 649 (−2PGU) 509 (−18), 483 (−44), 465 (−44−18), 437 (−90), 385 (−O–PGU), 261 (−bifuhalol), 401 (−PGU), 341 (−186), 491 (−36), 421 (−106), 455 (−72), 279 527 Phlorotannin derivative (−2PGU), 247 (−280) 17 12.5 635 575 (−60), 617 (−18), 335 (−300), 557 (−78), 369 (−bifuhalol), 509 (−PGU), 493 (−O–PGU), 457 (−178), 473 (−162), 273 (−2PGU−114), 229 (−406) Phlorotannin derivative 719 701 (−60), 553 (−166), 478 (−241), 460 (−259), 496 (−223), 683 (−36), 319 (−400), 331 (−388), 371 (−348), 249 (−3PGU−96) Phlorotannin derivative 723 677 (−60), 695 (−28), 705 (−18), 659 (−64), 583 (−140), 356 (−367), 339 (−384), 477 (−246) Unidentified 18 13.0 587 507 (−80) Unidentified 19 13.7 837 789 (−48), 747 (−90), 619 (−218), 581 (−256), 453 (−384), 265 (−572) Unidentified 950 904 (−46), 696 (−254) Unidentified 20 14.4 667 649 (−18), 635 (−32), 605 (−44−18), 379 (−288), 521 (−146), 507 (−160), 451 (−216), 317 (−350), 297 (−370), 271 (−396) Unidentified 21 14.6 587 507 (−80) Unidentified * Fragments are arranged in descending order of relative abundance with bold values highlighting the most abundant fragment.

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2.4 Conclusions

In this task, a single-factor experimental approach followed by a response surface methodology was carried out for determination of the optimum conditions that maximize the extraction of phlorotannins from F. vesiculosus. The optimal extraction conditions established were: X1 = 67%

(v/v), X2 = 70 mL/g and X3 = 25 °C. Under the optimized conditions, the experimental values agreed with the values predicted by each regression equation, allowing the validation of the accuracy and predictive capacity of the model. After a liquid-liquid partitioning step, the EtOAc fraction, which had the highest content of phlorotannins was analyzed using UHPLC-DAD-ESI-MSn allowing to disclose new structural features of F. vesiculosus phlorotannins through a detailed interpretation of their fragmentation patterns. Overall, this purified fraction was composed of fucols, fucophlorethols and fuhalols, together with several other phlorotannin derivatives of variable degrees of polymerization, ranging from 3 to 22 phloroglucinol units. Additionally, the appearance of possible new phlorotannin compounds, tentatively identified as fucofurodiphlorethol, fucofurotriphlorethol and fucofuropentaphlorethol was also observed in this extract of F. vesiculosus, although further research is necessary to verify their structural arrangements. In summary, with this section we were able to determine an extraction procedure that maximizes the recovery of phlorotannins from F. vesiculosus, contributing with valuable insights on the phlorotannin profile of this species and unveiling possible structures that have not been described yet.

Chapter 3 .

This chapter includes data from the paper:

Catarino, M.D.; Silva, A.; Cruz, M.T.; Mateus, N.; Silva, A.M.S.; Cardoso, S.M. Phlorotannins from Fucus vesiculosus: Modulation of Inflammatory Response by Blocking NF-κB Signaling Pathway. International Journal of Molecular Sciences 2020, 21, 6897.

CHAPTER 3

3.1 Antioxidant and anti-inflammatory properties of F. vesiculosus phlorotannins

Inflammation is the central feature of many pathophysiological conditions including coeliac disease, ulcerative colitis, Chron’s disease and colon cancer. In these conditions several pro-

●– ● inflammatory mediators, including ROS and RNS, such as O2 and NO , prostaglandins, cytokines among others are released abnormally in a vicious circle that result in damage to the host tissue itself [256,257]. The regulation of such pro-inflammatory molecules is mediated by several transcription factors of which NF-κB is perhaps one of the most important. Therefore, the inhibition of NF-κB transcriptional activity as well as its down-stream molecules may represent a viable therapeutic strategy for intervention in pathologies presenting a strong inflammatory component [258]. In this context, the knowledge of phytochemicals’ molecular mechanisms became an appropriate strategy in the search for novel anti-inflammatory compounds, and phlorotannins are emerging as potential candidates capable of modulating inflammation through the inhibition of the expression and/or activity of multiple inflammatory markers. The existing literature on the potential anti-inflammatory capacity of F. vesiculosus-derived phlorotannins remains, however, quite underexplored. The following section aims to explore the molecular mechanisms and signaling pathways involved in the anti-inflammatory properties of F. vesiculosus extract and subsequent phlorotannin purified fractions.

3.2 Materials and methods

3.2.1 Chemicals

Grounded F. vesiculosus samples from July 2017 were purchased from Algaplus Lda. Acetone, ethanol, methanol, n-hexane, ethyl acetate, acetonitrile, dimethyl sulfoxide and hydrochloric acid were acquired from Fisher (Pittsburgh, PA, USA). Fluorescein, 2,2’-azobis(2-amidinopropane)dihydrochloride (AAPH), sodium nitroprusside (SNP) and sulfanilamide were purchased from Acros Organics (Hampton, NH, USA). Formic acid, phosphate buffer saline (PBS) reagents (sodium salt, sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate), trolox, ascorbic acid, gallic acid, reduced nicotinamide adenine dinucleotide disodium salt hydrate (NADH), nitro blue tetrazolium (NBT), phenazine methosulfate (PMS), xanthine oxidase, allopurinol, Dulbecco’s modified Eagle’s medium (DMEM), Tween® 20, penicillin G sodium salt, streptomycin sulfate salt, sodium bicarbonate, D-glucose, lipopolysaccharide (LPS) from Escherichia coli—serotype 026:B6—and anti-β-actin antibody were purchased from Sigma (St. Louis, MO,USA). Anti-COX-2, anti-pro-IL-1β and alkaline phosphatase-conjugated anti-mouse antibodies were purchased from Abcam (Cambridge, UK). Anti-IκBα and anti-pIκBα (Serine 32/36) antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-iNOS

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

antibody was acquired from R&D Systems (Minneapolis, MN, USA) and alkaline phosphatase- conjugated anti-rabbit antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, Dallas, TX, USA). Fetal bovine serum (FBS) was purchased from Gibco (Paisley, UK), xanthine from AlfaAesar (Ward Hill, MA, USA), and sodium di-hydrogen phosphate was acquired from Panreac (Barcelona, Spain). All reagents were of analytical grade or of the highest available purity.

3.2.2 Extraction, purification and UHPLC-DAD-ESI/MS analysis of phlorotannins from F. vesiculosus

Extraction and solvent partitioning were performed as depicted in Figure 3.1., following the optimized procedure described in chapter 2, section 2.2.4. In order to obtain subfractions of different molecular weights, this EtOAc-soluble fraction was further submitted to gel filtration on a Sephadex LH-20 column according to the procedure reported by Wang et al. [15], using solvents of decreasing polarity eluting stepwise, namely aqueous methanol 50% (v/v), aqueous methanol 75% (v/v), pure methanol, methanol and acetone 3:1 (v/v), methanol and acetone 1:1 (v/v) and finally methanol and acetone 1:3 (v/v). From this gel filtration, nine subfractions were recovered (F1—267.9 mg, F2— 30.8 mg, F3—82.3 mg, F4—10.1 mg, F5—10.1 mg, F6—9.4 mg, F7—72.1 mg, F8—40.5 mg and F9—9.0 mg), and the solvents were evaporated under reduced pressure prior to lyophilization and storage at −20 ºC.

Figure 3.1. Flowchart for extraction and fractionation of phlorotannins from F. vesiculosus. F1 to F9 represent nine subfractions obtained from the Sephadex LH-20 column chromatography with a solvent system of decreasing polarity. S/L—solid/liquid ratio (g/mL), RT—room temperature, MeOH—methanol, Act—acetone.

CHAPTER 3

The chromatographic analysis was carried out for the sample with the most promising anti- inflammatory activity, following the procedure described in the chapter 2, section 2.2.6.

3.2.3 Antioxidant assays

3.2.3.1 Oxygen radical absorbance capacity (ORAC)

The ORAC assay was performed according to the method previously described by Catarino et al. [259]. In a 96-well plate, 150 µL of fluorescein (10 nM), diluted from the stock solution of 250 µM, with 75 mM sodium dihydrogen phosphate buffer (pH 7.4), were placed together with 25 µL of different trolox concentrations (3.13–25 µM). The same process was repeated for the samples. After 10 min incubation at 37 ºC, 25 µL of AAPH (153 mM) solution was added to each well and the plate was immediately placed in the plate reader (Biotek, Vienna, Austria), for monitoring the fluorescence (excitation 485 nm and emission at 528 nm) every min over 60 min at 37 ºC. Using the calibration curve of trolox, the ORAC value was calculated and expressed as µmol of trolox equivalents (TE) per g of extract.

●– 3.2.3.2 Superoxide anion (O2 ) scavenging assay

●– The O2 scavenging method was carried out according to the method described by Pereira et al. [260]. In a 96-well plate, 75 µL of six different sample concentrations (0.0–2.0 mg/mL) were mixed with 100 µL of β-NADH (300 µM), 75 µL of NBT (200 µM) and 50 µL of PMS (15 µM). After 5 min, the absorbances at 560 nm were recorded and the inhibition calculated as the concentration

●− capable of scavenging 50% of O2 (IC50). Gallic acid was used as the reference compound.

3.2.3.3 Xanthine oxidase (XO) assay

Inhibition of xanthine oxidase activity was carried out following the method described by Pereira et al. [260], with slight modifications. In a 96-well plate, 40 µL of sample (0–2 mg/mL) was mixed with 45 µL of sodium dihydrogen phosphate buffer (100 mM, pH 7.5) and 40 µL of enzyme (5 mU/mL). After 5 min incubation at 25 ºC, the reaction was initiated by adding 125 µL of xanthine (0.1 mM dissolved in buffer) and the absorbance at 295 nm was measured every 45 s over 10 min at 25 ºC. The inhibitory capacity was then calculated as the concentration of the sample capable of inhibiting 50% of the enzyme’s activity. Allopurinol was used as a reference inhibitor.

3.2.3.4 Nitric oxide (NO●) scavenging assay

The NO● scavenging method was adapted from Pereira et al. [260]. For this, 100 µL of six different sample concentrations (0–1 mg/mL) were mixed with 100 µL of sodium nitroprusside (3.33

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

mM in 100 mM sodium phosphate buffer pH 7.4) and incubated for 15 min under a fluorescent lamp (Tryun 26 W). Next, 100 µL of Griess reagent (0.5% sulfanilamide and 0.05% N-(1-naphthyl)-

ethylenediamine dihydrochloride in 2.5% H3PO4) were added to the mixture, which was incubated for another 10 min at RT in the dark. The absorbance was then measured at 562 nm, and the NO● scavenging capacity was calculated as the concentration of sample capable of scavenging 50% of the radical. Ascorbic acid was used as the reference compound.

3.2.4 Anti-inflammatory experiments

3.2.4.1 Cell culture

Raw 264.7 cells, a mouse leukemic monocyte macrophage cell line (ATCC TIB-71), were cultured in DMEM media supplemented with 10% inactivated fetal bovine serum, 100 U/mL

penicillin and 100 µg/mL streptomycin, at 37 ºC in a humidified atmosphere of 95% air and 5% CO2. Along the experiments, cells were monitored by microscopy in order to detect any morphological change.

3.2.4.2 Assessment of cell viability

The effect of each sample on cell viability/metabolic activity was evaluated according to the resazurin assay previously described [261]. For this assay, cells (6 × 104 cells/well) were plated in 96-well plates and allowed to stabilize overnight. Cells were then exposed to serial concentrations of each sample reconstituted in DMEM with 0.5% DMSO, which has been previously shown to have minimal impact on Raw 264.7 viability [262]. After 24 h incubation, resazurin (50 µM) was added to the cells 3 h prior recording absorbance at 570 nm, with a reference wavelength of 620 nm, using a standard spectrophotometer. The results were expressed relative to untreated cells viability/metabolic capacity.

3.2.4.3 Inhibition of LPS-stimulated NO●

The effect of each sample on the nitrite production in LPS-stimulated Raw 264.7 cells was measured using a colorimetric reaction with Griess reagent as described elsewhere [261]. For this, cells were plated and treated, as described above in 3.2.4.2 section. The LPS stimulus (50 ng/mL) was added 1 h post samples treatment, and 24 h later, the cell-free supernatants were collected and diluted with equal volumes of Griess reagent in the dark. After 30 min, the absorbance was registered in an automatic microplate reader at 550 nm. The extent of the inhibition of nitrite production was evaluated based on the comparison with untreated cells.

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3.2.4.4 Preparation of total protein extracts and western blotting

Raw 264.7 cells (1.2 × 106 cells/well) were seeded in 12-well plates and allowed to stabilize overnight. Cells were either maintained in culture medium (negative control), or pre-incubated with the highest concentrations of samples that inhibited NO● generation and without decreasing cell viability below 90%, for 1 h before the addition of LPS (50 ng/mL) for different time points, according to the proteins studied. The protein extraction was then carried out as described before [32]. Western blotting was subsequently performed for evaluation of protein levels of iNOS, COX- 2 and pro-IL-1β. For that, equivalent amounts of protein were electrophoretically separated on a 10% (v/v) sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% (w/v) fat-free dry milk in TBS-T, for 1 h at RT. Blots were then incubated overnight at 4 ºC with the primary antibody against: COX-2 (1:10,000), iNOS (1:500), pro-IL-1β (1:500), IκBα (1:1000) and pIκBα (1:1000). After washing 3 times during 10 min with TBS-T, the membranes were further incubated for 1 h at RT with alkaline phosphatase- conjugated anti-mouse (for iNOS and pIκBα) and anti-rabbit (for COX-2, pro-IL-1β and IκBα) secondary antibodies. The detection of the immune complexes was followed by scanning for blue excited fluorescence on the Typhoon imager (GE Healthcare) after 5 min of membrane exposure to the ECF reagent. The generated signals were analyzed using the ImageQuant TL software. Thereafter, the membranes were stripped, and the same process was repeated with mouse anti-β-actin (1:5000) antibody, which was used as a loading control and for the normalization of the results. The results exhibited correspond to the ratio of target protein/β-actin.

3.2.5 Statistical analysis

Data was expressed as mean ± standard deviation (SD) of three similar and independent experiments in the antioxidant experiments, whereas for the cellular experiments, data was expressed as mean ± standard error of the mean (SEM) of three similar and independent experiments. One-way ANOVA followed by Tukey’s post hoc test was performed for all the antioxidant assays with the exception of ORAC, for which a two-tailed unpaired t-test was used. In turn, one-way ANOVA followed by Dunnet’s post hoc test was performed for the experiments carried out on cells. The statistical tests were applied using GraphPad Prism, version 7.00 (GraphPad Software, San Diego, CA, California) and the significance level was p < 0.05.

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

3.3 Results and discussion

3.3.1 Antioxidant properties of F. vesiculosus crude extract and phlorotannin- rich fraction

The antioxidant activities of F. vesiculosus CRD and subsequent EtOAc were screened for their

● ●– ● ability to scavenge different free radicals, namely RCOO , O2 and NO , as well as to inhibit the activity of xanthine oxidase, which is known to catalyze the oxidation of hypoxanthine and xanthine

●– to uric acid with the concomitant production of O2 , making this enzymatic system an important biological source of ROS with key roles in several pathogenic processes. Overall, for each assay, a dose-dependent activity was observed in crude and EtOAc samples

(data not shown) and the corresponding IC50 values are shown in Table 3.1. Interestingly, even though the EtOAc had higher concentration in phlorotannins, the best radical scavenging activity

●– ● was observed for the CRD, particularly in O2 and NO , for which the IC50 values were two and three times stronger, suggesting that, in addition to phlorotannins, the CRD might contain other compounds with scavenging activity, such as fucoxanthin or tocopherols that are possibly contributing to these results. On that basis, since the EtOAc is a phlorotannin-rich fraction resultant from solvent partitioning of the CRD, it lacks several compounds that were retained in the other fractions, and therefore the possible synergistic effects were lost, causing a decrease in the

scavenging activity of the EtOAc. Nevertheless, it is worth noting that the IC50 values of the samples against NO● were comparable or even lower than that of the standard compound, which is a particularly relevant result since this free radical plays a pivotal role in the signaling and pathogenesis of inflammation and inflammation-related diseases, thus constituting a potential target for developing anti-inflammatory therapeutics [259]. Therefore, the good scavenging capacity demonstrated by these samples against NO● suggests that they may also display a promising anti-inflammatory potential. Interestingly, the antioxidant potential of the samples herein studied, considering the values

●– ● observed for oxygen radical absorbance capacity (ORAC), O2 and NO , are far more promising than those reported in previous studies on different Fucus species. For instance, in the work of Wang and co-workers [240], among 24 extracts of 12 different seaweeds, the 70% acetone extract of F. vesiculosus exhibited the highest ORAC value of 2567 μmol TE/g extract which is still considerably below compared to the results herein observed. It is also worth noting that, although these results fall short compared to a methanol extracts of rosemary, turmeric or cloves (4360, 5440 and 6150 μmol TE/g extract, respectively) [263], they are still comparable and even higher than the ORAC values reported for some of the highest antioxidant fruits and spices, such as cinnamon (3130 μmol TE/g extract methanol) [263], strawberry (540 μmol TE/g extract, acetone 50%) [264] or coffee (2463–

●– 2828 μmol TE/g extract, water) [265]. Regarding the O2 scavenging capacity, the F. vesiculosus

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crude extract produced in this work revealed approximately two- and seven-times stronger activity compared to those previously reported for 30% and 70% ethanol extracts of the same species, respectively [106]. In a different work, a purified phlorotannin extract from F. vesiculosus was not

●– able to achieve 10% of O2 scavenging for a concentration of 5 mg/mL [266]. According to Lopes et al. [233], the scavenging of 50% of the SNP-generated NO● was achieved only for concentrations between 2 and 4 mg/mL of a purified fraction of F. spiralis. More recently, a similar study reported that, among four different Fucus species, the phlorotannin-purified extracts of wild and cultivated F. vesiculosus showed IC50 values of 1330.6 and 2072.3 μg/mL, respectively, while the highest NO● scavenging capacity was achieved with F. guiryi purified extract, which presented an IC50 of 451.9 μg/mL, approximately two- and six-times higher compared to those of the EtOAc and CRD herein studied, respectively. It is well known that the phlorotannin profile and content in seaweeds are highly susceptible to variability, depending on several factors, such as environmental conditions, geographical origin, harvest season, post-harvest processing and others. This variability could explain the differences between the presented results and other previous studies. Other possible explanation is that Fucus vesiculosus and Ascophyllum nodosum are usually blended in the wild, so they are commonly harvested and processed together. The extraction of phlorotannins can therefore result in a mix of both species, which may impact on the biological properties of the extracts [18].

Table 3.1. Antioxidant activities of F. vesiculosus CRD, EtOAc and the respective reference compounds.

● ●– ● RCOO O2 NO XO Sample (1) (2) (3) (4) (μmol TE/g ext) (IC50 μg/mL) (IC50 μg/mL) (IC50 μg/mL) CRD 3395.04 ± 211.4 a 98.7 ± 11.1a 75.2 ± 5.1a 2.8 ± 0.4a EtOAc 2986.04 ± 338.7 b 268.0 ± 20.1b 235.9 ± 19.5b 1.2 ± 0.2b Standard - 7.8 ± 0.5c 212.1 ± 9.7b 0.1 ± 0.01c

(1) (2) ●– (3) ● TE—Trolox equivalent, Standard compound for O2 is gallic acid, Standard compound for NO is ascorbic acid, (4) Standard compound for xanthine oxidase (XO) is allopurinol. CRD – Crude extract, EtOAc— ●– ● Ethyl acetate fraction. IC50 value was determined as the concentration at which O2 , NO and XO activity were reduced by 50%. Mean values ± SD; statistical analysis was performed by two-tailed unpaired t-test for ORAC, and one-way ANOVA followed by Tukey’s test for the remaining assays. In each column, different letters mean significant differences (p < 0.05).

In contrast, the inhibitory effect of EtOAc against xanthine oxidase (XO) was considerably higher compared to that of the CRD, with the former showing an activity two-times higher than the latter, indicating that, in this case, phlorotannins might be important contributors for the inhibitory effects observed. While the effects and structure-relation between flavonoids and several other terrestrial phenolics with XO are well established [267,268], a handful number of authors have reported the effects of phlorotannins towards this enzyme. Notwithstanding, evidences point that, similar to the

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

terrestrial phenolics, the XO inhibitory capacity seems to be positively correlated with the amount of phlorotannins in the samples, since, according to the work Tanniou et al. [269], from the five different S. muticum sampled along the European coast, the highest XO inhibition was observed for those with the highest content in phlorotannins. Likewise, a recent study described that among four different species of Fucus, the extract from F. serratus, which had the highest concentration of phlorotannins (2.76 ± 0.20 μg phloroglucinol equivalents/mg dry extract), exhibited the best inhibitory capacity against XO, followed by F. guiryi (2.15 ± 0.22 μg PGE/mg DE), F. spiralis (1.56 ± 0.10 μg PGE/mg DE) and F. vesiculosus (1.45 ± 0.10 μg PGE/mg DE), thus supporting the evidence that the phlorotannin content of the extracts is indeed correlated with their inhibitory capacity towards XO

[266]. Interestingly, although the IC50 values obtained for the CRD and EtOAc herein studied were considerably higher compared to allopurinol (approximately 30 and 10 times, respectively), their inhibitory capacity is much stronger compared to that described by Lopes et al. [266] who reported

IC50 values more than 310 times that of allopurinol for F. vesiculosus phlorotannin purified extract and of 60–300 times higher for extracts of other Fucus species. Such differences might be related to variations in the harvesting season and location, and extraction procedures.

3.3.2 Anti-inflammatory properties of F. vesiculosus in Raw 264.7 cells

3.3.2.1 Effects on cell viability and LPS-induced NO● production

Since both F. vesiculosus samples displayed good NO● scavenging capacity, the next step was to evaluate whether these compounds could effectively display anti-inflammatory capacity in a biological system of inflammation—i.e., in Raw 264.7 cells stimulated with the TLR-4 agonist, LPS. As expected, control cells (untreated) produced insignificant NO● levels, while the culture medium of the cells treated with the LPS for 24 h showed a markedly elevated NO● content (Figure 3.2.). As shown in Figure 3.2., both crude and EtOAc samples inhibited the LPS-induced NO● production in a dose-dependent manner, decreasing the NO● production to 14.08 ± 3.68 and 16.80 ± 6.32% (at 100 μg/mL), respectively, comparing with the LPS. However, at 200 μg/mL a decrease in cell viability indicates a cytotoxic effect. These results are in line with those reported by Zaragoza and co-workers [106], who demonstrated that a phlorotannin-rich extract from F. vesiculosus (30–35% ethanol) could effectively inhibit the production of NO● by LPS-exposed macrophages, although their extracts only achieved 50% of inhibition for a concentration of 95 μg/mL. Notably, the activities herein observed for the F. vesiculosus CRD and EtOAc are also much more significant than those reported by Barbosa et al. [68] for a purified extract of phlorotannins obtained from cultivated F. vesiculosus, which at 317 μg/mL only achieved 25% of inhibition. Other Fucus species assayed in the same work exhibited

more promising IC25 values with F. guirii and F. spiralis, showing identical activity (IC25 = 97.7 and

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95.9 μg/mL), followed by F. serratus (IC25 = 77.0 μg/mL) and wild F. vesiculosus (IC25 = 56.5 μg/mL) harvested in the north coast of Portugal. In any case, inhibitions of 50% or close were only achieved at the concentration of 500 μg/mL for all the samples.

Figure 3.2. Effects of F. vesiculosus crude extract (CRD), ethyl acetate fraction (EtOAc) and subsequent subfractions (F1– F9) on the NO● production (grey bars) and viability (◼) of LPS-stimulated Raw 264.7 cells. Data represent the mean ± SEM from at least 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, indicate that NO● production is significantly different from the positive control (with LPS) and # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 indicate that cells viability are statistically different from the negative control (CTRL, without LPS), as determined by one-way ANOVA followed by Dunnet’s post hoc test.

In an attempt to further explore possible relations between the phlorotannins’ molecular sizes and their anti-inflammatory effects, fractionation of EtOAc was carried out in a Sephadex LH-20 gel following the procedure previously described by Wang et al. [15] which, as demonstrated by the authors, allows for the separation of compounds into subfractions of crescent molecular weights,

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

based on hydrogen-bonding between the phenolic OH groups with the matrix and a proper eluent system. According to Figure 3.2., it is noticeable that phlorotannins with more complex structures (fractions F7, F8 and F9) exhibited neglectable inhibitory effects on LPS-induced NO● production, showing a certain cytotoxic effect at the highest concentrations. In turn, exceptional effects were observed for the subfractions F2, F3, F4 and F6, all revealing inhibitions over 80% without affecting cell viability below 90% for any of the concentrations tested. Interesting results were obtained as well for F1 and F5, although both displayed accentuated toxicity for concentrations above 12.5 μg/mL, particularly F5. Nevertheless, at this concentration, both subfractions were able to inhibit NO● production to approximately 50% (compared to LPS positive control) while maintaining the cell viability above 90%, compared to the control untreated cells (Ctrl). Barbosa et al. [68] has previously suggested that the qualitative composition of the extracts could determine the effect of phlorotannins on the production of NO● in Raw 264.7 exposed to LPS. They observed that samples with distinct phlorotannin contents such as F. guirii and F. spiralis (288.4 and

165.9 μg PGE/100 mg, respectively) displayed identical inhibitory capacities (IC25 = 97.7 and 95.9 μg/mL, respectively), while samples with similar phlorotannin contents from cultivated and wild F. vesiculosus (110.3 and 144.5 μg PGE/100 mg, respectively), evoked different effects on NO●

production (IC25 = 317.7 and 56.5 μg/mL, respectively). Following this logic and considering that our results demonstrate that phlorotannins with higher polymerization degrees and complexity are less active than simpler phlorotannins, it is feasible to hypothesize that the balance between polymeric and oligomeric compounds in a phlorotannin extract may determine its potential to inhibit the production of NO● by LPS-stimulated macrophages. The involvement of phlorotannins in the inflammatory signaling cascades and, in particular, on the modulation of NO● levels has been already reported by several authors, although there are few studies focusing on the anti-inflammatory activity of phlorotannin extracts from Fucales [107,231,270,271]. In the particular case of phlorotannin extracts from F. vesiculosus, the existing studies only focused on the inhibition of NO● release by LPS and/or PMA-stimulated macrophages [68,106,233] and, to the best of our knowledge, this is the first study evidencing a relation between their complexity and anti-inflammatory activity, although further research must be carried out to better understand the structure–activity relation of phlorotannins in inflammation.

3.3.2.2 Effects on the expression of iNOS, COX-2 and IL-1β

Upon a pro-inflammatory stimulus, the enzyme iNOS converts L-arginine to L-citrulline with the concomitant release of NO● [267]. Therefore, in addition to the scavenging mechanism, phlorotannins may also interfere with the production of NO● in LPS-stimulated cells by the downregulation of iNOS expression. In fact, when comparing the results from the previous in

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chemico and in vitro assays regarding the samples’ activity towards NO●, it is notable that, although the CRD exhibited higher NO● scavenging capacity over EtOAc, such discrepancy did not occur in the cellular based system. These observations suggest that phlorotannins might not act solely as NO● scavengers but also as inhibitors of the iNOS activity and/or expression, and even against other inflammatory markers, such as COX-2 or IL-1β. Therefore, in order to deeply explore the possible involvement of the F. vesiculosus phlorotannins in the modulation of key inflammatory proteins expressed by Raw 264.7 in the presence of a pro- inflammatory stimulus, we further performed Western blotting with the samples that showed at least 25% inhibition of LPS-induced NO● production without affecting cell viability below 90% (200 μg/mL of F2 and F3, 100 μg/mL of CRD, EtOAc, F4 and F6, and 12.5 μg/mL of F1 and F5). As depicted in Figure 3.3., under normal conditions, the expression of iNOS, COX-2 and IL-1β is very residual, while the stimulation with LPS triggered the expression of these pro-inflammatory proteins, indicating that the cells entered into an inflammatory stage.

iN O S C O X -2

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L S D c 1 2 3 4 5 6 L S c 1 2 3 4 5 6 F F F F F F D R P R A R P R A F F F F F F T L C tO T L C O C C t E E

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L S D c 1 2 3 4 5 6 R P R A F F F F F F T L C O C t E

L P S

Figure 3.3. The effects of F. vesiculosus extract and partitioned fractions on the expression of pro IL-1β, iNOS and COX- 2 in LPS-stimulated Raw 264.7 cells. The immunoblots presented are representative of three independent blots; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicate significant differences from the positive control (LPS), as determined by one-way ANOVA, followed by Dunnett’s post hoc test. CRD—crude extract, EtOAc—ethyl acetate fraction.

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

Upon treatment with the different F. vesiculosus samples, it was clear that iNOS was the most affected marker. Since this enzyme is the major one responsible for the production of NO● during inflammation, the inhibition of its expression is expected to cause a decrease in the NO● produced by LPS-stimulated cells. Therefore, the results observed in Figure 3.3. for iNOS are in agreement with those from Figure 3.2., indicating that the reduction in NO● release was caused by the downregulation of the LPS-induced iNOS expression in presence of CRD, EtOAc and further subfractions. Following iNOS, pro-IL-1β was the second most affected marker, being inhibited by almost every sample. This cytokine is also an important marker in the inflammatory response, with a determinant role in the establishment of chronic inflammation and autoimmune diseases [272]. Therefore, targeting this interleukin would be a possible approach to alleviate the severity of chronic inflammation and attenuate autoimmune diseases. Overall, the F. vesiculosus samples markedly inhibited the expression of this interleukin after a pro-inflammatory stimulus, with F2 exhibiting the strongest inhibitory capacity. In turn, F1 and F5 did not exhibit significant inhibition of LPS- stimulated pro-IL-1β expression, although this does not necessarily mean that they do not affect IL- 1β release. For the pro-IL-1β to become active, it has firstly to be cleaved proteolytically by interleukin-1 converting enzyme (ICE). The inhibition of the ICE activity would therefore block the maturation of pro-IL-1β into active IL-1β and consequently prevent its release and the sustenance of the inflammatory stimulus. This mechanism of action is also described for pralnacasan, a potent orally bioavailable drug that inhibits the maturation of IL-1β by reversibly inhibiting ICE [273]. However, to verify if F1 and F5 could display a similar mechanism of action, further studies are necessary in order to evaluate their effect against ICE. Conversely, with exception of F2, none of the samples were able to significantly inhibit the expression COX-2. In fact, cells treated with F5 and F6 revealed a tendentially exacerbated expression of this enzyme, although this does not imply an intensification of the pro-inflammatory response but rather a possible post-translational inhibitory effect that simultaneously favors the accumulation of COX-2 due to the absence of the negative feedback of prostaglandins. This means that, if the synthesis of endogenous prostaglandins is blocked, the cell does not receive the feedback signal to stop expressing the COX-2 and will continue to accumulate the enzyme. Such mechanism of action is actually described for the non-steroidal anti-inflammatory drug indomethacin, a well- known COX inhibitor commercialized under the trade name of Indocin. Through a strong inhibition of the LPS-induced COX-2 activity, indomethacin decreases the synthesis of prostaglandins that favor COX-2 degradation and at the same time stabilizes the protein structure. As a result, the accumulation of the enzyme increases to levels significantly higher than the LPS control [274]. To

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confirm this possibility, it would be necessary to perform further experiments to explore whether F5 and F6 could inhibit COX-2 activity and consequent prostaglandin synthesis. The capacity of phlorotannin extracts of different seaweeds, as well as of purified phlorotannin fractions and/or compounds to modulate the expression of pro-inflammatory markers, such as iNOS, COX-2, interleukins, and several others, has been largely described [275–277]. Studies focusing on phlorotannins from Fucales, however, are much less numerous. Bahar et al. [278], reported that the treatment of ex vivo porcine colonic tissue either with Ascophyllum nodosum 80% ethanol extract or Fucus serratus water extract significantly inhibited the expression of the genes IL-8, IL-6 and TNFA, encoding for the cytokines IL-8, IL-6 and TNF-α, respectively. Further studies from this research group revealed that both A. nodosum 80% ethanol extract and F. vesiculosus water extract inhibited, more than two-fold, the expression of several cytokines, chemokines, cell adhesion molecules, TLRs and other pro-inflammatory mediators in LPS-stimulated porcine colonic tissue [231,279]. In Raw 264.7 macrophages, the treatment with a F. distichus purified fraction rich in fucophlorethol oligomers potently inhibited the expression of genes encoding for iNOS, COX-2, TLR4 and 9, IL- 1β, IL-6 and IL-17, TNF-α and other pro-inflammatory mediators [270]. However, to the best of our knowledge, such studies have not been carried out yet for phlorotannin purified fractions from F. vesiculosus.

3.3.2.3 Effects on the NF-κB signaling pathway

According to the previous results, F2 exhibited the best overall inhibitory effect, being the only sample that significantly decreased the expression of all the three pro-inflammatory proteins analyzed. This was followed by CRD and F3, which markedly inhibited the expression of pro-IL-1β and iNOS as well. Notably, although not statistically significant, the expression of COX-2 on cells treated with these two samples was still halved compared to LPS-stimulated cells. Note that the expression of these three markers are mediated by several transcription factors from which NF-κB is one of the most important. In fact, a strict relation between iNOS expression and NF-κB transcriptional activity in the presence of LPS has been well established [280], thus the strong inhibitions these three samples displayed against iNOS indirectly indicates that NF-κB transcriptional activity was affected as well. To further evaluate whether CRD, F2 and F3 could interfere with NF-κB activity, the samples were tested for their capacity to inhibit the phosphorylation and degradation of IκBα, and consequent translocation of NF-κB to the nucleus. In order to determine the peak of phosphorylation and degradation of IκBα, a time course was primarily carried out with LPS-stimulated cells at distinct time points (5, 15, 25, 30, 40, 60 and 70 min). Since the peak of phosphorylation and degradation of IκBα occurred at 15 and 25 min, respectively (data not shown), these were the time points selected for testing the samples.

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

As depicted in Figure 3.4., the presence of F2 completely abrogated the effects of LPS, maintaining the levels of phosphorylated and total IκBα at the basal levels. As this protein is responsible for maintaining NF-κB sequestered and inactive in the cytoplasm, the inhibition of its phosphorylation and degradation indirectly indicates that the NF-κB translocation to the nucleus was blocked and, consequently, that its transcriptional activity was inhibited. By contrast, the treatment with both CRD and F3 did not significantly affect the phosphorylation of IκBα, although a decreasing tendency could be noted. Nevertheless, none of these two samples were able to prevent the decrease in the total IκBα levels, which means that this protein was still being degraded by the proteasome, and therefore the NF-κB was being freed to translocate to the nucleus.

Figure 3.4. Effects of crude extract, F2 and F3, on the activation of the NF-κB signaling in LPS-stimulated Raw 264.7 cells after 15 min (pIκBα) and 25 min (IκBα) of incubation. The immunoblots presented are representative of 3 independent blots. ** p < 0.01 and **** p < 0.0001 indicate significantly differences from the positive control (with LPS) for pIκBα and negative control (without LPS) for IκBα, as determined by one-way ANOVA followed by Dunnett’s post hoc test. CRD—Crude extract

The involvement of phlorotannins in the activation of NF-κB has been already described by several authors. However, the majority of these works were performed with extracts and/or compounds typical from the genus Ecklonia. Indeed, Wei et al. [281] observed that the treatment of LPS-stimulated macrophages with an ethyl acetate fraction obtained from the 95% ethanol extract of Ecklonia stolonifera was capable of suppressing NF-κB transcriptional activation not only by preventing IκBα phosphorylation and degradation but also through the inhibition of its translocation to the nucleus. These findings corroborate those previously described by Lee et al. [150], who also reported similar results using a crude ethanol extract of the same species. In turn, an E. cava 95% ethanolic extract was shown to suppress the NF-κB translocation and DNA-binding in LPS- stimulated murine BV2 microglia via the inhibition of IκBα degradation [148], while a phlorotannin-

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rich commercial E. cava extract was shown to block the LPS-induced septic shock in C57BL/6 mice through the suppression of the NIK/TAK1/IKK/IκB/NF-κB pathway [174]. Some studies also evidenced promising anti-inflammatory effects through the suppression of NF-κB in different cell lines, for phlorotannin extracts and/or compounds of species from Eisenia [159,282,283], Sargassum [154,155,283] and other genera [231,275,276,283]. Studies focusing on the effects of Fucus phlorotannins in NF-κB transcriptional activity are, however, rather scarce. In fact, to our knowledge, only one study has shown that F. vesiculosus cold-water extract was capable of inhibiting the NF-κB gene expression in ex vivo porcine colonic tissue [279]. Therefore, the present work provides new insights into the potential of F. vesiculosus to modulate inflammation, showing that this seaweed may also be a source of relevant phlorotannin compounds capable of suppressing the NF-κB transcriptional activity through the inhibition of the phosphorylation and proteosomal degradation of the IκBα.

3.3.3 Characterization of F2

To elucidate the compounds present in the most active F. vesiculosus subfraction—i.e., F2—an UHPLC-MS analysis was carried out. Overall, the chromatogram of this subfraction (Figure 3.5.) was characterized by the presence of a major peak eluting at 13.4 min that showed a deprotonated molecular ion at m/z 507. Although, to our knowledge, no phlorotannin with such molecular weight has been described before, the MS/MS spectrum of this compound revealed a base peak at m/z 489, corresponding to the loss of water, which is a common characteristic of phlorotannins, and two major peaks at m/z 277 and 229, the former indicating the loss of fucol moiety ([M−H−230]−) and the latter corresponding to the fucol moiety itself. Additionally, other minor product ions also evidenced a fragmentation pattern coherent with those previously described for phlorotannins. This is the case of the product ions at m/z 463, which results from internal cleavage of benzene ring structures and loss of 44 Da, at m/z 445, corresponding to the loss of 44 Da plus an additional water molecule, and at m/z 479, corresponding to the loss of CO or ethylene (28 Da), also resultant from cross ring cleavage [66,253]. The product ion at m/z 245 is indicative of a phloroglucinol dimer, while the one at m/z 261 (−246 Da) indicates the loss of a phloroglucinol dimer [66,284]. Therefore, although the precise identification of this compound was not achieved, this evidence allows us to conclude that it may correspond to a phlorotannin derivative.

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

100 5 A 100 B

80 80

60 60 1 40 2 40 3 4

20 20 Relativeabundance(%) 0 Relativeabundance(%) 0 0 5 10 15 20 0 5 10 15 20 Time (min) Time (min)

Figure 3.5. Chromatographic profile at 280 nm (A) and total ion chromatogram (B) of F2. Peaks marked with numbers correspond to the tentatively identified compounds represented in Table 3.2.

Interestingly, peak 4, eluting at 13.1 min, showed a [M–H]− at m/z 587 and further fragmented into the main product ion at m/z 507, also yielding other minor product ions that were common in with those observed in the MS/MS spectrum of the compound described above—namely at m/z 277 and 229. Due to these similarities, it is possible that this compound may not only be a phlorotannin derivative but also a derivative of the compound with the [M–H]− at m/z 507, and, even though it has not been identified yet, previous studies have already reported its presence in extracts of F. vesiculosus. This is the case of our previous work described on chapter 2, in which the deprotonated molecular ion at m/z 587 detected on the F. vesiculosus EtOAc purified extract originated the same major product ion at m/z 507. Other authors also reported the presence of a compound with the same [M − H]− and MS2 base peak in a water extract of F. vesiculosus from Camariñas, Spain, although they were also unable to identify it [255]. Apart from these two major compounds, eluting in peaks 4 and 5, others were detected in F2 with lower intensities, including two isomers with [M–H]− at m/z 497 and two other compounds with [M − H]− at m/z 511 and 529. The first two compounds eluted at 1.4 and 2.0 min (peak 1 and 2) and correspond to two isomers of a phloroglucinol tetramer, both showing MS/MS spectra coherent with previous works [66,253]. Although it is not possible to ensure their exact structural arrangements, the absence of phloroglucinol moieties in the MS2 spectrum of the isomer eluting at 1.4 min suggest it might correspond to a tetrafucol—i.e., a tetramer composed exclusively of C-C linked phloroglucinols. In turn, the presence of the product ions at m/z 371 (−126 Da) and 353 (−126−18 Da) on the MS2 spectrum of the isomer eluting at 2.0 min indicates the loss of a phloroglucinol moiety and a phloroglucinol combined with water, respectively, suggesting that this compound contains at least one C-O-C linkage [66]. The compound with [M−H]− at m/z 529 (eluting at 2.4 min, peak 3) also showed an MS spectrum coherent with that of a phloroglucinol tetramer, although containing two additional OH groups, which is a characteristic feature of fuhalols. Therefore, based on these findings, as well as on literature data, this compound was attributed to hydroxytetrafuhalol [182]. Co-eluting at the same retention time, the [M−H]− at m/z 511 exhibited a fragmentation pattern

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with losses that typically occur in phlorotannins, including −18 Da, −44 Da and -126 Da, as well as their combinations (Table 3.2.). Moreover, the product ion at m/z 493 appears as the main product ion of this compound, which is in agreement with what Mezghani et al. [285] previously described for diphlorethohydroxycarmalol, although their work was carried out using extracts of Ulva rigida, a species belonging to the Chlorophyta, which contradicts the supposed exclusivity of phlorotannins to the Phaeophyta. Notably, the presence of phloroglucinol tetramers and hydroxytetrafuhalol have already been described in F. vesiculosus before, including in a previous work from our research group [65,66,255,286]. However, until now, with exception of the work reported by Mezghani et al. [285], the appearance of diphlorethohydroxycarmalol has been exclusive to the species Ishige okamurae [287,288]. Therefore, this is the first work reporting the presence of diphlorethohydroxycarmalol in a brown algae species other than Ishige okamurae, although further spectroscopic analysis would be necessary to ensure this hypothesis.

Table 3.2. Tentative assignment of the compounds detected in the F2 analyzed by UHPLC-ESI-MS/MS.

RT [M–H]- Peak MS/MS Ions (-loss)* Tentative Assignment (min) (m/z) 479 (-18), 331 (-PGU-44), 461 (-18-18), 435 (-44-18), 1 1.4 497 453 (-44), 413 (-84), 395 (-84-18), 347 (-150), 305 (- Tetrafucol 192), 165(-2PGU-84), 315 (-PGU-44-18) 479 (-18), 331 (-PGU-44), 461 (-18-18), 435 (-44-18), 2 2.0 497 453 (-44), 395 (-84-18), 305 (-192), 165 (-2PGU-84), 315 Fucophlorethol (-PGU-44-18), 371 (-PGU), 353 (-PGU-18) 493 (-18), 449 (-44-18), 475 (-18-18), 467 (-44), 439 (- 72), 411 (-84-16), 405 (-106), 345 (-PGU-44), 301 (- 511 210), 331 (-180), 347 (-164), 385 (-PGU), 395 (-98-18), Diphlorethohydroxycarmalol 3 2.4 369 (-PGU-16), 351 (-PGU-84), 329 (-PGU-44-18), 313 (-PGU-72) 511 (-18), 493 (-18-18), 411 (-84-16-18), 467 (-44), 429 529 Hydroxytetrafuhalol (-84-16), 439 (-90), 347 (-PGU-16-44) 507 (-80), 523 (-64), 229 (-PGU-108-80-44), 277 (-230- 4 13.1 587 80), 489 (-80-18), 383 (-PGU-80), 399 (-108-80), 463 (- Phlorotannin derivative PGU), 275 (-232-80), 569 (-18) 489 (-18), 277 (-230), 229 (-PGU-108-44), 461 (-46), 463 5 13.4 507 (-44), 479 (-28), 445 (-44-18), 275 (-232), 261 (-246), Phlorotannin derivative 245 (-262), 421 (-86), 297 (-PGU-84) * Product ions are arranged in descending order of relative abundance with bold values highlighting the most abundant fragment. PGU—phloroglucinol unit (-126/124).

3.4 Conclusions

The work developed in this section provides evidence of the antioxidant and anti-inflammatory activities of F. vesiculosus phlorotannin extract and subsequent purified fractions, as reflected by their ability to scavenge chemically generated ROS and to inhibit inflammatory response on LPS- stimulated macrophages. Both crude extract and EtOAc displayed good scavenging activity against

ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF F. VESICULOSUS PHLOROTANNINS

the different radicals tested, especially NO●, a free radical that plays a pivotal role in the signaling and pathogenesis of inflammation, indicating that these extracts may not only exhibit good antioxidant potential but also a possible anti-inflammatory capacity. This hypothesis was further confirmed with biological experiments using LPS-stimulated Raw 264.7 cells. At 100 µg/mL, both phlorotannin-rich samples decreased the NO● production to approximately 85% of the untreated cells. The inhibitory effects observed for the subsequent subfractions further allowed us to demonstrate a possible relation between phlorotannins’ complexity and anti-inflammatory activity, with lower molecular weights evidencing stronger effects. The expression of iNOS was the most affected, being susceptible to every sample tested, followed by IL-1β, inhibited by all samples except F1 and F5, and COX-2 which was only inhibited by F2. The potent inhibitory effects observed for this subfraction were finally demonstrated to be resultant from its capacity to inhibit IκBα phosphorylation and degradation, which translates to the blockage of the NF-κB signaling pathway. This subfraction was characterized by the presence of some minor phlorotannin tetramers and a major compound with [M − H]− at m/z 507 which could be attributed to a phlorotannin derivative based on the fragmentation products present in its MS/MS spectrum. Overall, this study contributes for a better understanding of the mechanisms behind the anti-inflammatory properties of F. vesiculosus phlorotannins extract and purified fractions. However, it must be considered that natural products are always susceptible to great variability. Therefore, further research is necessary not only to verify the reproducibility of these results but also to elucidate the structural features of the compound detected in F2 and evaluate whether it is the responsible for the remarkable anti-inflammatory activity displayed by this subfraction.

Chapter 4 .

This chapter includes data from the paper:

Catarino, M.D.; Fernandes, I.; Oliveira, H.; Carrascal, M.; Cruz, M.T.; Ferreira, R; Mateus, N.; Silva, A.M.S.; Cardoso, S.M. Antitumor Activity of Fucus vesiculosus Phlorotannins Through Activation of Apoptotic Signals. In preparation.

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4.1 Antitumor activity of F. vesiculosus phlorotannins through activation of apoptotic signals

Cancer is the second global leading cause of death and, according to WHO, in 2018 it was responsible for an estimated 9.6 million of deaths. Among the most common cancers, colorectal and stomach cancers occupy the 3rd and 5th places of the leading types of cancer in 2018, respectively. However, when it comes to the most frequent causes of cancer death, they appeared in the 2nd and 3rd places in the same year, respectively [3]. Poor behavioral and dietary habits including tobacco use, alcohol use, and lack of fruit and vegetables intake, or physical inactivity are the most common risk factors that will lead to the development of these two types of cancer. Moreover, infection by Helicobacter pylori is one of the most important risk factors for the development of stomach cancer, while inflammatory bowel disease, including Chron’s disease and ulcerative colitis, is also a major factor in the development of the colorectal cancer [289,290]. In this field, phlorotannins have drawn much attention during the recent years since they have shown promising antitumor potential by acting as anti-proliferative, anti-metastatic and anti-angiogenic agents on different types of cancer [291]. The existing literature on the potential antitumor capacity of F. vesiculosus-derived phlorotannins remains, however, quite underexplored. Moreover, since the digestive tract is the first organ exposed to components of the diet, it is the most probable target that will benefit from the potential bioactivities of phlorotannins, in particular their antitumor effects, after their ingestion. Therefore, this work aimed to explore the cytotoxic properties as well as the possible mechanisms by which F. vesiculosus extract and further phlorotannin purified fractions may activate cell death on different tumor cell lines of the gastrointestinal tract. In addition, characterization of the sample showing the strongest antitumor activity was carried out in an attempt to unveil the possible compounds responsible for such effects.

4.2 Materials and methods

4.2.1 Chemicals

Grounded F. vesiculosus samples from July 2017 were purchased from Algaplus Lda. Acetone, ethanol, methanol, n-hexane, ethyl acetate, acetonitrile, dimethyl sulfoxide and Nonidet P-40 were acquired from Fisher (Pittsburgh, PA, USA). Formic acid, PBS reagents (sodium salt, sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate), sodium citrate, antibiotic/antimycotic solution (100 units /mL of penicillin, 10 mg/mL of streptomycin and 0.25 mg/mL of amphotericin B), trypsin, Roswell Park Memorial Institute (RPMI) 1640 and DMEM mediums were purchased from Sigma (St. Louis, MO, USA). Propidium iodide (PI), RNAse and annexin V‑FITC were acquired from Immunostep (Salamanca, Spain), while FBS was purchased from Lonza (Belgium) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

ANTITUMOR ACTIVITY OF F. VESICULOSUS PHLOROTANNINS THROUGH ACTIVATION OF APOPTOTIC SIGNALS

bromide (MTT) from Himedia Laboratories, (Einhausen, Germany). All reagents were of analytical grade or of the highest available purity

4.2.2 Extraction, purification and UHPLC-DAD-ESI/MS analysis of phlorotannins from F. vesiculosus

The preparation of crude, phlorotannin-rich fraction and phlorotannins subfractions F1-F2 from F. vesiculosus phlorotannins was performed as described previously in chapter 3, section 3.2.2. The chromatographic analysis was carried out for the sample with the most promising antitumor activity, following the procedure described in the chapter 2, section 2.2.6

4.2.3 Cell culture

Three human cancer cell lines, MKN-28 (human stomach adenocarcinoma, Cell Bank, Riken BioResource Center), HT-29 (human colorectal adenocarcinoma, INSERMU178, Villejuif, France) and Caco-2 (human colorectal adenocarcinoma, ATCC, USA), were grown as monolayers from

passage number 30-50 and maintained at 37 ºC in an atmosphere of 5% CO2. For routine maintenance, the cells were cultured in 22.1 cm2 plates as monolayers and maintained in RPMI-1640 (MKN-28) or DMEM (HT-29 and Caco-2), supplemented with 10% (MKN-28) or 15% (Caco-2 and HT-29) FBS and 1% of antibiotic/antimycotic solution (100 units /mL of penicillin, 10 mg/mL of streptomycin and 0.25 mg/mL of amphotericin B). The medium was replaced every two days and the cells were harvested every two weeks.

4.2.4 Assessment of cell viability

The effect of each sample on cell viability was evaluated according to the MTT assay as previously described [292]. For this assay, cells (1.5 × 105 cells/well) were plated in 96-well plates

and allowed to stabilize overnight at 37 °C under a humidified atmosphere with 5% CO2. The effect of the vehicle solvent (DMSO) was evaluated in all experiments by exposing untreated control cells to the maximum concentration (0.1%) of DMSO used in each assay. A stock solution of the studied samples was prepared in DMSO and kept at -20 ºC, and appropriate dilutions of each sample were freshly prepared just prior to every assay. Cells were then exposed to the respective samples, with a maximal solvent concentration of 0.1% DMSO, and incubated for 48 h. Afterwards, the medium was discarded, and the cells were washed with PBS prior to the addition of MTT solution at 0.45 mg/mL. The crystals of formazan were then allowed to form for 1.5 h and subsequently solubilized with DMSO prior to recording the absorbance at 570 nm on a standard spectrophotometer (Biotek PowerWave XS). The results were expressed relative to untreated cells viability.

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4.2.5 Flow cytometric analysis of the cell cycle and apoptosis via annexin V-PI double staining

Caco-2 and MKN-28 cells at exponential growth were obtained by plating 1.5 × 106 cells/mL in a 6-multiwell plate followed by 24 h incubation. Afterwards, the medium was replaced with fresh medium (control) or fresh medium supplemented with F. vesiculosus samples at the IC50 concentration previously determined through the MTT assay. After 48 h of incubation cells were trypsinized, collected and centrifuged for 5 min at 500 g and the pellets were washed twice with PBS. For the cell cycle analysis, cells were fixed with ice cold 70% ethanol for 30 min followed by the staining with PI buffer (0.05 mg/mL PI, 0.02 mg/mL of RNAse, 0.2% m/v of Nonidet P40 and 0.1% m/v sodium citrate in water) for 4 h at 4 ºC temperature. Finally, the samples were analyzed on a BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA). In all experiments performed, at least 10×103 events were acquired, and the experimental data analyzed using the BD Cytometric Software (version 16 2.1). For apoptosis analysis cells were incubated with 5 μg/mL annexin V‑FITC (Immunostep) and 1 μg/mL PI for 15 min at room temperature in the dark, and then analyzed using the same apparatus.

4.2.6 Statistical analysis

Data was expressed as mean ± SEM of three similar and independent experiments and analyzed using one-way ANOVA followed by Dunnet’s post hoc test was. The statistical tests were applied using GraphPad Prism, version 7.00 (GraphPad Software, San Diego, CA, California) and the significance level was p < 0.05.

4.3 Results and discussion

4.3.1 Effect of CRD, EtOAc and purified fractions on tumor cells viability

To determine the effects of F. vesiculosus CRD, EtOAc and subsequent subfractions on the viability of gastric and colon tumor cells, serial dilutions of each were prepared and added to the culture medium of MKN-28, Caco-2 and HT-29 cells. To verify the specificity of the cytotoxic effect of the samples, the same procedure was also carried out on a normal cell line of human fibroblast (HFF-1). Overall, CRD, EtOAc and following subfractions presented a dose-dependent cytotoxic effect against the three tumor cell lines tested (Figure 4.1.). Among them, F7, F8 and F9 were the less active since none of these three were able to reduce the cell viability below 50%. On the contrary, EtOAc, F1 and F5 displayed the best overall cytotoxic effect against all the three tumor cell lines, consistently presenting the lowest IC50 values (161.7 ± 16.1, 173.0 ± 33.8 and 56.3 ±14.7 µg/mL for MKN-28, 257.2 ± 32.3, 219.5 ± 17.1 and 97.4 ± 11.6 µg/mL for Caco-2, and 170.0 ± 2.8, 146.8 ± 13.1 and 118.8 ± 19.7 µg/mL for HT-29, respectively). When verified for their cytotoxic effect on

ANTITUMOR ACTIVITY OF F. VESICULOSUS PHLOROTANNINS THROUGH ACTIVATION OF APOPTOTIC SIGNALS

HFF-1 cells, none of the samples affected cell survivability below 50% for any of the concentrations tested, suggesting that these F. vesiculosus samples, particularly EtOAc, F1 and F5, display specific cytotoxic effects against tumor cells without critically affecting the viability of normal cells.

Figure 4.1. Effect of F. vesiculosus samples on the cell viability of Caco-2, HT-29, MKN-28 and HFF-1 cells after 48 h. Data is expressed as percentage of survival compared to the negative control and are given as the means ± SEM of at least three independent experiments.

The antiproliferative properties of phlorotannins and phlorotannin-rich extracts is a subject that has already been approached by several authors. Indeed, phlorotannin extracts from Alaria esculenta, Ascophyllum nodosum, Laminaria japonica, Sargassum muticum, Bifurcaria bifurcata and several others were shown to dose-dependently reduce the cell proliferation of numerous tumor cell lines such as Caco-2, HT-29, human hepatoma (BEL-7402), mouse leukemia (P388) and mouse teratocarcinoma (ATDC5) cells [177,182,293–295]. Studies focusing the antitumor activity of phlorotannins from the genus Fucus are, however, quite scarce, although some authors have already evidenced promising results. According to Geisen et al. [188], a strong dose-dependent cytotoxic effect of a hydrophilic fraction isolated from a F. vesiculosus 70% acetone extract was found on four

different pancreatic cancer cell lines (Panc1, Panc89, PancTU1 and Colo357), with IC50 ranging from 17.35 to 28.9 µg/mL after 72 h of exposure. Additional research from this group revealed that, after further fractionation of a F. vesiculosus acetone extract, the two most active fractions against Panc89 and PancTU1 cells presented a characteristic 1H NMR fingerprint of two molecules belonging to the polyphenols group, although they could not achieve their full chemical structure [296]. In turn, a

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phlorotannin-rich ethyl acetate fraction from a dichloromethane/methanol (1:1) extract of F. spiralis was reported to induce significant decrease on the survival rate of human cervical adenocarcinoma (HeLa), colorectal adenocarcinoma (LS-174T), and lung carcinoma (A549) cells, without affecting the survivability of the normal human lung fibroblasts (MRC-5) [297]. Likewise, a study carried out with a crude polyphenolic fraction of E. cava revealed that the cytotoxic effects observed on two human leukemia cell lines (THP-1 and U-937), a murine colon cancer cell line (CT-26) and a mouse melanoma cell line (B-16) were not present on the normal Chinese hamster fibroblast cell line (V79- 4) [298]. These studies are therefore in agreement with the results herein attained, thus supporting the hypothesis that the F. vesiculosus samples obtained in this work, particularly EtOAc, F1 and F5, may exert a directed cytotoxic effect against tumor cells only without affecting normal cells.

4.3.2 Effects on cell cycle after exposure to F. vesiculosus samples

Defects in cell cycle checkpoints are associated with an uncontrolled cellular proliferation, which is a reason why targeting cell cycle represents an important strategy for cancer therapy. In this context, considering the promising cytotoxic effects of EtOAc, F1 and F5 observed in MTT assay, these three samples were selected for further evaluation of the possible alterations in the cell cycle of either gastric (MKN-28) or colon (Caco-2) cancer cells. For that, cells were incubated with each sample at their IC50 concentrations as previously determined in the MTT assay, and subsequently analyzed for the distribution of G0/G1 (resting/growth phase), S (DNA synthesis phase) and G2/M (preparation for mitosis/mitosis phase). As depicted in Figure 4.2., an arrest of the cell cycle at the S phase was observed in both cell lines when treated with F1, being this effect more pronounced on MKN-28. On the contrary, EtOAc did not affect the cell cycle distribution of Caco-2 cells, although it revealed the capacity to interfere with that of MKN-28 by significantly decreasing the number of cells at the G0/G1 (43.0 ± 0.6 %) phase and concomitant increasing the cell counts at G2/M phase (50.3 ± 0.4 %) comparing to the control (57.9 ± 1.1 and 36.2 ± 0.7 %, respectively). Interestingly, F5 did not cause significant changes on the cycle of MKN-28 and Caco-2 cells. Based on these results it is feasible to suggest that the capacity of EtOAc and F1 to inhibit cell proliferation occurs, at least partly, through cell cycle arrest, although the former only exhibited this capacity on MKN-28 cells. On the other hand, despite F5 displayed the most promising cytotoxic effect in MTT assay, it does not seem to affect cells cycle, suggesting that it might be promoting cell death via other mechanisms.

ANTITUMOR ACTIVITY OF F. VESICULOSUS PHLOROTANNINS THROUGH ACTIVATION OF APOPTOTIC SIGNALS

Figure 4.2. Effect of F. vesiculosus samples on the cell cycle distribution of Caco-2 and MKN-28 cells after 48 h. Data is expressed as percentage of PI+ cells and are given as the means ± SEM of at least three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 when compared to control; the symbols *, # and × were used for G0/G1, S, G2/M, respectively).

Previous studies have reported interesting results regarding the effect of brown algae phlorotannins on the cell cycle of tumor cell lines. Indeed, dieckol isolated from E. cava was shown to induce the cell cycle arrest of both MCF-7 and SK-BR-3 breast cancer cells at the G2/M phase [299], while the addition of phloroglucinol to HT-29 cells over 24 h caused a dose-dependent increase of the cell counts at the phase G0/G1 [191]. Moreover, Geisen et al. [188] addressed the effect of a hydrophilic fraction isolated from a F. vesiculosus 70% acetone extract on two different pancreatic cancer cell lines (Colo357 and Panc89), demonstrating that after 24 h of exposure, both cell lines evidenced a significant increase on the cell counts at the G2/M phase. No previous studies have, however, addressed the effect of F. vesiculosus phlorotannins on the cell cycle of gastric or colon cancer cell lines.

4.3.3 Apoptosis/necrosis detection on cells treated with F. vesiculosus samples

In order to evaluate cell death and discriminate whether the exposure to F. vesiculosus samples triggers apoptosis and/or necrosis, cells were incubated under the same conditions used for the evaluation of the cell cycle and then stained with annexin V-FITC/PI. As illustrated in the Figure

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4.3., after 48 h of exposure to EtOAc or F5, Caco-2 cells exhibited a significant increase in the percentage of the annexin V+/PI+, representing the late apoptotic cells (82.3 ± 0.42 and 86.1 ± 1.56 %, respectively, compared to 23.9 ± 1.70 % in the control), followed by a significant increase of annexin V-/PI+, representing the necrotic cells (10.7 ± 1.09 and 8.2 ± 0.87 %, respectively, compared to 1.5 ± 0.22 % in the control). Identical observations were recorded in MKN-28 cells, which also showed a great increase on the levels of late apoptotic cells exposed to EtOAc and F5 (60.8 ± 7.06 and 73.7 ± 2.40 %, respectively compared to 11.2 ± 1.3 % in the control) and an even more pronounced increase of the percentage of necrotic cells (35.4 ± 6.77 and 23.3 ± 2.55 %, respectively compared to 0.8 ± 0.03 % in the control).

Figure 4.3. Detection of apoptosis/necrosis after the 48 h treatment with F. vesiculosus samples via annexin V-FITC/PI labelling. The populations of early apoptotic cells, late apoptotic cells, necrotic cells and viable cells were evaluated as a percentage of total cells and are given as the means ± SEM of at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 when compared to control; the symbols *, # and × were used for viable, apoptotic and necrotic cells, respectively).

These results are in line with previous works that highlighted the ability of phlorotannins and/or phlorotannin extracts from other seaweeds to induce cell death on multiple tumor cell lines [179,300,301] and even in vivo [185]. Indeed, dieckol isolated from E. cava was shown to trigger early and late apoptosis in SKOV3 ovarian cancer cells [179], MCF-7 breast cancer cells [299], and A549 non‐small lung cancer cells [190], while induction of both early and late apoptosis was reported in Panc89 pancreatic cancer cells exposed to a hydrophilic fraction isolated from a F. vesiculosus

ANTITUMOR ACTIVITY OF F. VESICULOSUS PHLOROTANNINS THROUGH ACTIVATION OF APOPTOTIC SIGNALS

70% acetone extract for 24 h [188]. Stimulation of necrosis is, however, a mechanism that is not usually observed for these compounds, although two studies carried out with Cystoseira seaweeds reported a similar behavior. In more detail, the exposure of MCF-7 breast cancer cells to C. barbata 70% acetone extract during 48 h caused a dose-dependent increase not only in the number of apoptotic cells (both early and late) but also in the necrotic cells [301], while in HL60 and THP-1 cells, the treatment with C. tamariscifolia methanol extracts significantly increased the percentages of late apoptotic and necrotic cells but not the percentages of the early apoptotic cells [302].

4.3.4 Characterization of F5

The subfraction F5, which exhibited the best overall activity evidencing the highest cytotoxic effect and the highest induction of apoptosis/necrosis in the different cell lines tested, was analyzed additionally through UHPLC-DAD-ESI-MS in order to elucidate the compounds that might be contributing for such effects. Figure 4.4. shows the UV chromatogram recorded at 280 nm as well as the extracted ion chromatogram (EIC) of the major compounds detected. Overall, 7 different compounds were detected in this subfraction, presenting well-defined and abundant ions in their respective EICs, although in the chromatogram recorded at 280 nm they did appear with low intensity. From these compounds, it was possible to identify one phloroglucinol trimer (peak 5), one tetramer (peak 4), one pentamer (peak 1), two hexamers (peaks 2 and 3), and two other phlorotannin derivatives (peaks 6 and 7). In more detail, the proposed structure assigned to the trimer eluting at 8.5 min was eckstolonol, according to its deprotonated molecular ion at m/z 369, followed by a fragmentation pattern typical of phlorotannins, with common losses of −18 Da, −44 Da and −124 Da, and consistent with that previously reported in other works [253]. The appearance of this compound (also known as dioxinodehydroeckol) is more frequently associated to species from the genus Ecklonia [112,303,304], although it has been previously detected in Fucales, including in two species of the genus Fucus, namely F. spiralis and F. guiryi [124,253]. To our knowledge, eckstolonol was not previously reported in F. vesiculosus, although one eckstolonol derivative (MW 464 Da) was described in an aqueous extract of this macroalgae species [255].

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Figure 4.4. UV chromatograms recorded at 280 nm and extracted ion chromatograms (EIC) of the major compounds detected in F5. Peaks marked with numbers correspond to the tentatively identified compounds listed in Table 4.1.

ANTITUMOR ACTIVITY OF F. VESICULOSUS PHLOROTANNINS THROUGH ACTIVATION OF APOPTOTIC SIGNALS

The compound eluting at 7.0 min presented a deprotonated molecular ion at m/z 479, indicating a dehydroxylated phloroglucinol tetramer. This further fragmented into the product ions at m/z 461, corresponding to the loss of water, at m/z 435 which results from the internal cleavage of benzene ring structures and elimination of 44 Da, at m/z 353 as a consequence of the elimination of a PGU, and other fragments such as m/z 417 or m/z 313 which are resultant from the combination of the previous losses (−44−18 and −PGU−44, respectively). A compound presenting a similar retention time as well as an identical MS spectrum is described in chapter 2 as fucofurodiphlorethol due to its structural resemblance to fucofuroeckol ([M − H]− at m/z 477), except for the absence of the dioxin ring between the two inner phloroglucinol moieties, which would explain the 2 Da difference between their deprotonated ions. Although this compound is not usually described in literature, recent works of Amarante et al. [305] reported its presence in two different F. vesiculosus extracts, namely a microwave-assisted extract (using 57% ethanol) and a conventional solid-liquid extract (using 70% acetone). The compounds eluted at 3.1 min ([M − H]− at m/z 621) and 4.9 min ([M − H]− at m/z 745) also presented retention times and MS spectra similar to those of trifucophlorethol and hexafucol, previously assigned in chapter 2, respectively. Interestingly, the isomer of the compound with a [M − H]− at m/z 745 that eluted at 3.9 min, revealed a fragmentation pattern slightly different from the one eluting at 4.9 min. In this case, the formation of the product ions such as m/z 619 and 601 denote the loss of a phloroglucinol moiety and a phloroglucinol plus water, respectively, which is indicative of the presence of an ether linkage in the its structure [253]. Therefore, this hexamer (eluting at 3.9 min) was tentatively assigned to a tetrafucophlorethol. Two additional compounds were detected at 12.2 and 13.3 min. The later showed a [M − H]− at m/z 507, presenting an MS/MS spectrum that was very similar to that described in chapter 3, evidencing several product ions such as m/z 277, 229, 245 or 261 (corresponding to the loss of fucol moiety, a fucol moiety itself, a phloroglucinol dimer and loss of a phloroglucinol dimer, respectively), that strongly suggest that this is a phlorotannin derivative, even though its precise identification was not possible to achieve. Interestingly, although such compound has not been described in the literature yet, recent works of our research group found a compound with the same deprotonated molecular ion and fragmentation pattern on a microwave-assisted ethanolic extract from F. vesiculosus [305]. Likewise, although the identification of the compound eluting at 12.2 min ([M − H]− at m/z 611) was not achieved, its MS/MS spectrum presented some product ions including m/z 593 (−18), 567 (−44) and 469 (−O−phloroglucinol) that are typical of phlorotannin compounds, thus allowing to suggest this might be a phlorotannin derivative as well.

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Table 4.1. Tentative assignment of the compounds detected in the F5 analyzed by UHPLC-ESI-MS/MS.

[M - H]- Peak RT (min) MS/MS Ions (-loss)* Tentative assignment (m/z) 603 (-18), 585 (-36), 455 (-166), 331 (-290), 559 (-44-18), 577 (-44), 519 (-102), 289 (- 1 3.1 621 Trifucophlorethol 332), 429 (-192), 495 (-126), 477 (-126-18), 411 (-126-84) 727 (−18), 709 (−36), 455 (−PGU−166), 579 (−166), 289 (−3PGU−84), 701 (−44), 683 2 3.9 745 Tetrafucophlorethol (−44−18), 643 (−84−18), 661 (-84), 331, 437 (−PGU−166−18), 601 (-126-18), 619 (-126) 727 (−18), 455 (−PGU−166), 579 (−166), 709 (−36), 701 (-44), 289 (−3PGU−84), 683 3 4.9 745 (−44−18), 643 (−84−18), 437 Hexafucol (−PGU−166−18), 411 (-334), 429 (-316), 553 (-192), 433 (-2PGU-44-18) 461 (−18), 435 (−44), 417 (-44-18), 391 4 7.0 479 Fucofurodiphlorethol (−88), 313 (−166), 349 (-130), 353 (-126) 325 (-44), 341 (-28), 299 (-70), 351 (-18), 297 5 8.5 369 Eckstolonol (-72), 281 (-88), 245 (-124) 565 (-46), 593 (-18), 567 (-44), 579 (-32), 469 6 12.2 611 Phlorotannin derivative (-142), 356 (-255), 551 (-60), 243 (-368) 489 (-18), 277 (-230), 229 (-PGU-108-44), 461 (-46), 463 (-44), 479 (-28), 445 (-44-18), 7 13.3 507 Phlorotannin derivative 275 (-232), 261 (-246), 245 (-262), 421 (-86), 297 (-PGU-84) * Fragments are arranged in descending order of relative abundance with bold values highlighting the most abundant fragment. RT – retention time, PGU – phloroglucinol unit.

Notably, among the compounds detected, eckstolonol has been described as a good antitumor agent against MCF-7 breast cancer cells causing a dose-dependent anti-proliferative effect that was resultant from the induction of apoptosis via activation of the expression of p53, Bax, caspase-3 and caspase-9, alongside with the decreased expression of Bcl-2 [189]. Moreover, phlorofucofuroeckol- A, a derivative of fucofuroeckol that has close structural similarities to fucofurodiphlorethol, was also reported to induce apoptosis in several human colorectal cancer cell lines (HCT116, SW480, LoVo and HT-29 cells) via activation of the expression of ATF3, a protein that is tightly correlated to the induction of apoptosis in colorectal cancer [192]. Therefore, the presence of these two compounds in F5 could be one of the possible explanations for the remarkable increase of the levels of apoptotic cells observed in the annexin V-FITC/PI-stained cells (section 4.3.3.).

4.4 Conclusions

In this section, the Fucus vesiculosus phlorotannins were shown to hold potential as antitumor agents against both gastric and colon cancer since some of its fractions were capable of inducing selective cytotoxic effects towards tumor cell lines but not on normal cell lines. Among the 11 samples tested, those presenting the most promising effects were EtOAc, F1 and F5. The collected data suggests that F1 antitumor activity might result in part from induction of cell cycle arrest at S

ANTITUMOR ACTIVITY OF F. VESICULOSUS PHLOROTANNINS THROUGH ACTIVATION OF APOPTOTIC SIGNALS

phase, while F5 exerts its effect via a remarkable stimulation apoptosis and necrosis. Likewise, EtOAc antiproliferative effects on MKN-28 cells were resultant of both cell cycle arrest (on G2/M phase) and induction of apoptosis/necrosis, while on Caco-2 cells, it did not interfere with the cell cycle but strongly triggered apoptosis and necrosis. The UHPLC-DAD-ESI-MSn analysis of the most active sample, i.e., F5, allowed to detect seven phlorotannin compounds, of which eckstolonol and fucofurodiphlorethol might be important contributors for the anti-proliferative effects displayed by this sample. In summary, the results herein reported contribute for a better understanding of the mechanisms behind the antitumor properties of F. vesiculosus phlorotannins although further work would be necessary to better comprehend the intracellular mechanisms and proteins affected by the presence of these samples.

Chapter 5 .

This chapter includes data from the paper:

Catarino, M.D.; Marçal, C.; Bonifácio, T.; Campos, D.; Mateus, N.; Silva, A.M.S.; Pintado, M.; Cardoso, S.M. Impact of Phlorotannin Extracts from Fucus vesiculosus on Human Gut Microbiota. In preparation.

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5.1 Impact of phlorotannin extracts from F. vesiculosus on human digestive enzymes and gut microbiota

The digestive process includes a large number of components responsible for the breakdown of the complex molecules of the food matrix into their basic and absorbable forms. Among these components are enzymes such as α-amylase, α-glucosidase and pancreatic lipase which are key elements in the conversion of carbohydrates and triacylglycerols into their simpler and absorbable molecules, making them potential therapeutic targets for the treatment of diabetes and obesity [306,307]. The food components that are not absorbed in the small intestine end up reaching the colon where they will be exposed to the bacterial activity of the gut microbiota. Some of these food components have the capacity to stimulate the growth of certain beneficial gut microorganisms as well as the production of several bacterial metabolites with health-promoting properties such as short-chain fatty acids, being categorized as prebiotics. According to the current scientific definition developed by ISAPP, a prebiotic is a substrate that is selectively utilized by host microorganisms conferring a health benefit. Some dietary fibres, such as inulin, FOS and GOS, are well-recognized prebiotics [308]. Additionally, over the last years, several studies have proven the existence of interactions between polyphenols and gut microbiota, and even the occurrence of phenolic derivatives, with relevant bioactive properties, resultant from the bacterial metabolization of some dietary polyphenols which is the case of ellagitannin-derived urolithins [309]. In contrast, the fate of phlorotannins when crossing the gastrointestinal tract is still a poorly understood subject. In this context, the aim of this task was to evaluate whether F. vesiculosus phlorotannin-rich extracts could display anti-obesity and anti-diabetic properties through inhibition of α-amylase, α- glucosidase and pancreatic lipase activities as well as their stability and bioaccessibility when crossing the gastrointestinal tract, and possible modulatory effects towards the gut microbiota and short-chain fatty acids production.

5.2 Material and methods

5.2.1 Chemicals

Grounded Fucus vesiculosus harvested in July 2017 was purchased from Algaplus Lda. Acetone, methanol, n-hexane, ethyl acetate, DMSO, glacial acetic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, sodium and potassium tartarate, tris-HCl, and starch were acquired from Fisher (Pittsburgh, PA, USA). Sodium nitroprusside and sulfanilamide were acquired from Acros Organics (Hampton, NH, USA). Ascorbic acid, gallic acid, NADH, NBT, PMS, FOS, DMBA, α-amylase, α- glucosidase, pancreatic lipase, 4-nitrophenyl α-D-glucopyranoside (PNPG), 4-nitrophenyl butyrate

(PNPB), paraffin, bile salts, pancreatin, pepsin, sodium hydrogen carbonate, D-glucose, organic acids (succinate, lactate, propionate butyrate and acetate) and sulphuric acid were obtained from Sigma

IMPACT OF PHLOROTANNIN EXTRACTS FROM F. VESICULOSUS ON HUMAN DIGESTIVE ENZYMES AND GUT MICROBIOTA

(St. Louis, MO, USA). Man-Rogosa-Sharp (MRS) medium and L-cysteine-HCl were purchased from Biokar (Allonne, France) and Merck (Darmstadt, Germany), respectively, while trypticase soya broth (TSB) without dextrose and bactopeptone were acquired from BBL (Lockeysville, USA) and

Amersham (Buckinghamshire, UK), respectively. Salt solution A (100.0 g/L NH4Cl, 10.0 g/L

MgCl2·6H2O, 10.0 g/L CaCl2·2H2O), salt solution B (200.0 g/L K2HPO4·3H2O) and resazurin solution were ordered from ATCC (Virginia, USA). Sodium di-hydrogen phosphate and potassium di-hydrogen phosphate were acquired from Panreac (Barcelona, Spain). Dinitrosalycilic acid and acarbose were purchased from Acros Organics (Hampton, NH, USA), calcium chloride from ChemLab (Eernegem, Belgium) and orlistat from AlfaAesar (Ward Hill, MA, USA). Finally, the Bifidobacterium animalis BB0 were acquired from CSK (Ede, Netherlands), Bifidobacterium animalis spp. lactis Bb12 and Lactobacillus casei 01 from Chr. Hansen (Hørsholm, Denmark) and Lactobacillus acidophillus La-5 from Lallemand (MontReal, Canada).

5.2.2 Extraction procedure

The preparation of CRD and EtOAc were carried out according to the optimized procedure described in chapter 2, section 2.2.4.

5.2.3 Effects on the metabolic enzymes

5.2.3.1 α-Amylase inhibition assay

Inhibition of α-amylase activity was measured according to Pereira et al. [260], with slight modifications. The experiment started by adding 200 µL of six different extract concentrations (0– 0.06 mg/mL for CRD and 0–0.005 mg/mL for EtOAc fraction) dissolved in 20 mM phosphate buffer (pH 6.9, containing 6 mM of NaCl) to 400 µL of a 0.8% (w/v) starch solution prepared in the same phosphate buffer. After 5 min at 37 ºC, the reaction was initiated with the addition of 200 µL of α- amylase solution. Following another incubation of 5 min, 200 µL of the reaction mixture were collected and immediately mixed with 600 µL of DNS reagent (10 g/L of 3,5-dinitrosalicylic acid, 300 g/L of potassium and sodium tartrate tetrahydrate and 0.4 M NaOH) to stop the reaction. A second aliquot of 200 µL was collected 15 min later and mixed with DNS reagent as well. Samples were then boiled for 10 min and once they had cooled, 250 µL were transferred to the wells in a 96- well microplate for absorbance reading at 450 nm. Blank readings (no enzyme) were then subtracted from each well and the inhibitory effects towards α-amylase activity was calculated as follows:

∆Absc − ∆Abse % inhibiton = × 100 ∆Absc

where ∆Absc is the variation in the absorbance of the negative control and ∆Abse is the variation in the absorbance of the extract. Acarbose was used as a positive control of inhibition.

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5.2.3.2 α-Glucosidase inhibition assay

Inhibition of α-glucosidase was measured according to the method previously described by Neto et al. [233]. This consisted in the addition of 50 µL of different extract concentrations (0–0.006 mg/mL for CRD and 0–0.001 mg/mL for EtOAc fraction) prepared in 50 mM phosphate buffer pH 6.8) to 50 µL of 6 mM pNPG, dissolved in deionized water. The reaction was started with the addition of 100 µL of α-glucosidase solution and the absorbance was monitored at 405 nm every 60 s for 20 min at 37 ºC. Acarbose was used as positive control of inhibition.

5.2.3.3 Pancreatic lipase inhibition assay

Inhibition of lipase activity was measured following the procedure described by Pereira et al. [260]. The reaction mixture was prepared in a microtube by mixing 55 µL of five different concentrations of extract (0–0.4 mg/mL for CRD and 0–0.07 mg/mL for EtOAc fraction) dissolved in Tris buffer 100 mM (pH 7.0) with 467.5 µL of Tris-HCl (100 mM, pH 7.0, containing 5 mM of

CaCl2) and 16.5 µL of enzyme. The reaction was started by adding 11 µL of 20 mM pNPB diluted in DMSO. The final DMSO concentration in the reaction mixture did not exceed 2%. The reaction mixture was then quickly transferred to a 96-well plate and incubated for 35 min at 37 ºC while the absorbance was being measured every 60 s at 410 nm. Orlistat was used as a positive control of inhibition.

5.2.4 Gastrointestinal digestion simulation

The simulation of the gastrointestinal digestion of the F. vesiculosus sample extracts was performed according to the method described by Campos et al. [310]. The oral digestion was started by suspending 1g of dried sample (CRD or EtOAc) in 20 mL of distilled water followed by the adjustment of the pH between 5.6 and 6.9 with NaHCO3 prior to the addition of 0.6 mL/min of α- amylase at 100 U/mL. The enzymatic digestion was carried out during 2 min of mastication, at 37 °C and 200 rpm. Before the gastric digestion, pH was adjusted to 2.0 using 1M HCl and then mixed with a simulated gastric juice consisting of pepsin 25 mg/mL added at a ratio of 0.05 mL/mL of mouth digest. Incubation was carried out over 60 min at 37 ºC and 130 rpm, after which an aliquot of 2 mL was taken for evaluation of its biological capacities. Finally, for the intestinal digestion the pH of the gastric digest was adjusted to 6.0 using 1M NaHCO3 prior to the addition of a simulated intestinal juice consisting of 2 g/L of pancreatin and 12 g/L bile salts at a ratio of 0.25 mL/mL of gastric digest. The samples were then incubated during 120 min, at 37 °C and 45 rpm, to mimic a long intestine digestion. In the final step of the intestinal digestion samples were submitted to a dialysis process during 48 h at room temperature using a membrane with a molecular pore size of 3 kDa, to reproduce the natural absorption step in the small intestine. At the end of this process, the

IMPACT OF PHLOROTANNIN EXTRACTS FROM F. VESICULOSUS ON HUMAN DIGESTIVE ENZYMES AND GUT MICROBIOTA

permeate represented the bioaccessible fraction while the retentate represented the non-absorbable fraction which were then used for the fermentation experiments. An aliquot of 2 mL was collected before the digestion simulation and after at each step of digestion, i.e., mouth digest, gastric digest, intestinal digest, permeate and retentate, and stored at −80 °C until further use for phlorotannins quantification and antioxidant experiments.

5.2.5 Determination of the phlorotannin content and antioxidant activities

The quantification of the total phlorotannin content was carried out according to the procedure

●– described in the chapter 2, section 2.2.5. while the antioxidant activities, namely O2 scavenging and NO● scavenging assays were performed according to the methods described in chapter 3, section 3.2.3.2. and 3.2.3.4., respectively.

5.2.6 Prebiotic potential

Potential prebiotic effects of F. vesiculosus phlorotannin-rich samples were determined for Bifidobacterium animalis B0, Bifidobacterium animalis spp. lactis BB12, Lactobacillus casei 01 and Lactobacillus acidophilus LA-5. Strains were stored at −80 °C in de MRS broth with 30% (v/v) glycerol. L. casei 01 and L. acidophilus LA-5 inocula were prepared by suspending each bacterial colony into MRS broth, achieving a turbidity equivalent to 0.5 McFarland standard, and then diluting to reach the recommended concentration of probiotic bacteria in wells, 5 x 105 CFU/mL. Twenty microliters of each inoculum were transferred to a 96-well microplate and every well was fulfilled (to the final volume of 200 µL) with each F. vesiculosus sample, diluted in basal MRS broth without glucose at concentrations of 1, 1.5 and 2% (w/v). Microplate was incubated (Multiskan GO, Thermo Scientific) at 37 °C for 48 h with agitation. Similarly, B. animalis B0 and B. lactis BB12 inocula were prepared under anaerobic atmosphere, by suspending each bacterial colony into MRS broth

supplemented with 0.05% (v/v) L-cysteine-HCl, achieving a final turbidity equivalent to 0.5 McFarland standard, and then diluted to reach the recommended concentration of probiotic bacteria in wells, 5 x 105 CFU/mL. Twenty microliters of each inoculum were transferred to a 96-well microplate and every well was fulfilled (to final volume of 200 µL) with each F. vesiculosus sample, diluted in basal MRS broth without glucose at concentrations of 1, 1.5 and 2% (w/v). Microplate was sealed with paraffin and incubated at 37 °C for 48 h with agitation. In all plates, OD measurements at 620 nm were registered every hour. Three controls were also performed: the first one containing inoculum and MRS broth with glucose (positive control), the second one containing inoculum and FOS in MRS broth without glucose (FOS control) and the third one containing only inoculum and MRS broth (negative control).

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5.2.7 In vitro fermentation assays

The human faeces were collected into sterile plastic vases and kept under anaerobic conditions, until further notice (maximum of 2 h after collection). The samples were obtained fresh, from healthy human donors, with the premises of not having any known metabolic and gastrointestinal disorder. Moreover, the donors confirmed not to be taking any probiotic or prebiotic supplements, as well as any form of antibiotics for the last 3 months. The basal medium was prepared as described previously [310], consisting of a nutrient base medium containing 5.0 g/L trypticase soya broth (TSB) without dextrose (BBL, Lockeysville, USA), 5.0 g/L bactopeptone (Amersham, Buckinghamshire, UK), 0.5 g/L L-cysteine-HCl (Merck, Germany), 1.0% (v/v) of salt solution A (100.0 g/L NH4Cl, 10.0 g/L

MgCl26H2O, 10.0 g/L CaCl22H2O), 0.2% (v/v) of salt solution B (200.0 g/L K2HPO43H2O) and 0.2% (v/v) of 0.5 g/L resazurin solution, prepared in distilled water and with pH adjustment at 6.8. The basal medium was dispensed into airtight glass anaerobic bottles, sealed with aluminum caps before sterilization by autoclave. Stock solutions of Yeast Nitrogen Base (YNB) were sterilized with 0.2 μm syringe filters (Chromafils, Macherey-Nagel, Düren, Germany) and inserted into the bottles. The serum bottles were incorporated with CRD and EtOAc extract retentate from the in vitro GIT simulation at a final concentration of 2% (w/v) and inoculated with faecal slurries of 2% (v/v) at 37 °C for 48 h without shaking nor pH control. Samples were taken at 0, 12, 24 and 48 h of fermentation.

All the experiments were carried out inside an anaerobic cabinet with 5% of H2, 10% of CO2 and

85% of N2 and performed in compliance with the institutional guidelines.

5.2.8 Gut microbiota evaluation

5.2.8.1 DNA extraction

Genomic DNA was extracted and purified from stool samples as previous described [310] using NZY Tissue gDNA Isolation Kit (Nzytech, Lisboa, Portugal) with some modifications. Samples were centrifuged at 11000 g during 10 min, to separate the supernatant from the pellet. Around 170–200 mg of pellet was taken from control and test samples for all times. After, the pellets were homogenized in TE buffer (10 mM Tris/HCl; 1 mM EDTA, pH 8.0) and centrifuged again at 4000 g for 15 min. Supernatant was discarded and the pellet was resuspended in 350 μL of buffer NT1. After an incubation step at 95 °C for 10 min, samples were centrifuged at 11000 g for 1 min. Then, 25 μL of proteinase K was added to 200 μL of supernatant and incubated at 70 °C for 10 min. The remaining steps followed the manufacturer’s instructions. The DNA purity and quantification were assessed with a NanoDrop spectrophotometer (ThermoScientific, Wilmington, DE, USA).

5.2.8.2 Real-time PCR for microbial analysis at stool

IMPACT OF PHLOROTANNIN EXTRACTS FROM F. VESICULOSUS ON HUMAN DIGESTIVE ENZYMES AND GUT MICROBIOTA

Real-time PCR was performed as described before by [310] in sealed 96-well microplates using a LightCycler FastStart DNA Master SYBR Green kit and a LightCycler instrument (Roche Applied Science, Indianapolis, ID, USA). PCR reaction mixtures (total of 10 μL) contained 5 μL of 2 × Faststart SYBRGreen (Roche Diagnostics Ltd), 0.2 μL of each primer (final concentration of 0.2 μM), 3.6 μL of water and 1 μL of DNA (equilibrated to 20 mg). Primer sequences (Sigut microbiota- Aldrich, St. Louis, MO, USA) used to target the 16S rRNA gene of the bacteria and the conditions for PCR amplification reactions are reported in Table 5.1.

Table 5.1. Primer sequences and real-time PCR conditions used for gut microbiota analysis.

Maximum growth rate (µmax.h-1) Target group PCR product AT Primer sequence (5’-3’) Genomic DNA standard size (bp) (ºC) AAA CTC AAA GGA ATT GAC GG Bacteroides vulgatus Universal 180 45 ACR RCA CGA GCT GAC ATCC 8482 (DSMZ 1447) ATG TGG TTT AAT TCG AAG CA Lactobacillus gasseri Firmicutes 126 45 AGC TGA CGA CAA CCA TGC AC ATCC 33323 (DSMZ 20243) CCC TTA TTG TTA GTT GCC ATC ATT Enterococcus gilvus Enterococcus spp. 144 45 ACT CGT TGT ACT TCC CT TGT ATCC BAA-350 (DSMZ 15689) GAG GCA GCA GTA GGG AAT CTT C Lactobacillus gasseri Lacctobacillus spp. 126 55 GGC CAG TTA CTA CCT CTA TCC TTC TTC ATCC 33323 (DSMZ 20243) CAT GTG GTT TAA TTC GAT GAT Bacteroides vulgatus Bacteroidetes 126 45 AGC TGA CGA CAA CCA TGC AG ATCC 8482 (DSMZ 1447) ATA GCC TTT CGA AAG RAA GAT Bacteroides vulgatus Bacteroides spp. 495 45 CCA GTA TCA ACT GCA ATT TTA ATCC 8482 (DSMZ 1447) CGC GTC YGG TGT GAA AG Bifidobacterium longum subsp. Infantis Bifidobacterium spp. 244 50 CCC CAC ATC CAG CAT CCA ATCC 15697 (DSMZ 20088) AT – annealing temperature; bp – base pairs; PCR – polymerase chain reaction

To verify the specificity of the amplicon, a melting curve analysis was performed via monitoring SYBR Green fluorescence in the temperature ramp from 60 to 97 °C. Data was processed and analyzed using the LightCycler software (Roche Applied Science). Standard curves were constructed using serial tenfold dilutions of bacterial genomic DNA, according to the following webpage http://cels.uri.edu/gsc/cbdna.html. Bacterial genomic DNA used as a standard (Table 5.1.) was obtained from DSMZ (Braunschweig, Germany). Genome size and the copy number of the16S rRNA gene for each bacterial strain used as a standard was obtained from NCBI Genome database (http://www.ncbi.nlm.nih.gov). Data is presented as the mean values of duplicate PCR analyses. The F:B ratio was obtained by dividing the number of copies of Firmicutes divisions by the number of copies of Bacteroidetes divisions. Moreover, the relative differences to negative control percentage (only faeces fermentation) were calculated using the following equation: 푆푀퐶 − 퐶푀퐶 푅푒푙푎푡푖푣푒 푑푖푓푓푒푟푒푛푐푒 푡표 푐표푛푡푟표푙 % = × 100 퐶푀퐶

where SMC is the mean copy numbers of the sample at a certain time (12 or 24 or 48 h) and CMC is the mean copy numbers of the control sample at the same time as SMC. Positive % values mean

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the occurrence of an increase in the number of copies relative to the control sample at that certain time. The higher the value, the higher increase.

5.2.8.3 Determination of organic acids

Supernatants from the batch cultures were filtered through 0.20 μm cellulose acetate membranes. The chromatographic analysis was performed using a Beckman & Coulter 168 series HPLC system with refractive index - RI detector (Knauer, Berlin, Germany). The separation was performed using Aminex HPX-87H column (BioRad, Hercules, CA) operated at 50 °C; mobile phase, 0.003 mol/L

H2SO4; flow, 0.6 mL/min. Aliquots of the filtered samples were assayed for organic acids (lactic, acetic, succinic, propionic and butyric) using an Agilent 1200 series HPLC system with an RI detector (Agilent, Germany) and with UV detector.

5.2.9 Statistical analysis

Data was expressed as mean ± SD of three similar and independent experiments and analyzed using one-way ANOVA followed by Tukey’s post hoc test. The statistical tests were applied using GraphPad Prism, version 7.00 (GraphPad Software, San Diego, CA, California) and the significance level was p < 0.05.

5.3 Results and discussion

5.3.1 Effects on the metabolic enzymes

α-Amylase, α-glucosidase and pancreatic lipase are key enzymes in the digestive system, catalyzing the hydrolysis of complex food ingredients such as carbohydrates and triacylglycerols into simple and easily absorbable molecules. In this context, the inhibition of the activity of these enzymes promotes a delay in the carbohydrate and lipid digestion, and a consequent reduction of postprandial plasma glucose levels and overall bodyweight, thus contributing to the amelioration of type-II diabetes mellitus and obesity symptoms. As shown in Table 5.2., the highest inhibitory activity of both CRD and EtOAc was observed against α-glucosidase, followed by α-amylase and pancreatic lipase. Notably, the inhibitory capacities of CRD and EtOAc against α-glucosidase were respectively 45 and 250 times stronger than that of acarbose (206.6 ± 25.1 µg/mL), the latter being a medication for type-II diabetes mellitus. Interestingly, a significant increment of the inhibitory activity was noticed compared with previous data from our research group, in which the 70% acetone extracts of

F. vesiculosus showed an IC50 of 32 µg/mL, which is likely to be related to factors such as the differences in the harvesting batch, as well as the differences in the extraction conditions [232]. EtOAc also exhibited better inhibitory activity than crude extract against α-amylase (2.8 ± 0.3 and 28.8 ± 1.2 µg/mL, respectively) and close to that of acarbose (0.7 ± 0.2 µg/mL), which is coherent

IMPACT OF PHLOROTANNIN EXTRACTS FROM F. VESICULOSUS ON HUMAN DIGESTIVE ENZYMES AND GUT MICROBIOTA

with its higher content in phlorotannins. Likewise, although the effects of these samples against pancreatic lipase were far from matching that of orlistat (1.8 ± 0.5 ng/mL), an identical tendency was observed, i.e., EtOAc, which is higher in phlorotannins, presented better inhibitory activity than the crude extract (19.0 ± 1.8 and 45.9 ± 3.4 µg/mL, respectively). In fact, this outcome was already expected since phenolic compounds, particularly those with polymeric arrangements such as tannins, are well-known for their capacity to interact with proteins and form soluble or insoluble complexes [311]. Moreover, previous studies have already indicated that phlorotannin-rich extracts from F. vesiculosus and other species from the same genus are generally good inhibitors of α-glucosidase and α-amylase [312,313]. Interestingly, the inhibitions of approximately 470 and 305 times stronger than acarbose have been reported for F. vesiculosus aqueous and hydroethanolic extracts, respectively, against α-glucosidase, which is substantially higher than those herein reported for the EtOAc [314]. In this case, the inhibitory effects might be resultant from other non-phenolic compounds such as fucoidans and sulfated polysaccharides, which have been previously reported to display promising inhibitory activity against α-glucosidase [315]. In turn, the inhibitory activity of

those extracts against α-amylase showed IC50 values of 63.5 and 59.1 µg/mL (for aqueous and hydroethanolic extracts, respectively), corresponding to twice or more the values herein observed [314].

Table 5.2. Inhibition of α-glucosidase, α-amylase and pancreatic lipase by F. vesiculosus CRD, EtOAc and the respective reference compounds.

IC50 Value (μg/mL) Sample α-amylase α-glucosidase Pancreatic Lipase CRD 28.8 ± 1.2 a 4.5 ± 0.7 a 45.9 ± 3.4 a EtOAc 2.8 ± 0.3 b 0.82 ± 0.05 a 19.0 ± 1.8 b Acarbose 0.7 ± 0.2 c 206.6 ± 25.1 b - Orlistat * - - 1.8 ± 0.5 c

CRD – Crude extract, EtOAc – ethyl acetate fraction from F. vesiculosus extract. IC50 value was determined as the concentration at which α-amylase, α-glucosidase, and pancreatic lipase were inhibited by 50%. All values are expressed as mean ± SD. Different letters within a column mean significantly differences at p < 0.05 using

student’s t test. * IC50 value for orlistat is expressed in ng/mL.

To our knowledge, in addition to our previous works, only Chater et al. [316], reported the inhibitory activity of Fucus against pancreatic lipase. According to their study, from the four different F. vesiculosus preparations tested, namely homogenate, ethanol extract, ethanol pellet and sodium

carbonate extract, good pancreatic lipase inhibitory effects were observed only for the first two (IC50

= 0.119 and 0.159 mg/mL, respectively), while sodium carbonate extract only presented an IC50 of approximately 1 mg/mL. Interestingly, previously results from our research group, showed that several F. vesiculosus ethanol extracts did not exert any inhibition of pancreatic lipase and only mild

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inhibition was observed for 70% acetone and aqueous extracts [232]. Once again, differences in the extraction procedures and experimental protocols might be the explanation for the observation of such discrepancies, as well as other non-considered factors. Overall, these results suggest that phlorotannins from F. vesiculosus are important contributors to the antidiabetic and anti-obesity properties claimed for this species and that these compounds hold the potential to control blood glucose levels and overall energy intake through inhibition of α-glucosidase, α-amylase and pancreatic lipase. In fact, the active doses herein reported may be within physiological range to affect these three enzymes at a digestive level, since their action takes place in the digestive tract and, therefore, they are more exposed to phlorotannin interactions and less dependent of bioavailability- related issues. Nevertheless, it should still be considered that, during their passage through the digestive tract, phlorotannins are susceptible to different environmental conditions including pH variations, enzymatic activity, or the presence of bile salts, which can significantly affect their stability and bioactivity.

5.3.2 Stability, bioaccessibility and antioxidant activity of F. vesiculosus extracts throughout the simulated GIT

To evaluate the stability of F. vesiculosus phlorotannins throughout the digestive tract, both CRD and EtOAc were submitted to a simulated GIT digestion and evaluated for their total phlorotannin content and antioxidant activity after each gut compartment. The results presented in Table 5.3. clearly demonstrate that the total phlorotannin content of the EtOAc fraction progressively decreased after each step from the GIT simulation. Interestingly, in the case of CRD, after the initial decrease in the mouth, the total phlorotannin content increased again after the stomach digestion, followed by another decrease in the intestine. The reduction of the TPhC of the samples after the mouth digestion could be explained by possible interactions occurring between phlorotannins and the salivary proteins. In fact, such interactions are very well described for plant tannins and very important for the development of important sensory characteristics from certain foods and beverages such as wine [317]. The extreme pH conditions in the stomach can also explain why the TPhC of the EtOAc kept decreasing in this compartment. However, in the case of CRD, because this sample is more complex and contains other non-phlorotannin compounds, it is possible that such compounds might be interacting with phlorotannins and protecting them from reacting with the mouth proteins and degrading with the low stomach pH. In turn, the stomach pH may also promote the degradation of those non-phlorotannin compounds, promoting the release of the phlorotannins, making them more available to be quantified by the DMBA assay. In fact, similar observations have been previously reported for plant phenolics [318,319] and are on the basis of delivery strategies in which phenolic compounds are encapsulated in order to resist the gastrointestinal conditions and reach intact for absorption in the intestines [320]. Additionally, it was noticed that even though undigested EtOAc

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IMPACT OF PHLOROTANNIN EXTRACTS FROM F. VESICULOSUS ON HUMAN DIGESTIVE ENZYMES AND GUT MICROBIOTA

had higher TPhC compared to the undigested CRD, after the stomach and intestine digestions, the TPhC of the later was slightly higher compared with the EtOAc, which is in agreement with the hypothesis that the EtOAc phlorotannins were more exposed to the GIT degradation while CRD phlorotannins were more protected.

Table 5.3. Total phlorotannin content and antioxidant activity of F. vesiculosus CRD and EtOAc through the different stages of the gastrointestinal digestion.

(1) ● (2) ●– TPhC NO O2 Sample GIT stage (mg PGE/g ext) (IC50 μg/mL) (IC50 μg/mL) Undigested 9.93 ± 1.48 a 161 ± 8.8 a 417 ± 164.5 a Mouth 6.33 ± 2.96 b 309 ± 105.2 b 745 ± 88.2 b Stomach 8.52 ± 1.16 ab 171 ± 27.1 a 378 ± 26.6 a CRD Intestine 5.17 ± 0.70 b 287 ± 27.2 ab 1105 ± 421.3 b Retentate* 4.60 ± 0.26 b 141 ± 9.1a 294 ± 19.3 a Permeate* 1.40 ± 0.19 c 2551 ± 30.7 c 2580 ± 75.2 c Undigested 17.39 ± 1.77 a 45 ± 2.5 a 118 ± 17.6 a Mouth 13.83 ± 0.74 b 73 ± 11.0 ab 221 ± 1.1 ab EtOAc Stomach 5.67 ± 0.91 c 109 ± 7.1 ab 244 ± 0.4 ab Intestine 3.28 ± 0.55 c 195 ± 38.5 bc 564 ± 19.9 c Retentate* 2.97 ± 0.62 cd 281 ±16.1 c 383 ± 18.2 bc Permeate* 0.37 ± 0.10 d 1531 ± 52.2 d 3074 ± 32.3 d Standard Compound - 36 ± 0.9 6 ± 0.5 CRD – crude extract, EtOAc – Ethyl acetate fraction, GIT – gastrointestinal tract, TPhC – total phlorotannin content. (1) Standard compound for NO• is ascorbic acid, (2) Standard compound for •− * O2 is gallic acid, results for DMBA expressed in mg PGE/g intestine digest. For each sample, different letters indicate significant differences within the same column (p < 0.05).

At the end of the simulated GIT, only a small portion of the total phlorotannins loaded in the system was bioaccessible, much likely to what happens with plant polyphenols. At this point, it is important to clarify the terms “bioavailability” and “bioaccessibility”. The former expresses the fraction of an ingested compound/nutrient that reaches the systemic circulation to be distributed to organs and tissues and to manifest its bioactivity. However, before becoming bioavailable, the target compound/nutrient must be released from the food matrix and made available for bloodstream absorption, which is what defines of bioaccessibility [321]. Interestingly, despite the undigested CRD exhibited lower TPhC compared to the undigested EtOAc, the bioaccessible index of the former was 14.1% while the later was only 2.0%. Once again, this outcome might be in part explained by the fact that EtOAc experienced higher phlorotannin degradation than CRD, and therefore, when the compounds reach the intestine to be absorbed, the TPhC of the matrix is already lower.

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In regard to the antioxidant activity of the samples after each step of the GIT simulation, both NO● and SO●– results were in line with the TPhC of the respective samples, i.e., the samples with higher phlorotannin concentrations exhibited the lowest IC50 values and vice versa. Indeed, strong negative correlations were found between the TPhC and the antioxidant assays with CRD showing R2 of -0.82 and -0.94, and EtOAc showing R2 of -0.91 and -0.82 in NO● and SO●–, respectively, thus indicating a clear association between the phlorotannin content after each step of the simulated digestion and the antioxidant activities observed.

5.3.3 Prebiotic effect

The prebiotic activity of digested F. vesiculosus CRD and EtOAc was studied on 4 strains in basal MRS medium without glucose, at concentrations of 1 - 2% (w/v). Figure 5.1. presents the growth curves of the evaluated Lactobacillus and Bifidobacteria strains, respectively, until 24 h since no further alterations were observed between 24 and 48 h. All the probiotic microorganisms were affected by the presence of the F. vesiculosus samples in different manners. Lactobacillus casei exhibited a growth behavior identical for almost all the conditions tested, with no differences observed on the maximum OD, although the seaweed samples seem to slightly delay their growth during the first 10 hours. The only notable exception was EtOAc at 1%, which caused a slight decrease of the growth curve of this strain. In turn, the incubation of L. acidophillus either with CRD or EtOAc presented a growth curve considerably higher than that of FOS, for all the concentrations tested, thus indicating that both CRD and EtOAc stimulate the growth of this strain. In contras, B. animalis growth was the least pronounced of all the strains tested, either in presence of CRD or EtOAc, indicating that they might exert a bacteriostatic effect on this strain. The results for B. animalis spp. lactis demonstrated that the CRD at 1% displayed better stimulatory effects than FOS, although the bacterial growth was completely abolished for higher concentrations, indicating that in such conditions, this extract impairs the growth of this strain. Positive stimulatory effects were noticed for EtOAc at 1 and 1.5% as well, which demonstrated growth curves identical to that of FOS. However, for the concentration of 2%, this sample also exhibited inhibitory effects towards this strain. The potential prebiotic effect of seaweeds is a subject that has already been studied by some authors. Indeed, Martelli et al. [322] recently showed that four strains of probiotic bacteria (L. casei, L. paracasei, L. rhamnosus and B. subtilis) all exhibited good capacity to grow in a broth medium containing Himanthalia elongata flour (5%), which is in line with previous works that demonstrated the capacity of different brown algae species (Sargassum siliquanstrum, Laminaria digitata, Laminaria saccharina) to stimulate the growth several probiotic bacteria including Weissella spp., Lactobacillus spp., Leuconostoc spp., L. plantarum and L. rhamnosus [323–325]. Seaweeds are

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however a very complex matrix and the contribute of phlorotannins for the effects observed in these studies are much likely to be neglectable. In fact, current knowledge regarding the fate of seaweed polyphenols in the human gastrointestinal tract is very scarce. In the work developed by Corona et al. [176], after submitting a polyphenol-rich extract from A. nodosum to a simulated gastrointestinal digestion followed by fecal fermentation, they were able to find seven phlorotannin-derived metabolites, and although the microbiota composition was not assessed, the presence of these metabolites suggest that phlorotannins might have been used by the colonic bacteria. In turn, in a 24 h in vitro fermentation carried out using Ecklonia radiata phlorotannin extract, a significantly increase of the populations of Bacteroidetes, Clostridium coccoides, E. coli, and Fecalibacterium prausnitzii was observed, although the levels of Bifidobacterium and Lactobacillus populations were found decreased [224]. With these results, we demonstrate for the first time that F. vesiculosus extract and phlorotannin-enriched fraction can stimulate the growth of some probiotic strains in a similar way to that of FOS.

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L . c a s e i_ C R D L . c a s e i_ E tO A c

3 3 C + C +

F O S 1 % F O S 1 % )

C R D 2 % ) E tO A c 2 % m

2 m 2

C R D 1 .5 % E tO A c 1 .5 %

0 2 C R D 1 % 2

6 E tO A c 1 %

(

1 D 1

O O

0 0 0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 2 0 2 5 tim e (h ) tim e (h )

L . a c id o p h ilu s _ C R D L . a c id o p h ilu s _ E tO A c

3 3 C + C +

F O S 1 % F O S 1 % )

C R D 2 % ) E tO A c 2 % m

2 m 2

C R D 1 .5 % E tO A c 1 .5 %

0 2

C R D 1 % 2 E tO A c 1 %

(

1 D 1

O O

0 0 0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 2 0 2 5 tim e (h ) tim e (h )

B . a n im a lis _ C R D B . a n im a lis _ E tO A c

3 3 C + C +

F O S 1 % F O S 1 % )

C R D 2 % ) E tO A c 2 % m

2 m 2

C R D 1 .5 % E tO A c 1 .5 %

0 2

C R D 1 % 2 E tO A c 1 %

(

1 D 1

O O

0 0 0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 2 0 2 5 tim e (h ) tim e (h )

B . a n im a lis s p p . la c tis _ C R D B . a n im a lis s p p . la c tis _ E tO A c

3 3 C + C +

F O S 1 % F O S 1 % )

C R D 2 % ) E tO A c 2 % m

2 m 2

C R D 1 .5 % E tO A c 1 .5 %

0 2

C R D 1 % 2 E tO A c 1 %

(

1 D 1

O O

0 0 0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 2 0 2 5 tim e (h ) tim e (h )

Figure 5.1. Growth curves of L. casei, L. acidophilus, B. animalis and B. animalis spp. lactis in presence of different concentrations of digested crude (CRD) and ethyl acetate fraction (EtOAc).

5.3.4 Evolution of the gut microbiota profile groups

After GIT simulation, the digested F. vesiculosus CRD and EtOAc were applied to human faeces fermentation during 48 h, to study the effect upon the human microbiota. To understand the

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IMPACT OF PHLOROTANNIN EXTRACTS FROM F. VESICULOSUS ON HUMAN DIGESTIVE ENZYMES AND GUT MICROBIOTA

modulation of the microorganisms’ growth and metabolic activities during fermentation, aliquots at 0, 12, 24 and 48 h were taken and analyzed. Three of the four dominant phyla in the human gut were evaluated, namely Firmicutes, Bacteroidetes and Actinobacteria, and the compositional average of copy numbers obtained by PCR real time of these main groups are depicted in the Table 5.4.

Table 5.4. Fecal microbiota composition of volunteer participants.

Division (genus) Number of copies (n = 5) a Universal 7.52 ± 0.38 Firmicutes 4.76 ± 0.20 Enterococcus spp. 2.07 ± 0.63 Lactobacillus spp. 3.27 ± 0.72 Bacteroidetes 5.46 ± 0.63 Bacteroides spp. 3.76 ± 0.55 Bifidobacterium spp. 4.42 ± 0.45 F:B ratio 0.97 ± 0.23 a Values are presented as mean ± SD and expressed as log10 16S rRNA gene copies per 20 ng of DNA Numbers were in accordance with the ones found in healthy human volunteers’ feces, with Bacteroides and Bifidobacterium comprising the dominant genera while Lactobacillus spp. and Enterococcus appeared as the subdominant genera [310,326,327]. In Figure 5.2. are represented the relative differences (in %) between the microbiota groups of the tested samples and control feces along the 12, 24 and 48 h of fermentation. Overall, both CRD and EtOAc promoted a modest positive effect on the gut microbiota growth as noticed by the increment of the universal microorganisms compared to the control over time, while FOS exerted a positive effect on the initial 12 h that reversed for the following 24 and 48 h. The EtOAc fraction showed a positive effect overtime on the phyla Firmicutes and Bacteroidetes, which are representative of a healthy microbiota [328], while CRD and FOS exhibited null or negative effect on these two groups. In turn, as expected, FOS exerted a very positive effect on Lactobacillus spp. and Bifidobacterium spp., two genera that are the markers of prebiosis par excellence. Likewise, despite not having an effect as sharp as FOS, EtOAc fraction also positively stimulated the growth of these two probiotic groups over time, although in the case of Lactobacillus spp., the effect lasted only until 24 h, becoming null at the end of the fermentation (48 h). Identical behavior was noticed for CRD on Bifidobacterium spp., promoting their growth only during the first 24 h. Curiously, no effect was observed on Lactobacillus spp., contrarily to what was expected since L. casei, L. acidophilus responded with a very positive growth behavior in presence of this sample on the prebiotic studies (section 5.3.2.).

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Interestingly, the group of Enterococcus spp. was the most beneficiated by CRD and EtOAc, although the levels of these organisms progressively decreased over time, contrarily to FOS which promoted their growth at each time point. Poor gut health outcomes have generally been linked to this genus [329], although this is a controversial subject since not all enterococcal strains cause health problems. In fact, strains such as E. faecium SF68® and E. faecalis Symbio-flor® have been marketed as probiotics for two decades without incidence and with very few reported adverse events [330]. Moreover, Enterococcal probiotics have been shown to be effective in limiting gastrointestinal infectious burden and in the treatment of gastrointestinal infections and diarrhea [331]. In turn, even though all the samples promoted an increment of Bacteroides spp. during the first 12 h, only EtOAc maintained this positive effect throughout the fermentation course. Instead, FOS and CRD turned out to negatively affect the growth of these bacteria after 24 h and until the end of the fermentation. Like with Enterococcus spp., there is some controversy around the probiotic potential of the genus Bacteroides. If in one hand this group has been associated to the development of intestinal dysfunctions such as diarrhea, inflammatory bowel disease, and colorectal cancer, on the other hand it has been recently considered as a next generation probiotic candidate due to its potential role in promoting host health through regulation of the intestinal redox levels or production of important short chain fatty acids such as acetate, propionate and butyrate that in turn can contribute for the regulation of toxin transport from the gut lumen to blood, prevention of colon cancer and prevention of inflammatory conditions [332]. Another important aspect to consider is the ratio between Firmicutes and Bacteroidetes (F:B) which are the most predominant phyla in the human colon. Together they comprise 90% of the total gut microbiota and thus, their proportion can give us a global idea of the total effect of F. vesiculosus samples on the intestinal flora. Commonly, healthy individuals display a nearly 1:1 ratio of Firmicutes to Bacteroidetes, and significant alterations of this ratio have been associated to pathological states [333]. For instance, increased F:B ratios have been linked to the pathophysiology of obesity [334], while patients of type-II diabetes mellitus were found to have their levels of Firmicutes significantly reduced compared to their non-diabetic counterparts, and consequently decreased F:B ratios [335]. In this work, a slight increase of the F:B ratio was noticed for FOS and EtOAc (1.36 ± 0.10 and 1.24 ± 0.14, respectively) compared to the control (1.09 ± 0.05) during the first 12 h of fermentation which then returned to normal levels over the next 24 and 48 h. On the contrary, CRD did not cause any significant alterations of this parameter maintaining the F:B ratio values stable and close to one over the course of the fermentation.

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IMPACT OF PHLOROTANNIN EXTRACTS FROM F. VESICULOSUS ON HUMAN DIGESTIVE ENZYMES AND GUT MICROBIOTA

Figure 5.2. Evolution of the gut microbiota groups (relative differences to negative control in %) and Firmicutes:Bacteroidetes (F:B) ratio along the fermentation.

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Very few studies focusing the prebiotic potential of phlorotannin-rich extracts have been conducted so far, although there are already some insights on this matter. Interestingly, Charoensiddhi et al. [224] reported that, after the 24 h fermentation period of a phlorotannin-rich extract of E. radiata with human fecal samples, only the group of Bacteroidetes showed an increased growth compared to the negative control, while Firmicutes and Bifidobacterium spp. remained unchanged and Lactobacillus spp. and Enterococcus spp. actually decreased. However, these authors also observed a stimulation of the growth of Faecalibacterium prausnitzii and Clostridium coccoides which were not analyzed in this study but are two important groups associated to SCFAs production, particularly butyrate, and health promoting effects [206,336]. In a different work, the administration of a polyphenol-rich extract from the brown algae Lessonia trabeculata to streptozotocin-induced diabetic rats under a high-fat diet significantly restored the levels of the three dominant phyla, i.e., Firmicutes, Bacteroidetes and Proteobacteria, as well as the F:B ratio to values identical of the negative control [222]. However, to the authors knowledge, this work was the first assessing the potential modulatory effects of F. vesiculosus phlorotannin extracts on human gut microbiota and allowed to disclose valuable information on how F. vesiculosus phlorotannins may impact on the human gastrointestinal microflora.

5.3.5 Organic acids profile and pH variation

The changes in the concentration of short-chain fatty acids along the fermentation of FOS, CRD and EtOAc with human feces in basal media were analyzed by HPLC and presented in Table 5.5. SCFAs such as acetate, propionate and butyrate are volatile fatty acids that are produced by the gut microbiota in the colon as a result from the fermentation and metabolization of food components that are undigested/unabsorbed in the upper GIT. In this study, fermentation with FOS produced a remarkable increase of the production of the total organic acids, while on the fermentations carried out with F. vesiculosus samples, a tendential increase of the total organic acid levels was noticed despite not statistically significant when comparing with the negative control. These results are also reflected in the pH changes registered during the fermentation (Figure 5.3.), with FOS producing a significant decrease of the pH values, while the pH registered for CRD and EtOAc remained similar to that of the control, at least for the time window tested. Differences on the SCFAs profiles, however, were detected between samples. One of the most evident differences is the case of lactate which was the main metabolite produced over the entire fermentation of FOS. This outcome is also coherent with the high stimulatory effects that FOS produced on Lactobacillus spp. and Bifidobacterium spp. validated above with the 16S rRNA gene analysis (section 5.3.3.). On the other hand, similarly to the negative control, the lactate production on fermentations carried out with CRD and EtOAc were nearly null, and even

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undetectable at 48h. An identical pattern was found for acetate production, which was remarkably stimulated in presence of FOS but not affected by CRD or EtOAc. Under normal conditions this acid together with propionic and butyric acids comprise the three major SCFAs normally produced in the gut and are important for the maintenance of the intestinal homeostasis [337]. Particularly, acetate plays a very important role on the energy homeostasis, contributing for the appetite regulation, promoting fat oxidation, improving insulin sensitivity and glucose homeostasis, and enhancing the

inflammatory status [338]. D a ta 1

8 C - F O S 6 C R D E tO A c

H 4 p

0 0 2 0 4 0 6 0 tim e (m in )

Figure 5.3. Variation of the pH throughout fermentation of digested FOS, CRD and EtOAC with human microbiota.

Interestingly, all the three samples promoted an increase of the succinate levels, which reached its maximum at 12 h and kept constant for FOS until the end of the fermentation while for CRD and EtOAc it decreased overtime. In the one hand, the accumulation of this organic acid in the gut lumen is usually associated with microbiota disturbances commonly linked to poor gut health states such as antibiotic-induced dysbiosis, motility disturbances and specially IBD [339]. On the other hand, succinate is also a key intermediate in the production of propionate that in turn is responsible for modulating lipogenesis, controlling appetite and preventing colon cancer [340]. In fact, the levels of propionate production herein noted seem to follow an identical behavior compared with that of succinate, showing an accentuated increase during the initial phase of the fermentation and a decrease at the end, only for CRD and EtOAc. Indeed, high correlation coefficients between these two organic acids were obtained (R2 = 0.99, 0.88 and 0.97 for FOS, CRD and EtOAc, respectively), which confirms that the production of propionate is indeed associated with the production of succinate. One of the most important SCFAs produced in the gut is butyrate, which has been repeatedly reported for its positive health promoting effects. In addition to its function as the primary energy source for colonocytes, butyrate also importantly contributes for the improvement of the gut barrier function, exerts anti-inflammatory and regenerative effects, prevents the formation of colon cancer and helps reducing both type II diabetes and obesity [333]. Therefore, stimulating the production of

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high levels of this SCFA is of great interest for promoting a healthy function of the gut. The results herein obtained revealed that only the EtOAc led to a significant increase of butyric acid while in CRD and FOS, the levels of this SCFA did not differ much from the negative control. The main butyrate-producing bacteria are Faecalibacterium prausnitzii, Clostridium spp., Eubacterium spp., Roseburia spp. and Anaerostipes spp., which belong to the Firmicutes phylum [337] and despite these genera were not measured in the 16S rRNA gene analysis, an increase of the Firmicutes was indeed noticed on the EtOAc fermentation which is in line with the increased levels of butyrate registered for this sample. In turn, the lack of production of butyrate that was not expected on FOS fermentation could be possibly explained by the absence of the common cross-feeding effect among intestinal bacteria that produce acetate, propionate or butyrate as the final product of the lactate metabolization [341]. Indeed, the fact that lactate has accumulated so much throughout the fermentation of FOS indicates that it has not been utilized as substrate by other bacteria. Nevertheless, it must be considered that this experiment was performed without pH control, and thus, it is much likely that the sharp decrease of the pH may have impaired the growth of certain lactate- utilizing bacteria and favor the growth of the lactate-producing ones, therefore contributing for the increasing accumulation of this organic acid at the expense of other SCFAs [342]. When comparing with the results published by Charoensiddhi et al. [224], the stimulatory effects of F. vesiculosus phlorotannin samples herein tested on the production SCFAs was much more promising than those of the E. radiata phlorotannin-rich extract used on their study. In fact, the authors reported that the fermentation of this phlorotannin extract caused a reduction on the levels of total SCFAs with a remarkable decrease of the concentration of acetic acid in comparison with the negative control. Contrarily, Yuan et al. [222] found that the administration of a polyphenol-rich extract from the brown algae Lessonia trabeculata to streptozotocin-induced diabetic rats under a high-fat diet significantly restored the levels of acetate and butyrate that were depleted in the diabetic control groups. Notably, the levels of butyrate in the treated rats were even superior to those of the control group, i.e., healthy rats. In our study, despite the total SCFA production was much lower than that observed for FOS, both CRD and EtOAc exhibited interesting SCFA profiles stimulating the production of propionate and in the case of EtOAc, butyrate as well. These SCFAs could exert interesting beneficial health properties not only in the colon and gut microbiota but also in other organs, which could partly explain the health benefits attributed to phlorotannins.

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Table 5.5. Concentration of organic acids (succinic, lactic, acetic, propionic and butyric) throughout fermentation of digested FOS, CRD and EtOAC with human microbiota (mg/mL). Organic acids Time (h) Ctrl FOS CRD EtOAc

a;A a;A a;A a;A Total 0 2.38 ± 0.63 2.38 ± 0.63 2.38 ± 0.63 2.38 ± 0.63 a;A b;B b;A b;A,B 12 5.24 ± 1.98 10.89 ± 2.79 7.43 ± 2.09 7.76 ± 1.92 a;A b,c;B b;A b;A 24 4.90 ± 1.59 12.63 ± 2.37 7.08 ± 2.45 7.55 ± 1.75 a;A c;B a,b;A b;A 48 4.10 ± 2.01 14.78 ± 4.00 5.04 ± 1.57 6.38 ± 1.98 a;A a;A a;A a;A Succinic acid 0 0.45 ± 0.20 0.45 ± 0.20 0.45 ± 0.20 0.45 ± 0.20 a;A b;B c;B b;B 12 0.77 ± 0.75 1.85 ± 0.92 2.29 ± 1.39 2.15 ± 1.15 a;A b;B b,c;A,B a,b;A,B 24 1.12 ± 0.53 1.97 ± 0.58 2.02 ± 0.93 1.35 ± 0.50 a;A b;B a,b;A,B a,b;A,B 48 0.74 ± 0.71 2.03 ± 0.85 1.12 ± 0.20 1.40 ± 0.89 Lactic acid 0 ND ND ND ND a;A a;B a;A a;A 12 1.21 ± 0.93 3.91 ± 1.94 0.87 ± 0.22 0.87 ± 0.23 a;A a,b;B a;A a;A 24 0.34 ± 0.14 4.81 ± 0.75 0.76 ± 0.58 0.26 ± 0.16 b 48 ND 5.49 ± 2.14 ND ND a;A a;A a;A a;A Acetic acid 0 0.16 ± 0.04 0.16 ± 0.04 0.16 ± 0.04 0.16 ± 0.04 b;A b;A b;A b;A 12 0.81 ± 0.10 1.36 ± 0.75 1.03 ±0.09 1.02 ± 0.19 b;A b;B b;A b;A 24 0.82 ± 0.17 1.65 ± 0.52 0.92 ± 0.22 0.96 ± 0.30 b;A c;B b;A b;A 48 0.78 ± 0.20 2.77 ± 1.21 0.78 ± 0.20 0.93 ± 0.28

a;A a;A a;A a;A Propionic acid 0 0.34 ± 0.09 0.34 ± 0.09 0.34 ± 0.09 0.34 ± 0.09 a;A b;B b;B b;B 12 0.53 ± 0.23 1.48 ± 0.32 1.14 ± 0.49 1.43 ± 0.87 a;A b;C b;B a,b;A,B 24 0.65 ± 0.35 1.89 ± 0.75 1.25 ± 0.58 0.85 ± 0.27

a;A b;B a,b;A a,b;A 48 0.50 ± 0.24 1.64 ± 0.60 0.77 ± 0.20 0.90 ± 0.24

a;A a;A a;A a;A Butyric acid 0 1.41 ± 0.25 1.41 ± 0.25 1.41 ± 0.25 1.41 ± 0.25 a;A a;A a;A a;A 12 1.92 ± 0.69 2.29 ± 0.99 2.10 ± 0.79 2.71 ± 0.94 a;A a;A a;A b;B 24 2.24 ± 0.67 2.23 ± 0.86 2.54 ± 1.05 4.12 ± 0.37 a;A a;A a;A b;B 48 2.23 ± 1.35 2.70 ± 1.43 2.31 ± 0.85 4.31 ± 0.62 Ctrl – negative control, FOS - fructo-oligosaccharides, CRD – crude extract, EtOAc – ethyl acetate fraction, ND – Not detected. Different letters indicate significant differences (p < 0.05). The capital letters indicate the differences among Ctrl, FOS, CRD and EtOAc for organic acids concentration at the same time (same row), and small letters indicate the differences for the same sample over time for each organic acid concentration (same column within an organic acid).

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5.4 Conclusions

Overall, the work developed in this section provides a great contribution for the understanding of F. vesiculosus phlorotannins potential to act against key metabolic enzymes as well as their stability along the digestive tract, bioaccessibility, and stimulatory effects towards gut microbiota and SCFAs production. Promising inhibitory effects were observed against the metabolic enzymes, particularly against α-glucosidase, which was more sensitive to these compounds than the pharmaceutical drug acarbose. In turn, similar to plant polyphenols, phlorotannins seem to be susceptible to the gut environmental conditions leading to a decrease of their concentration and antioxidant activity along the digestive tract. Moreover, from the portion of phlorotannins that can reach intact to the intestinal lumen, only a small fraction of less than 15 % will become bioaccessible and available for absorption, which indicates that the majority of these compounds will accumulate in the large intestine where they will be exposed to the metabolic activity of the gut microbiota. Meanwhile, the fermentation of the digested CRD and EtOAc revealed a slight positive effect on the growth of certain commensal bacteria from the human gut, with Enterococcus spp. showing the most relevant growth. Moreover, both samples demonstrated an interesting capacity to enhance the production of propionate, while EtOAc caused a notable increase of butyrate levels which are two important SCFAs known for their health promoting status. In summary, the data herein gathered provides valuable information regarding the behavior of F. vesiculosus phlorotannins along their passage throughout the gastrointestinal tract and although the results obtained do not allow to claim F. vesiculosus phlorotannin extracts as prebiotics, they present clear evidence that these compounds can still positively contribute for the maintenance of good gastrointestinal health. Additional studies would, however, be necessary not only to evaluate whether the digestive conditions could interfere with phlorotannins’ inhibitory effects towards the digestive enzymes but also to evaluate if the fermentation with the human colonic bacteria could affect the antioxidant and other bioactive properties of F. vesiculosus CRD and EtOAc. Moreover, it would be particularly relevant to disclose the possible formation of phlorotannin metabolites resultant from the biotransformation and bacterial metabolization in the colon.

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Chapter 6

CHAPTER 6

Conclusions and future perspectives The work developed in this thesis aimed to determine the optimum conditions for the extraction of phlorotannins from Fucus vesiculosus and further characterize the phlorotannin profile of these extracts as well as to evaluate the bioactive properties that could be relevant from a point of view of performance throughout the gastrointestinal tract, namely antioxidant, anti-inflammatory, antitumor and prebiotic effects. Through application of a response surface methodology, it was possible to determine the optimal extraction conditions for the maximum recovery of phlorotannins from grounded F. vesiculosus which consisted in acetone 67% (v/v) in a proportion of 70 mL/g of seaweed powder at 25 °C. After a liquid-liquid partitioning step, the phlorotannin-rich fraction was analyzed using UHPLC-DAD- ESI-MSn, allowing to detect the presence of fucols, fucophlorethols, fuhalols and several other phlorotannin derivatives, as well as to disclose new structural features of F. vesiculosus phlorotannins and even identify three possible new phlorotannin derivatives including fucofurodiphlorethol, fucofurotriphlorethol and fucofuropentaphlorethol, although further research would be necessary to properly verify the structural features of these compounds and confirm their tentative assignment. From a broadened perspective, the results herein obtained allow to conclude that F. vesiculosus phlorotannins could contribute for the improvement of gut health acting on different fronts. As antioxidant and anti-inflammatory agents, these compounds could enhance the inflammatory status of the gut, and importantly contribute for the amelioration of pathophysiological conditions in which inflammation has a major prevalence including coeliac disease, ulcerative colitis or Chron’s disease. Since inflammation is also an important risk factor for the development of gastric and colon cancer, it means that, in a way, the anti-inflammatory activity of F. vesiculosus phlorotannins for itself may already constitute one mechanism of antitumor activity. However, the results herein obtained also demonstrated that these compounds selectively trigger the cell death of tumor cells, indicating that F. vesiculosus phlorotannins could contribute for the prevention of gastric and colon cancer acting on two distinct flanks. Additionally, F. vesiculosus phlorotannin extracts revealed potential anti-diabetic effects as inhibitors of α-glucosidase activity, and regardless exerting only modest modulatory effects on the gut microbiota, the data herein gathered allows to conclude that these compounds promote stimulatory effects on the SCFAs production, particularly on propionate and butyrate which have important functions as antitumor, anti-inflammatory, and regenerative agents in the gut, as well as intermediates in the management of type-II diabetes and obesity. As such, in addition to the previous mechanisms mentioned, F. vesiculosus phlorotannins can also exert indirect anti-inflammatory, antitumor and other health promoting effects by stimulating the production of benefic metabolites by the gut microbiota.

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CONCLUSION AND FUTURE PERSPECTIVES

Overall, this work could be the basis for the development of exploitation strategies using phlorotannins from F. vesiculosus to produce novel functional foods, nutraceuticals, and other ingredients for application in food industry aiming to improve the health status of the consumers. As such, it also contributes for the valorization of F. vesiculosus that grows profusely along the Portugal coasts, raising awareness for the possible applications of this seaweed and boosting its commercial interest. Some questions remain, however, unanswered whereas others have been raised, giving ground for new research. In this sense, further research would be necessary to better elucidate the following aspects: • Isolation and full structural characterization (via NMR) of the compounds tentatively assigned, particularly fucofurodiphlorethol, fucofurotriphlorethol and fucofuropentaphlorethol, and those for which a conclusive assignment could not be attributed (compound with [M − H]− at m/z 507). • Modulatory properties of the samples with the most promising antitumor activity (EtOAc, and subfractions F1 and F5) on the expression of pre- and proapoptotic proteins in tumor cells as well as the expression of proteins involved in necrosis. • The antioxidant, anti-inflammatory, antitumor and anti-enzymatic activities of phlorotannins after their passage throughout the digestive tract and fermentation by the gut microbiota. • Identification of possible phlorotannin-derived metabolites resultant from the bacterial activity in the gut and screening of their bioactive properties. • Evaluation of the bioavailability and tissue accumulation of F. vesiculosus phlorotannins orally ingested.

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