Multimodal Actions of Brown Seaweed (Ochrophyta)

Mariana Nunes Barbosa

MULTIMODAL ACTIONS OF BROWN SEAWEED (OCHROPHYTA)

BIOACTIVE COMPOUNDS IN INFLAMMATION AND ALLERGY NETWORK

Thesis for Doctor Degree in Pharmaceutical Sciences Phytochemistry and Pharmacognosy Specialty

Work performed under the supervision of Professor Doctor Paula Cristina Branquinho de Andrade

and co-supervision of Professor Doctor Patrícia Carla Ribeiro Valentão

May 2018

Study nature, love nature, stay close to nature. It will never fail you.

– Frank Lloyd Wright

To my beloved family and my dear friends

Work financially supported through the attribution of a Doctoral Grant (SFRH/BD/95861/2013) by the Fundação para a Ciência e a Tecnologia (FCT) under the framework of POPH – QREN – Type 4.1 – Advanced Training, funded by the Fundo Social Europeu (FSE) and by National funds of Ministério da Educação e Ciência (MEC), and by Programa de Cooperación Interreg V-A España–Portugal (POCTEP) 2014–2020 (project 0377_IBERPHENOL_6_E).

VII IT IS AUTHORIZED THE REPRODUCTION OF THIS THESIS ONLY FOR RESEARCH PURPOSES,

UNDER THE WRITTEN STATEMENT OF THE INTERESTED PARTY, COMMITTING ITSELF TO DO

IT.

VIII PUBLICATIONS

PUBLICATIONS

The data contained in the following works make part of this thesis.

PUBLICATIONS IN INTERNATIONAL PEER-REVIEWED JOURNALS INDEXED AT THE JOURNAL CITATION REPORTS (JCR) OF THE ISI WEB OF KNOWLEDGE:

1. Barbosa M, Valentão P, Andrade PB. Bioactive compounds from macroalgae in the new millennium: Implications for neurodegenerative diseases. Mar Drugs. 2014 Sep; 12 (9): 4934–4972.

2. Barbosa M, Collado-González J, Andrade PB, Ferreres F, Valentão P, Galano JM, Durand T, Gil-Izquierdo Á. Nonenzymatic α-linolenic acid derivatives from the sea: Macroalgae as novel sources of phytoprostanes. J Agric Food Chem. 2015 Jul ;63 (28): 6466–6474.

3. Barbosa M, Valentão P, Andrade PB. Biologically active oxylipins from enzymatic and nonenzymatic routes in macroalgae. Mar Drugs. 2016 Jan; 14 (1): 23.

4. Fernandes F, Barbosa M, Oliveira AP, Azevedo IC, Sousa-Pinto I, Valentão P, Andrade PB. The pigments of kelps (Ochrophyta) as part of the flexible response to highly variable marine environments. J Appl Phycol 2016 Dec; 28 (6): 3689–3696.

5. Barbosa M, Fernandes F, Pereira DM, Azevedo IC, Sousa-Pinto I, Andrade PB, Valentão P. Fatty acid patterns of the kelps Saccharina latissima, Saccorhiza polyschides and Laminaria ochroleuca: Influence of changing environmental conditions. Arab J Chem 2017 (in press). DOI: 10.1016/j.arabjc.2017.01.015.

6. Barbosa M, Lopes G, Ferreres F, Andrade PB, Pereira DM, Gil-Izquierdo Á, Valentão P. Phlorotannin extracts from Fucales: Marine polyphenols as bioregulators engaged in inflammation-related mediators and enzymes. Algal Res 2017 Dec; 28: 1–8.

7. Lopes G, Barbosa M, Vallejo F, Gil-Izquierdo Á, Andrade PB, Valentão P, Pereira DM, Ferreres F. Profiling phlorotannins from Fucus spp. of the Northern Portuguese coastline: Chemical approach by HPLC-DAD- ESI/MSn and UPLC-ESI-QTOF/MS. Algal Res 2018 Jan; 29: 113–120.

IX PUBLICATIONS

8. Barbosa M, Lopes G, Valentão P, Ferreres F, Gil-Izquierdo Á, Pereira DM, Andrade PB. Edible seaweeds’ phlorotannins in allergy: a natural multitarget approach. (Under review)

9. Barbosa M, Lopes G, Andrade PB, Valentão P. Inflammation and allergy network: The multimodal actions of brown seaweed phlorotannins. (Manuscript in preparation)

BOOK CHAPTER:

1. Barbosa M, Valentão P, Andrade PB. Astaxanthin and fucoxanthin: Promising marine xanthophylls with therapeutic potential. Accepted for publication in Encyclopedia of Marine Biotechnology, Kim SK (Ed.). Wiley- Blackwell, New Jersey, USA.

ORAL COMMUNICATION:

1. Barbosa M, Fernandes F, Pereira DM, Valentão P, Ferreres F, Gil- Izquierdo Á, Andrade PB. UHPLC-QqQ-MS/MS method for phytoprostane profiling in macroalgae. 11th National Meeting of Organic Chemistry and 4th Meeting of Therapeutic Chemistry. December 1–3, 2015. Porto, Portugal.

POSTER COMMUNICATIONS:

1. Andrade PB, Lopes G, Barbosa M, Weber GM, Pinto E, Valentão P. Exploring seaweeds: the potential of phlorotannins. 8th ISANH Congress on Polyphenols Applications. June 5–6, 2014. Lisboa, Portugal.

2. Barbosa M, Collado-González J, Ferreres F, Valentão P, Fernandes F, Pereira DM, Gil-Izquierdo Á, Andrade PB. Non-enzymatic α-linolenic acid derivatives in macroalgae: Phytoprostane profiling. 2nd EuCheMS Congress on Green and Sustainable Chemistry (EuGSC). October 4–7, 2015. Lisboa, Portugal.

X PUBLICATIONS

3. Andrade PB, Barbosa M, Lopes G, Ferreres F, Gil-Izquierdo Á, Pereira DM, Valentão P. Marine algal polyphenols: Phlorotannin-targeted extracts from Fucus spp. and their anti-inflammatory potential. XXIX International Conference on Polyphenols and 9th Tannin Conference. July 16–20, 2018. Madison, USA.

XI AUTHOR STATEMENT

The author declares to have afforded a major contribution to the technical execution, interpretation of the results and manuscript preparation of all works included in this thesis, with the collaboration of other coauthors.

XII ACKNOWLEDGMENTS

ACKNOWLEDGMENTS

Accomplishing this PhD thesis would not have been possible without the contribution of several people and institutions to whom I would like to thank:

To “Fundação para a Ciência e a Tecnologia” (FCT) for granting me a Doctoral scholarship (SFRH/BD/95861/2013) under the POPH – QREN – Type 4.1 – Advanced Training, funded by the European Social Fund (FSE) and by National funds from the “Ministério da Educação e Ciência”, and by Programa de Cooperación Interreg V-A España–Portugal (POCTEP) 2014–2020 (project 0377_IBERPHENOL_6_E).

To Prof. Doctor Paula Cristina Branquinho de Andrade, my supervisor, for the continuous support of my PhD. I am gratefully indebted to her for accepting me in the Laboratory of Pharmacognosy of the Faculty of Pharmacy of the University of Porto and for all the years of guidance and encouragement. With her, I began my humble path in research, always as her dedicated student. I have always admired her professional journey and her charisma, which incented me to pursue the PhD. Prof. Paula consistently allowed this thesis to be my own work but steered me in the right direction whenever she thought I needed. In fact, without her valuable inputs this thesis could not have been successfully conducted. I could not have imagined having a better supervisor and mentor for my PhD. Thank you.

To Prof. Doctor Patrícia Carla Ribeiro Valentão, co-supervisor of this thesis, for her uninterrupted patience and insightful recommendations. Whenever I ran into a trouble spot or had a question about my research or writing, her door was always open. Prof. Patrícia’s attention to detail drove me to be better and her hard questions incented me to widen my research from various perspectives. For all this, I would like to express my very great appreciation.

To Prof. Doctor Federico Ferreres, from Centro de Edafología y Biología Aplicada del Segura (CEBAS), of Consejo Superior de Investigaciones Científicas (CSIC), Murcia, Spain, for his availability and essential contribution for the identification of phlorotannins by HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS. His willingness to give his time so generously has been very much appreciated.

To Prof. Doctor Ángel Gil-Izquierdo, also from CEBAS-CSIC, for his assistance in conducting the UHPLC-QqQ-MS/MS analysis of phytoprostanes.

XIII ACKNOWLEDGMENTS

To my PhD colleagues and to all the staff of the Laboratory of Pharmacognosy of the Faculty of Pharmacy of the University of Porto, for their companionship, for the stimulating discussions and for contributing to the normal functioning of the lab and the development of this thesis.

To my dear friends that even under the toughest circumstances always made me laugh. I sincerely appreciate your support.

To Lara Reis, my friend of a lifetime, for her precious encouragement and unceasing support.

To my family, particularly my grandmother Belmira, my uncle Serafim, my cousin Ana, my brother, and my nephew Francisco, for their care and support.

To Hugo Santos, for all his dedication, understanding and care. Thank you for always being there.

To my parents, for their never-ending support and unconditional love. None of this would have been possible without them.

XIV

ABSTRACT

Among the wealth of biodiversity characterizing the marine environment, macroalgae, commonly addressed as seaweeds, have proved their auspicious ecological roles, as well as their chemical and biological potential. Seaweeds are an abundant and heterogenous group of photosynthetic organisms, distributed worldwide and endowed of unique molecules with high impact in food science, pharmaceutical industry, and public health. Within seaweed groups, the brown ones (Ochrophyta) stand out, as one of the most prolific producers of functional compounds. To harness the biotechnological potential of the Portuguese marine flora, several seaweed species were analyzed and explored for their chemical composition and biological activities. Moreover, as seaweed cultivation has become more widespread, there is a need to expand the knowledge on this material. Therefore, seaweeds grown in integrated multi-trophic aquaculture (IMTA) systems were also studied.

A complex fatty acid profile, characterized mainly by the presence of medium and long fatty acyl chains (14–22 carbon atoms), with different degrees of unsaturation, was observed in Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saunders, Saccorhiza polyschides (Lightfoot) Batters, and Laminaria ochroleuca Bachelot de la Pylaie tissues subjected to seasonal variations, from different sources (wild and aquaculture), and cultivated at different depths at sea. The specimens of S. latissima, S. polyschides, and L. ochroleuca also exhibited a variable composition in terms of carotenoids and chlorophylls. In these works, a major contribution of surrounding environmental conditions, as well as of species-specific factors, was observed for both fatty acid and pigment composition.

The free phytoprostane composition was assessed, for the first time, in seaweed material through advanced mass spectrometry-based analysis. The profile of phytoprostanes varied greatly, F1t-phytoprostanes and L1-phytoprostanes being the predominant and the minor classes, respectively. No correlation was observed between the phytoprostane content and the amounts of α-linolenic acid, their known precursor, reinforcing the influence of intra- and inter-species interactions on chemical signatures.

Different species of Fucus, the most prominent and species-rich genus within the order Fucales, were analyzed for their phlorotannin composition. Isomers of fucophlorethol, dioxinodehydroeckol, difucophlorethol, fucodiphlorethol, bisfucophlorethol, fucofuroeckol, trifucophlorethol, fucotriphlorethol, tetrafucophlorethol, and of fucotetraphlorethol were tentatively identified in purified extracts of Fucus guiryi Zardi, Nicastro, E.S. Serrão & G.A. Pearson, Fucus serratus Linnaeus, Fucus spiralis

XVII ABSTRACT

Linnaeus, and Fucus vesiculosus Linnaeus. The characterized phlorotannins exhibited generally low degree of polymerization (3–6 phloroglucinol units) and belonged mainly to the class of fucophloretols, suggesting a relationship between taxon and the structural type of phlorotannins.

In addition to the chemical profiling, the anti-inflammatory and anti-allergic potential of the phlorotannin-targeted extracts was addressed.

The extracts were able to dose-dependently inhibit lipoxygenase (LOX) activity and to scavenge nitric oxide (NO) radical in cell-free assays, both being correlated with the total phlorotannin content. The overproduction of NO induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages was also efficiently surmounted by non-cytotoxic concentrations of phlorotannin extracts, evidencing their potential benefits in inflammation-related conditions.

Likewise, phlorotannin-targeted extracts from Fucus spp. strongly inhibited allergy- related enzymatic systems. In particular, F. guiryi and F. serratus extracts (the ones with higher phlorotannin amounts) inhibited hyaluronidase (HAase) more efficiently than the reference drug disodium cromoglicate (DSCG). The purified extracts were also able to reduce RBL-2H3 basophils’ degranulation induced by either antibody-antigen complex or by calcium ionophore and, again, strong correlations were found between the above- mentioned effects and the amount of phlorotannins in the extracts.

The outcomes of these studies point to the potential interest of the use of the selected seaweed species as both food and for nutraceutical and/or pharmaceutical applications. In particular, the multi-target capacity addressed to phlorotannin-targeted extracts supports the potential of these polyphenols as valuable naturally occurring pharmacological alternatives with a large spectrum of activity, making high-purity extracts potential candidates for functional foodstuffs.

Keywords: Seaweeds; Fatty acids; Pigments; Phytoprostanes; Phlorotannins; Inflammation; Allergy.

XVIII

RESUMO

Entre a incalculável biodiversidade que caracteriza o ambiente marinho, as macroalgas têm provado a sua importância ecológica e o seu promissor potencial biotecnológico. A distribuição destes organismos fotossintéticos é ubiquitária e a heterogeneidade de espécies traduz-se numa vasta diversidade química, com particular relevância na indústria farmacêutica e alimentar. As espécies de macroalgas castanhas (Ochrophyta) destacam-se como fontes prolíficas de compostos funcionais. No sentido de valorizar o potencial biotecnológico da flora algal Portuguesa, diferentes espécies de macroalgas foram exploradas no que respeita à sua composição química e atividades biológicas. Tendo em conta o aumento da produção de macroalgas em aquacultura e a necessidade de aprofundar o conhecimento deste material, foram também objeto de estudo espécies cultivadas em sistemas de aquacultura multi-trófica integrada (IMTA).

Foi observado um perfil de ácidos gordos complexo, caracterizado principalmente pela presença de ácidos gordos de cadeia média e longa (14–22 átomos de carbono) e com diferentes graus de insaturação, em tecidos de Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saunders, Saccorhiza polyschides (Lightfoot) Batters, e Laminaria ochroleuca Bachelot de la Pylaie, desenvolvidos sob a influência de diferentes fatores. Também ao nível da composição em carotenoides e clorofilas, S. latissima, S. polyschides e L. ochroleuca apresentaram perfis variáveis. A variabilidade observada no perfil químico das diferentes algas demonstrou não só a expressão da individualidade de cada espécie, como também a complexa influência de fatores ambientais na distribuição e acumulação de compostos.

Pela primeira vez determinou-se o perfil de fitoprostanos de diferentes espécies de macroalgas, por espetrometria de massa. Nas amostras analisadas, F1t-fitoprostanos e L1- fitoprostanos foram, respetivamente, os compostos maioritários e minoritários. Não se observou qualquer correlação entre os níveis de fitoprostanos nas amostras e o conteúdo do seu percursor (ácido α-linolénico), reforçando, uma vez mais, a relevância de fatores intra e interespecíficos para a composição química.

Diferentes espécies de algas do género Fucus, o mais proeminente e rico em espécies dentro da ordem Fucales, foram analisadas relativamente à sua composição em florotaninos. Isómeros de fucofloretol, dioxinodesidroecol, difucofloretol, fucodifloretol, bisfucofloretol, fucofuroecol, trifucofloretol, fucotrifloretol, tetrafucofloretol, e de fucotetrafloretol foram tentativamente identificados nos extratos purificados de Fucus guiryi Zardi, Nicastro, E.S. Serrão & G.A. Pearson, Fucus serratus Linnaeus, Fucus spiralis Linnaeus e Fucus vesiculosus Linnaeus. Os florotaninos aqui caracterizados

XXI RESUMO

apresentaram, em geral, um baixo grau de polimerização (3–6 unidades de floroglucinol), sendo a maioria pertencente à classe dos fucofloretóis, sugerindo a existência de uma relação quimiotaxonómica.

Para além do estudo da composição química, foi também explorado o potencial anti- inflamatório e anti-alérgico dos extratos purificados de florotaninos.

Em sistemas não celulares, os extratos inibiram a atividade da enzima lipoxigenase (LOX) e sequestraram o radical óxido nítrico (NO), de forma dependente da concentração. Ambos os efeitos mostraram estar correlacionados com o teor de florotaninos dos extratos. Concentrações não citotóxicas dos extratos foram capazes de reduzir o NO produzido pela linha celular de macrófagos RAW 264.7, quando estimulados com lipopolissacarídeo bacteriano (LPS), evidenciando os seus potenciais benefícios em patologias com um processo inflamatório associado.

De igual modo, os extratos purificados de florotaninos inibiram enzimas relevantes em condições alérgicas. Os extratos obtidos de F. guiryi e F. serratus, aqueles com maior conteúdo de florotaninos, foram particularmente promissores e mais eficientes na inibição da hialuronidase (HAase) do que o fármaco de referência, cromoglicato dissódico (DSCG). Os extratos purificados reduziram também a desgranulação de basófilos RBL-2H3 quando estimulados por um complexo anticorpo-antigénio ou por um ionóforo de cálcio e, uma vez mais, foi observada a existência de forte correlação entre os efeitos descritos e o teor de florotaninos nos extratos.

Os principais resultados destes estudos apontam para o potencial promissor do uso das espécies de algas selecionadas, quer como alimento, quer para aplicações nutracêuticas e/ou farmacêuticas. Em particular, a capacidade de atuação em múltiplos alvos dos extratos purificados de florotaninos reforça o potencial destes polifenóis como alternativas farmacológicas de origem natural com um grande espetro de atividade, tornando extratos de elevado grau de pureza candidatos interessantes para o desenvolvimento de formulações farmacêuticas e/ou de alimentos funcionais.

Palavras-chave: Macroalgas; Ácidos gordos; Pigmentos; Fitoprostanos; Florotaninos; Inflamação; Alergia.

XXII

TABLE OF CONTENTS

PUBLICATIONS ...... IX ACKNOWLEDGMENTS ...... XIII ABSTRACT ...... XVII RESUMO ...... XXI LIST OF FIGURES ...... XXXI LIST OF TABLES ...... XXXVII LIST OF ABBREVIATIONS ...... XLI

THESIS OUTLINE ...... 1

CHAPTER I – INTRODUCTION AND OBJECTIVES

1. Introduction ...... 5 1.1. The marine biome: A treasure of biological and chemical diversity ...... 5 1.1.1. Bioprospecting seaweeds for human health: Challenges and opportunities ...... 6 1.1.2. Seaweeds and the Portuguese seascape ...... 8 1.2. Brown seaweeds: A special focus ...... 10 1.2.1. The order Laminariales ...... 10 1.2.2. The order Fucales ...... 12 1.2.3. Functional compounds ...... 14 1.2.3.1. Fatty acids and oxidation derivatives ...... 15 1.2.3.1.1. Non-enzymatically-derived algal oxylipins: The phytoprostanes ...... 18 1.2.3.1.2. Extraction and profiling of fatty acids and phytoprostanes ...... 21 1.2.3.2. Pigments: Chlorophylls and carotenoids ...... 22 1.2.3.2.1. Pigment extraction and profiling ...... 27 1.2.3.3. Phlorotannins ...... 28 1.2.3.3.1. Extraction, purification, and profiling ...... 32 1.3. General overview into inflammation and allergy network ...... 35 1.3.1. Inflammation and multimodal actions of phlorotannins on inflammatory responses ...... 36 1.3.2. Allergy and modulation of allergic events by phlorotannins ...... 46

2. Objectives ...... 53

CHAPTER II – EXPERIMENTAL SECTION

3. Experimental Section ...... 57 3.1. Standards and reagents ...... 57 3.2. Sampling...... 59

XXV TABLE OF CONTENTS

3.3. Fatty acids and pigments ...... 60 3.3.1. Algal material ...... 60 3.3.2. Fatty acid extraction and derivatization ...... 62 3.3.3. GC/IT-MS qualitative analysis of fatty acids ...... 62 3.3.4. Fatty acid quantification by GC-FID ...... 63 3.3.5. Pigment extraction ...... 64 3.3.6. HPLC-DAD analysis of pigments ...... 65 3.4. Phytoprostanes ...... 65 3.4.1. Algal material ...... 65 3.4.2. Phytoprostane extraction ...... 66 3.4.3. UHPLC-QqQ-MS/MS analysis of free phytoprostanes ...... 68 3.5. Phlorotannin purified extracts: Composition and biological activity ...... 68 3.5.1. Preparation of phlorotannin purified extracts ...... 69 3.5.2. Phlorotannin quantification ...... 70 3.5.3. Phlorotannin qualitative profiling ...... 71 3.5.3.1. HPLC-DAD-ESI/MSn analysis...... 71 3.5.3.2. UPLC-ESI-QTOF/MS analysis ...... 71 3.5.4. Biological effects of phlorotannin purified extracts ...... 72 3.5.4.1. Anti-inflammatory activity ...... 72 3.5.4.1.1. Cell assays ...... 72 3.5.4.1.1.1. Cell culture conditions and treatments ...... 72 3.5.4.1.1.2. Cell viability ...... 72 3.5.4.1.1.3. Nitric oxide determination ...... 73 3.5.4.1.2. Cell-free assays ...... 74 3.5.4.1.2.1. Nitric oxide radical scavenging capacity ...... 74 3.5.4.1.2.2. Lipoxygenase inhibition ...... 74 3.5.4.2. Anti-allergic activity ...... 75 3.5.4.2.1. Cell assays ...... 75 3.5.4.2.1.1. Cell culture conditions and assays ...... 75 3.5.4.2.1.2. A23187-mediated cell degranulation ...... 76 3.5.4.2.1.3. IgE/antigen-mediated cell degranulation ...... 76 3.5.4.2.1.4. MTT reduction assay ...... 76 3.5.4.2.1.5. Crystal violet staining assay ...... 76 3.5.4.2.1.6. Determination of β-hexosaminidase released ...... 77 3.5.4.2.1.7. Determination of histamine released ...... 77 3.5.4.2.2. Cell-free assays ...... 78 3.5.4.2.2.1. β-Hexosaminidase inhibition ...... 78 3.5.4.2.2.2. Hyaluronidase inhibition...... 78

XXVI TABLE OF CONTENTS

CHAPTER III – RESULTS AND DISCUSSION

4. Results and Discussion ...... 83 4.1. Influence of changing environmental conditions on fatty acid and pigment composition of the kelps L. ochroleuca, S. latissima, and S. polyschides ...... 83 4.1.1. Fatty acids ...... 83 4.1.1.1. General overview ...... 83 4.1.1.2. Seasonal variation vs fatty acid profile ...... 94 4.1.1.3. Wild natural stocks vs IMTA ...... 95 4.1.1.4. Cultivation depth vs fatty acid profile ...... 98 4.1.2. Pigments ...... 100 4.1.2.1. General overview ...... 100 4.1.2.2. Harvesting period and origin vs pigment profile ...... 104 4.1.2.3. Pigment distribution within thalli ...... 105 4.1.2.4. Pigment composition of S. latissima tissues vs cultivation depth ...... 105 4.2. Non-enzymatic α‑linolenic acid derivatives from the sea: Macroalgae as novel sources of phytoprostanes ...... 107 4.2.1. Occurrence of α‑linolenic acid in macroalgae ...... 107 4.2.2. Occurrence of free phytoprostanes in macroalgae ...... 107 4.2.3. Free phytoprostanes vs α‑linolenic acid ...... 113 4.3. Phlorotannins ...... 114 4.3.1. Phlorotannin profile of Fucus spp...... 114 4.3.1.1. Phlorotannin trimers ...... 120 4.3.1.2. Phlorotannin tetramers ...... 122 4.3.1.3. Phlorotannin pentamers ...... 123 4.3.1.4. Phlorotannin hexamers ...... 124 4.3.2. Phlorotannin extracts from Fucales as bioregulators engaged in inflammation- related mediators and enzymes...... 125 4.3.2.1. Quantitative overview ...... 125 4.3.2.2. Anti-inflammatory activity ...... 127 4.3.2.2.1. Lipoxygenase inhibitory potential ...... 127 4.3.2.2.2. Effect on inflammatory mediators ...... 129 4.3.3. Phlorotannin extracts from Fucales in allergy network...... 134 4.3.3.1. Effect of phlorotannin purified extracts on cell degranulation ...... 134 4.3.3.1.1. A23187-mediated cell degranulation ...... 138 4.3.3.1.2. IgE/antigen-mediated cell degranulation ...... 139 4.3.3.2. Allergy-related enzymes ...... 143 4.3.3.2.1. β-Hexosaminidase inhibition ...... 143 4.3.3.2.2. Hyaluronidase inhibition ...... 145

XXVII TABLE OF CONTENTS

CHAPTER IV – CONCLUSIONS

5. Conclusions ...... 149

CHAPTER V – REFERENCES

6. References ...... 153

XXVIII

LIST OF FIGURES

Figure 1. Schematic representation of an IMTA system ...... 8

Figure 2. Laminaria ochroleuca (A), Saccharina latissima (B), and a young specimen of Saccorhiza polyschides attached to rocks (C) ...... 12

Figure 3. Fucus vesiculosus (A) and its air-filled bladders (B) ...... 13

Figure 4. Chemical structures of the main polyunsaturated fatty acids found in macroalgae ...... 16

Figure 5. Non-enzymatic formation of phytoprostanes from α-linolenic acid ...... 20

Figure 6. Conversion of chlorophyll a to pheophytin a catalyzed by Mg-dechelatase under acidic conditions...... 23

Figure 7. Chemical structure of the main c-type chlorophylls in seaweeds ...... 24

Figure 8. Hypothetical fucoxanthin biosynthetic pathway in brown macroalgae ...... 26

Figure 9. Proposed biosynthetic pathway of phloroglucinol and oxidative coupling between pairs of phloroglucinol free radical units to form the first dimeric phlorotannins……………………………………………………………………………………….……………….. 29

Figure 10. Structures of representatives of each phlorotannin class, highlighting their distinctive chemical features ...... 31

Figure 11. 2,4-Dimethoxybenzaldehyde (DMBA) colorimetric reaction ...... 34

Figure 12. Schematic representation of the main allergy and inflammation targets of phlorotannins ...... 39

Figure 13. Studied Portuguese waters with the sampling sites marked with numbers .... 59

Figure 14. Schematic representation of the protocol for phytoprostane extraction from macroalgae samples and their profiling by UHPLC-QqQ-MS/MS ...... 67

Figure 15. Schematic representation of the general procedure for obtaining phlorotannin purified extracts from Fucus spp. and their profiling by HPLC-DAD-ESI/MSn and UPLC- ESI-QTOF/MS ...... 69

Figure 16. Schematic representation of the general procedure for obtaining phlorotannin purified extracts from Fucus spp. for biological assessment...... 70

Figure 17. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, by mitochondrial dehydrogenases of metabolically active cells ...... 73

- Figure 18. Nitrite (NO2 ) determination by Griess assay ...... 74

Figure 19. Linoleic acid oxidation to 13-hydroperoxy-linoleic acid catalyzed by lipoxygenase (LOX) ...... 75

XXXI TABLE OF CONTENTS

Figure 20. β-Hexosaminidase-catalyzed conversion of p-nitrophenyl N-acetyl-β-D- glucosaminide into N-acetyl-β-D-glucosaminide and the yellow p-nitrophenolate product……………………………………………………………………………………………………………………77

Figure 21. Morgan-Elson reaction applied to the determination of hyaluronidase (HAase) activity ...... 79

Figure 22. Representative GC/IT-MS chromatogram of the fatty acid profile of whole- specimen samples of L. ochroleuca (Lo_W_Am_Apr13)...... 84

Figure 24. Projection of S. latissima (A1), S. polyschides (B1) and L. ochroleuca (C1), under the influence of different parameters and loadings (A2, B2 and C2) by fatty acid composition into the plane composed by the principal components PC1 and PC2 containing 63.0, 100.0 and 69.7% of the total variance for S. latissima, S. polyschides and L. ochroleuca, respectively...... 93

Figure 25. n-3 and n-6 PUFA content (mg/kg dry algae), and n-6/n-3 ratio of L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts ...... 97

Figure 27. HPLC-DAD carotenoid and chlorophyll profiles of acetone extracts from L. ochroleuca (Lo_W_IMTA_Jan13), S. latissima (Sl_W_IMTA_Apr13) and S. polyschides (Sp_W_N_Jan13) ...... 101

Figure 28. Representative UHPLC-QqQ-MS/MS chromatogram of detected phytoprostanes (C. tomentosum) (A), presumed fragmentation and MRM transitions for quantification of 9-F1t-phytoprostane (B), 9-epi-9-F1t-phytoprostane (C), 16-B1- phytoprostane (D), and 9-L1-phytoprostane (E) ...... 111

Figure 29. Projection of macroalgae (A) and loadings (B) by phytoprostane composition into the plane composed by principal components PC1 and PC2 containing 92.9% of the total variance ...... 112

Figure 30. Extracted Ion Chromatograms (EIC) obtained from HPLC-DAD-ESI/MSn of purified phlorotannin extracts from wild-sourced and aquaculture-grown F. vesiculosus (Fves-w and Fves-a, respectively), F. guiryi (Fg), F. serratus (Fser), and F. spiralis (Fspi)………………………………………………………………………………………………………………………117

Figure 31. Proposed fragmentation patterns of the structures tentatively identified in phlorotannin purified extracts from Fucus spp...... 121

Figure 32. Mass spectra analysis of the pentamer 18 detected in wild-sourced F. vesiculosus (Fves-w)...... 124

Figure 33. Phlorotannin content in the purified extracts from Fucus spp...... 126

XXXII TABLE OF CONTENTS

Figure 34. Lipoxygenase (LOX) inhibition of phlorotannin purified extracts in cell-free assay ...... 127

Figure 35. Effect of phlorotannin purified extracts on the viability and NO levels of RAW 264.7 cells pre-treated for 2 h with the extracts, followed by 22 h co-treatment with LPS (1 μg/mL) or vehicle (culture medium) ...... 130

Figure 36. Nitric oxide radical (•NO) scavenging of phlorotannin purified extracts in cell- free assay ...... 133

Figure 37. Effect of IgE/antigen (A) and calcium ionophore A23187 (B) on the cell viability (MTT reduction), and on β-hexosaminidase and histamine released from RBL- 2H3 cells ...... 136

Figure 38. Effect of phlorotannin purified extracts on the viability of RBL-2H3 cells with and without stimulation by the calcium ionophore A23187 or by IgE/antigen ...... 137

Figure 39. Effect of phlorotannin purified extracts on β-hexosaminidase and histamine released from RBL-2H3 cells when stimulated with the calcium ionophore A23187 ...... 138

Figure 40. Effect of phlorotannin purified extracts on β-hexosaminidase and histamine released from RBL-2H3 cells when stimulated with IgE/antigen ...... 140

Figure 41. β-Hexosaminidase inhibition of phlorotannin purified extracts in cell-free systems ...... 144

Figure 42. Hyaluronidase (HAase) inhibition of phlorotannin purified extracts in cell- free systems ...... 145

XXXIII

LIST OF TABLES

Table 1. Anti-inflammatory effects of phloroglucinol (basic unit) and phlorotannins isolated from brown seaweeds ...... 40

Table 2. Anti-inflammatory effects of phlorotannin-rich extracts/fractions obtained from brown seaweeds ...... 43

Table 3. Anti-allergic effects of phloroglucinol (basic unit) and phlorotannins isolated from brown seaweeds ...... 49

Table 4. Anti-allergic effects of phlorotannin-rich extracts/fractions obtained from brown seaweeds ...... 51

Table 5. Characterization of macroalgae material used for fatty acid and pigment analysis………………………………………………………………………………………………………………….. 61

Table 6. Regression equations, r2, linearity, limit of detection (LOD), and limit of quantification (LOQ) for FAME with the employed analytical conditions ...... 64

Table 7. Characterization of macroalgae samples used for phytoprostane analysis ...... 66

Table 8. Characterization of macroalgae samples used for phlorotannins analysis and biological studies ...... 69

Table 9. Saturated fatty acid content of L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts (mg/Kg dry algae) ...... 88

Table 10. Unsaturated fatty acid content of L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts (mg/Kg dry algae) ...... 90

Table 11. Carotenoids and chlorophylls content in L. ochroleuca, S. latissima and S. polyschides extracts (mg/kg dry algae) ...... 102

Table 12. α-Linolenic acid (g/Kg dry algae) and phytoprostane (ng/100 g dry algae) content in the analyzed macroalgae species ...... 109

Table 13. Retention times (Rt), molecular formula and mass spectrometric data of molecular ions and main observed fragments of phlorotannins in the extracts of wild- sourced F. vesiculosus (1, 7, 14–19, 21, 22), aquaculture-grown F. vesiculosus (5, 7, 13, 20, 22), F. guiryi (4–6, 8, 10–12), F. serratus (2, 5, 9, 11) and F. spiralis (2, 3, 7, 10)………………………………………………………………………………………………………………….……. 118

Table 14. IC50 values found for the phlorotannin purified extracts on LOX inhibition .. 129

Table 15. IC50 values found for the phlorotannin purified extracts on NO levels in RAW 264.7 cell culture medium ...... 131

• Table 16. IC50 values found for the phlorotannin purified extracts on NO scavenging……………………………………………………………………………………………………………. 134

Table 17. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase and histamine released by A23187-stimulated RBL-2H3 cells ...... 139

XXXVII LIST OF TABLES

Table 18. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase and histamine released by IgE/antigen-challenged RBL-2H3 cells ...... 139

Table 19. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase inhibition ...... 144

Table 20. IC50 values found for the of phlorotannin purified extracts on hyaluronidase (HAase) inhibition ...... 146

XXXVIII

LIST OF ABBREVIATIONS

β-Hex β-Hexosaminidase AA Arachidonic acid AD Alzheimer’s disease ALA δ-Aminolevulinic acid AP-1 Activator protein-1 APC Antigen presenting cells BHA Butylated hydroxyanisole BHT Butylated hydroxytoluene BIS-TRIS Bis(2-hydroxyethyl)-amino-tris-(hydroxymethyl)-methane BMCMC Bone marrow-derived cultured mast cells BSA Bovine serum albumin CD23 Low-affinity IgE receptor CNS Central nervous system COX Ciclooxygenase CRA-1 Anti-human FcεRI antibody CVS Crystal violet staining DAD Diode array detection DE Dry extract DMAB 4-Dimethylaminobenzaldehyde DMBA 2,4-Dimethoxybenzaldehyde DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl sulfoxide DNP Dinitrophenyl DOXP/MEP 1-Deoxyxylulose 5-phosphate/2-C-methylerithrytol 4-phosphate DSCG Disodium cromoglicate EBSS Earle’s Balanced Salt Solution EI Electron impact EIC Extracted Ion Chromatogram ERK Extracellular signal-regulated kinase ESI Electrospray ionization FAME Fatty acid methyl esters FBS Fetal bovine serum FcεRI High-affinity IgE receptor GC/IT-MS Gas chromatography/ion trap-mass spectrometry GC-FID Gas chromatography-flame ionization detector

XLI LIST OF ABBREVIATIONS

GGPP Geranyl geranyl pyrophospate HA Hyaluronic acid HAase Hyaluronidase HBSS Hanks' Balanced Salt Solution HFD High-fat diet HMBG High mobility group box protein HPLC High performance liquid chromatography HUVEC Human umbilical vein endothelial cells ICAM Intercellular adhesion molecule ICR Institute of Cancer Research IgE Immunoglobulin E IL Interleukin IMTA Integrated multi-trophic aquaculture iNOS Inducible nitric oxide synthase IκB-α Inhibitor κB-α JNK c-Jun N-terminal kinase LC Liquid chromatography LLE Liquid-liquid extraction LLP Liquid-liquid partition LOD Limit of detection LOQ Limit of quantification LOX Lipoxygenase LPS Lipopolysaccharide

LTB4 Leukotriene B4 MAPK Mitogen-activated protein kinases MCP-1 Monocyte chemoattractant protein-1 MDA Malondialdehyde MMP Matrix metalloproteinase MRM Multiple reaction monitoring MS Mass spectrometry MTBE Methyl tert-butyl ether MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MUFA Monounsaturated fatty acids MVA Mevalonate NF-κB Nuclear factor-κB NP Normal phase Nrf2/HO-1 Nuclear factor erythoid-2-related factor 2 /heme oxygenase-1

XLII LIST OF ABBREVIATIONS

OPA o-Phthalaldehyde OXA Oxazolone PAR Photosynthetically active radiation PC Phosphatidylcholine PCA Principal component analysis PG Prostaglandin PGE Phloroglucinol equivalents

PI3K/Akt Phosphatidylinositol 3-kinase/protein kinase B PKS Polyketide synthase

PLA2 Phospholipase A2 PMA Phorbol 12-myristate 13-acetate PPAR Peroxisome proliferator-activated receptor PS Photosystem PTFE Polytetrafluoroethylene PUFA Polyunsaturated fatty acids QTOF Quadrupole time-of-flight rhIL-1α Recombinant human interleukin-1α ROS Reactive oxygen species RP Reverse phase SFA Saturated fatty acids SLE Solid-liquid extraction SNP Sodium nitroprusside dehydrate SPE Solid-phase extraction SST Sea surface temperature TQD Tandem quadrupole detector Th T helper THF Tetrahydrofuran TLR Toll-like receptor TNF Tumor necrosis factor TPA 12-O-tetradecanoyl-phorbol-13-acetate UAE Ultrasound-assisted extraction UCP-1 Uncoupling protein-1 UFA Unsaturated fatty acids UHPLC-QqQ- Ultra-high performance liquid chromatography coupled to triple- MS/MS quadrupole mass spectrometry UV-vis Ultraviolet-visible VCAM Vascular cell adhesion molecule

XLIII LIST OF ABBREVIATIONS

WHO World Health Organization ZO-1 Zona occludens-1 protein

XLIV

THESIS OUTLINE

This thesis is composed of five main sections:

CHAPTER I – INTRODUCTION AND OBJECTIVES

This chapter provides a broad and state-of-the art overview of the main topics from the workplan of this thesis, including information required to understand the experimental findings. The first part of the Introduction addresses the raising global interest in the use of marine functional ingredients, with special focus on those from brown seaweeds (Ochrohpyta), notwithstanding the main challenges that hinder the full exploitation of these bioresources of the oceans. Furthermore, the occurrence, biosynthesis, chemistry, and biotechnological challenges of some of the most promising functional compounds of Ochrophyta are explored in this section. The last part of this chapter encompasses a general description of the inflammatory and allergic processes, and a review of the literature concerning phlorotannins and their capacity to act upon different critical steps of both inflammatory and allergic response. The main objectives of this thesis are listed at the end of Chapter I.

CHAPTER II – EXPERIMENTAL SECTION

This section contains information on sampling procedures and experimental protocols employed for the assessment of the parameters under study.

CHAPTER III – RESULTS AND DISCUSSION

This chapter displays the main outcomes of the different works, and the approached subjects are discussed in light of the current knowledge.

CHAPTER IV – CONCLUSIONS

The main conclusions drawn from this thesis are summarized in this chapter.

CHAPTER V – REFERENCES

This section contains the bibliographic references used in the writing of this thesis.

CHAPTER I

INTRODUCTION

OBJECTIVES

INTRODUCTION

1. Introduction

1.1. The marine biome: A treasure of biological and chemical diversity

Oceans dominate the earth’s surface (71%) and harbor a wide variety of living organisms, rendering them prolific reservoirs of chemical diversity. Marine organisms thrive in a complex seawater environment, characterized by broad fluctuations of light (from complete darkness to extensive photic zones), and pressure (1 to over 1 000 atmospheres), also facing huge ranges of temperature (from -1.5 °C in ice sea to 350 °C in deep hydrothermal systems), and nutrients, in cohabitation with a high number of different species (1).

The ability to endure and adapt to a subset of surrounding factors (biotic and abiotic) relies on different adaptation mechanisms of organisms, such as the production of biologically active secondary metabolites. Unlike primary metabolism, that furnishes intermediates for the synthesis of macromolecules directly involved in growth and development of an organism, secondary metabolism is of restricted distribution and an expression of the individuality of the species (2). Secondary metabolites fulfill pivotal functions, maintaining an intricate balance with the multivariate ecological changes and mediating interactions with other organisms. Nevertheless, evidence suggests that many compounds, once considered to be strict secondary metabolites, are now known to act in both primary and secondary roles (e.g., brown seaweed phlorotannins) (3). The chemical diversity of bioactive compounds mirrors the genetic diversity of organisms and the complex habitat in which they are discovered (4). It can then be argued that the chemical profile of an organism, population or community represents an alternative source of information, providing a broad perspective of how environmental changes or pressures may influence the synthesis and activity of primary and secondary metabolites (5). Notwithstanding the eco-physiological roles, what makes marine natural products of great relevance for humans is the uniqueness of their molecular structures and the potency of their biological effects, providing important chemical scaffolds for the discovery of new drugs for the management of numerous diseases.

The marine ecosystem has then raised great curiosity and interest in several scientific fields and industries, as a prospective source for new potential drug leads. Nevertheless, research on the pharmacological properties of marine organisms is limited, and most of it remains unexplored (6). The ﬁrst serious effort on studying marine natural products dates from 1951 with the pioneering work of Bergman and Feeney (7) that resulted on the isolation of two nucleoside derivatives (spongothymidine and spongouridine) from the

5 INTRODUCTION

sponge Tectitethya crypta de Laubenfels (formely known as Cryptotethya crypta de Laubenfels). This finding led to the synthesis of cytarabine or Ara-C, a spongothymidine analogue clinically approved in 1969 and still currently used to treat different types of leukemia (8). In fact, marine natural products have their stronghold in anticancer chemotherapy: of the seven marine pharmaceuticals in current clinical use, four are anticancer drugs (8). However, the prospects of yielding novel marine-derived compounds with other valuable clinical applications are promising, as the number of compounds isolated from marine organisms reaches now almost 30 000, with hundreds of new ones being discovered every year (9).

1.1.1. Bioprospecting seaweeds for human health: Challenges and opportunities

Although it is true that bioprospecting of marine sources has provided some structurally unique marine products, the search for new biologically active compounds can be considered an almost unlimited field. The increasingly consumer awareness and demand for bio-based products has also redirected efforts towards marine bioprospecting activities and, alongside the pharmaceutical applications, both nutraceutical and cosmeceutical industries have been devoting many resources into the incorporation of marine natural products as functional ingredients (8,10). Contrary to the high-risk/high- reward pharmaceutical market, nutraceuticals and cosmeceuticals have, in general, a rapid route to commercialization, offering low risk and quicker potential return on investment (8). In fact, the global cosmetic market is now estimated to reach 675 billion dollars by 2020 (www.researchandmarkets.com/research/f2lvdg/global_cosmetics), and the global nutraceutical market, comprised of functional foods and beverages and dietary supplements, should reach 285 billion dollars by 2021 (www.researchandmarkets.com/research/8ltg7l/nutraceuticals), with marine-based products progressively occupying a large market share.

Interestingly, this renewed focus on marine bioresources relies on the ancient knowledge and empirical use of indigenous communities that have been dependent on these resources for food, medicine, and livelihood (11,12). Examples of early utilization of seaweeds (also addressed as marine algae or macroalgae) for medicinal purposes comprise the Chinese use of some species to treat thyroid-related diseases, such as goiter (16th century, Chinese herbal, ‘Pen Tsae Kan Mu’), Gelidium spp. for intestinal afflictions and dehydrated Laminaria spp. stipes for the dilation of the cervix in difficult childbirths (13). Seaweeds, in particular, have been an important part of the human diet all around the globe: in Pacific and Asian cultures, seaweeds have long been consumed in a variety of

6 INTRODUCTION

dishes; in Europe, the traditional consumption of seaweed-based foods was limited to some few countries, like Iceland, Wales and France, but recent trends have shown an increasing acceptance of seaweeds in the Western diet (10,14). Although still largely dominated by Asian countries, seaweed exploitation accounts now for a billionaire market (15). In 2014, about 28.5 million tons of seaweeds and microalgae were harvested for various purposes, including direct consumption or further processing for food, as well as for use as fertilizers and in pharmaceuticals and cosmetics (15). Over the years, promising insights into the bioactivities of extracts, fractions, and isolated compounds from seaweeds have been detailed in numerous reviews (16–25), opening doors for the development of seaweed-derived products with commercial potential.

Despite this global growing interest in the use of marine functional ingredients, there are still many challenges ahead that must be overcome for the full exploitation of marine resources. In general, the supply problem has hampered the research of marine bioactive compounds, mainly because secondary metabolites occur at relatively low concentrations, and their production varies in the same species and even within different parts of a single specimen, as result of variable environmental conditions (26,27). As means of ensuring and improving the supply of these high-value chemicals, different biotechnological approaches have been developed and optimized (28,29). Large-scale cultivation of seaweeds has been a tremendous case of success, having nearly tripled between 2000 and 2014, from 9.3 to almost 27 million tons, and providing more than 95% of the harvested seaweeds around the globe (15). Seaweeds can grow with little or no demand on fresh water in production cycles, and their growth rates exceed those of terrestrial plants (30). However, intensive aquaculture practices of single-species farms have raised some concerns, especially regarding the discharge of nutrients to coastal areas, potentially responsible for deterioration of local marine communities (31,32).

The use of ecological engineering tools, such as integrated multi-trophic aquaculture (IMTA) systems, has arisen as a practical approach for mitigating the environmental impact of monocultures (33–35), but there is still a need for regulation and establishment of ‘‘good practices” for seaweed harvesting, management, and cultivation to enhance the sustainability in the use of ecological goods and services that coastal zones provide (36). IMTA is a flexible system that aims to replicate a small-scale ecosystem, where species from different trophic or nutritional levels are incorporated, and that can be executed in both open water and land-based systems (37). In IMTA systems, seaweeds function as extractive components within a cultivation food web, assimilating fish-excreted ammonia

3- (NH3), phosphate (PO4 ) and carbon dioxide (CO2), and converting them into potentially valuable biomass (Figure 1) (38). Furthermore, in land/on-shore-based IMTA systems it

7 INTRODUCTION

is possible to manipulate key factors (e.g., light intensity and nutrient loading), allowing a high control over biomass traceability, quality, and security of supply, which are major requisites of the emergent market of functional products from seaweeds for human use (39,40).

Light Feed Clean water Water discharge

O2 O2

CO2

3- 3- PO4 PO4 NH NH3 3 CO Solid 2 wastes Seaweed pond Fish pond

Mechanical filter

Figure 1. Schematic representation of an IMTA system.

1.1.2. Seaweeds and the Portuguese seascape

Seaweeds are abundant and potentially renewable bioresources of the oceans. They drive the biodiversity and functioning of many shallow benthic ecosystems, accounting for up to 10% of the global oceanic primary production (41). Seaweeds comprise a heterogenous group of photosynthetic eukaryotic organisms, with more than 10 000 species worldwide (42). There are three macroalgae phyla, conventionally established according to their morphological pigmentation: Rhodophyta (red seaweeds), Chlorophyta (green seaweeds), and Ochrophyta (brown seaweeds). The color of Chlorophyta is due to the presence of chlorophylls a and b in the same proportions as in terrestrial higher plants. The greenish brown color of Ochrophyta is essentially attributed to the presence of fucoxanthin, combined with chlorophylls a and c. Phycobilins, such as phycoerythrin and phycocyanin, are responsible for the color of Rhodophyta (16). Besides their thallus color, seaweeds differ considerably in many ultrastructural components (e.g., presence/absence, number, and position of flagella) and biochemical features (e.g., composition of cell walls, and storage compounds) (4).

Unlike green algae, commonly found in freshwater, and even in terrestrial locations, red and brown algae are both almost exclusively marine (43). Global distribution of seaweed species is highly dynamic and physiologically constrained by nutrient availability, temperature, and light (44). Moreover, climate-related stressors, intensive fishing and

8 INTRODUCTION

other anthropogenic activities have deeply impacted nearshore ecosystems for centuries, leading to significant changes in the structure and functioning of seaweed natural beds (45).

The coast of mainland Portugal is one of the longest in the European Union, with approximately 830 km long, and a hotspot of marine diversity (46). The Portuguese coastline is subjected to particular biogeographic circumstances, receiving climatic influences from both the Atlantic Ocean and the Mediterranean Sea, which determine unique combinations of species forming macroalgal communities (46,47). In fact, a marked gradient in the distribution of macroalgal flora is evidenced: the flora of the Northern plateau is similar to that found in Central Europe (Brittany and South of the British Isles), whereas in the south, the algal flora is quite different, with a clear influence of the Mediterranean and of the North zone of the West African coast (48). Despite its biogeographic importance, the macroalgal flora of this region has not been thoroughly studied; the latest updated checklist of the benthic marine macroalgal dates back to 2009, reporting the presence of 320 species (200 Rhodophyta, 70 Ochrophyta, and 50 Chlorophyta) in the Northern Portuguese coast (47).

In Portugal, the major economic activities related to the sea are shipbuilding, shipping, and fishing; however, and, despite the high diversity of species, macroalgae are still one of the least studied and exploited aquatic resources (49). Back in the seventies, Portugal was one of the leading producers of agar (a hydrophilic colloid extracted from certain seaweeds of Rhodophyta) in the world, but the situation has changed considerably, Indonesia and China emerging as the largest producers of agar-bearing seaweeds and agar manufacturers, achieved by means of in- and offshore cultivation (50). The country’s recent focus on aquaculture development opened doors for macroalgal cultivation (38,51), and the need to expand the knowledge on this material. Recently, some young companies, together with research groups from Academia, have been promoting numerous initiatives to harness the biotechnological potential of the Portuguese marine flora (52), and considerable efforts were made on the search of functional compounds from native macroalgae species during the last decade (53–68).

Among macroalgae, Rhodophyta and Ochrophyta are known to be the most prolific producers of secondary metabolites (9,69). The latest annual “Marine Natural Products” review (9) reported the discovery of 33 new compounds from both red and brown algae, highlighting the current interest in Ochrophyta phylum, for which a large number of studies concerning biological activities is available.

9 INTRODUCTION

1.2. Brown seaweeds: A special focus

Ochrophyta represents the second largest group of seaweeds, comprising approximately 1 800 species (42). Both red and green algae originated from a primary endosymbiosis between a prokaryotic photosynthetic cyanobacterium and a non- photosynthetic eukaryotic protist host, whilst brown algae developed from a secondary endosymbiotic process involving a non-photosynthetic eukaryote and a unicellular red alga (70). Therefore, brown algae (kingdom Chromista) belong to a lineage phylogenetically distant from the red and green algae (kingdom Plantae), holding quite distinctive characteristics, such as wall composition (alginates and sulfated fucans) (71), carbon storage compounds (laminarin and mannitol) (72), the ability to synthetize both plant-like (C18) and animal-like (C20) oxylipins (73), as well as a number of lateral gene transfer events that have shaped their metabolism (74,75).

In general, brown seaweeds exhibit pronounced spatiotemporal variability and a wide range of sizes, including the largest of all algae. The large size of much brown seaweed and their distribution along the rocky intertidal and subtidal zones have made them very suitable subjects for human study, i.e. the access to most components is easily accomplished, and the large size allows the extraction, in large amounts, of the associated bioactive compounds (4).

Among the most prominent constituents of the seaweed belt on rocky shores, brown algae of the orders Fucales and Laminariales dominate the intertidal and the subtidal regions, respectively (76).

1.2.1. The order Laminariales

To date, the order Laminariales comprises 142 species (42) of large-sized brown seaweeds, commonly known as kelps. Kelps can reach up to 60 m in length and display a strong morphological thallus differentiation into holdfasts, stipes, and blades, which has been shown to translate into biochemical gradients (76–80). Though not considered taxonomically diverse, kelps are highly distinct structurally and functionally (44), and the only marine algae known to possess specialized cells for the transport of nutrients (81). Kelp species often co-exist within forests, which lie along temperate and polar coastlines, representing some of the most diverse and productive habitats on Earth, providing shelter and serving as food for a great number of associated organisms (44). They are also an economically important resource for humans, with a vast array of applications in different industrial branches, such as food, textiles, and pharmaceuticals. Owing to their rich

10 INTRODUCTION

polysaccharide composition and the current demand for clean, non-fossil fuel-based energy production, kelps have been thrown into the limelight as potential sources of biofuels (bioethanol) (45,82).

Within kelp forests, the members of the family Laminariaceae (e.g., Laminaria hyperborea (Gunnerus) Foslie and Laminaria ochroleuca Bachelot de la Pylaie), are generally the dominant canopy formers of the North East (NE) Atlantic subtidal rocky reefs (83). In the coastal waters of Portugal, kelps are only found associated with areas of intense upwelling, in which Saccorhiza polyschides (Lightfoot) Batters, L. hyperborea, L. ochroleuca and, sporadically, Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Drueh, are the most important ones (84).

L. ochroleuca is a warm-temperate kelp species, morphologically characterized by a large heavy holdfast that gives rise to a rigid stipe, which tapers as it approaches the blade (Figure 2A) (85). The genus Laminaria J.V. Lamouroux was taxonomically re-organized by Lane et al. (81), reinstating the genus Saccharina Stackhouse to embrace 18 species formerly included in Laminaria. As with most kelps, S. latissima (formerly Laminaria saccharina (Linnaeus) J.V. Lamouroux) has a strong morphological differentiation, exhibiting a long undivided frond with a frilly undulating margin (Figure 2B) (45). This kelp is biogeographically widespread and usually found from the sublittoral fringe down to a depth of 30 m (85,86). Often found at the margins of dense Laminaria forests, S. polyschides can also be dominant canopy-forming macroalgae along large stretches of the NE Atlantic coastline (87). Although S. polyschides is not a true kelp of the order Laminariales, but rather a pseudo-kelp of the order Tilopteridales, it serves a similar ecological function, being commonly treated as a kelp (45). Contrary to L. ochroleuca and S. latissima, two perennial kelp species, S. polyschides is an annual species, also displaying distinctive morphological features (a large warty holdfast, a flattened stipe with a frilly margin and a large blade divided into ribbon-like sections) (Figure 2C) (85).

11 INTRODUCTION

A B C

Figure 2. Laminaria ochroleuca (A), Saccharina latissima (B), and a young specimen of Saccorhiza polyschides attached to rocks (C). Photographs provided by Pereira, L. (2018). MACOI – Portuguese Seaweeds Website (MARE, University of Coimbra), available online at http://macoi. ci.uc.pt/.

From a human health perspective, extracts of L. ochroleuca have been found to act as a central nervous system (CNS) depressants with slight analgesic activity (88), also displaying anti-inflammatory effects (89). S. latissima extracts have also demonstrated several promising bioactivities, including anti-hyperglycemic (90), anti-inflammatory (91), anti-obesity (91), anticoagulant (92), antimicrobial (93), and antioxidant (94,95). Extracts of S. polyschides have shown antiprotozoal (96), antioxidant (58), anti-hyperglycemic (58), and more recently, anti-obesity (97) potential.

1.2.2. The order Fucales

Brown macroalgae of the order Fucales thrive across the rocky intertidal shores all around the world, particularly in cold-temperate regions, producing almost monospecific belts (4). Although to a different extent than Laminariales, brown algae of the order Fucales are also key structuring species, adjusting physical and biological factors within the colonized habitat and promoting biological diversity (98). Besides the ecological roles, Fucales is also pointed out as an economically promising group, with a vast array of applications (fertilizers, food products, drugs, cosmetics).

12 INTRODUCTION

In the cold-temperate waters of the Northern hemisphere, the genus Fucus Linnaeus is widely spread, and it is, undoubtedly, the most prominent and species-rich genus within the order Fucales. It currently comprises 71 taxonomically accepted species (42), among which Fucus vesiculosus Linnaeus (Figure 3) is, by far, the most well-known and the lectotype species (42).

Morphologically, thallus differentiation of Fucales is generally less pronounced than that of Laminariales, most sections of the thalli being photosynthetically active. In Fucus species, however, a clear distinction into holdfast, stipe, and blade also occurs, and intra- thallus variation in photosynthetic activity and pigmentation has been documented (76). The overall structure of Fucus spp. consists of a flattened, dichotomously-branched thallus that has a small stipe and a holdfast. The blades usually exhibit a central-thickened area (the midrib), and air-filled bladders can be found next to the midrib to keep the seaweed floating upright in its rocky anchorages (Figure 3), as it happens in F. vesiculosus (85).

Although the taxonomy of the Fucus genus is fairly established, new species are occasionally described; this is the case of Fucus guiryi Zardi, Nicastro, E.S. Serrão & G.A. Pearson that was elevated from variety (Fucus spiralis var. platycarpus (Thuret) Batters) to species level few years ago (99).

A B

Figure 3. Fucus vesiculosus (A) and its air-filled bladders (B). Photographs of Mariana Barbosa.

Fucus is one of the oldest genera of macroalgae described and its uses are vast: species of Fucus are edible and their extracts have been incorporated into cosmetic preparations, and used in seaweed baths, for hundreds of years (85,100). Fucus spp. have also been described as an alternative to chemical pesticides for the management of plant diseases (101,102), but their most popular application, particularly of F. vesiculosus, one of the strongest iodine accumulators of this algae genus, is in the treatment of underactive thyroid glands (hypothyroidism) and goiter, a swelling of the thyroid gland caused by

13 INTRODUCTION

iodine deficiency (103). Several other bioactivities have been ascribed to Fucus spp. extracts, including their well-recognized antioxidant effects (58,104–106), as well as anti- hyperglycemic (58,90), anti-inflammatory (107,108), anti-cholinesterase (58), antiprotozoal (96), anti-hypertensive (66), and anti-tumor (109–111) potential.

1.2.3. Functional compounds

The discovery and development of marine bioactive compounds is a relatively new area when compared to the discovery of those from terrestrial sources. However, research in marine natural products of seaweeds has experienced significant advances in recent years, and screening of multimodal acting compounds to improve human health is indeed at the forefront of scientific innovation. In general, bioactive compounds obtained from seaweeds are structurally and chemically diverse, making their isolation, purification, and subsequent biological testing of single bioactive compounds often difficult (108). Obtaining pure compounds is an expensive process, and isolated compounds rarely have the same degree of activity as an extract, at comparable concentrations or dose of the isolated single constituents (112). The superior effectiveness of many herbal drug extracts used in traditional medicine, in comparison to the single components thereof, can be the result of synergism phenomena between the overall constituents of the extract (113). Besides, a shift in the “one drug, one target” paradigm to multitarget approaches has been encouraged as potential strategies for the management of diseases of multifactorial etiology and complex pathophysiology (114). The multitarget capacity of natural compounds can be, therefore, a promising therapeutic asset.

The employment of new, faster, and environmental-friendly technologies is becoming a primary concern in laboratories devoted to the extraction of natural compounds; however, when working with marine organisms, the conventional solvent- and time- consuming extraction techniques are still the most common practice around the world. In general, it is more challenging to obtain large quantities of bioactive compounds from marine organisms than from terrestrial species. It is therefore understandable that exhaustive extraction procedures, like Soxhlet extraction and maceration, are still used to extract marine compounds (115). Extraction from the algal biomass is also technically challenging and is usually not selective, resulting in complex mixtures of major compounds, namely fatty acids, photosynthetic pigments, phenolic compounds (i.e., phlorotannins), among others (116).

14 INTRODUCTION

1.2.3.1. Fatty acids and oxidation derivatives

The ability to synthesize a variety of lipids is essential to all organisms and, among the known algal functional compounds, lipids have indeed raised considerable interest. Fatty acids, in particular, are important components of cell membranes that play a key role in growth and development of organisms (e.g., as sources of energy) (117). They are carboxylic acids with long aliphatic chains that may be straight or branched, saturated or unsaturated (118). In general, the first committed step of fatty acid synthesis is the condensation of acetyl-CoA and malonyl-CoA units to form an aceto-acetyl group, which undergoes reduction of the carbonyl at C-3 to a methylene group, resulting in the formation of a saturated C4 (butyryl) group. Successive rounds of condensation and reduction yield palmitic acid (C16:0), which can then undergo separate elongation and/or unsaturation, being the precursor of other long-chain fatty acids (119).

Fatty acids are commonly classified as monounsaturated (MUFA) and polyunsaturated (PUFA), according to the number of double bonds present in the fatty acyl chain. Within PUFA, four series (n-3, n-6, n-7, and n-9) can be recognized, depending on the position of the first double bond from the methyl end. PUFA of n-3 and n-6 series have opposing physiological functions and their balance is important for normal growth and development (116). In humans, both n-3 and n-6 PUFA are metabolized into important biologically active mediators, including prostaglandins, thromboxanes, and leukotrienes, collectively termed eicosanoids, which have been implicated in various pathological processes, such as chronic inflammation (120) and cancer (121). While eicosanoids derived from n-6 PUFA are generally pro-inflammatory, those from n-3 PUFA are anti-inflammatory (122), and their biological effects depend on the maintenance of a proper balance between total n-6 and n-3 PUFA, rather than the absolute amount of each single molecule (123). In fact, a low ratio of n-6/n-3 PUFA is more desirable in reducing the risk of several emerging chronic diseases (124); however, a high n-6/n-3 PUFA ratio (of around 20-30:1) is currently found in modern Western societies, when compared to that found in human diets many years ago (125,126). Despite no specific recommendation for the maximum dietary n-6/n-3 ratio, the World Health Organization (WHO) advocates the replacement of saturated fatty acids (SFA) with PUFA in the diet (127).

Although marine fish oils are the current main PUFA sources for human nutrition, seaweeds have shown to have a preferable fatty acid pattern. The fatty acid composition of numerous seaweed species has been reported worldwide; however, several others remain to be characterized, not only to better understand their metabolism, but also to allow the manipulation of the algal lipid profile to develop sustainable, high-quality commercial

15 INTRODUCTION

products for nutraceutical application (128). When compared with other major nutrients, macroalgae usually exhibit a relatively low percentage of lipid components (1–5% per dry weight) (129); however, their PUFA content can be as high as that of land plants, or even higher (116). The distribution of fatty acids in seaweeds has shown to be specific of taxonomic groups: species of Chlorophyta contain high levels of C18 PUFA, particularly of the human-essential linoleic (C18:2n-6c) and α-linolenic (C18:3n-3c) acids; Rhodophyta are generally rich in C20 PUFA, eicosapentaenoic acid (C20:5n-3c) being the dominating one, followed by arachidonic acid (C20:4n-6c); species of Ochrophyta exhibit both C18 and C20 PUFA in appreciable amounts (Figure 4) (118,130).

Linoleic acid α-Linolenic acid

Stearidonic acid Eicosapentaenoic acid

Docosahexaenoic acid Arachidonic acid

Figure 4. Chemical structures of the main polyunsaturated fatty acids found in macroalgae.

Apart from differences between species, studies have also demonstrated variations in fatty acid qualitative and quantitative profiles, under changing environmental/growth conditions (80,86,131–133), and within algal organs (76,80). For instance, an increase in fatty acid unsaturation has been broadly accepted as a general acclimation response of macroalgae to cold-water conditions, as means of retaining membrane fluidity and to protect photosynthetic machinery from low temperature photoinhibition (118,134). In fact, PUFA are active participants in the signal transduction chain following elicitor recognition, serving as precursors for the biosynthesis of a myriad of secondary metabolites involved in algal defense and promoting their survival, the so-called oxylipins (reviewed by (22)).

The biosynthesis of oxylipins, oxygenated derivatives of PUFA, is highly dynamic and occurs as both a developmentally regulated mode (catalyzed by enzymatic systems) and as

16 INTRODUCTION

a response to abiotic and biotic stresses (chemical (auto)oxidation) (135). Both processes often occur concomitantly and may influence each other, even producing structurally related molecules. Combined enzymatic and non-enzymatic peroxidation builds the natural peroxide status of membranes. It is the further rearrangement or metabolism of membrane lipid peroxides, by enzymatic and non-enzymatic mechanisms, that results in the accumulation of a far greater variety of secondary oxidation products (22).

The primary hydroperoxide products are converted into a large variety of oxylipin classes, through an array of alternative and subsequent reactions, having crucial signaling functions in different organisms (136–144). In fact, oxylipins’ cellular functions are as diverse as oxylipins themselves (135). This family of structurally diverse metabolites is ubiquitously distributed in nature, being found in plants, animals, bacteria, mosses, and algae (136,145). In algal systems they appear to be involved in systemic defense mechanisms, accumulating in response to wounds (146,147), pathogen infection (148), metal toxicity (73,149–151), desiccation (152,153), and other kinds of stress (154–156).

Due to the wealth of novel oxylipin structures encountered in marine organisms, the uniqueness of their biosynthetic pathways, and the potency of their biological effects, marine oxylipins have been recent targets of lipid research. The overwhelming majority of marine oxylipins arise from lipoxygenase (LOX) metabolism of PUFA precursors, having a variety of carbon lengths (C16 to C22) and unsaturation patterns (n-3, n-6, n-9) (137). On the other hand, before it became possible for enzymatic oxylipin signaling pathways to evolve, another reaction sequence that gives rise to a great variety of oxylipins was already present in all aerobic PUFA-containing organisms: free-radical-catalyzed non-enzymatic lipid peroxidation. This early chemical process, which has prevailed throughout the evolution of the oxylipin pathways, can be catalyzed by reactive oxygen species (ROS), generated continuously during normal aerobic metabolism (157). However, a massive production of ROS can likely represent a hallmark of defense responses to a variety of abiotic and biotic stresses. Non-enzymatic reactions are therefore widespread in organisms, even in healthy ones, and because they often evade genetic studies, their relevance can be difficult to estimate. So far, most studies focused on the non- enzymatically-derived oxylipins, the so-called phytoprostanes, are from higher terrestrial plants and information regarding the occurrence of this large family of biologically active oxidized lipids in macroalgae is still scarce. However, the presence of α-linolenic acid, the known phytoprostane precursor, in macroalgae, along with the broad fluctuations of environmental conditions that characterize the marine ecosystem, suggest that they could be valuable sources of phytoprostanes (158).

17 INTRODUCTION

1.2.3.1.1. Non-enzymatically-derived algal oxylipins: The phytoprostanes

The occurrence and distribution of algal oxylipins from non-enzymatic reactions is highly unpredictable, differing between species and as a consequence of the surrounding growth conditions. However, the biosynthesis of phytoprostanes (Figure 5) is proposed to be initiated by the attack of ROS to α-linolenic acid, yielding a linolenate radical that readily oxidizes and cyclizes to complex regio- and stereoisomeric prostaglandin-like compounds (159). Two regioisomeric series (16- and 9-series) can be generated according to the position where the hydrogen abstraction occurs, and the oxygen atoms are inserted into the PUFA backbone (160). G1-phytoprostanes can spontaneously decay, forming malondialdehyde (MDA), or be the precursors of different classes of phytoprostanes, named in analogy with the prostaglandin nomenclature system as A1-, B1-, D1-, E1-, F1-, dJ1-, and L1-phytoprostanes, the latter being the regioisomer of B1-phytoprostanes (Figure 5) (161). Thus, a myriad of oxygenated lipids is generated, some of which remain anchored in membranes, while others are released. Ritter et al. (151) have described the accumulation of A1-phytoprostanes in the brown macroalgae Ectocarpus siliculosus (Dillwyn) Lyngbye subjected to copper stress, thus supporting the occurrence of ROS- mediated lipid peroxidation processes (151). These results also suggest the involvement of phytoprostanes in macroalgae defense responses. In fact, previous reports in land plants have shown that phytoprostanes exert a wide range of biological activities, inducing the biosynthesis of secondary metabolites, the expression of genes involved in detoxification processes, and the regulation of the oxidative stress-related mitogen-activated protein kinase (MAPK)-dependent signaling pathway (162–164). Despite these, the exact role and physiological function of phytoprostanes have not been yet fully elucidated. Moreover, and because these metabolites also play a crucial role in both mammalian physiology and disease, interest in the structural chemistry, biosynthesis, and pharmacological activities of these marine products has increased.

The interest in phytoprostanes targets two general areas: their use as biomarkers of oxidative stress in plant-derived foodstuffs and as bioactive mediators with potential benefits in different biological systems. Evidence points to the involvement of certain phytoprostane classes in the regulation of immune function in humans. E1- phytoprostanes, previously identified in pollen, inhibited dendritic cell interleukin (IL)-12 production and increased T helper (Th) type 2 cell polarization in vitro (165). In contrast,

Gutermuth et al. (166) found that both E1- and F1-phytoprostanes partially inhibited Th1 and Th2 cytokine production in vivo (166). The immunomodulatory effects of E1- phytoprostanes were found to occur via peroxisome proliferator-activated receptor (PPAR)-γ and nuclear factor-κB (NF-κB)-dependent mechanisms (167,168). Karg and co-

18 INTRODUCTION

workers (169) reported that A1- and dJ1-phytoprostanes displayed anti-inflammatory effects in human embryonic kidney cells and RAW 264.7 murine macrophages, by down- regulating NF-κB and inhibiting nitric oxide (NO) synthesis, respectively (169). Minghetti et al. (170) showed that B1-phytoprostanes were biologically active in experimental models of immature cells of the central nervous system, exhibiting neuroprotective effects against oxidant injury induced by hydrogen peroxide (H2O2) and promoting myelination through PPAR-γ activation (170). Still, further studies are required to decipher the real importance of natural dietary sources of phytoprostanes, including marine macroalgae, in human health.

Considering the key roles in different biological systems, it comes as no surprise that detection, identification, and proper quantification of lipid components are prerequisites for macroalgal potential utilization and exploitation (118,171).

19 INTRODUCTION

α-Linolenic acid ROS -H11 -H14

G1-Phytoprostanes

Reduction Reduction Reduction MDA

F1-Phytoprostanes E1-Phytoprostanes D1-Phytoprostanes

Dehydration Dehydration

16-Phytoprostane series

R: C7 H14COOH ' R : C2H5

9-Phytoprostane series

R: C2H5 A1-Phytoprostanes dJ1-Phytoprostanes ' R : C7 H14COOH

B1-Phytoprostanes

Figure 5. Non-enzymatic formation of phytoprostanes from α-linolenic acid. ROS, reactive oxygen species; MDA, malondialdehyde.

20 INTRODUCTION

1.2.3.1.2. Extraction and profiling of fatty acids and phytoprostanes

The fatty acid composition of several natural matrices, including marine macroalgae, is conventionally determined by assessing the corresponding methyl esters (FAME) through gas chromatography (GC). Prior to instrumental analysis, typical sample- processing protocols, comprising an extraction step and a derivatization process, are therefore required (172). The general procedures for macroalgae lipid research employ different organic solvents, often consisting of solvent mixtures, to allow higher recovery yields. The most common approach uses a binary system of chloroform-methanol (2:1, v/v), based on the extraction protocol previously described by Folch et al. (173). Briefly, the sample is brought into contact with the solvent mixture that dissolves the fat (chloroform), breaks down the lipid protein bonds, and inactivates the lipases (methanol), creating a mono-phase system that extracts the lipids from the sample matrix (174). Despite some disadvantages (e.g., time consuming procedures using hazardous solvents), conventional methods of lipid extraction are still the first choice to ascertain fatty acid composition in macroalgae (175).

GC has become a powerful platform and a reliable tool for the analysis of fatty acids in complex sample matrices (172,176–178). Metabolites need to be volatile and thermally stable at temperatures around 300 ºC (179). Derivatization of fatty acids increases their volatility, enhancing detection sensitivity and improving chromatographic resolution (172). Transmethylation of fatty acids usually esterified to triacylglycerols, phospholipids and glycolipids involves acid- or alkaline-catalyzed reactions. Anhydrous methanol with an acid catalyst [e.g., boron trifluoride (BF3)] is a common reagent used to generate FAME; for alkaline-catalyzed transesterification, the use of a potassium hydroxide (KOH) methanol solution is one of the simplest and most widespread approach (172,174,180). If, on the one hand, quantification of FAME is achievable by GC with flame ionization detection (FID), the qualitative analysis of FAME, especially when applied to complex sample matrices, demands for more selective and sensitive methods (181). GC coupled to mass spectrometry (MS) has proved to be especially appropriate for these analyses, as it allows the selective detection of both saturated and unsaturated fatty acids, as well as their positional isomers, thus being routinely utilized in lipidomic research (178,182). Nonetheless, a combination of both chromatographic systems may facilitate fatty acid analysis with great accuracy and precision.

The analysis of phytoprostanes in natural matrices is extremely challenging, requiring highly sensitive and specific tools for their profiling and characterization (183). The first

21 INTRODUCTION

main concern lies in sample preparation that depends greatly on the investigated matrix, as well as the subsequently selected methodological procedure used for analysis (160,184).

The first reports on phytoprostane analysis employed GC-MS methods, which, as mentioned above, require extensive and laborious sample preparation together with derivatization processes (162,183,185–187). Moreover, the great diversity granted by the presence of racemic mixtures of phytoprostanes increases the complexity of these analyses and the need for fast, robust, selective, and highly specific techniques. Liquid chromatography (LC) devices coupled to a tandem mass spectrometer, such as ultra-high- performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ-MS/MS), operating in multiple reaction monitoring (MRM) mode, have been successfully carried out for profiling all phytoprostane classes in simple methanol extracts from several natural matrices, including macroalgae, without the need of a derivatization procedure (158,188–193).

As phytoprostanes are structurally characterized by the presence of a free carboxylic group in their backbone, the choice for negative electrospray ionization (ESI) mode in MS is appropriate for their direct analysis, resulting in an abundant [M–H]- carboxylated ionic environment, which helps the detection of low concentrations of phytoprostanes (194). The indubitable identification and quantification of the target compounds is further accomplished by additional complementary MRM transitions (194). Although there is still room for improvement (e.g., the possibility for enantiomer separation), this advanced MS- based analytical method allows the detection of low levels of phytoprostanes in complex sample matrices, as well as the separation and identification of regio- and stereoisomers, involving fewer sample preparation steps, and thus being lesser prone to artifact generation (188,194).

The most recent protocols for the extraction of naturally occurring phytoprostanes involve solid-phase extraction (SPE) with different types of cartridges, as means of cleaning-up, concentrating, and decreasing the sample matrix effect for MS analysis (158,188–194).

1.2.3.2. Pigments: Chlorophylls and carotenoids

Among the great algal chemical diversity, chlorophylls and carotenoids are the main classes of photosynthetic pigments found in brown seaweeds (195). The photosynthetic machinery of algae usually hosts two photosystems (PS I and PS II), which are connected via the electron transport chain (196). Chlorophylls are the major light-harvesting

22 INTRODUCTION

pigments of the algal photosynthetic systems, containing a network of alternating single and double bonds, in their molecular backbone that delocalizes the electrons along the polyene structure. The delocalized polyene exhibits very strong absorption bands within the visible regions of the spectrum, allowing algae to absorb energy from light (197,198). Chemically, chlorophylls are defined as a group of cyclic tetrapyrrole pigments coordinated to a central magnesium atom (Figure 6). In a biological context, on the other hand, pheophytins (derivatives which lack the central magnesium) (Figure 6) are also included within this definition because they are active in photosynthetic electron transport (198).

Chlorophyll a

H+ Mg-dechelatase -Mg2+

Pheophytin a

Figure 6. Conversion of chlorophyll a to pheophytin a catalyzed by Mg-dechelatase under acidic conditions.

There are various types of chlorophylls and derivatives, but their distribution in macroalgae appears to be phylum specific (199). Chlorophyll a, biosynthesized from δ- aminolevulinic acid (ALA) through multiple steps, is known to occur in all algal phyla, serving as a primary photoreceptor. Chlorophyll c (Figure 7), of which there are several

23 INTRODUCTION

distinct forms, is found only in members of Ochrophyta, acting as an accessory pigment to chlorophyll a, not only by enhancing the light-harvesting properties, but also by replacing chlorophyll a in PS II (200). Chemically, members of chlorophyll c family (Figure 7) differ from other chlorophylls as their macrocycle is structurally not a chlorin, but a porphyrin (i.e., they have a fully unsaturated tetrapyrrole macrocycle). In addition, instead of the propionic acid side chain of chlorophylls a and b, chlorophylls c have a trans acrylic (propenoic) acid substituent at C-17 (ring D), which is not esterified to phytol or other aliphatic long-chain alcohol in all polar chlorophyll c pigments (201). Although the biosynthetic origin of these pigments is still unclear, it has been proposed that chlorophyll c biosynthesis may occur as a closely related branch of the chlorophyll a formation (201).

Chlorophyll c1 (R: CH2-CH3) Chlorophyll c2 (R: CH=CH2)

Figure 7. Chemical structure of the main c-type chlorophylls in seaweeds.

Besides chlorophylls, the light-harvesting complexes of brown algae also contain carotenoids, which play important roles as accessory pigments or as structural molecules that stabilize protein folding in the photosynthetic apparatus (202). Besides their key roles in oxygenic photosynthesis, both chlorophylls and carotenoids have attracted much attention as they are described as powerful antioxidants (203,204), with a vast potential to be incorporated in food products, as well as into nutraceutical and pharmaceutical preparations for human health (25,205–207).

Carotenoids are chemically divided into xanthophylls (oxygenated compounds) and carotenes (nonpolar hydrocarbons). They derive from the polymerization of isoprene units to form regular and highly conjugated C40 structures (tetraterpenes) via the mevalonate (MVA) and 1-deoxyxylulose 5-phosphate/2-C-methylerithrytol 4-phosphate (DOXP/MEP) pathways (118,206): it begins with the head-to-head condensation of two C20 geranyl geranyl pyrophospate (GGPP) molecules to produce C40 phytoene, which is sequentially modified to ζ-carotene, neurosporene and lycopene by a series of membrane-localized

24 INTRODUCTION

enzymes and, finally, cyclized to form β-carotene, an end product for photosynthesis in plants and algae, as well as a precursor for ketocarotenoids in the chloroplast and cytosol. This large family of compounds encompasses more than 750 members ubiquitously distributed in nature, 250 of which are from marine origin (208). The major carotenoids that occur in seaweeds include β-carotene, lutein, violaxanthin, neoxanthin and zeaxanthin in Chlorophyta; α- and β-carotene, lutein, and zeaxanthin in Rhodophyta; β- carotene, violaxanthin and fucoxanthin in Ochrophyta (28).

Fucoxanthin, firstly isolated from marine brown macroalgae of the genera Fucus, Dictyota and Laminaria (209), represents more than 10% of the total carotenoids in nature (210) and is considered the chemotaxonomic marker of the phylum Ochrophyta. Although its chemical structure was elucidated decades ago by Englert et al. (211), some points remain unclear regarding the biosynthetic pathway of fucoxanthin, at either the biochemical or molecular biological levels (212). The first committed step of the still putative fucoxanthin biosynthesis in Ochrophyta follows the general carotenoid pathway up to β-carotene (Figure 8). Then, and based on the knowledge in diatoms, two different pathways are proposed for fucoxanthin biosynthesis: the diadinoxanthin hypothesis and the neoxanthin hypothesis. The first involves a sequential conversion of violaxanthin to diadinoxanthin, which is the precursor of fucoxanthin. The neoxanthin hypothesis, on the other hand, establishes a branching of the pathway from neoxanthin to both diadinoxanthin and fucoxanthin (212).

Fucoxanthin has a unique carotenoid structure, including an unusual allenic bond, nine conjugated double bonds, a 5,6-monoepoxide, and some oxygenated functional groups, such as hydroxyl (OH), carbonyl and carboxyl moieties. The structure of fucoxanthin is, in fact, closely related to its biological properties: the allenic bond is responsible for the potent antioxidant activity of fucoxanthin (213), the presence of intramolecular oxygen atoms makes fucoxanthin more sensitive to radicals, especially under anoxic conditions (214), and the α,β-unsaturated carbonyl group may function as a Michael’s acceptor, which can react with import macromolecules (215). Even though the antioxidant properties of this xanthophyll have been implicated in its biological activities, it is now established that the realm of fucoxanthin potential is wider and involves other biological processes as well (216).

25 INTRODUCTION

MVA DOXP/MEP

β-carotene β-carotene hydroxylase

β-cryptoxanthin

β-carotene hydroxylase

Zeaxanthin

Zeaxanthin epoxidase Violaxanthin deepoxidase

Antheraxanthin

Zeaxanthin epoxidase Violaxanthin deepoxidase

Violaxanthin

Neoxanthin synthase

Neoxanthin

* *

Diadinoxanthin Fucoxanthin

Diadinoxanthin deepoxidase Diatoxanthin epoxidase

Diatoxanthin

Figure 8. Hypothetical fucoxanthin biosynthetic pathway in brown macroalgae. MVA, mevalonate; DOXP/MEP, 1-Deoxyxylulose 5-phosphate/2-C-methylerithrytol 4-phosphate. *Enzymes that remain unidentified in macroalgae. Adapted from (212).

The presence or absence of certain pigments and their relative content provide useful information regarding the possible acclimation or photoprotection responses of an organism (217). In response to tidal cycles and seasonal variations, brown seaweeds are able to adjust their metabolism by optimizing their pigment composition (218–220).

26 INTRODUCTION

Additionally, the strong morphological thallus differentiation exhibited by most kelps (detailed above) has already been shown to be translated into significant variability in photosynthetic pigments (76,221,222). Although it is not surprising that pigment levels vary considerably according to light environment (223), the combined influence of several other external and species-specific factors (e.g., thallus age, metabolic activity, and physiologic function) may also influence the overall pigment composition (224,225), which, by its turn, can affect the biological potential of seaweed extracts/fractions and their end uses for commercial applications. Therefore, quantifying and understanding the drivers of the observed variability in biochemical profiles may underpin more targeted and sustainable seaweed selection (226), also reinforcing that algal cultivation systems may provide an interesting approach to optimize the production of some high-value metabolites.

1.2.3.2.1. Pigment extraction and profiling

The extraction of algal pigments is technically challenging, usually requiring the combination of conventional solid-liquid extraction (SLE) procedures with mechanical disruption techniques. In a general way, pigment extraction is hampered by differences in cell wall composition and in pigment polarity (227), being also conditioned by the stability of these compounds. In fact, it has been shown that long duration extractions can increase the formation of pigment degradation products and isomerization (228,229).

Despite the diversity of protocols found in literature, the extraction of both chlorophylls and carotenoids from seaweeds has been carried out with organic polar solvents (e.g., acetone), because they have high polarity and allow their solubilization (230). Mechanical disruption techniques, including ultrasound-assisted extraction (UAE), have proved to be very effective for seaweed pigment recovery (231,232). UAE is a green and economically feasible technology, suitable for the extraction of thermolabile compounds (233), offering greater penetration of the solvent into the cellular matter and yielding higher pigment recovery rates (231). In addition, UAE provides shorter extraction times with high reproducibility, also reducing solvent and energy consumption (233). Though considerable efforts have been made to obtain reliable and more consistent pigment recovery yields, this still represents a significant challenge for pigment analysis. High performance liquid chromatography (HPLC) combined with an ultraviolet-visible (UV-vis) diode array detector (DAD) is amongst the most popular instrumentation-based methodologies for pigment identification and quantification (234,235). Although pigment separation can be carried out using both normal phase (NP)- and reverse phase (RP)- HPLC, the latter usually provides better peak resolution (235). Among the stationary

27 INTRODUCTION

phases employed for pigment analysis, polymeric, non-endcapped columns with C30 ligands provide optimal separation of carotenoids and their isomers, when compared to classical C18 stationary phases (236). Other analytical features are also determinant to ensure the correct identification of compounds in complex sample matrices. For instance, a stable column temperature is important for the reproducibility of compounds’ retention times.

1.2.3.3. Phlorotannins

Within the algal chemical diversity already unveiled, phlorotannins have been pointed out as high-value bioactive compounds (23,237).

From a chemical point of view, an extremely variable and often complex molecular assemblage from the polymerization of phloroglucinol (1,3,5-trihydroxybenzene) monomeric units is at the root of this almost inexhaustible family of naturally occurring polyphenolic entities. Although the exact phlorotannin biosynthesis in macroalgae is not yet fully understood, it is known that the aromatic precursor, phloroglucinol, is biosynthesized by the acetate-malonate pathway, also known as polyketide pathway (238), under the catalysis of polyketide synthase (PKS)-type enzyme complexes (239–242). Knowledge on phloroglucinol biosynthesis has been mostly deepen either in microorganisms capable of producing phloroglucinol compounds (e.g., Pseudomonas spp.), or through the heterologous expression of encoding genes in Escherichia coli model (239,240). Only in 2013, Meslet-Cladiere et al. (241) identified and characterized a type III PKS (PKS1) in the brown macroalgae E. siliculosus, which was found to be responsible for catalyzing the synthesis of phloroglucinol monomers from malonyl-CoA (241). The first committed step in the proposed biosynthesis of phloroglucinol (Figure 9) encompasses the decarboxylative condensation of acetyl CoA (starter) and malonyl CoA (extender) units, similar to the synthesis of fatty acids (238,243). A highly reactive methylene (acetoacetyl-CoA) is formed, assisting the polymerization process to occur without the need for high energy investment (244). A carboxyl group or its decarboxylated product (carbanion group) is necessary to be retained at the end position of the resulting linear polyketide intermediate to carry out the final C6-C1 Claisen condensation for ring closure (242). The cyclization product is an unstable triketide, which is converted to phloroglucinol, thermodynamically more stable, by tautomerization (244). The subsequent polymerization of phloroglucinol units has been proposed to occur through C- C and/or C-O-C oxidative coupling (Figure 9) (245), giving rise to a myriad of compounds.

28 INTRODUCTION

Polymerization

C-C oxidative C-O-C oxidative coupling coupling

Difucol

Diphlorethol

Figure 9. Proposed biosynthetic pathway of phloroglucinol and oxidative coupling between pairs of phloroglucinol free radical units to form the first dimeric phlorotannins. Adapted from (241,245).

29 INTRODUCTION

Structurally, phlorotannins can be divided into six different categories (phlorethols, fuhalols, fucols, fucophlorethols, eckols and carmalols), according to the nature of the linkages between phloroglucinol units, as well as to the number and distribution of OH groups (Figure 10). Within each class, it is still possible to have both structural and conformational isomers, increasing the complexity and variability of these molecules (246). In a general way, phlorethols and fuhalols are characterized by the presence of aryl- ether (C-O-C) linkages between phloroglucinol units, the latter exhibiting a regular sequence of para- and ortho- ether bonds, as well as additional OH groups in every third ring, and lack of one or more OH groups in the whole molecule. The generally low molecular weight of eckols and the presence of a phenoxyl substitution at C4 are key- structural motifs that differentiate eckols from carmalols, both being characterized by the presence of dibenzodioxin linkages (245,247). Fucols, on the other hand, consist of phloroglucinol monomers connected only by aryl-aryl (C-C) bonds, and fucophlorethols exhibit a mixture of ether and phenyl linkages between the basic unit (247) (Figure 10).

More than 150 phlorotannins were already identified (245), but the noteworthy structural heterogeneity and complexity of these compounds makes their profiling an almost unlimited field of research. These polyphenolic entities have a wide range of molecular sizes, which spans from 126 Da for the non-polymerized form to 650 kDa. However, the most common observed range is from 10 to 100 kDa (248). Phlorotannins are biosynthesized exclusively by brown seaweeds (Ochrophyta), where they can be stored in specialized membrane-bound vesicles, the physodes, or bound to cell wall. The physode-associated phlorotannins, i.e., the soluble ones, are known to occur at generally higher levels than those cell wall-bound, representing up to 25% of algal dry weight (249). Phlorotannins have been suggested to play important roles in brown seaweeds, as integral components of cell wall and acting as chemical defenses to changes in environmental conditions (e.g., salinity level, nutrient and light availability, ultraviolet radiation, and intensity of herbivory) (250–254). In fact, the amount of phlorotannins, particularly of the soluble ones, is in a state of flux, varying among different algal tissues (255), showing phenotypic plasticity in response to biotic and abiotic factors, and high genotypic variation (256). Due to their structural complexity and polymeric nature there is currently limited understanding of the array of phlorotannins in marine algae, and of their distribution within specific algal species (20).

30 INTRODUCTION

Phlorethols and fuhalols

Triphlorethol A

Trifuhalol B Tetrafuhalol A

Eckols and carmalols

Eckol Diphlorethohydroxycarmalol

Fucols Fucophlorethols

Tetrafucol A Fucophlorethol A

Fucophlorethol B

Trifucol

Figure 10. Structures of representatives of each phlorotannin class, highlighting their distinctive chemical features.

Over the last decade, studies on the biological activities of phlorotannins have increased exponentially, and a growing commercial interest on their potential application in a range of therapeutics has arisen (20,21,237). Under experimental conditions phlorotannins and phlorotannins-rich extracts/fractions have displayed numerous positive health-related effects, including antioxidant (257–259), antimicrobial (56,260),

31 INTRODUCTION

anti-hyperglycemic (261,262), antiproliferative (263,264), hepatoprotective (265,266), anti-hypertensive (66,267), photoprotective (268,269), somnogenic (270,271), neuroprotective (272,273), anti-inflammatory (54,274,275) and anti-allergic (276–278). These many outcomes support the potential of polyphenol components as valuable naturally occurring pharmacological alternatives with a large spectrum of activity and point to a multitarget capacity of phlorotannins and phlorotannin-rich extracts/fractions. The magnitude of the activity of the extracts/fractions has been shown to be significantly different from that of the individual compounds contained thereof, which makes high- purity extracts potential candidates for functional foodstuffs. Nevertheless, the possibility of designing new functional foods and pharmaceuticals is challenging, and the incorporation of phlorotannin-derived products needs further insights to ensure the relation between efficacy and safety, and in vivo studies for a more detailed understanding of the action mechanisms underlying the documented effects.

1.2.3.3.1 Extraction, purification, and profiling

Despite the high potentialities endowed to phlorotannins, some obstacles (e.g., difficulties in separation and purification steps and subsequent characterization, due to their similar polarity and polymeric structure) have hindered the development and commercialization of phlorotannin-based products.

Different approaches have been recently employed to optimize the extraction of phlorotannins, considering the putative influence of experimental conditions (e.g., pre- treatment, type of solvent, drying temperature, particle size, etc.) (279). The employment of a set of extraction variables that generate high yields of phlorotannins is then of crucial matter. The procedures used for phlorotannin extraction are widely variable, but most of them rely on SLE with either pure or water-mixed organic solvents (280). As the solubility of phenolic compounds is generally higher in polar organic solvents than in water (244), methanol and acetone aqueous mixtures have found to provide high phlorotannin extraction efficacy (54,249,281,282). When using water alone as extraction solvent, other compounds, such as polysaccharides that increase viscosity, complicating work-up (e.g., filtration), are co-extracted. On the other hand, the presence of organic polar solvents, particularly acetone, in the extraction mixture has been considered to increase the total yield, either by inhibiting protein-polyphenol complex formation (283), or by breaking down hydrogen bonds between protein-polyphenol complexes (284).

Although the type of solvent has a great impact on the amount of total extracted substances, other variables, such as temperature, must also be considered. For instance,

32 INTRODUCTION

Wang et al. (282) found that hot water extraction resulted in a significantly lower total phlorotannin content than did cold water extraction, suggesting the occurrence of thermal decomposition of some phlorotannin compounds at elevated temperatures, and a significant increase in the extraction of undesired concomitant components (282).

The procedures employed for obtaining phlorotannin purified extracts/fractions usually involve liquid-liquid partitioning (LLP) and/or SPE separation, both based on the polarity of the molecules (248,285–288), as well as discrimination of molecular size through dialysis, and/or ultrafiltration steps (246,282,289,290). Alternatively, macroporous adsorption resins have also been successfully applied for separating and purifying phlorotannins (291,292). These resins are polar, non-polar or slightly hydrophilic polymers which can absorb target components from both aqueous and non- aqueous systems, through electrostatic forces, hydrogen bonding, interaction and size sieving action (293,294). Microcrystalline cellulose is a stable, nontoxic, and chemically inactive form of hydrolyzed cellulose, consisting of extensive cellulose microcrystals together with amorphous regions (295). Although microcrystalline cellulose has been utilized as reinforcing agent in thermoplastic and thermosetting resins (295), it has also been directly applied for selective adsorption and separation of polyphenols, including phlorotannins (54,56,57,286,296,297). Benefiting from the natural adherence capacity of phlorotannins to cell wall polymers, these polyphenols, in aqueous solutions, are adsorbed on the microcrystalline cellulose, removing them from solution (57). The adsorbed phlorotannins are later desorbed using a mixture of water with an organic solvent. These adsorption-desorption processes allow the separation and subsequent purification of phlorotannins from undesirable co-extracted compounds, making the extracts of suitable purity for further analysis.

Notwithstanding the structural heterogeneity of phlorotannins and the varying degrees of polymerization of phloroglucinol basic unit, the chemical properties of these marine polyphenols are similar, making the quantitative determination of single phlorotannins in complex algal extracts hardly feasible (298). Phlorotannins have been commonly determined on crude extracts as the total amount of phenolic compounds, using non-specific spectrophotometric-based assays like Folin-Ciocalteu (248). This method relies on the transfer of electrons in alkaline medium from phenolic compounds to form a blue chromophore constituted by a phosphotungstic/phosphomolybdenum complex, which is spectrophotometrically detectable in the range of 690 to 710 nm (299). Because it measures not only the phenolic compounds, but also reacts with any reducing substance, more specific methods, such as the one employing 2,4-dimethoxybenzaldehyde (DMBA) have been considered more appropriate for phlorotannin quantification (54,245).

33 INTRODUCTION

DMBA reacts specifically with 1,3- and 1,3,5-substituted phenols, such as phloroglucinol and phlorotannins, minimizing the occurrence of interferences. The reaction is based on an electrophilic attack by the aldehyde in a strong acidic solution (300), leading to the formation of a colored product that can be spectrophotometrically measured (Figure 11). Overall, colorimetric methods are quite simple to use, giving a general estimation of the amount of phlorotannins in the extract; however, they provide no information on the qualitative phlorotannin profile, and none of the currently available methods for the quantitative determination of brown seaweed polyphenols is entirely devoid of disadvantages. For instance, the main drawback of DMBA assay is the fact that the reaction is both time- and temperature-dependent (299,300). Still, this method has been employed with good repeatability and high precision (54). Conventional colorimetric assays can measure reactive phenolics, corresponding to the physode-bound phlorotannins, but not those with structural functions (i.e., the cell-wall bound phlorotannins), which require more drastic analytical conditions (e.g., alkaline hydrolysis) to release the esterified phlorotannins (301).

+ H+ DMBA

Orange chromogen Phloroglucinol

Figure 11. 2,4-Dimethoxybenzaldehyde (DMBA) colorimetric reaction.

Chromatographic techniques arise then as powerful tools for the analysis of these algal constituents, and recent studies have achieved the tentative identification and structural characterization of phlorotannins through the employment of advanced LC-MS methods, using ESI as interfaces under positive and negative ionization modes (57,91,246,248,257,263,279,282,285,286,289,290,302–306). The MS data obtained can be compared with theoretical mono-isotopic masses corresponding to known phlorotannin oligomers (302). The Extracted Ion Chromatograms (EIC) of protonated ([M+H]+) or deprotonated ([M-H]-) molecular ions from the most common phlorotannins found in literature have been used for profiling phlorotannins in brown seaweed extracts/fractions. Furthermore, the application of sensitive methods with high mass accuracy and rapid full mode scanning, such as methods with QTOF (quadrupole time-of- flight) detector, have shown to be of great value for identification of phlorotannins

34 INTRODUCTION

(257,286,289,304,307). A QTOF analyzer has high resolution, sensitivity, and mass accuracy, allowing to establish the elemental composition of all product ions obtained, in a rapid an efficient way, which is very helpful in the elucidation process of unknowns (308,309).

For algal phlorotannins to be further exploited is necessary to develop advanced chromatographic and mass spectrometric techniques, capable of resolving extremely polar complex polymer mixtures, and elucidating the metabolite profile both in terms of their molecular weights and level of isomerization (246). Although some works have successfully employed UHPLC-tandem quadrupole detector (TQD)-MS analysis to investigate the isomeric complexity of phlorotannins present in different algal extracts (246,290,309), the full characterization of their structures was not achieved. Furthermore, efforts were made to develop and validate analytical methodologies for the commercialization of standardized phlorotannin preparations using, for instance, dieckol, as reference (310).

1.3. General overview into inflammation and allergy network

Though the pace of aging has not been uniform, it is estimated that up to 20% of the globe will be over 60 years old in the near future, and that the proportion of people at very old ages will also increase (311). As the average life expectancy continues to rise in developed countries, the health span (i.e., the functional and disease-free period of life) has become an emergent matter in modern medicine (312,313). Though most medical research is targeted at isolated diseases, a multifactorial approach should be considered, since a growing body of evidence has demonstrated that physiological changes associated with aging underlie a vast majority of chronic complex disease states (313). In the last decades, inflammatory processes have been pointed out as pathophysiological hallmarks in several age-related diseases and in the major chronic diseases of developed populations, including cardiovascular diseases, type 2 diabetes mellitus, asthma, neurodegenerative disorders, and many types of cancer (314–320). While the practical benefit of acute inflammation is to reinstall tissue homeostasis, chronic inflammation can lead to life-long debilitation, accompanied by loss of tissue function and organ failure (321). A plethora of biological entities (e.g., cells, enzymes, growth factors, cytokines, leukotrienes, prostaglandins, thromboxanes, among others) is known to participate in tightly regulated signaling pathways and critical events underlying the complex, yet coordinated, inflammation scenario. In parallel, the allergic process has an important inflammatory component, in which mast cell activation and degranulation are the first events taking place (322). Although most emphasis has been given to the inflammatory mechanism in

35 INTRODUCTION

allergy, the complex interplay between mediators and/or cell types is still not clear (323). It comes then as no surprise that targeting individual molecules, cell types or a single signaling pathway, is unlike to produce very effective therapies for the management of emerging chronic diseases.

1.3.1. Inflammation and multimodal actions of phlorotannins on inflammatory response

Inflammation has been widely acknowledged as the first line of host defense mechanism to harmful stimuli, consisting of a tightly regulated cascade of events orchestrated by both pro- and anti-inflammatory components, to ensure a quick resolution and restoration of normal tissue architecture (324). A disruption of this complex network can lead to chronic inflammation. The onset of chronic inflammation is often characterized by the replacement of neutrophils by macrophages and other cells (325). Macrophages play a pivotal role in all stages of the inflammatory response, producing pro-inflammatory cytokines, such as IL-1β and IL-6, and tumor necrosis factor

(TNF)-α, as well as inflammatory mediators like NO and prostaglandin (PG) E2 (326). An overproduction of these components is linked to the pathogenesis of several chronic diseases (324), suggesting that their modulation can constitute an attractive strategy to avoid inflammation onset and progression, as reviewed in Sugimoto et al. (327). Inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, the key-enzymes responsible for the synthesis of NO and PGE2, respectively, are primarily controlled at transcriptional levels (328,329). Transcriptional induction of iNOS and COX-2 is largely dependent on the activity of the NF-κB. In response to pro-inflammatory stimuli, the activation of NF-κB leads to the degradation of the inhibitor κB (IκB)-α, resulting in the translocation of NF- κB into the nucleus and consequent transcription of inflammatory mediators and pro- inflammatory cytokines (330). The activation of NF-κB is also regulated by MAPK, including the extracellular signal-regulated kinase (ERK), the p38 MAPK, and the c-Jun N-terminal kinase (JNK), as well as by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways (331,332).

During the inflammatory process, the biosynthesis of eicosanoids, locally acting bioactive signaling molecules, is considerably increased. Eicosanoid biosynthesis is initiated by the activation of phospholipase A2 (PLA2) and the release of arachidonic acid from membrane phospholipids, which is then transformed by COX and LOX pathways to prostaglandins, thromboxanes and leukotrienes, potent molecules that exert their effects via binding to specific membrane and nuclear receptors (333). Hence, these enzymatic

36 INTRODUCTION

systems may also constitute important targets for the development of anti-inflammatory agents.

Furthermore, the accumulation of ROS is widely acknowledged to support the inflammatory process. Although ROS play important roles in assuring life processes, an improper balance between the production of ROS and the biological system’s ability to detoxify the reactive intermediate or to repair the resulting damage can result in lipid peroxidation, protein oxidation, DNA damage and, ultimately, in cell death (334). Such injurious actions have been implicated in the pathogenesis of many chronic diseases including atherosclerosis, cancer, diabetes, rheumatoid arthritis, post-ischemic perfusion injury, myocardial infarction, cardiovascular diseases, chronic inflammation, stroke and septic shock, aging and other degenerative disorders in humans (335). A currently accepted hypothesis points to the existence of a direct interaction between ROS and pro- inflammatory markers (320), and the unveiling of this strong overlapping link has been crucial for expanding the knowledge on novel strategies to dwindle the emerging ROS- and inflammation-associated conditions. Antioxidants are believed to prevent the onset and/or to slow down the progression of numerous pathological conditions in which an imbalance between pro-oxidant and antioxidant homeostasis is involved.

Considering the multifactorial etiology of several chronic diseases, the multi-target capacity of natural compounds can be a crucial asset (336). Several mechanisms of action have been proposed to explain the anti-inflammatory effects of phytoconstituents, comprising i) antioxidant and radical scavenging activities, ii) modulation of the activity of pro-inflammatory enzymes, such as PLA2, COX and LOX, and the NO producing enzyme (iNOS), iii) modulation of the production of other pro-inflammatory molecules, and iv) modulation of pro-inflammatory gene expression (as reviewed in Bellik et al. (337)).

Within the range of marine chemical diversity, phlorotannins have been widely acknowledged as potent modulators of several biochemical processes linked to the breakdown of homeostasis in major chronic diseases (Figure 12, Tables 1 and 2) (23,237). Several studies have looked at the capacity of phlorotannins to act upon different critical steps of inflammatory response, generally using the in vitro model of RAW 264.7 macrophages following activation with the well-known bacterial endotoxin lipopolysaccharide (LPS), a potent elicitor of pro-inflammatory cytokines and mediators production (53,54,91,274,275,338–348). Nevertheless, other in vitro cell systems (349– 354), as well as cell-free (53,54,355–357) and in vivo (306,348,352,356,358) models of inflammation have been used to screen the anti-inflammatory potential of marine polyphenols.

37 INTRODUCTION

The great majority of the published data addressing the anti-inflammatory potential of phlorotannins focus essentially on the ones isolated from seaweeds of Eisenia and Ecklonia genera (Tables 1 and 2), providing an exciting perspective for further works to be developed with species that remain unexplored.

38 INTRODUCTION

Release of IgE degranulation ↑ cytokines markers ↑ growth factors ↑ chemotactic factors FCεRI IgE binding ANTIGEN β-Hexosaminidase Propagation of Histamine allergic and ↑ Ca2+ Degranulation inflammatory responses

Mast cells Basophils CD23 Influx APC 2+ Ca LEGEND Epithelial MAPK PLA NO 2 Myeloid + B cell Receptor ERK p38 L-citrulline IκB-α JNK PC NF-ΚB iNOS Calcium channel AP-1 AA Translocation ROS L-arginine C iNOS O RNA Calcium m AA X Transcription L IL-6 O X Thromboxanes Granules containing IL-1β PGE degranulation markers TNF-α Leukotrienes 2 Growth factors Antibody Matrix components Cell membrane Monocyte activation HA Phlorotannin targets HAase ↑ [HA] at the site of inflammation

CONNECTIVE TISSUE

Figure 12. Schematic representation of the main allergy and inflammation targets of phlorotannins. AA, arachidonic acid; AP-1, activator protein-1; APC, antigen presenting cells; CD23, low-affinity IgE receptor; COX, cyclooxygenase; ERK, extracellular signal-regulated kinase; FcεRI, high-affinity IgE receptor; HA, hyaluronic acid; HAase, hyaluronidase; IgE, immunoglobulin E; IL, interleukin; IκB-α, inhibitor κB-α ; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; LOX, lipoxygenase; MAPK, mitogen-activated protein kinases; NF-κB, nuclear factor-κB; NO, nitric oxide; PC, phosphatidylcholine; PG, prostaglandin; PLA2, phospholipase A2; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α.

39 INTRODUCTION

Table 1. Anti-inflammatory effects of phloroglucinol (basic unit) and phlorotannins isolated from brown seaweeds.1

Compound Species Proposed mechanism of action Model Reference

Phloroglucinol Eisenia bicyclis ↓ NO, PGE2, TNF-α, IL-6 and IL-1β LPS-stimulated RAW 264.7 cells (275,352,359,360) (Kjellman) production LPS- and HMGB-1-stimulated Setchell ↓ Barrier disruption HUVEC ↓ NF-κB and AP-1 activation PMA-stimulated HT1080 cells ↓ p38 MAPK phosphorylation LPS-stimulated HepG2 cells ↓ iNOS, COX-2, MMP-2, MMP-9, VCAM-1, ICAM-1 and E-selectin expression ↑ ZO-1 and occludin expression ↓ Leukocyte adhesion and migration

Dieckol Ecklonia cava ↓ NO, PGE2, IL-1β, TNF-α and HMBG-1 LPS-stimulated BV-2 cells (275,344,347– Kjellman production LPS- and PMA-stimulated MG- 350,352,355) Ecklonia ↓ iNOS, COX-2, TLR-4, VCAM-1, ICAM-1, E- 63 cells stolonifera selectin, MMP-1, MMP-3 and MMP-13 LPS-induced septic mice Okamura expression LPS-stimulated RAW 264.7 cells E. bicyclis ↓ NF-κB activation LPS- and HMGB-1-stimulated ↓ JNK and p38 MAPK phosphorylation HUVEC ↑ Survival rate Cell-free enzymatic systems ↓ Barrier disruption ↑ ZO-1 and occludin expression ↓ Leukocyte adhesion and migration

PLA2, COX and LOX inhibition Eckol E. stolonifera ↓ NO and HMGB-1 production LPS-stimulated RAW 264.7 cells (275,344,347,352,354– Eisenia arborea ↓ MMP-2, MMP-9, TNF-α, COX-2, iNOS, LPS- and HMGB-1-stimulated 356) Areschoug TLR-4, VCAM-1, ICAM-1 and E-selectin HUVEC E. bicyclis expression Propionibacterium acnes- ↓ Barrier disruption induced HaCaT cells ↓ NF-κB activation Mouse (ICR strain) ear edema ↓ p38 MAPK and Akt phosphorylation Cell-free enzymatic systems ↑ ZO-1 and occludin expression ↓ Leukocyte adhesion and migration ↓ AA-, TPA- and OXA-induced ear edema PLA2, COX and LOX inhibition

40 INTRODUCTION

Table 1. Cont.

Compound Species Proposed mechanism of action Model Reference Dioxinodehydroeckol E. stolonifera ↓ NO production LPS-stimulated RAW 264.7 cells (275,347) E. bicyclis

Phlorofucofuroeckol A E. stolonifera ↓ NO, PGE2, TNF-α, IL-1β and IL-6 LPS-stimulated RAW 264.7 cells (275,342– E. arborea production Mouse (ICR strain) ear edema 344,347,355,356) E. bicyclis ↓ iNOS and COX-2 expression and promoter Cell-free enzymatic systems activity ↓ NF-κB and AP-1 activation ↓ AA-, TPA- and OXA-induced ear edema

PLA2, COX and LOX inhibition 1-(3',5'-Dihydroxyphenoxy)-7- E. cava ↓ NO production LPS- and PMA-stimulated MG- (350) (2'',4'',6''-trihydroxyphenoxy)- ↓ MMP-1, MMP-3, MMP-13, iNOS and COX- 63 cells 2,4,9-trihydroxydibenzo-1,4- 2 expression dioxin ↓ JNK and p38 MAPK phosphorylation

6,6'-Bieckol E. cava ↓ NO, PGE2 and IL-6 production LPS-stimulated primary (345,347,358) E. stolonifera ↓ iNOS and COX-2 expression macrophages E. arborea ↓ NF-κB activation LPS-stimulated RAW 264.7 cells ↓ NF-κB binding to TNF-α and IL-6 Mouse (ICR strain) ear edema promoters ↓ AA-, TPA- and OXA-induced ear edema

8,8'-Bieckol E. cava ↓ NO, PGE2, IL-6 and TNF-α production LPS-stimulated primary (274,355,356) E. arborea ↓ iNOS expression macrophages E. bicyclis ↓ NF-κB activation LPS-stimulated RAW 264.7 cells ↑ Survival rate LPS-induced septic mice ↓ AA-, TPA- and OXA-induced ear edema Mouse (ICR strain) ear edema Cell-free enzymatic systems PLA2, COX and LOX inhibition 7-Phloroeckol E. bicyclis ↓ NO production LPS-stimulated RAW 264.7 cells (275)

41 INTRODUCTION

Table 1. Cont.

Compound Species Proposed mechanism of action Model Reference

Phlorofucofuroeckol B E. stolonifera ↓ NO, PGE2, TNF-α, IL-1β and IL-6 LPS-stimulated BV-2 cells (344,347,353,356) E. arborea production LPS-stimulated RAW 264.7 cells ↓ iNOS and COX-2 expression Mouse (ICR strain) ear edema

↓ NF-κB activation ↓ Akt, ERK, and JNK phosphorylation ↓ AA-, TPA- and OXA-induced ear edema 2-Phloroeckol E. stolonifera ↓ NO production LPS-stimulated RAW 264.7 cells (347) 974-B

Fucofuroeckol A E. bicyclis ↓ NO, PGE2, TNF-α, IL-6 and MCP-1 LPS-stimulated RAW 264.7 cells (346) production ↓ iNOS and COX-2 expression ↓ NF-κB activation ↓ JNK and p38 MAPK phosphorylation 6,8'-Bieckol E. arborea ↓ AA-, TPA- and OXA-induced ear edema Mouse (ICR strain) ear edema (358) Fucophlorethol C Colpomenia LOX inhibition Cell-free enzymatic system (357) bullosa (D.A. Saunders) Yamada

1 AA, arachidonic acid; Akt, protein kinase B; AP-1, activator protein-1; COX, cyclooxygenase; ERK, extracellular signal-regulated kinase; HMBG, high mobility group box protein; HUVEC, human umbilical vein endothelial cells; ICAM, intercellular adhesion molecule; ICR, Institute of Cancer Research;IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; LOX, lipoxygenase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP, monocyte chemoattractant protein; MMP, matrix metalloproteinase; NF-κB, nuclear factor-κB; NO, nitric oxide; OXA, oxazolone; PG, prostaglandin; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13- acetate; TLR, toll-like receptor; TNF-α, tumor necrosis factor-α; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; VCAM, vascular cell adhesion molecule; ZO-1, zona occludens-1 protein.

42 INTRODUCTION

Table 2. Anti-inflammatory effects of phlorotannin-rich extracts/fractions obtained from brown seaweeds.1

Species Extract/fraction Proposed mechanism of action Model Reference Ascophyllum Marketed extract with 18% ↓ TNF-α and IL-6 production LPS-stimulated U937 cells (351) nodosum (Linnaeus) phlorotannins Le Jolis Cladostephus Acetone:water (7:3, v/v) ↓ NO production LPS-stimulated RAW 264.7 cells (54) spongiosus (Hudson) extract purified with •NO scavenging activity Cell-free non-enzymatic system C. Agardh microcrystalline cellulose Cystoseira nodicaulis (Withering) M. Roberts Cystoseira tamariscifolia (Hudson) Papenfuss Cystoseira usneoides (Linnaeus) M. Roberts

Ecklonia cava Water extract ↓ PGE2 production LPS-stimulated RAW 264.7 cells (340) Kjellman Ethanol (30%) extract ↓ rhIL-1α-induced proteoglycan degradation Rabbit cartilage explant culture LAD103 [ethanol (30%) extract partitioned with ethyl ether]

Fermented processing by- ↓ NO, PGE2, IL-1β and IL-6 production LPS-stimulated RAW 264.7 cells (338) product extract ↓ iNOS and COX-2 expression Processing by-product ethyl ↓ NO production LPS-stimulated RAW 264.7 cells (292) acetate fraction ↓ iNOS and COX-2 expression LPS-stimulated zebrafish embryos ↑ Survival rate Ethanol (70%) extract ↓ MCP-1, TNF-α, IL-1β production HFD-induced obese mice (306) partitioned with ethyl acetate ↓ NF-κB activation

43 INTRODUCTION

Table 2. Cont.

Species Extract/fraction Proposed mechanism of action Model Reference

Ecklonia cava Ethanol (30%) extract ↓ NO, PGE2, TNF-α, IL-6 and HMBG-1 LPS-stimulated primary macrophages (348) Kjellman partitioned with ethyl ether production LPS-stimulated RAW 264.7 cells ↓ iNOS and COX-2 expression LPS-induced septic mice ↓ NF-κB activation ↑ Nrf2/HO-1 activation ↑ Survival rate

Ecklonia stolonifera Ethanol (96%) extract ↓ NO, PGE2, TNF-α, IL-1β and IL-6 production LPS-stimulated RAW 264.7 cells (344) Okamura ↓ iNOS and COX-2 expression ↓ NF-κB activation ↓ Akt, ERK, JNK and p38 MAPK phosphorylation

Ethanol (95%) extract ↓ NO, PGE2, TNF-α, IL-1β and IL-6 production LPS-stimulated RAW 264.7 cells (347) partitioned with ethyl acetate ↓ iNOS and COX-2 expression ↓ NF-κB activation Eisenia arborea Methanol:chloroform (1:2, ↓ AA-, TPA- and OXA-induced ear edema Mouse (ICR strain) ear edema (356,358) Areschoug v/v) extract Eisenia bicyclis Methanol extract ↓ NO production LPS-stimulated RAW 264.7 cells (275,354) (Kjellman) Setchell Methanol extract partitioned Propionibacterium acnes -induced with ethyl acetate HaCaT cells Fucus distichus Methanol (80%) extract ↓ IL-1β, IL-6, IL-17, TNF-α, MCP-1, iNOS, COX- LPS-stimulated RAW 264.7 cells (91) Linnaeus partitioned with ethyl acetate 2, ICAM, TLR-4, TLR-9, UCP-1 and leptin 3T3-L1 cells and sub-fractionated in flash expression chromatography ↑ Adiponectin expression Fucus guiryi Zardi, Acetone:water (7:3, v/v) ↓ NO production LPS-stimulated RAW 264.7 cells (53,54) Nicastro, E.S. Serrão extract purified with •NO scavenging activity Cell-free enzymatic and non- & G.A. Pearson microcrystalline cellulose LOX inhibition enzymatic systems Fucus serratus Linnaeus Fucus spiralis Linnaeus

44 INTRODUCTION

Table 2. Cont.

Species Extract/fraction Proposed mechanism of action Model Reference Fucus vesiculosus Hydroethanol (30-35% and ↓ NO production LPS- and PMA-stimulated RAW 264.7 (341) Linnaeus 50-70%) extracts cells Rat plasma and erythrocytes (ex vivo) Acetone:water (7:3, v/v) ↓ NO production LPS-stimulated RAW 264.7 cells (53) extract purified with •NO scavenging activity Cell-free enzymatic and non- microcrystalline cellulose LOX inhibition enzymatic systems Halopteris filicina Acetone:water (7:3, v/v) ↓ NO production LPS-stimulated RAW 264.7 cells (54) (Grateloup) Kützing extract purified with •NO scavenging activity Cell-free non-enzymatic system microcrystalline cellulose Padina pavonica (Linnaeus) Thivy Saccorhiza polyschides (Lightfoot) Batters Sargassum vulgare C. Agardh Stypocaulon scoparium (Linnaeus) Kützing

1 AA, arachidonic acid; Akt, protein kinase B; COX, cyclooxygenase; ERK, extracellular signal-regulated kinase; HFD, high-fat diet; HMBG, high mobility group box protein; ICAM, intercellular adhesion molecule; ICR, Institute of Cancer Research; IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; LOX, lipoxygenase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; NO, nitric oxide; Nrf2/HO-1, nuclear factor erythoid-2-related factor 2 /heme oxygenase-1; OXA, oxazolone; PG, prostaglandin; PMA, phorbol 12-myristate 13-acetate; rhIL-1α, recombinant human interleukin-1α; TLR, toll-like receptor; TNF-α, tumor necrosis factor-α; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; UCP-1, uncoupling protein-1.

45 INTRODUCTION

1.3.2. Allergy and modulation of allergic events by phlorotannins

Allergic reactions, specifically type I hypersensitivity disorders, are the most common immunological diseases, representing one of the most widespread and fast growing chronic human health problems in developed countries (361,362). Allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, and food allergy are the most prevalent worldwide and, even though their symptoms may be different, they are caused by similar cellular mechanisms (362). Functionally, allergy is an abnormal adaptive immune response to generally innocuous substances commonly found in the environment (antigen). Allergic responses may involve, or not, immunoglobulin (Ig) E; however, IgE- mediated reactions are the most prevalent (363). IgE is responsible for organ-specific allergic disorders, locally produced in the affected anatomical sites, consequently being the immunoglobulin isotype found at the lowest concentration in blood circulation (364). The biological functions of IgE are mediated by its binding to specific receptors: the low- affinity IgE receptor (CD23) and the high-affinity IgE receptor (FcεRI), expressed by different cell types. CD23 can be expressed in the surface of epithelial, myeloid and B cells. On the other hand, FcεRI is expressed by mast cells and basophils (in the αβγγ form), as well as by antigen presenting cells (APC), such as macrophages and dendritic cells (in αγγ form) (365). The binding of IgE to its receptors triggers the beginning of the degranulation process, featuring a cascade of intracellular events, which include the increase of intracellular Ca2+ levels and the exocytosis of membrane-bound granules, resulting in the release of a range of preformed and newly synthesized pharmacologically active molecules, such as biogenic amines (e.g., histamine), serine proteases (e.g., tryptases) and various other enzymes, along with certain cytokines and growth factors that evoke a potent immune allergic response (322). Among the plethora of mast cell mediators, histamine is certainly the most studied one. Histamine results from the decarboxylation of histidine through the action of L-histidine decarboxylase, and is the most potent vasoactive mediator implicated in immediate hypersensitivity (366). It can be produced by cell types presenting a high L-histidine decarboxylase activity, like myeloid and lymphoid cells, being stored in the granules of mast cells and basophils, together with other important mediators (367). During the degranulation process, the release of histamine contributes to the progression of allergic and inflammatory responses, mediating interactions between different cells and leading to an increase of cytokines, growth and chemotactic factors that are responsible for the common clinical manifestations of allergic diseases (368).

Currently, the management of allergic diseases relies on patient education, allergen avoidance, pharmacotherapy, and allergy immunotherapy (369). If on the one hand the drugs used to treat allergies have no effect on the progression of the disease and need to be

46 INTRODUCTION

administered repeatedly, as long as symptoms prevail, which often means life-long, on the other hand, adherence to immunotherapy is one of the foremost issues. Therapeutic approaches to control allergic diseases generally focus on the decrease of total IgE production, regulation of FcεRI receptor expression, and inhibition of bioactive mediator production, as well as of their release from mast cells (322,370). Although the knowledge about the pathophysiology of allergic diseases has substantially increased, the rising number of allergic threats makes the understanding of molecular and cellular events of pivotal relevance to target the actual cause(s) of allergic disorders and to counteract the side effects from continued use of anti-allergic drugs, particularly by children. The chronic nature and lack of preventive and curative therapy of allergic diseases is leading patients in Western societies to seek complementary and alternative medicines (371). Nature- derived bioactive compounds, particularly those from non-conventional environments, such as marine organisms, have attracted much attention and may provide valuable candidates of new generation anti-allergic therapeutics. Within the range of marine chemical diversity, phlorotannins are thought to be among the most promising candidates as anti-allergic compounds (19).

Compared to anti-inflammatory assessment, anti-allergenicity studies focused on seaweed-derived products are scarcer. The anti-allergic effects of phlorotannins, mainly isolated from Eisenia and Ecklonia genera, are known to occur via inhibition of FcεRI expression, Ca2+ influx, and cell degranulation (276,278,372–375), as well as by the interaction with allergy-related enzymatic systems (e.g., HAase) (376) (Figure 12, Table 3). Some works have also screened the anti-allergic activity of crude seaweed extracts, which was mainly attributed to the presence/content of phlorotannins (374,377–383), and only few surveys have highlighted the interplay between inflammation and allergy and the key-role of phlorotannins and phlorotannin-rich extracts/fractions in this complex network (277,383) (Table 4).

The rat basophilic leukaemia cell line (RBL-2H3) and the human immature pre- basophilic cells (KU812) have been widely employed in allergic and immunological research, particularly in the screening of the anti-allergic properties of isolated compounds and extracts from natural sources (55,276–278,372–374,377,382–384). These cell models have been used to evaluate the anti-allergic activity related to cell degranulation, following immunological (with IgE/antigen) or non-immunological (e.g., calcium ionophore A23187) elicitation (385). Artificial activation of basophils by the calcium ionophore A23187 is routinely used to shuttle Ca2+ directly across cell membrane, bypassing all signal transduction steps prior to Ca2+ influx, which allows the identification of target pathways, i.e. upstream or downstream Ca2+ signaling (386). By contrast,

47 INTRODUCTION

stimulation with IgE/antigen activates the FcεRI receptor, triggering a cascade of tyrosine phosphorylation events, accompanied by efflux of Ca2+ from internal stores and influx of Ca2+ into the cell (387,388). The exocytosis of membrane-bound granules results in the release of a range of pharmacologically active mediators that evoke a potent immune allergic response (385). The degree of cell degranulation can be rapidly and effectively measured by monitoring the release of substances stored in the secretory granules, like histamine and β-hexosaminidase, into cell supernatant.

48 INTRODUCTION

Table 3. Anti-allergic effects of phloroglucinol (basic unit) and phlorotannins isolated from brown seaweeds.1

Compound Species Proposed mechanism of action Model Reference Phloroglucinol Eisenia bicyclis HAase inhibition Cell-free enzymatic system (376) (Kjellman) Setchell Dieckol Ecklonia cava ↓ β-hex and histamine release A23187- and IgE/antigen-stimulated RBL-2H3 and (373,375,376) Kjellman ↓ FcεRI expression KU812 cells E. bicyclis ↓ IgE-FcεRI binding IgE/antigen-stimulated-BMCMC and LAD2 cells ↓ IL-4, IL-6, IL-13 and TNF-α IgE/antigen-sensitized mice production Cell-free enzymatic system ↓ Mast cell degranulation ↓ passive cutaneous anaphylaxis reaction HAase inhibition Eckol E. cava ↓ β-hex release A23187- and IgE/antigen-stimulated RBL-2H3 and (278,372,376,381) KU812 cells Eisenia arborea ↓ LTB4 and PGD2 production Cell-free enzymatic systems Areschoug PLA2, COX, LOX and HAase inhibition E. bicyclis Dioxinodehydroeckol Ecklonia ↓ Histamine release CRA-1-stimulated KU812 cells (384) stolonifera ↓ Ca2+ influx Okamura ↓ FcεRI expression Phlorofucofuroeckol A E. cava ↓ β-hex and histamine release A23187-, IgE/antigen-stimulated RBL-2H3 and (278,372,376,384) 2+ KU812 cells E. stolonifera ↓ Ca influx ↓ FcεRI expression CRA-1-stimulated KU812 cells E. arborea ↓ IgE-FcεRI binding Cell-free enzymatic system E. bicyclis HAase inhibition 6,6'-Bieckol E. cava ↓ β-hex and histamine release A23187- and IgE/antigen-stimulated RBL-2H3 and (372,373) KU812 cells E. arborea ↓ FcεRI expression ↓ IgE-FcεRI binding 8,8'-Bieckol E. arborea ↓ β-hex release A23187- and IgE/antigen-stimulated RBL-2H3 and (372,376,381) KU812 cells E. bicyclis ↓ LTB4 and PGD2 production PLA2, COX, LOX and HAase inhibition Cell-free enzymatic systems

49 INTRODUCTION

Table 3. Cont.

Compound Species Proposed mechanism of action Model Reference Phlorofucofuroeckol B E. arborea ↓ β-hex release A23187- and IgE/antigen-stimulated RBL-2H3 cells (280,376) 6,8'-Bieckol E. arborea ↓ β-hex release A23187- and IgE/antigen-stimulated RBL-2H3 and (372,381)

↓ LTB4 and PGD2 production KU812 cells PLA2, COX and LOX inhibition Cell-free enzymatic systems Fucodiphlorethol G E. cava ↓ β-hex and histamine release A23187- and IgE/antigen-stimulated RBL-2H3 and (278) ↓ FcεRI expression KU812 cells ↓ IgE-FcεRI binding

1 BMCMC, bone marrow-derived cultured mast cells; COX, cyclooxygenase; CRA-1, anti-human FcεRI antibody; FcεRI, high-affinity IgE receptor; HAase, hyaluronidase; β-hex, β-hexosaminidase; IL, interleukin; IgE, immunoglobulin E; LTB4, leukotriene B4; LOX, lipoxygenase; PLA2, phospholipase A2; PG, prostaglandin; TNF-α, tumor necrosis factor- α.

50 INTRODUCTION

Table 4. Anti-allergic effects of phlorotannin-rich extracts/fractions obtained from brown seaweeds.1

Species Extract/fraction Proposed mechanism of action Model Reference Cystoseira nodicaulis Acetone:water (7:3, v/v) HAase inhibition Cell-free enzymatic system (57) (Withering) M. Roberts extract purified with microcrystalline cellulose Cystoseira tamariscifolia (Hudson) Papenfuss Cystoseira usneoides (Linnaeus) M. Roberts Ecklonia cava Kjellman Methanol (1:10, w/v) ↓ Histamine release CRA-1-stimulated KU812 cells (374) extract ↓ FcεRI expression Patient serum (allergic to dust mites) ↓ IgE-FcεRI binding Methanol (80%) extract ↓ Histamine release IgE/antigen-stimulated RBL-2H3 cells (377) Ecklonia kurome Methanol:chloroform (1:2, ↓ β-hex release IgE/antigen-stimulated RBL-2H3 cells (277) Okamura v/v) extract partitioned PLA2, COX and LOX inhibition Cell-free enzymatic systems with diethyl ether Ecklonia stolonifera Methanol:chloroform (1:2, ↓ Histamine release A23187- and IgE/antigen-stimulated RBL- (382)

Okamura v/v) extract ↓ LTB4 and PGD2 production 2H3 and KU812 cells PLA2, COX, LOX and HAase inhibition Eisenia arborea Methanol:chloroform (1:2, ↓ Histamine release A23187- and IgE/antigen-stimulated RBL- (381) Areschoug v/v) extract ↓ LTB4 and PGD2 production 2H3 and KU812 cells PLA2, COX, LOX and HAase inhibition Methanol:chloroform (1:2, ↓ β-hex release IgE/antigen-stimulated RBL-2H3 cells (277) v/v) extract partitioned PLA2, COX and LOX inhibition Cell-free enzymatic systems with ethyl acetate Methanol (80%) extract ↓ Histamine release IgE/antigen-stimulated RBL-2H3 cells (377)

51 INTRODUCTION

Table 4. Cont.

Species Extract/fraction Proposed mechanism of action Model Reference Fucus guiryi Zardi, Acetone:water (7:3, v/v) ↓ β-hex and histamine release A23187- and IgE/antigen-stimulated RBL- (55,57) Nicastro, E.S. Serrão & extract purified with HAase and β-hex inhibition 2H3 cells G.A. Pearson microcrystalline cellulose Cell-free enzymatic systems Fucus serratus Linnaeus Fucus spiralis Linnaeus Fucus. vesiculosus Linnaeus Ishige foliacea Methanol (80%) extract ↓ Histamine release IgE/antigen-stimulated RBL-2H3 cells (377) Okamura Sargassum fusiforme Methanol:chloroform (1:2, ↓ β-hex release IgE/antigen-stimulated RBL-2H3 cells (383) (Harvey) Setchell v/v) extract partitioned PLA2, COX, LOX and HAase Cell-free enzymatic systems with diethyl ether inhibition Sargassum Methanol (80%) extract ↓ Histamine release IgE/antigen-stimulated RBL-2H3 cells (377) micracanthum (Kützing) Endlicher Sargassum ringgoldianum Harvey Sargassum thunbergii (Mertens ex Roth) Kuntze

1 COX, cyclooxygenase; CRA-1, anti-human FcεRI antibody; FcεRI, high-affinity IgE receptor; HAase, hyaluronidase; β-hex, β-hexosaminidase; IgE, immunoglobulin E; LTB4, leukotriene B4; LOX, lipoxygenase; PLA2, phospholipase A2; PG, prostaglandin.

52 OBJECTIVES

2. Objectives

The objectives of this thesis are referred below:

1) Comparison of the fatty acid and pigment profiles of the kelp species L. ochroleuca, S. latissima, and S. polyschides under changing environmental conditions.

2) Determination of the free phytoprostane composition of macroalgae and establishment of possible relationships between the phytoprostane and the α-linolenic acid content.

3) Characterization of Fucus spp. extracts in terms of phlorotannins.

4) Evaluation of the anti-inflammatory potential of phlorotannin-targeted extracts from Fucus spp. and establishment of possible relationships between the bioactivity and the amount of phlorotannins thereof.

5) Assessment of the anti-allergenicity of phlorotannin-targeted extracts from Fucus spp. and establishment of possible relationships between the bioactivity and the amount of phlorotannins in each extract.

CHAPTER II

EXPERIMENTAL SECTION

3. Experimental Section

3.1. Standards and reagents

Phytoprostanes (9-F1t-phytoprostane, 9-epi-9-F1t-phytoprostane, ent-16-F1t- phytoprostane, ent-16-epi-16-F1t-phytoprostane, 9-D1t-phytoprostane, 9-epi-9-D1t- phytoprostane, 16-B1-phytoprostane, ent-16-B1-phytoprostane, 9-L1-phytoprostane, and ent-9-L1-phytoprostane) were synthesized by Institut des Biomolécules Max Mousseron

(Montpellier, France). d4-15-F2t-Isoprostane (8-isoPGF2α-d4) [molecular weight (MW):

358.2; C20H30D4O5] was purchased from Cayman Chemicals (Ann Arbor, MI, USA).

The mixed standard solution of fatty acid methyl esters (FAME) (CRM47885), used in gas chromatography/ion trap-mass spectrometry (GC/IT-MS) and gas chromatography- flame ionization detector (GC-FID) analyses and composed of butanoic acid (C4:0) (CAS

Number: 623-42-7), hexanoic acid (C6:0) (CAS Number: 106-70-7), octanoic acid (C8:0)

(CAS Number: 111-11-5), decanoic acid (C10:0) (CAS Number: 110-42-9), undecanoic acid

(C11:0) (CAS Number: 1731-86-8), dodecanoic acid (C12:0) (CAS Number: 111-82-0), tridecanoic acid (C13:0) (CAS Number: 1731-88-0), tetradecanoic acid (C14:0) (CAS Number:

124-10-7), cis-9-tetradecenoic acid (C14:1n-5c) (CAS Number: 56219-06-8), pentadecanoic acid (C15:0) (CAS Number: 7132-64-1), cis-10-pentadecenoic acid (C15:1n-5c) (CAS Number:

90176-52-6), hexadecanoic acid (C16:0) (CAS Number: 112-39-0), cis-9-hexadecenoic acid

(C16:1n-7c) (CAS Number: 1120-25-8), heptadecanoic acid (C17:0) (CAS Number: 1131-92-6), cis-10-heptadecenoic acid (C17:1n-7c) (CAS Number: 75190-82-8), octadecanoic acid (C18:0)

(CAS Number: 112-61-8), cis-9-octadecenoic acid (C18:1n-9c) (CAS Number: 112-62-9), trans-9-octadecenoic acid (C18:1n-9t) (CAS Number: 1937-69-8), cis-9,12-octadecadienoic acid (C18:2n-6c) (CAS Number: 112-63-0), trans-9,12-octadecadienoic acid (C18:2n-6t) (CAS

Number: 2566-97-4), cis-6,9,12-octadecatrienoic acid (C18:3n-6c) (CAS Number: 16326-32-

2), cis-9,12,15-octadecatrienoic acid (C18:3n-3c) (CAS Number: 301-00-8), eicosanoic acid

(C20:0) (CAS Number: 1120-28-1), cis-11-eicosenoic acid (C20:1n-11c) (CAS Number: 2390-09-

2), cis-11,14-eicosadienoic acid (C20:2n-6c) (CAS Number: 2463-02-7), cis-8,11,14- eicosatrienoic acid (C20:3n-6c) (CAS Number: 21061-10-9), cis-11,14,17-eicosatrienoic acid

(C20:3n-3c) (CAS Number: 55682-88-7), cis-5,8,11,14-eicosatetraenoic acid (C20:4n-6c) (CAS

Number: 2566-89-4), cis-5,8,11,14,17-eicosapentaenoic acid (C20:5n-3c) (CAS Number:

2734-47-6), heneicosanoic acid (C21:0) (CAS Number: 6064-90-0), docosanoic acid (C22:0)

(CAS Number:929-77-1), cis-13-docosenoic acid (C22:1n-9c) (CAS Number: 1120-34-9), cis-

13,16-docosadienoic acid (C22:2n-6c) (CAS Number: 61012-47-3), cis-4,7,10,13,16,19- docosahexaenoic acid (C22:6n-3c) (CAS Number: 2566-90-7), tricosanoic acid (C23:0) (CAS

Number: 2433-97-8), tetracosanoic acid (C24:0) (CAS Number: 2442-49-1), and cis-15-

57 EXPERIMENTAL SECTION

tetracosenoic acid (C24:1n-9c) (CAS Number: 2733-88-2), was obtained from Supelco (Bellefonte, PA, USA).

Fucoxanthin (≥ 95.0%), β-carotene (≥ 95.0%) and chlorophyll a (≥ 85.0%), as well as acetone, butylated hydroxytoluene (BHT), ethanol and methyl tert-butyl ether (MTBE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Violaxanthin (95.0%) and zeaxanthin (97.0%) were obtained from CaroteNature (Lupsinggen, Switzerland), and chlorophyll c2 (99.9%) and pheophytin a (90.0%) were from LGC Standards (Manchester, NH, USA).

Phloroglucinol (≥ 99.0%), quercetin (≥ 95.0%), disodium cromoglicate (DSCG) (≥ 95.0%), linoleic acid (≥ 99.0%), dexamethasone (≥ 97.0%), 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), crystal violet staining (CVS), trypan blue solution, sodium nitroprusside dehydrate (SNP), sulfanilamide, 4- dimethylaminobenzaldehyde (DMAB), 2,4-dimethoxybenzaldehyde (DMBA), p- nitrophenyl N-acetyl-β-D-glucosaminide, toluene, dimethyl sulfoxide (DMSO), butylated hydroxyanisole (BHA), bis(2-hydroxyethyl)-amino-tris-(hydroxymethyl)-methane (BIS-

TRIS), chloroform, isooctane, KOH, anhydrous sodium sulfate, BF3-methanol solution, sodium hydroxide (NaOH), potassium chloride (KCl), o-phthalaldehyde (OPA), o- phosphoric acid (H3PO4), citric acid, LPS from Escherichia coli, sodium formate, albumin from bovine serum (BSA), BSA fraction V (7.5% solution), lipoxygenase from Glycine max (L.) Merr. (type V-S; EC 1.13.11.12), hyaluronic acid (HA) sodium salt from Streptococcus equi, HAase from bovine tests (type IV-S; EC 3.2.1.35), as well as anti-2,4-dinitrophenyl monoclonal antibody (anti-DNP IgE) produced in mouse, 2,4-dinitrophenyl albumin (DNP-BSA), and calcium ionophore A23187 (≥ 98.0%) were purchased from Sigma- Aldrich (St. Louis, MO, USA).

Chromabond C18 columns (1000 mg/6 mL) were obtained from Macherey-Nagel

(Düren, Germany), Sep-Pak C18 Plus Short Cartridge [360 mg sorbent per cartridge, 55- 105 μm particle size, 50/pk (WAT020515)] from Waters (Milford, MA, USA), and Strata X-AW (500 mg/3 mL) columns from Phenomenex (Torrance, CA, USA).

Microcrystalline cellulose for thin-layer chromatography, N-(1-napthyl) ethylenediamine, glacial acetic acid, hydrochloride acid (HCl), methanol, acetonitrile, tetrahydrofuran (THF), sodium chloride (NaCl), disodium tetraboratedecahydrate

(Na2B4O7·10H2O), disodium hydrogen phosphate (Na2HPO4), disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O), potassium dihydrogen phosphate (KH2PO4), and formic acid were acquired from Merck (Darmstadt, Germany). n-Hexane was purchased from Panreac (Barcelona, Spain).

58 EXPERIMENTAL SECTION

Dulbecco's Modified Eagle Medium (DMEM)-GlutaMAX™-I, Hanks' Balanced Salt Solution (HBSS), Earle’s Balanced Salt Solution (EBSS), heat inactivated fetal bovine serum (FBS), and Pen Strep solution (penicillin 5000 units/mL and streptomycin 5000 μg/mL) were obtained from Gibco® (Life Technologies, Invitrogen™; Grand Island, NY, USA). Both the murine macrophage-like cell line RAW 264.7 and the rat basophilic leukaemia cell line RBL-2H3 were from the American Type Culture Collection (ATCC®) (LGC Standards S.L.U., Spain).

Water was deionized using a Milli-Q water purification system (Millipore, Bedford, MA, USA) and all reagents and solvents were of analytical grade.

3.2. Sampling

Macroalgae samples were randomly collected during low-tide periods from different places of the Western coast of Portugal, and from IMTA systems (Figure 13).

N ATLANTIC OCEAN 1 2 3 4 5

Figure 13. Studied Portuguese waters with the sampling sites marked with numbers. 1 – Praia da Amorosa (N 41°38'51.44, W 8°49'32.53); 2 – Praia Norte (N 41°41'49.75, W 8°51'3.52); 3 – Praia de São Bartolomeu do Mar (N 41º34'19.66, W8º47'52.80); 4 – IMTA system Estela (N 41°27'11.20, W 8°46'28.29); 5 – Sea cultivation in vertical longlines off Póvoa de Varzim (N 41°22'21.00, W 8°46'13.00); 6 – IMTA system AlgaPlus® (N 40°36'45.27, W 8°40'26.88); 7 – Praia do Quebrado (N 39°22'0.91, W 9°22'25.86).

59 EXPERIMENTAL SECTION

To prevent sample alterations, after collection they were immediately placed on ice and transported to the laboratory in insulated, sealed ice boxes to protect them from heat, air, and light exposure. Macroalgae were then carefully and quickly washed with NaCl aqueous solution (3.5%, w/v) to remove epiphytes and encrusting material, at room temperature, without exposure to direct light. Each sample corresponds to a pool of ten individuals in the same stage of development: five were studied as whole individuals and the other five were split into blades and stipes to assess both fatty acid and pigment composition in those tissues separately. For phytoprostane and phlorotannin analyses, only the whole individuals were considered. All samples were kept at –20 °C prior to their lyophilization in a Virtis SP Scientific Sentry 2.0 apparatus (Gardiner, NY, USA). The dried material was powdered (< 910 μm) and kept in the dark, in a desiccator, before use.

3.3. Fatty acids and pigments

3.3.1. Algal material

Wild specimens of L. ochroleuca, S. latissima and S. polyschides were collected in the rocky shores of Praia Norte, Amorosa and São Bartolomeu do Mar, at different seasons (Figure 13, Table 5).

The specimens cultivated at sea (S. latissima) were grown in vertical longlines off Póvoa de Varzim. These sites are located in Northern Portugal, where the sea surface temperature (SST) ranges from around 11 °C in the winter to around 22 °C in the summer and coastal upwelling events are present, with maxima from July to September (389,390). S. latissima samples collected at 5, 10 and 15 m deep, in May, were subjected to light intensities around 272.2, 137.8 and 66.5 µmol/m2/s, respectively, and SST of 16.3, 15.2 and 14.7 ºC, respectively, at sampling time (around 1:00 pm). The S. latissima specimens deployed at sea in February and sampled in July were subjected to SST of 15.2 °C, decreasing to 15, 14.4 and 13.8 °C at 5, 10 and 15 m depths, respectively. Light intensities measured at those depths were around 780, 550 and 360 μmol photons/m2/s, respectively. These values were retrieved from vertical profiles of photosynthetically active radiation (PAR) and temperature, obtained using an instrument to measure the conductivity, temperature, and depth of the ocean (CTD90M, Sea & Sun Technology), from 0.4 m to approximately 40 m deep (Table 5).

Specimens of L. ochroleuca, S. latissima and S. polyschides cultivated in a pilot land- based installation (IMTA system) were grown in outdoor tanks, tumbling in the water column, as in Abreu et al. (38) and Azevedo et al. (51), from January to April at an initial

60 EXPERIMENTAL SECTION

density of 2 kg/m2, under water temperatures ranging from 13–14 °C in January to 13.5– 16 °C in April. Light intensity at the tanks surface ranged from 500 to 2000 μmol photons/m2/s over the cultivation period, depending on cloud cover (Table 5).

Table 5. Characterization of macroalgae material used for fatty acid and pigment analysis.

Date of Species Tissue Origina Sample collection Laminaria ochroleuca Whole 2 April 2013 Lo_W_N_Apr13 Bachelot de la Pylaie Whole 2 June 2013 Lo_W_N_Jun13 Whole 1 February 2013 Lo_W_Am_Feb13 Whole 1 April 2013 Lo_W_Am_Apr13 Whole 1 September 2013 Lo_W_Am_Sep13 Whole 1 November 2013 Lo_W_Am_Nov13 Whole 1 December 2013 Lo_W_Am_Dec13 Whole 4 April 2013 Lo_W_IMTA_Apr13 Whole 2 October 2012 Lo_W_N_Oct12 Blade 2 December 2012 Lo_B_N_Dec12 Stipe 2 December 2012 Lo_S_N_Dec12

Whole 2 December 2012 Lo_W_N_Dec12 Blade 3 January 2013 Lo_B_SBM_Jan13 Stipe 3 January 2013 Lo_S_SBM_Jan13 Whole 3 January 2013 Lo_W_SBM_Jan13 Blade 3 June 2013 Lo_B_SBM_Jun13 Blade 1 April 2013 Lo_B_Am_Apr13 Whole 4 January 2013 Lo_W_IMTA_Jan13 Blade 4 April 2013 Lo_B_IMTA_Apr13 Saccharina latissima Whole 4 January 2013 Sl_W_IMTA_Jan13 (Linnaeus) C. E. Lane, C. Mayes, Druehl & G. Whole 4 April 2013 Sl_W_IMTA_Apr13 W. Saunders Whole 4 July 2013 Sl_W_IMTA_Jul13 Blade 4 April 2013 Sl_B_IMTA_Apr13 Whole 1 October 2012 Sl_W_Am_Oct12 Whole 1 January 2013 Sl_W_Am_Jan13 Whole 1 July 2013 Sl_W_Am_Jul13 Whole 1 September 2013 Sl_W_Am_Sep13 Whole 1 December 2013 Sl_W_Am_Dec13 Stipe (5 m deep) 5 May 2012 Sl_S(5m)_PV_May12 Stipe (10 m deep) 5 May 2012 Sl_S(10m)_PV_May12 Stipe (15 m deep) 5 May 2012 Sl_S(15m)_PV_May12 Blade (5 m deep) 5 May 2012 Sl_B(5m)_PV_May12 Blade (10 m deep) 5 May 2012 Sl_B(10m)_PV_May12 Blade (15 m deep) 5 May 2012 Sl_B(15m)_PV_May12 Blade (5 m deep) 5 July 2012 Sl_B(5m)_PV_Jul12 Blade (10 m deep) 5 July 2012 Sl_B(10m)_PV_Jul12 Blade (15 m deep) 5 July 2012 Sl_B(15m)_PV_Jul12

61 EXPERIMENTAL SECTION

Table 5. Cont.

Date of Species Tissue Origina Sample collection Saccorhiza Whole 2 January 2013 Sp_W_N_Jan13 polyschides (Lightfoot) Batters Whole 4 January 2013 Sp_W_IMTA_Jan13 Whole 4 April 2013 Sp_W_IMTA_Apr13 Blade 4 April 2013 Sp_B_IMTA_Apr13 Stipe 4 April 2013 Sp_S_IMTA_Apr13 a As in Figure 13.

3.3.2. Fatty acid extraction and derivatization

The complete extraction and derivatization procedure was performed as previously described (391), with slight modifications. Briefly, 0.25 g of the dried macroalgae was extracted with 25 mL of chloroform:methanol (2:1, v/v), under magnetic stirring at 500 rpm, for 10 min, at 40 ºC. The extraction procedure was repeated five times and the resulting extracts were pooled and concentrated to dryness under reduced pressure (40 ºC).

The residue was then hydrolyzed with 1 mL of KOH methanol solution (11 g/L), at 90 ºC, for 10 min. The free fatty acids originally present and those resulting from the alkaline hydrolysis were derivatized to their methyl esters with 1 mL of BF3-methanol solution (10%), at 90 ºC, for 10 min. FAME were purified with 2 × 10 mL of isooctane, and anhydrous sodium sulfate was added to ensure the total absence of water. The derivatized extract was evaporated to dryness under a stream of nitrogen, dissolved in isooctane, and filtered through a 0.45 μm pore size hydrophobic polytetrafluoroethylene (PTFE) membrane (Millipore, Bedford, MA, USA) for GC/IT-MS and GC-FID analysis.

3.3.3. GC/IT-MS qualitative analysis of fatty acids

GC/IT-MS analysis was performed following a previously established method (391). Derivatized extracts (1 μL) were analyzed using a Varian CP-3800 gas chromatograph (Walnut Creek, CA, USA) equipped with a VarianSaturn 4000 mass selective detector and a Saturn GC-MS work station software version 6.8. Analyses were carried out using a 30 m × 0.25 mm, i.d., 0.25 μm film column VF-5 ms (Varian). The injector port was heated to 250 ºC and the injections were performed in split mode, with a ratio of 1/40. High purity helium C-60 (Gasin, Portugal) was the carrier gas at a constant flow rate of 1 mL/min. The oven temperature was set at 40 ºC for 1 min, then increased 5 ºC/min to 250 ºC, 3 ºC/min to 300 ºC and held for 15 min. All mass spectra were acquired in electron impact (EI)

62 EXPERIMENTAL SECTION

mode. Ionization was maintained off during the first 4 min, to avoid solvent overloading. The detection was performed using an ion trap detector set as follows: the transfer line, manifold and trap temperatures were 280, 50 and 180 ºC, respectively. The mass ranged from m/z 50 to 600, with a scan rate of 6 scans/s. The emission current was 50 μA, and the electron multiplier was set in relative mode to auto tune procedure. The maximum ionization time was 25 000 μs, with an ionization storage level of m/z 35. The analyses were performed in full scan mode. The compounds were identified according to Ribeiro et al. (391), by comparison of their retention time and mass spectra with those from pure standard solution analyzed under the same conditions, and from NIST 05 MS Library Database (392).

3.3.4. Fatty acid quantification by GC-FID

Quantitative analysis was performed using a Finnigan Focus GC apparatus (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a FID. Chromatographic conditions were the same described above for GC/IT-MS analysis. Each derivatized extract (1 μL) was injected in triplicate. The amount of FAME was achieved from the calibration curve of the respective standard prepared in isooctane and analyzed under the same conditions (Table 6). The linearity range of the method was assessed by building calibration curves using, at least, six different concentration levels of the analytes, according to the range of concentrations found in the extracts. The limit of detection (LOD) and limit of quantification (LOQ) were determined from the residual standard deviation (σ) of the regression curve and the slope (S), according to the following equations:

LOD = (3 ×σ)/S LOQ = (10 ×σ)/S

63 EXPERIMENTAL SECTION

Table 6. Regression equations, r2, linearity, limit of detection (LOD), and limit of quantification (LOQ) for FAME with the employed analytical conditions.

Linearity LOD LOQ Compounda Regression equation r2 (µg/mL) (µg/mL) (µg/mL)

5 C12:0 y=1.8×10 x - 2924625.7 0.9976 26.1–417.0 5.0 16.7

5 C13:0 y=1.5×10 x - 620566.5 0.9992 6.5–207.5 0.3 0.9

5 C14:0 y=1.3×10 x - 774229.5 0.9999 12.9–411.8 1.6 5.2

5 C14:1n-5c y=1.9×10 x - 1796514.1 0.9973 6.5–206.6 0.4 1.4

5 C15:0 y=1.7×10 x - 969472.1 0.9963 6.5–206.5 0.1 0.3

5 C16:0 y=1.6×10 x - 2244823.2 0.9991 19.4–1239.6 2.9 9.5

5 C16:1n-7c y=1.8×10 x - 951350.0 0.9991 6.4–205.8 0.9 3.1

5 C17:0 y=1.3×10 x - 434370.6 0.9989 6.6–209.8 1.7 5.6

5 C18:0 y=1.5×10 x - 1538027.1 0.9989 12.9–412.0 1.4 4.6

5 C18:1n-9c y=2.8×10 x - 3663111.0 0.9993 12.9–413.3 0.7 2.2

5 C18:1n-9t y=1.1×10 x + 1245590.5 0.9977 13.0–208.3 3.3 11.0

5 C18:2n-6c y=1.7×10 x - 1016817.0 0.9985 6.5–207.1 1.1 3.6

5 C18:2n-6t y=1.4×10 x - 962411.0 0.9993 6.5–209.0 0.3 1.1

5 C18:3n-6c y=1.4×10 x - 750372.1 0.9992 6.4–204.5 1.4 4.6

5 C18:3n-3c y=2.1×10 x - 1464445.6 0.9981 6.4–205.8 0.1 0.4

5 C20:0 y=1.4×10 x - 1192068.1 0.9993 12.9–411.9 0.8 2.6

5 C20:2n-6c y=1.5×10 x - 992170.7 0.9994 6.5–207.1 1.2 4.1

5 C20:3n-6c y=1.2×10 x - 787645.9 0.9989 6.8–218.8 0.5 1.7

5 C20:4n-6c y=1.3×10 x - 709213.4 0.9993 6.7–425.8 0.7 2.5

5 C20:5n-3c y=1.3×10 x - 738853.9 0.9992 6.3–200.7 0.7 2.4

5 C21:0 y=1.4×10 x - 598527.1 0.9993 6.4–206.2 0.2 0.6

5 C22:0 y=1.5×10 x - 1423248.2 0.9991 12.9–411.8 0.6 2.0

5 C22:1n-9c y=1.4×10 x - 260693.1 0.9994 6.4–205.7 0.9 3.1

5 C22:6n-3c y=1.2×10 x - 752067.3 0.9988 6.6–210.4 0.4 1.2

5 C23:0 y=1.5×10 x - 645275.1 0.9993 6.4–206.0 0.2 0.7

5 C24:0 y=1.8×10 x - 831206.8 0.9999 12.9–413.5 3.0 10.1

5 C24:1n-9c y=1.5×10 x - 875485.1 0.9989 6.5–207.5 0.8 2.5 a Parent fatty acid.

3.3.5. Pigment extraction

Extracts were prepared with approximately 1 g of each dried algae material, using 20 mL of acetone with 0.05% BHT, under the following conditions: 15 min of sonication followed by 45 min of stirring maceration (600 rpm) at room temperature. Each sample was extracted three times. The obtained extracts were combined, filtered under vacuum, and evaporated at reduced pressure (Rotavapor R-215, Büchi Labortechnik, Switzerland) until complete dryness. The dried extracts were kept at –20 °C and protected from light until analysis.

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3.3.6. HPLC-DAD analysis of pigments

Briefly, the dried residue of each algae extract was reconstituted in methanol Lichrosolv, sonicated, filtered through a 0.45 μm size pore membrane (Millipore) and then analyzed on an analytical HPLC unit (Gilson Medical Electronics, France), as in Oliveira et al. (393). Separation was carried out using a C30 YMC column (5 µm, 250 × 4.6 mm i.d.; YMC, Kyoto, Japan), with oven temperature set at 25 ºC. The mobile phase consisted of two solvents: methanol (A) and MTBE (B). Elution started with 95% A and a gradient was used to obtain 70% A at 30 min, followed by 50% A at 50 min. The injection volume was 20 µL and the flow rate 0.9 mL/min. Spectral data from all peaks were collected in the range of 200–700 nm, and chromatograms were recorded at 450 nm. The data was processed on a Unipoint System software (Gilson Medical Electronics). The compounds were identified by comparing their retention times and UV-vis spectra in the range of 200–700 nm with those of authentic standards analyzed under the same chromatographic conditions.

Pigment quantification was achieved by the absorbance recorded in the chromatograms relative to external standards. Stock solutions of fucoxanthin, violaxanthin and zeaxanthin were prepared individually in ethanol; the solution of β- carotene was prepared in THF and those of chlorophyll a, pheophytin a and chlorophyll c2 in acetone. BHT was added to all standard solutions (final concentration, 0.05%), which were kept at –20 °C until use.

All compounds were quantified as themselves, excepting the cis isomers of fucoxanthin, which were quantified as fucoxanthin, and pheophytin a and chlorophyll c derivatives, determined as chlorophyll a. Peak purity was checked by the software contrast facilities.

3.4. Phytoprostanes

3.4.1. Algal material

Macroalgae samples consisted of three Chlorophyta, five Rhodophyta, and sixteen Ochrophyta species collected between 2010 and 2013 (Table 7).

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Table 7. Characterization of macroalgae samples used for phytoprostane analysis.

Date of Phylum Species Origina collection

Chlorophyta Codium tomentosum Stackhouse 7 April 2011 Ulva lactuca Linnaeus 7 August 2011 Ulva sp. 4 July 2012

Rhodophyta Asparagopsis armata Harvey 7 June 2010 Gigartina sp. 1 December 2013 Gracilaria vermiculophylla (Ohmi) Papenfuss 4 July 2012 Plocamium cartilagineum (Linnaeus) P. S. Dixo 7 July 2012 Sphaerococcus coronopifolius Stackhouse 7 June 2010

Ochrophyta Bifurcaria bifurcata R. Ross 1 December 2013 Cladostephus spongiosus (Hudson) C. Agardh 7 June 2010 Cystoseira tamariscifolia (Hudson) Papenfuss 7 July 2012 Cystoseira usneoides (Linnaeus) M. Roberts 7 August 2011 Fucus guiryi G. I. Zardi, K. R. Nicastro, E. S. Serrão & G. A. 1 December 2013 Pearson Fucus serratus Linnaeus 1 December 2013 Fucus spiralis Linnaeus 1 December 2013 Halopteris filicina (Grateloup) Kützing 7 July 2012 Laminaria ochroleuca Bachelot de la Pylaie 1 December 2013 Padina pavonica (Linnaeus) Thivy 7 July 2012 Pelvetia canaliculata (Linnaeus) Decaisne & Thuret 2 December 2013 Saccharina latissima (Linnaeus) C. E. Lane, C. Mayes, Druehl 4 December 2013 & G. W. Saunders Saccorhiza polyschides (Lightfoot) Batters 7 September 2012 Sargassum muticum (Yendo) Fensholt 1 December 2013 Sargassum vulgare C. Agardh 7 August 2011 Stypocaulon scoparium (Linnaeius) Kützing 7 June 2010 a As in Figure 13.

3.4.2. Phytoprostane extraction

Each pulverized macroalgae sample (1 g) was crushed with a mortar and pestle, with 5 mL of methanol (0.1% BHA). The mixture obtained was transferred to a polypropylene tube, which was vortexed during 5 min, and then centrifuged (2 000 g, 10 min). The supernatant was applied in a Chromabond C18 column, previously conditioned with methanol and Milli-Q water. The eluate (in methanol) was subjected to liquid-liquid extraction (LLE) and SPE, according to the procedure described by (188). Briefly, 1 mL of eluate was diluted in 10 mL of n-hexane and then rediluted in 2 mL of methanol and 2 mL

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of BIS-TRIS buffer (0.02 M, pH 7.0). The resulting emulsion was applied in a Strata-X- AW cartridge, conditioned and equilibrated with n-hexane, methanol and Milli-Q water. After the column had been loaded with the emulsion, a series of solvents were applied to remove undesired compounds (Figure 14). Target compounds were eluted with 1 mL of methanol and dried under nitrogen stream. The dried residue was reconstituted with 200 μL of a mixture of A/B solvents (90:10, v/v), solvent A being Milli-Q water/0.01% acetic acid and solvent B being methanol/0.01% acetic acid. Reconstituted extracts were sonicated, filtered through a 0.45 μm filter (Millipore), and further analyzed in an UHPLC-QqQ-MS/MS apparatus (Figure 14).

Extraction Purification

CHROMA BOND®

Conditioning (10 mL MeOH, 10 mL H2O) Methanol Centrifugation Loading (supernatant) mixture Elution (10 mL MeOH) 1 g lyophilized seaweed 5 mL MeOH (0.1% BHA)

Eluate + 10 mL n-hexane 2 mL MeOH 2 mL BIS TRIS buffer

STRATA X-AW®

Conditioning (2 mLn-hexane, 2 mLMeOH, 2 mL H2O) Loading (emulsion)

Washing (2 mL n-hexane, 2 mL H2O, 2 mL MeOH/H2O (1:3, v/v), 2 mL ACN) Elution (1 mLMeOH)

Solvent evaporation Reconstitution

Phytoprostane profiling (UHPLC-QqQ-MS/MS)

Figure 14. Schematic representation of the protocol for phytoprostane extraction from macroalgae samples and their profiling by UHPLC-QqQ-MS/MS. H2O, water; MeOH, methanol; ACN, acetonitrile.

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3.4.3. UHPLC-QqQ-MS/MS analysis of free phytoprostanes

Separation of phytoprostanes present in the purified extracts was achieved through an UHPLC coupled to 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany), applying a validated method for the qualitative and quantitative determination of free phytoprostanes in foodstuffs (188). The column used was a 50 × 2.1 mm i.d., 1.7 μm, BEH

C18 (Waters, Milford, MA, USA). The column temperature was 6 °C. The mobile phases employed were solvent A [Milli-Q water/acetic acid (99.99:0.01, v/v)] and solvent B [methanol/acetic acid (99.99:0.01, v/v)]. The elution was performed at a flow rate of 0.2 mL/min using the following gradient: 60% B at 0 min, 62% B at 2 min, 62.5% B at 4 min, reaching 65% B at 8 min, and returning to the initial conditions at 8.01 min. The MS analysis was applied in the MRM negative ESI mode. ESI conditions and ion optics were as previously described (188): 325 °C gas temperature, 8 L/min gas flow, 30 psi nebulizer pressure, 350 °C sheath gas temperature, 12 L/min jetstream gas flow, 3000 V capillary voltage, and 1750 V nozzle voltage. Data acquisition and processing were performed using MassHunter software version B.04.00 (Agilent Technologies). The quantification of phytoprostanes detected in each algal extract (20 μL) was performed using authentic standards of 9-F1t-phytoprostane, 9-epi-9-F1t-phytoprostane, 16-B1-phytoprostane, and 9-

L1-phytoprostane. Due to the lack of commercially available deuterated phytoprostane standards, the synthetic isoprostane 8-isoPGF2α-d4 (containing four deuterium atoms at positions 3, 3', 4, and 4') was used as internal standard.

3.5. Phlorotannin purified extracts: Composition and biological activity

Macroalgae used for phlorotannin analysis and biological assessment consisted of four Ochrophyta species of the order Fucales: F. guiryi, F. serratus, F. spiralis, and F. vesiculosus (Table 8). With the exception of F. vesiculosus, collected in 2015, all the other Fucus specimens were collected in the year 2013. Of the selected species, F. vesiculosus was also grown in an IMTA system, and supplied by ALGAPlus (Ílhavo, Portugal) in July 2016 (Table 8).

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Table 8. Characterization of macroalgae samples used for phlorotannins analysis and biological studies.

Date of Species Origina Sample collection Fucus guiryi G. I. Zardi, K. R. 1 December 2013 Fg Nicastro, E. S. Serrão & G. A. Pearson

Fucus serratus Linnaeus 1 December 2013 Fser

Fucus spiralis Linnaeus 1 December 2013 Fspi

Fucus vesiculosus Linnaeus 2 May 2015 Fves-w

6 July 2016 Fves-a

a As in Figure 13.

3.5.1. Preparation of phlorotannin purified extracts

For HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS analyses, the extracts were prepared with approximately 1 g of powdered lyophilized algae material, using 10 mL of methanol:water (1:1, v/v), under the following conditions: 1 h of sonication, 24 h maceration at room temperature, followed by 1 h of sonication. Afterwards, the extracts were centrifuged (10 000 rpm, 10 min) and the methanol present in each supernatant was removed in a Savant™ SPD121P SpeedVac™ Concentrator (Thermo Scientific,

Alcobendas, Spain). The remaining aqueous mixture was loaded onto a Sep-Pak C18 Plus Short Cartridge, which had been pre-conditioned with methanol followed by water, and then washed with water. Phlorotannins were eluted with methanol and the solvent was evaporated under reduced pressure until complete dryness (30 ºC). The resulting phlorotannin-rich fraction was resuspended in a mixture of methanol:water (1:1, v/v) and filtered through a 0.45 μm pore size membrane (Millipore) before analysis (Figure 15).

Extraction Purification

Centrifugation Aqueous Conditioning (10 mL MeOH, 10 mL H2O) MeOH mixture evaporation Loading (aqueous mixture) Phlorotannins + Washing (10 mL H2O) co-extracted 1 g lyophilized seaweed compounds 10 mL MeOH:H2 O (1:1, v/v) (e.g., carbohydrates) No phlorotannins detected Elution (10 mL MeOH)

Solvent evaporation

Phlorotannin profiling Redissolution in MeOH:H2O (1:1, v/v) (HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS)

Figure 15. Schematic representation of the general procedure for obtaining phlorotannin purified extracts from Fucus spp. and their profiling by HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS.

H2O, water; MeOH, methanol.

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For biological determinations, high-purity phlorotannin extracts were prepared following a well-established protocol (Figure 16) (54,56,57). Briefly, ca. 1 g of powdered lyophilized algal material was extracted with 20 mL of aqueous acetone mixture, for 1 h under stirring maceration (400 rpm), at room temperature, followed by centrifugation for 5 min at 4 000 rpm. This procedure was repeated four times, and the resulting organic fractions were gathered and evaporated to dryness under reduced pressure (30 ºC). The resulting dried extracts were resuspended in 30 mL of methanol and adsorbed into microcrystalline cellulose. The suspension was dried under reduced pressure (30 ºC), until cellulose powder detachment. Cellulose was thoroughly washed with toluene (3 × 25 mL) to remove lipophilic components, and then rinsed with 30 mL of acetone:water (7:3, v/v) to release the adhered phlorotannins. After centrifugation (5 min at 4000 rpm), the supernatant was evaporated until complete dryness under reduced pressure (30 ºC). To prepare stock phlorotannin working solutions, the obtained extracts were reconstituted in DMSO at a final concentration of 100 mg of dry purified extract (DE)/mL. All stock solutions were properly stored at –20 °C until cell-free and cell-based assays.

Extraction Purification

Supernatant Redissolution Solvent gathering Dried crude with MeOH evaporation Dried Solvent extract suspension evaporation

Pigment removal Adsorption in with toluene 1 g lyophilized seaweed cellulose 20 mL acetone:H2O (7:3, v/v)

Cellulose rinsing

Acetone:H2O (7:3, v/v)

Dried purified Centrifugation phlorotannin extract Solvent Phlorotannin evaporation release

Biological assessment (cell-free and cell-based assays)

Figure 16. Schematic representation of the general procedure for obtaining phlorotannin purified extracts from Fucus spp. for biological assessment. H2O, water; MeOH, methanol.

3.5.2. Phlorotannin quantification

The phlorotannin content of the purified Fucus spp. extracts was spectrophotometrically determined by the specific reaction between DMBA and 1,3- and 1,3,5-substituted phenols to form a colored product, as before (54). The working reagent was prepared, just prior to use, by mixing equal volumes of DMBA (2%, w/v) and HCl

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(6%, v/v), both dissolved in glacial acetic acid. Aliquots of each extract diluted in water (40 μL) were mixed with 200 μL of the working reagent, in a 96-well plate. The reaction was conducted at room temperature, in the dark, during 60 min. Thereafter the absorbance was measured at 515 nm against the blank in a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA). The amount of phlorotannins in each purified extract was determined from a standard calibration curve with serial dilutions of phloroglucinol and expressed as phloroglucinol equivalents (PGE).

3.5.3. Phlorotannin qualitative profiling

3.5.3.1. HPLC-DAD-ESI/MSn analysis

Chromatographic analyses were performed in an Agilent HPLC 1200 series equipped with a diode array and mass detectors in series (Agilent Technologies, Waldbronn, Germany), as previously described (394). The HPLC consisted of a binary pump (model G1376A), an autosampler (model G1377A) refrigerated at 4 °C (G1330B), a degasser (model G1379B), and a diode array detector (model G1315D). The HPLC system was controlled by ChemStation software (Agilent, v. B.01.03-SR2). The mass detector was a Bruker ion trap spectrometer (model HCT Ultra) equipped with an electrospray ionization interface and was controlled by LCMSD software (Agilent, v. 6.1). Briefly, extract (20 μL) elution was carried out at a flow rate of 0.8 mL/min, on a Kinetex column (5 μm, C18, 100 Å, 150 × 4.6 mm; Phenomenex, Macclesfield, UK), with formic acid (1%) in water (A) and acetonitrile (B), starting with 1% B and installing a gradient to obtain 10% B at 12 min, 30% B at 25 min, 50% B at 27 min, 50% B at 28 min, 1% B at 29 min, and 1% B at 33 min. The ionization conditions were adjusted at 350 °C and 4.0 kV for capillary temperature and voltage, respectively. The nebulizer pressure and flow rate of nitrogen were 65.0 psi and 11 L/min, respectively. The full scan mass covered the range from m/z 100 up to m/z 1200. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 up to 2 V. Mass spectrometry data were acquired in the negative ionization mode.

3.5.3.2. UPLC-ESI-QTOF/MS analysis

Exact mass determination was carried out in an Agilent 1290 Infinity LC system coupled to a 6550 Accurate-Mass QTOF (Agilent Technologies, Waldbronn, Germany) with an electrospray interface (Jet Stream Technology). Extracts (1 μL) were injected onto a reverse phase Luna Omega column (1.6 μm, PS C18, 100 Å, 50 × 2.1 mm; Phenomenex,

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Macclesfield, UK) with SecurityGuard ULTRA Cartridges of the same material, operating at 30 °C and a flow rate of 0.5 mL/min. The mobile phases used were acidified water (0.1% formic acid) (A) and acidified acetonitrile (0.1% formic acid) (B). Compounds were separated using the following gradient conditions: elution started with 1% B to obtain 10% B at 8 min, 30% B at 14 min, 50% B at 16 min, and 1% B at 20 min. The optimal conditions for the electrospray interface were as follows: 280 °C gas temperature, 11 L/min drying gas, 45 psi nebulizer pressure, 400 °C sheath gas temperature, and 12 L/min sheath gas flow. The MS system was operated in negative ion mode with the mass range set at m/z 50–1100 in full scan resolution mode, and data was processed using the Mass Hunter Qualitative Analysis software (version B.06.00 Agilent Technologies) (395).

3.5.4. Biological effects of phlorotannin purified extracts

3.5.4.1. Anti- inflammatory activity

3.5.4.1.1. Cell assays

3.5.4.1.1.1. Cell culture conditions and treatments

RAW 264.7 macrophages were routinely cultured using DMEM + GlutaMAX™-I supplemented with 10% FBS and 1% Pen Strep. Cells were kept in a humidified

3 atmosphere of 5% CO2, at 37 °C, in 25 cm flasks. At near-confluent stage, RAW 264.7 cells were detached from the flask with a countersink and seeded in 96-well plates. Solvent (DMSO) concentrations that did not affect cellular viability were determined and set at a maximum of 0.5% (v/v). Stock solutions of each phlorotannin purified extract were prepared with culture medium and sterilized through a 0.22 μm pore size membrane. To determine the effect of the purified phlorotannin extracts on RAW 264.7 cell viability and NO production, serial dilutions were tested in the range of 31.25 to 500 μg DE/mL.

3.5.4.1.1.2. Cell viability

The MTT assay was chosen to assess RAW 264.7 cell viability. RAW 264.7 cells were seeded at 2.5 × 104 cells/well and treated with serial dilutions of phlorotannin purified extracts for 24 h. Culture medium was then removed, and cells were incubated for 2 h, at 37 °C, with a 0.5 mg/mL MTT solution (in DMEM culture medium). MTT is reduced by mitochondrial dehydrogenases of metabolically active cells to form an insoluble purple

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formazan product (Figure 17). The formazan crystals were solubilized in a DMSO:isopropanol (3:1, v/v) mixture, and the extent of MTT reduction by the cells was quantified at 560 nm on a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA). The results of RAW 264.7 cell viability were expressed as the percentage of MTT reduction of treated cells relative to control. Four independent assays were performed in triplicate.

Mitochondrial dehydrogenases

MTT Formazan yellow tetrazolium salt purple dye

Figure 17. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, by mitochondrial dehydrogenases of metabolically active cells.

3.5.4.1.1.3. Nitric oxide determination

The effect on NO levels in the culture medium was evaluated after pre-treating RAW 264.7 cells (3.5× 104 cells/well) with non-toxic phlorotannin extract concentrations for 2

- h, followed by 22 h of co-exposition to 1 μg/mL LPS. Nitrite (NO2 ) accumulated in the culture medium was measured as an indicator of NO production. For this, equal volumes (75 µL) of culture supernatant and Griess reagent (1% sulfanilamide and 0.1% N-(1- napthyl) ethylenediamine in 2% H3PO4) were incubated for 10 min in the dark, at room

- temperature. Under acidic conditions, NO2 diazotize with sulfanilamide acid and couples with naphthyl ethylenediamine, resulting in a pink colored product, which was spectrophotometrically measured at 562 nm on a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA) (396) (Figure 18).

The effect of phlorotannin purified extracts on NO levels was calculated as 100× [1 − (absorbance of sample − absorbance of blank) / (absorbance of control − absorbance of blank)]. Cells in the control were grown in the absence of extracts, and blanks were made without the stimulation with LPS for each extract concentration tested. Four independent assays were performed in triplicate. Dexamethasone was used as positive control.

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+ - H N-(1-Naphthyl) NO2 + + ethylenediamine

Sulfanilamide

Diazonium product (pink)

- Figure 18. Nitrite (NO2 ) determination by Griess assay.

3.5.4.1.2. Cell-free assays

3.5.4.1.2.1. Nitric oxide radical scavenging capacity

The free radical scavenging activity of phlorotannin purified extracts was evaluated

• using SNP (6 mg/mL in phosphate buffer KH2PO4 100 mM, pH 7.4) as an NO donor in vitro, and measured by the Griess reaction (397). The •NO generated interacts with oxygen

- to form NO2 that can be estimated using Griess reagent (Figure 18). Different extract concentrations were incubated with the same volume (75 μL) of SNP, during 1 h, under light. Griess reagent was added and the absorbance was read at 562 nm on a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA), after 10 min incubation in the dark, at room temperature. The scavenging capacity was calculated according to the following equation: •NO scavenging (%) = 100× [1 − (absorbance of sample − absorbance of blank) / (absorbance of control − absorbance of blank)]. The percentage of scavenging activity was plotted against the sample concentration to obtain the IC50. Three independent assays were performed in triplicate.

3.5.4.1.2.2. Lipoxygenase inhibition

The inhibitory effect on LOX was assessed by monitoring the oxidation of linoleic acid to its conjugated diene, 13-hydroperoxy-linoleic acid, which was measured continuously at 234 nm, for 3 min, on a UV-vis Synergy™ HT plate reader (Biotek Instruments; Winooski,

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USA) operated by Gen5 Software (Figure 19) (398). Soybean LOX is widely accepted to model human 5-, 12-, and 15-LOX, given the high catalytic domain similarity between plant and mammalian LOX (399).

LOX

Linoleic acid 13-Hydroperoxyoctadecadienoic acid

Figure 19. Linoleic acid oxidation to 13-hydroperoxy-linoleic acid catalyzed by lipoxygenase (LOX).

Each algal extract (20 µL) was pre-incubated with 20 µL of soybean LOX 100 U in phosphate buffer (Na2HPO4·2H2O 100 mM, pH 9.0), for 5 min at room temperature, before adding 20 µL of substrate (linoleic acid) at 4.18 mM in ethanol to start the reaction. The maximum non-interfering DMSO concentration was determined, and 0.5% (v/v) was not exceeded. LOX inhibition was calculated as follows: LOX inhibitory activity (%) =100 × [1 − (mean V of sample / mean V of control)], where mean V corresponds to the mean velocity of kinetic well analysis. The percentage of inhibitory activity was plotted against the sample concentration to obtain the half maximal inhibitory concentration (IC50). Three independent assays were performed in triplicate. Quercetin was used as positive control.

3.5.4.2. Anti-allergic activity

3.5.4.2.1. Cell assays

3.5.4.2.1.1. Cell culture conditions and assays

RBL-2H3 cells were cultured in DMEM + GlutaMAX™ – I supplemented with 10% FBS and 1% Pen Strep solution and maintained at 37 °C in a humidified atmosphere with

5 5% CO2. At near confluence (80–90%), cells were sub-cultured and seeded at 3 × 10 cells/mL in 24-wells plates, as outlined below. The calcium ionophore A23187 and an immunologic stimulus referred herein as IgE/antigen were used to induce RBL-2H3 cell degranulation, which was assessed by the amount of β-hexosaminidase and histamine released. Solvent (DMSO) concentrations that did not affect cellular viability were determined and set at a maximum of 0.5% (v/v). The effect of the extracts (125, 250 and

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500 μg DE/mL) in the absence of stimuli was simultaneously determined for each experiment. Quercetin was used as positive anti-degranulation control.

3.5.4.2.1.2. A23187-mediated cell degranulation

Cells were pre-treated with serial dilutions of each purified phlorotannin extract (125, 250 and 500 μg DE/mL in EBSS + 0.1% BSA) 30 min prior to the addition of the ionophore (150 ng/mL), and maintained in culture for another 30 min. Afterwards, supernatants were collected and used for the determination of both β-hexosaminidase and histamine release, whereas MTT viability and CVS proliferation assays were performed in adherent cells.

3.5.4.2.1.3. IgE/antigen-mediated cell degranulation

RBL-2H3 cells were challenged with anti-DNP IgE (50 ng/mL in culture medium) during 16 h and further stimulated for 1 h with DNP-BSA (50 ng/mL in EBSS + 0.1% BSA). Throughout the assay, cells were co-incubated (16 h) with serial dilutions of each phlorotannin extract (125, 250 and 500 μg DE/mL). Supernatants were collected to quantify released β-hexosaminidase and released histamine, while viability assays were performed on adherent cells.

3.5.4.2.1.4. MTT reduction assay

RBL-2H3 cell viability was assessed by the mitochondrial dehydrogenase dependent reduction of MTT to formazan (Figure 17). Briefly, after cell treatment, the adhered cells were incubated with MTT (0.5 mg/mL in DMEM culture medium), for 30 min at 37 °C. Cell supernatants were further discarded and formazan crystals, solubilized in a DMSO:isopropanol (3:1, v/v) mixture, were spectrophotometrically quantified at 560 nm in a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA). Results were expressed as percentage of control. Four independent assays were performed in duplicate.

3.5.4.2.1.5. Crystal violet staining assay

CVS assay is a simple, non-enzymatic method in which the amount of dye absorbed depends on the total DNA content in the culture, allowing the estimation of RBL-2H3 cell proliferation (400). For this, RBL-2H3 cells were incubated with a 0.1% CVS solution (in

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0.9% NaCl) for 20 min at room temperature, on a bench rocker (Movil-Tub, J.P. Selecta, s.a., Barcelona, Spain). The cells were lysed with 10% glacial acetic acid solution and the absorbance was measured at 560 nm, using a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA). Four independent assays were performed in duplicate.

3.5.4.2.1.6. Determination of β-hexosaminidase released

The measurement of β-hexosaminidase released from stimulated-RBL-2H3 cells was conducted as described by Pinho et al. (398). To 30 μL of cell supernatant were added 50 μL of substrate solution (p-nitrophenyl N-acetyl-β-D-glucosaminide 1.3 mg/mL in citrate buffer, pH 4.5). After 1 h incubation at 37 °C, the reaction was stopped with NaOH 0.5 M and the reaction product, p-nitrophenolate, was spectrophotometrically determined at 405 nm, using a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA) (Figure 20). Four independent assays were performed in duplicate.

β-hexosaminidase +

p-Nitrophenolate (yellow) p-Nitrophenyl N-acetyl-β-D-glucosaminide N-Acetyl-β-D-glucosaminide

Figure 20. β-Hexosaminidase-catalyzed conversion of p-nitrophenyl N-acetyl-β-D-glucosaminide into N-acetyl-β-D-glucosaminide and the yellow p-nitrophenolate product.

3.5.4.2.1.7. Determination of histamine released

The quantity of histamine released from stimulated-RBL-2H3 cells was indirectly measured by the conversion of histamine into fluorescent histamine-OPA-products. After incubation of 250 μL of cell supernatant with 50 μL of NaOH 1 M and 12.5 μL of OPA 1% (w/v), 4 min at room temperature, the reactional mixture was stopped with HCl 3M. Precipitated proteins were removed by high-speed centrifugation (14 000 rpm, 3 min) and the fluorescent OPA-products were quantified in the supernatants (λex 360 nm; λem 450 nm), in a microplate spectrofluorimeter (Synergy™ HT, Biotek Instruments Winooski, USA) operated by Gen5 Software. The histamine content (µM) was calculated by the interpolation on the standard calibration curve (y = 20158x + 4803.8; r2 = 0.9993). Four independent assays were performed in duplicate.

77 EXPERIMENTAL SECTION

3.5.4.2.2. Cell-free assays

3.5.4.2.2.1. β-Hexosaminidase inhibition

The capacity of purified phlorotannin extracts to directly inhibit β-hexosaminidase enzymatic activity was assessed as follows: serial dilutions of each extract (5 μL) were incubated with supernatant of degranulated RBL-2H3 cells (25 μL), together with 50 μL of substrate solution (p-nitrophenyl N-acetyl-β-D-glucosaminide 1.3 mg/mL in citrate buffer, pH 4.5) for 1 h, at 37 °C. The reaction was stopped with NaOH 0.5 M and absorbance was read at 405 nm on a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA). In each experiment, the protein content of cell supernatant was determined spectrophotometrically at 595 nm with Bradford reagent, using BSA as standard, and adjusted to approximately 20.0 µg/mL. Four independent experiments were performed in duplicate.

3.5.4.2.2.2. Hyaluronidase inhibition

HAase inhibition assay was performed as in Ferreres et al. (57), with some modifications. Each reaction tube contained 25 μL of extract serial dilution (prepared in sodium formate buffer pH 3.68), 175 μL of HA (0.7 mg/mL in water:buffer, 5:2, v/v) and 25 μL of HAase (900 U/mL in NaCl 0.9%). After 30 min incubation at 37 °C, the enzymatic reaction was stopped with 25 μL of Na2B4O7·10H2O 0.8 M, followed by subsequent heating for 3 min in a boiling water bath (Morgan-Elson reaction) (Figure 21). To each cooled test tube, 375 μL of DMAB solution (prepared in a mixture of water:HCl:glacial acetic acid (2.1:10.4:87.5, v/v) and further diluted 1:2 (v/v) with glacial acetic acid immediately before use) were added. The tubes were incubated at 37 °C for another 20 min and the absorbance of the colored product was measured at 560 nm in Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA). Four independent assays were performed in duplicate. DSCG was used as positive control.

78 EXPERIMENTAL SECTION

Hyaluronidase

D-glucuronic D-Glucuronic acid acid N-Acetyl-D-glucosaminide N-Acetyl-D-glucosaminide

Hyaluronic acid

N-Acetyl-D-glucosaminide Morgan-Elson reaction at the reducing end 100 ºC pH 9.0 -ROH

HCl Glacial acetic acid

-H2O

Chromogen III Chromogens I and II

DMAB + H+

- H2O

Red colored product

Figure 21. Morgan-Elson reaction applied to the determination of hyaluronidase (HAase) activity.

DMAB, 4-dimethylaminobenzaldehyde; H2O, water; HCl, hydrochloric acid. Adapted from (401).

CHAPTER III

RESULTS AND DISCUSSION

4. Results and Discussion

4.1. Influence of changing environmental conditions on fatty acid and pigment composition of the kelps L. ochroleuca, S. latissima, and S. polyschides

Kelps are major primary producers in coastal ecosystems and well-known natural reactors with a huge metabolic plasticity. Unrevealing algal chemical signatures is therefore important for understanding the vital functions of algae and their adaptation to changing environmental conditions (402). Such chemical studies may also help identifying and systematizing the effects of different parameters in algal chemical profiles, encouraging the rational exploitation of coastal proliferative seaweed species when bioprospecting for valuable functional compounds.

The effect of cultivation at different depths and harvesting periods, as well as the distribution of fatty acids and pigments among algal tissues of both wild and cultivated specimens of L. ochroleuca, S. latissima, and S. polyschides, when available, was investigated (Table 5). Fatty acid qualitative and quantitative profiles of each analyzed material were achieved by GC/IT-MS and GC-FID, respectively. For pigment analysis, HPLC coupled to DAD was employed.

4.1.1. Fatty acids

4.1.1.1. General overview

Twenty-seven fatty acids, containing between 12 and 24 carbon atoms, were identified by GC/IT-MS, after saponification and derivatization to their respective methyl esters (Figure 22). S. latissima, S. polyschides and L. ochroleuca tissues subjected to seasonal variations, from different sources (wild and aquaculture), and cultivated at different depths at sea exhibited a complex fatty acid profile, characterized mainly by the presence of medium and long fatty acyl chains (14–22 carbon atoms), with different degrees of unsaturation.

Aside from species-specific variation, the overall qualitative profiles of the kelps studied herein are in agreement with previous reports (76,132,133,403–405).

83 RESULTS AND DISCUSSION

7 11 1000 1000 14

10 Milhares Milhares 800 800

600 600 9 12 4 13

400 KCounts 400 6 16 20 17 21 23 18 200 200 24 19 22 25 27 5 1 2 3 8

0 0 15 1520 2025 2530 3035 3540 4540 5045 50 2500 Time (min.)

Milhares 2000

1500

KCounts 1000

500

0 0 10 20 30 40 50 60 Time (min.)

Figure 22. Representative GC/IT-MS chromatogram of the fatty acid profile of whole-specimen samples of L. ochroleuca (Lo_W_Am_Apr13). C12:0 (1), C13:0 (2), C14:1n-5c (3), C14:0 (4), C15:0 (5),

C16:1n-7c (6), C16:0 (7), C17:0 (8), C18:3n-6c (9), C18:2n-6c (10), C18:1n-9c (11), C18:1n-9t (12), C18:0 (13), C20:4n-6c

(14), C20:5n-3c (15), C20:3n-6c (16), C20:2n-6c (17), C18:3n-3c (18), C20:0 (19), C21:0 (20), C22:6n-3c (21), C18:2n-

6t (22), C22:1n-9c (23), C22:0 (24), C23:0 (25), C24:1n-9c (26), C24:0 (27).

The quantification achieved by GC-FID revealed total fatty acid concentrations ranging between 1255.31 and 2662.85 mg/kg of dry algae. The lowest fatty acid amounts were found in the whole specimens of L. ochroleuca from Amorosa, collected in February 2013 (Lo_W_Am_Feb13), whereas samples of whole specimens of S. polyschides from Praia Norte, collected in January 2013 (Sp_W_N_Jan13), exhibited the highest content (Figure 23).

84 RESULTS AND DISCUSSION

3000 i,l

d,i,j 2500 d,k,l c,e,j,k,m,s,u h,q,t h,t,u h,m,t 2000 b,h,s,t a,b,g a,b,n,q a,b,n,r a,b,n,o f f,g,o,p,q,r d,f f,g,n,p f,g,o,p,q,r 1500 d,f f,p p b,h a,c,h,k, b,h b,h,i b,n b,k a,b a,b,m m,n a,i,k,m,n 1000 a,b,m j,k a,j,m a,l mg/Kg dryalgae mg/Kg l,m c,l,m,r,w g,j,l k,s m b,e,i, m b,e,i,t q,t,u,v g m,q r,s c,i,r,w b,i,j, b,i,l,v g,n a,d,n,u g,h,n, g,o a,d,p j j k,n,o,p n,o,q d,g k,q c,j,l d,o o,p 500 a,c,i,j a,c,j a,c,j a,c a,e a,e d,f,h d,e h

∑ FA ∑ SFA ∑ MUFA ∑ PUFA

Figure 23. Sum of saturated (ΣSFA), monounsaturated (ΣMUFA), polyunsaturated (ΣPUFA) and total fatty acid content (ΣFA) in L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts (mg/kg dry algae). Results are expressed as mean (standard deviation) of three determinations. Different letters denote statistically significant differences (p < 0.05) between samples (One-way ANOVA, Tukey's multiple comparison test). Identity of samples according to Table 5.

85 RESULTS AND DISCUSSION

The algal specimens were mainly composed by SFA, which represented a minimum of 59.8%, reaching up to 73.3% of total fatty acids. Similar to other Ochrophyta

(404,406,407), C16:0 was the dominant SFA, its absolute content ranging between 500.52 and 1022.72 mg/Kg of dry algae (Table 9). Although many brown seaweeds are described to contain higher amounts of unsaturated fatty acids (UFA) than SFA, species collected along the Atlantic coast of the Iberian Peninsula, like the ones investigated herein, and from other areas with warm waters, exhibit more SFA than that usually found for brown macroalgae grown in colder waters (404,407,408). Still, the overall UFA profile of the studied kelps is of particular interest, comprising relatively high-value compounds, of which some PUFA are highlighted.

Consistent with the typical fatty acid profile of Ochrophyta, the main PUFA across all the studied specimens were C18 and C20 fatty acids, namely C18:2n-6c, C20:4n-6c and C20:5n-3c.

Brown macroalgae use both C18 and C20 PUFA as substrates for LOX and other enzymatic systems, which catalyze the formation of several hydroxylated fatty acid derivatives, short chain aldehydes, and carboxylic oxylipins involved in their defense and innate immunity

(22). In this work, the concentrations of C20 PUFA were always generally higher than those of C18 PUFA. Among all algal specimens, n-3 PUFA accounted for 2.5–8.7% of total fatty acids, whereas the proportion of n-6 PUFA ranged between 8.7% and 27.3% of total fatty acids. These proportions were in part due to the high levels of C20:4n-6c (36.74–387.30 mg/kg of dry algae), in contrast with the low amounts of C20:5n-3c (28.12–151.79 mg/kg of dry algae) (Table 10). PUFA of n-3 and n-6 series have opposing physiological functions and their balance is important for normal growth and development (116). From a human health perspective, C20:5n-3c is an important dietary PUFA that has been linked to promising results in the prevention of cardiovascular events and to a low risk of development of Alzheimer’s disease (AD) (409). The n-6 PUFA C20:4n-6c, on the other hand, is the prototypal substrate for enzymatically-catalyzed biosynthesis of oxylipins in mammals, which operate in inflammatory processes (410). Although the relationship between n-3 and n-6 fatty acids, inflammation and disease pathogenesis continues to be the subject of extensive study, the biological effects of these fatty acids depend then on the maintenance of a proper balance between total n-6 and n-3 PUFA, rather than on the absolute amount of each single molecule (123).

Overall, the n-6/n-3 ratio found herein were higher (1.70–7.02) than those reported by others (132,403). Yet, the role of other compounds cannot be overlooked. For instance, the n-9 MUFA C18:1n-9c plays an important role in the metabolism of essential fatty acids, being also described to exert anti-inflammatory effects (411,412). As so, it is possible that the generally high amounts of C18:1n-9c may offset the levels of C20:4n-6c. Although some

86 RESULTS AND DISCUSSION

controversial data can be found in the literature, it is believed that C18:1n-9c exerts its anti- inflammatory effects through competitive substitution of membrane arachidonate, reducing the biosynthesis and subsequent release of arachidonic-derived metabolites (412,413).

87 RESULTS AND DISCUSSION

Table 9. Saturated fatty acid content of L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts (mg/Kg dry algae)1.

2 Sample C12:0 C13:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 C21:0 C22:0 C23:0 C24:0

304.55 14.65 512.36 11.63 86.17 21.55 Sl_S(5m)_PV_May12 nqa nqa nqa nqa nqa nqa (4.02)a (0.30)a (19.57)a,f,q (0.39)a,i (0.34)a,f,p (1.21)a,p 347.01 18.24 679.07 16.15 125.35 Sl_S(10m)_PV_May12 nqa nqa nqb nqa nqa nqa nqa (6.49)b (1.70)b,i,j (61.62)b,d (0.91)b,h (5.60)b 388.79 21.15 594.21 89.27 27.02 20.99 Sl_S(15m)_PV_May12 nqa nqa nqc nqa nqa nqa (19.06)c (0.41)c,h (8.89)a,b (5.54)a,c,f (1.13)c,f,g,n,o (1.87)b,h 263.34 28.67 662.49 43.51 339.26 30.13 Sl_B(5m)_PV_May12 nqa nqa nqa nqa nqa nqa (17.64)d,f (1.21)d (4.18)b,c (1.76)d (29.16)d (1.22)c,d 301.47 30.88 802.19 41.13 346.73 33.21 23.00 11.58 Sl_B(10m)_PV_May12 nqa nqa nqa nqa (0.18)a,d (1.97)d,e (78.50)d,k,i (2.48)d (10.07)d (1.81)d,e,j (0.05)b,c (0.04)b 292.53 31.54 790.05 31.76 300.52 32.88 23.00 11.21 Sl_B(15m)_PV_May12 nqa nqa nqa nqa (2.29)a,d,e (1.42)e (57.01)d,k,i (2.08)e (27.34)e (1.38)d,e,j (0.77)b,d (0.59)b 241.49 18.20 528.29 11.95 114.95 30.34 Sl_W_Am_Out12 nqa nqa nqa nqa nqa nqa (8.59)f (1.16)b,i,j (26.39)a,e,f,q (0.45)a,i (2.44)a,b (0.79)c,e 288.40 14.38 500.52 64.24 25.16 19.67 Sl_W_Am_Jul13 nqa nqa nqc nqa nqa nqa (3.10)a,d,p (0.03)a (14.17)a (1.75)f,p (0.57)a,f (1.46)b 254.52 15.02 608.18 12.90 97.74 24.81 Sl_W_Am_Sep13 nqa nqa nqa nqa nqa nqa (21.55)e,f,p (0.08)a (42.56)a,b (0.04)a,f,i (5.87)a,b,g,h,o (1.53)a,g,h 238.42 638.34 78.73 27.88 Sl_W_Am_Dec13 nqa nqa nqf nqc nqa nqa nqa nqa (16.93)f (22.19)b,f,g (5.08)f,g,i,p (0.41)c,f,g,i 608.27 24.35 740.34 17.34 102.92 36.23 25.12 Sl_W_IMTA_Apr13 nqa nqa nqa nqa nqa (10.37)g (0.42)g (71.95)c,d,g,h,k (0.96)b (7.07)a,b,i,j,l,m,n (2.80)j (1.68)c,d,e 430.93 21.30 789.36 20.85 118.64 41.18 Sl_W_IMTA_Jul13 nqa nqa nqa nqa nqa nqa (32.10)h (1.19)c,h (15.91)c,d,i,k (0.88)g (5.11)b,c,h,j (1.59)k 166.73 16.53 1022.72 17.28 159.73 34.12 25.27 Sp_W_N_Jan13 nqa nqa nqa nqa nqa (1.09)i,j,o (0.30)a,b (57.71)j (0.88)b (12.04)k (0.60)e,j (2.47)c,d,f 182.73 18.73 827.66 13.46 85.37 25.62 Sp_W_IMTA_Jan13 nqa nqa nqa nqa nqa nqa (9.32)i,k (0.49)b,c (24.26)k,i (1.12)a,h,i (1.58)a,f,p (0.26)f,g,l 192.20 16.61 718.01 14.52 74.86 67.22 23.22 20.22 Sp_W_IMTA_Apr13 nqa nqa nqa nqa (4.40)i (0.13)a,b (13.58)b,d,k (0.83)a,b (3.64)f,g,l,p (1.73)m (0.20)b,e,f,g (0.46)b

88 RESULTS AND DISCUSSION

Table 9. Cont.

2 Sample C12:0 C13:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 C21:0 C22:0 C23:0 C24:0

135.23 15.25 644.96 12.40 72.24 Lo_W_N_Apr13 nqa nqa nqb nqa nqa nqa nqa (0.14)k,l (0.32)a (21.80)b,e,h,l,m (0.31)a,i,j (0.21)f,g,m,p 144.92 22.25 760.97 22.52 84.60 24.42 24.79 Lo_W_N_Jun13 nqa nqa nqa nqa nqa (9.07)j,k,l,m (0.41)g,h (33.26)c,d,g,k,l,n (0.65)g (6.46)a,f,p (0.62)a,h,i,l,n (0.10)c,d,g 120.74 530.41 56.22 23.97 19.52 Lo_W_Am_Feb13 nqa nqa nqf nqc nqa nqa nqa (6.34)l (44.58)a,f,m,o (1.36)p (1.72)a,l,o,p (0.71)h 178.91 15.79 754.94 11.05 76.47 21.10 Lo_W_Am_Apr13 nqa nqa nqa nqa nqa nqa (7.15)i,m (0.05)a,i (31.17)c,d,g,k,l,p (0.78)i (0.26)f,g,n,p (0.88)h,p 167.00 15.75 889.43 70.42 21.72 Lo_W_Am_Sep13 nqa nqa nqc nqa nqa nqa nqa (3.08)i,j,n (0.49)a,j (44.83)i (7.01)f,o,p (1.28)a,p 150.30 14.01 633.31 66.96 22.63 Lo_W_Am_Nov13 nqa nqa nqc nqa nqa nqa nqa (12.51)k,l,m,n,o (0.50)a (37.86)b,h,o,p,q (5.36)f,g,p (0.73)a,l,p 127.35 15.45 645.68 63.56 Lo_W_Am_Dec13 nqa nqa nqc nqb nqa nqa nqa nqa (10.32)l (0.58)a (24.96)b,e,h,n,o,p (2.55)f,p 192.20 15.58 687.88 15.37 103.58 26.58 9.01 25.00 11.74 Lo_W_IMTA_Apr13 nqa nqa nqa (12.16)i (0.98)a (52.05)b,d (1.21)b,f,h,j (8.45)a,b,i,l,n (0.92)c,f,g,n,o (0.45)b (1.12)c,d,g (0.83)b

1 Results are expressed as mean (standard deviation) of three determinations. nd, not detected; nq, not quantified. Different superscript letters denote statistically significant differences (p < 0.05) in the same column (One-way ANOVA, Tukey's multiple comparison test). 2 Identity of samples according to Table 5.

89 RESULTS AND DISCUSSION

Table 10. Unsaturated fatty acid content of L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts (mg/Kg dry algae)1.

MUFA PUFA 2 Sample C14:1 C16:1 C18:1 C18:1 C22:1 C24:1 C18:2 C18:2 C18:3 C18:3 C20:2 C20:3 C20:4 C20:5 C22:6

n-5c n-7c n-9c n-9t n-9c n-9c n-6c n-6t n-6c n-3c n-6c n-6c n-6c n-3c n-3c 77.87 185.96 79.73 16.88 12.83 15.35 172.06 35.97 11.38 Sl_S(5m)_PV_May12 nqa nqa nqa nqa nqa nqa (2.26)a,o,p,t (16.57)a,d,n (7.53)a,c,f,p,w,x (1.11)a,g,q (0.12)a,d (0.54)a,l (15.53)a,o,p (0.76)a,j (0.30)a 106.35 193.03 66.46 19.22 12.27 15.30 212.48 36.41 12.11 Sl_S(10m)_PV_May12 nqa nqa nqa nqa nqa nqa (5.95)b,i (12.40)a,l,q (4.49)a,c (0.04)a,b,g (0.04)a,b,q,r (0.77)a,l (15.52)b (0.33)a,j (1.18)a,f 95.11 234.96 88.76 15.43 15.87 9.18 12.50 21.29 205.80 37.25 16.73 Sl_S(15m)_PV_May12 nqa nqa nqa nqa (4.37)b,c,k (13.76)b,f (0.80)a,b (0.14)b (0.54)a,c (0.56)b (0.66)a,c,q,r (1.61)b,r (7.16)b (1.27)a,j (0.89)b 81.25 120.96 27.33 15.71 61.15 11.90 17.35 19.16 14.73 16.53 36.74 28.12 11.39 Sl_B(5m)_PV_May12 nqa nqa (0.02)a,d,p,t (5.93)c (0.36)b (0.95)b (1.81)c (0.36)c (0.27)a,d,q (1.47)c (0.53)d,f (0.69)a,c,d,s (2.96)c (2.25)a (0.71)a 102.92 135.42 29.98 12.76 70.78 19.11 23.98 17.46 18.77 19.06 59.72 49.57 19.00 Sl_B(10m)_PV_May12 nqa nqa (4.11)b,i (11.84)c (0.83)b (0.05)c (5.35)a,c,d (0.04)d (1.86)e,h (0.84)d (0.31)e (0.41)b,d (3.91)c,d (3.25)b,l (0.25)c 78.96 206.47 25.97 15.45 105.14 11.77 44.48 19.17 15.84 21.02 156.70 151.79 37.68 Sl_B(15m)_PV_May12 nqa nqa (0.14)a,e (12.70)a,b,l,q (0.17)b (0.76)b (6.23)b,e,o,s (0.21)c,e (0.12)f,k,l (0.85)c (0.59)f,l,s (1.29)b,e,r (9.86)a,e,p (7.65)c (1.19)d 94.13 154.77 95.75 10.70 26.30 12.63 20.50 107.97 35.83 11.79 Sl_W_Am_Out12 nqa nqa nqa nda nqa (6.12)b,d,f (10.61)c,d (6.57)b,f,s (0.56)e,f (1.56)e (0.34)a,d,g (0.89)b,e,f,r (8.42)f,n (1.50)a,j (0.81)a 41.86 142.27 100.32 20.86 15.39 64.78 32.92 Sl_W_Am_Jul13 nqa nqa nqa nda nqa nda nqh nqe (0.31)g (1.23)c,e (5.52)b,g,o,x (1.41)d,g,h (0.05)a,g,l (0.68)c,g (2.95)a 97.65 263.58 123.57 32.07 10.35 16.32 142.87 57.65 Sl_W_Am_Sep13 nqa nda nqa nda nqa nqa nqe (1.79)b,h,m,r,s (3.36)f,i,o,p (10.01)e,g,h,q (0.96)i (0.46)b,c,i (0.65)a,d,m (7.50)a,h (4.67)b,d 111.72 373.79 132.60 10.67 47.78 15.25 31.38 252.74 99.42 Sl_W_Am_Dec13 nqa nda nqa nda nqa nqe (3.63)i (0.67)g (7.68)h,i,k,t (0.09)f (0.01)f (0.76)f,j,l,s (0.67)h (15.43)i (5.61)e,k 14.42 134.84 273.73 151.66 20.30 37.87 16.28 17.79 117.20 73.36 Sl_W_IMTA_Apr13 nda nqa nqa nqa nqe (0.48)b (3.85)j (16.44)i,r (12.76)i,j (1.31)g (2.94)j,m,s (1.59)f,l (1.26)a,d,f,i (6.26)f,h,j,n (1.42)f,i 16.70 107.75 219.65 122.12 41.22 10.45 103.80 127.03 Sl_W_IMTA_Jul13 nqa nqa nqa nqa nqa nqj nqe (0.71)c (4.29)h,i,k (6.28)a,b,h (3.20)e,g,k,q (0.17)j,k (0.09)b,c,g,k (5.23)f,n (5.17)g 59.88 379.11 58.61 172.29 41.12 10.27 17.16 32.10 370.11 79.82 Sp_W_N_Jan13 nqa nqa nda nqa nqe (2.07)l (3.55)g (0.37)c (4.32)j,l,n,v (0.08)j,l (0.33)b (1.15)e,l (0.97)h (16.61)k (2.60)f 82.10 290.13 96.19 180.25 35.21 15.53 29.39 387.30 106.90 Sp_W_IMTA_Jan13 nqa nqa nda nqa nqa nde (1.89)a,c,f,p,t (16.81)i (3.09)d (16.02)l,m (1.55)i,m (1.26)f,l,m (2.36)h (15.04)k (2.52)e 87.98 199.35 85.62 152.63 46.37 22.22 26.87 21.78 213.33 70.56 Sp_W_IMTA_Apr13 nqa nqa nda nqa nqe (5.03)a,c,f,m (16.95)a,j (6.99)e (13.67)i,n (0.54)h (0.11)b,h,n (1.14)n (0.42)b,k (16.11)b (4.54)f,h

90 RESULTS AND DISCUSSION

Table 10. Cont.

MUFA PUFA 2 Sample C14:1 C16:1 C18:1 C18:1 C22:1 C24:1 C18:2 C18:2 C18:3 C18:3 C20:2 C20:3 C20:4 C20:5 C22:6

n-5c n-7c n-9c n-9t n-9c n-9c n-6c n-6t n-6c n-3c n-6c n-6c n-6c n-3c n-3c 84.15 185.47 89.98 17.82 11.18 13.27 114.22 77.94 Lo_W_N_Apr13 nqa nqa nqa nda nqa nqa nde (6.52)a,c,f,n (2.87)a,d,k (1.71)a,f,o (0.49)a,g,o (0.68)a,i,k (0.75)l (0.42)f,h,l,n (0.35)f 66.89 174.44 99.48 16.35 11.74 14.95 125.99 62.63 Lo_W_N_Jun13 nqa nqa nqa nqa nqa nqa nde (3.57)e,l,o (2.98)d,e,j,l,m (1.54)b,o,p,q (0.93)a,p (0.72)a,i,k,o (0.16)l,m,s (9.09)e,f,h,m (3.11)d,h,i 73.36 151.51 74.58 11.84 14.47 13.01 15.63 92.43 44.15 13.47 Lo_W_Am_Feb13 nqa nqa nqa nda nqa (5.22)e,n,p,q (5.23)a,k,m,n (3.25)a,c,f,r (0.48)c (1.06)c,o,p,q (0.27)a,d (0.27)a,l,n (3.54)d,g,n (1.72)j,l (0.21)f 85.56 240.68 119.79 18.78 13.01 17.59 127.14 79.11 Lo_W_Am_Apr13 nqa nqa nqa nda nda nqa nqe (1.10)a,c,f,p,r (15.37)b,f,o,r (11.06)e,g,q,s,t (1.05)a,g,n,r (0.55)a,d (0.74)a,d,m,o (8.26)e,f,h (5.54)f 99.52 297.36 179.08 34.62 12.39 17.18 195.33 103.19 Lo_W_Am_Sep13 nqa nqa nqa nqa nqa nqa nqe (6.18)b,i,m (22.83)i (10.36)l (3.33)i,s (0.44)a,i,k,p (0.74)a,d,m,p (19.48)b,o (0.93)e,k 85.50 235.07 148.99 39.05 16.48 19.51 180.71 94.23 Lo_W_Am_Nov13 nqa nqa nqa nqa nqa nqa nde (5.93)a,c,f,p,s (0.61)b,p (3.41)i,v (0.39)j,m (0.20)f,l (0.44)b,i,o,p,q,r (10.64)b,p (5.28)k 71.92 181.52 93.67 17.01 13.56 18.20 167.40 62.16 Lo_W_Am_Dec13 nqa nqa nqa nda nqa nqa nqe (6.82)e,l,n,t (4.99)d,j,n,q (3.18)b,d,o,r,w (0.42)a,g,q (0.89)d,j,m,o,p,q (0.41)c,d,e,g,n,q (0.77)a,o,p (1.22)d,h 63.93 246.96 168.10 13.07 22.57 13.83 22.12 142.58 103.83 Lo_W_IMTA_Apr13 nqa nqa nqa nqa nqa nqe (4.49)l,q (5.34)f,h,p,r (10.26)j,l,m,n,u,v (0.26)i (1.03)b,e,h,r (0.52)d,m,o,p,r,s (0.98)k,r (6.50)a,h,j,l (0.78)e,k

91 RESULTS AND DISCUSSION

Both Pearson correlation test and principal component analysis (PCA) were performed to confirm the influence of different environmental factors on fatty acid distribution and accumulation in algal material. Multivariate statistics allowed the segregation of algal specimens according with their fatty acid content (Figure 24). PCA was performed for all species, individually, using the fatty acid content (mg/kg of dry algae) of each sample as variables.

For S. latissima, PCA of normalized fatty acid dataset explained 63.0% of total variations, PC1 accounting for 33.3% of the variance and PC2 for 29.7% (Figure 24A1 and A2). Regarding S. polyschides, the two scores obtained from the latent vectors accounted for all registered variations (100.0%) (Figure 24B1 and B2), while for L. ochroleuca, PCA explained 69.7% of total variations, PC1 accounting for 46.5% of the variance and PC2 for 23.2% (Figure 24C1 and C2).

In spite of qualitative similar profiles, S. latissima blades cultivated at sea (Sl_B(5m)_PV_May12, Sl_B(10m)_PV_May12, and Sl_B(15m)_PV_May12) were clearly distinguished from each other along PC2: samples from deeper locations (Sl_B(15m)_PV_May12) were placed in the more positive side of the plan due to their high loadings of UFA (Figure 24A1 and A2). The whole-specimen samples grown in IMTA (Sl_W_IMTA_Apr13 and Sl_W_IMTA_Jul13), as well as the ones from Amorosa collected in winter (Sl_W_Am_Dec13) (Figure 24A1), were closely positioned along the positive axis of PC2 due to their high amounts of the MUFA C16:1n-7c and C18:1n-9c, and to the high levels of PUFA, namely C18:3n-6c, C18:2n-6c, C20:4n-6c and C20:2n-6c (Figure 24A1 and A2). Sample positioning across PC1 and PC2 positive axes indicates that lipid changes throughout the year (from summer to winter), and with increasing depth, are often accompanied by high unsaturation indexes and accumulation of specific fatty acids. Due to both the low total fatty acid content and the relatively high n-6/n-3 ratio (> 5), the remaining S. latissima specimens were located along the negative region of PC1 (Figure 24A1), with a clear distinction of the sample collected in the summer at Amorosa

(Sl_W_Am_Jul13) based on its levels of C24:0.

92 RESULTS AND DISCUSSION

S. latissima Variables: PC1 and PC2 (63.0%) Observations: PC1 and PC2 (63.0%)

A1 A2

PC2 (29.7%) PC2 (29.7%) PC2 (29.7%)

PC1 (33.3%) PC1 (33.3%)

S. polyschides Variables: PC1 and PC2 (100.0%) Observations: PC1 and PC2 (100.0%)

B1 B2

PC2 PC2 (37.2%) PC2 PC2 (37.2%)

PC1 (62.8%) PC1 (62.8%)

L. ochroleuca Variables: PC1 and PC2 (69.7%) Observations: PC1 and PC2 (69.7%)

C1 C2

PC2 PC2 (23.2%) PC2 (23.2%)

PC1 (46.5%) PC1 (46.5%)

Figure 24. Projection of S. latissima (A1), S. polyschides (B1) and L. ochroleuca (C1), under the influence of different parameters (variables: all samples identified according with Table 5) and loadings (A2, B2 and C2) by fatty acid composition [variables: C14:0, C14:1n-5c, C15:0, C16:0, C16:1n-7c, C17:0, C18:0, C18:1n-9c, C18:1n-9t, C18:2n-6c, C18:2n-6t, C18:3n-6c, C18:3n-3c, C20:0, C20:2n-6c, C20:3n-6c, C20:4n-6, C20:5n-3c,

C21:0, C22:0, C22:1n-9c, C22:6n-3c, C23:0, C24:0, SFA (saturated fatty acids), MUFA (monounsaturated fatty acids), PUFA (polyunsaturated fatty acids), n-3, n-6, n-9, n-6/n-3 (n-6/n-3 ratio), and Total (total fatty acids)] into the plane composed by the principal components PC1 and PC2 containing 63.0, 100.0 and 69.7% of the total variance for S. latissima, S. polyschides and L. ochroleuca, respectively.

93 RESULTS AND DISCUSSION

The model was also able to discriminate S. polyschides samples richer in fatty acids, by positioning them in the positive region of PC1 (samples Sp_W_N_Jan13 and Sp_W_IMTA_Jan13) (Figure 24B1 and B2). The levels of SFA and PUFA, and the n- 6/n-3 ratio dictated their spatial separation along PC2. In fact, the high PUFA loadings, namely of C18:2n-6c, C20:4n-6c and C20:5n-3c, positioned the sample Sp_W_IMTA_Jan13 in the positive side of PC2. On the other hand, Sp_W_N_Jan13 was placed in the negative side of PC2 due to considerably high n-6/n-3 ratio and high levels of the SFA C16:0, C17:0, C18:0 and C22:0. In addition, Sp_W_N_Jan13 was the only sample exhibiting C18:3n-3c at quantifiable amounts (Figure 24B1 and B2). Among the analyzed specimens of S. polyschides, the one harvested in IMTA during April (Sp_W_IMTA_Apr13) showed lower total fatty acid amount, leading to its positioning in the negative plane of PC1. Moreover, this was the only S. polyschides sample in which C18:2n-6t and C24:0 levels could be measured (Figure 24B1 and B2).

As Lo_W_IMTA_Apr13, Lo_W_Am_Apr13, Lo_W_Am_Nov13, and Lo_W_Am_Sep13 were the L. ochroleuca samples with higher fatty acids content, they were placed in the positive plan of PC1 (Figure 24C1). The specimen grown in IMTA (Lo_W_IMTA_Apr13) clearly stood out for its SFA composition, particularly for the presence of C21:0, C22:0 and C23:0 at quantifiable amounts. The remaining L. ochroleuca samples were placed on the negative axis of PC1 due to their relatively low total fatty acid content. The whole specimens from Amorosa collected in February (Lo_W_Am_Feb13) was the only samples in which C22:6n-3c was quantifiable.

4.1.1.2. Seasonal variation vs fatty acid profile

One of the many outcomes of this study was the detection of significant variation in the quantitative fatty acid profile of S. latissima, S. polyschides and L. ochroleuca specimens, sampled from different sites, at distinct harvesting periods (Figure 23).

Total fatty acid content in whole individuals of S. latissima from Amorosa, collected between October 2012 and December 2013, was evaluated. The specimens harvested in colder months (Sl_W_Am_Oct12, Sl_W_Am_Sept13, and Sl_W_Am_Dec13) exhibited higher total fatty acid content (1515.59, 1757.23, and 2058.72 mg/kg of dry algae, respectively), than the one collected in summer (Sl_W_Am_Jul13) (1330.77 mg/kg of dry algae). A similar trend was observed in S. latissima specimens grown in the pilot land- based installation: total fatty acid content, as well as total MUFA and PUFA levels, were lower in specimens collected in the summer (Sl_W_IMTA_Jul13) than in those sampled in the beginning of the spring (Sl_W_IMTA_Apr13) (Figure 23). In agreement with

94 RESULTS AND DISCUSSION

previous reports, this study provides clear evidence of seasonal variation of fatty acids in S. latissima collected from both wild natural stocks and IMTA: higher total fatty acid content and a general trend for increasing unsaturation are found during the colder winter months (132,133). The levels of MUFA (Pearson correlation; r = 0.9158; p = 0.0103), PUFA (Pearson correlation; r = 0.9562; p = 0.0028), and the total fatty acid amounts (Pearson correlation; r = 0.9030; p = 0.0136) in S. polyschides samples (Sp_W_IMTA_Jan13 and Sp_W_IMTA_Apr13) also showed to be positively correlated with typically low temperatures and light availability of colder months (Figure 23).

Variations of total fatty acid profile can be a response to changes in environmental parameters, such as temperature and light availability, in the course of the seasons. An increase of unsaturation promoted in part by the regulated activity of fatty acid desaturases has been broadly accepted as an adaptation strategy of macroalgae to cold- water conditions, to retain cell membrane fluidity and to protect photosynthetic machinery from low temperature photoinhibition (118,134). Although environmental variation provides an explanation for the observed fatty acid patterns, species-specific factors, such as thallus age, metabolic activity and the overall physiological state of the algae, may also translate into strong chemical differentiation (76,405). This is supported by our study, as total fatty acid levels (Pearson correlation; r = –0.8895; p = 0.0177) of L. ochroleuca specimens collected in 2013 at Amorosa (Lo_W_Am_Feb13, Lo_W_Am_Apr13, Lo_W_Am_Sep13, Lo_W_Am_Nov13, and Lo_W_Am_Dec13), and at Praia Norte (Lo_W_N_Apr13 and Lo_W_N_Jul13) showed to be negatively correlated with colder sampling periods, also following a distinct fatty acid distribution pattern across seasons than that observed for the other analyzed kelp species (Figure 23). These contrasting results can be mainly due to differences in algal reproductive period: S. latissima, for instance, reaches a maximum reproductive activity during coldest months (414), whereas L. ochroleuca is at a maximum in the summer (415). Indeed, the involvement of specific fatty acids during sexual reproduction of several brown macroalgae, as substrates for pheromone production, has been widely described (reviewed by (140)).

4.1.1.3. Wild natural stocks vs IMTA

No significant differences (p > 0.05) were observed between the fatty acid amounts found in wild and cultivated samples of S. polyschides (Sp_W_N_Jan13 and Sp_W_IMTA_Jan13, respectively) and L. ochroleuca (Lo_W_Am_Apr13 and Lo_W_IMTA_Apr13, respectively) (Figure 23). However, S. latissima grown in the pilot land-based installation (Sl_W_IMTA_Jul13) exhibited almost 60% more fatty acids per

95 RESULTS AND DISCUSSION

dry weight than the wild whole-specimen samples (Sl_W_Am_Jul13), at equal harvesting times. Though significantly higher SFA, MUFA and PUFA (p < 0.05) amounts were found (Figure 23, Tables 9 and 10), the most noteworthy difference was observed in the content of the n-3 PUFA C20:5n-3c, which reached nearly 4 times the levels found in S. latissima from natural wild stocks (Figure 25). To the best of our knowledge, such variation was registered herein for the first time. In a study conducted by Marinho et al. (132) no differences in the lipid content and fatty acid composition were detected between S. latissima from IMTA and reference site. The results obtained herein with cultivated S. latissima specimens suggest that algal cultivation in a pilot-scale land-based system is responsible for higher fatty acid amounts than those from natural beds, but such assumption would prove to be tendentious. Most likely, the differences found for fatty acid levels and composition are the result of the combined influence of several ecological conditions under which a species persists in its natural habitat, and also of the small-scale conditions experienced within the IMTA system (e.g., nutrient loading, light penetration and local interactions between co-cultured species) (37,51). Nevertheless, the cultivation of macroalgae in IMTA systems may represent a sustainable approach for optimizing the recovery of natural valuable compounds with potential benefits on human health.

96 RESULTS AND DISCUSSION

800 10 n-3 n-6 9 700 n n-6/n-3 n 8 600 h,k,l,m 7 k k 500 k,r g,n,o,p 6 h,l,m l,r,s b,c,r,t b,c,g,l 400 a,c,s a,g,q,s,t 5 a,b,h,p a,c,g,h,j a,h,o,p h,o,p,q a,g,h,p e,i,m,o 4 e,h 300 h,m e,j,m,p

mg/Kg dry algae dry mg/Kg d,e,f e,i,m 3 e d,f,i 200 a,c,k d,f i 2 g,j j j,n j,p d,o d,g 100 d,l d,m c,h a,c c,h c,h 1 a a a,c a f 0 0

Figure 25. n-3 and n-6 PUFA content (mg/kg dry algae), and n-6/n-3 ratio of L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts. Results are expressed as mean (standard deviation) of three determinations. Different letters denote statistically significant differences (p < 0.05) between samples (One-way ANOVA, Tukey's multiple comparison test). Identity of samples according to Table 5).

97 RESULTS AND DISCUSSION

4.1.1.4. Cultivation depth vs fatty acid profile

The fatty acid composition of S. latissima blades and stipes collected in the longlines cultivated at sea, at 5, 10 and 15 m deep (samples Sl_B(5m)_PV_May12, Sl_B(10m)_PV_ May12, Sl_B(15m)_PV_ May12, and Sl_S(5m)_PV_ May12, Sl_S(10m) _PV_ May12, Sl_S(15m)_PV_ May12), was examined. As environmental parameters (e.g., temperature and light availability) change with depth, it is possible to predict that physiological responses of seaweeds at a given moment should vary along the gradient, resulting in significant metabolic changes. Total fatty acid content of stipes and blades increased by over 21.5 and 31.4% with increasing depth, respectively. Our results showed significant correlations between cultivation depths and MUFA (Pearson correlation; r = 0.9491; p < 0.0001), PUFA (Pearson correlation; r = 0.9497; p < 0.0001), and total fatty acids (Pearson correlation; r = 0.9235; p= 0.0004) in S. latissima blades. Likewise, S. latissima stipes MUFA (Pearson correlation; r = 0.8731; p= 0.0021), PUFA (Pearson correlation; r = 0.8733; p = 0.0021), and total fatty acid (Pearson correlation; r = 0.8202; p = 0.0068) amounts were positively correlated with increasing depths. The tendency towards higher levels of fatty acids, as well as higher degree of unsaturation, in algal tissues cultivated at deeper locations may represent a general environmental acclimation to decreasing temperature and to reduced light conditions, along the water column. The production of specific PUFA under stressful conditions (e.g., changes in temperature, desiccation, decreased solar radiation, and biotic interactions) may also trigger the activation of defense signaling pathways, promoting algal survival (41,416).

Aside the major contribution of changing environmental conditions for the variability of fatty acid patterns observed, a distinct intra-thallus variation was also found. S. latissima blades exhibited generally higher total fatty acid amounts (1829.72–2403.93 mg/kg of dry algae) than stipes (1558.94–1894.31 mg/kg of dry algae) (Figure 26), as well as more n-3 PUFA (Table 10).

Noteworthy was the occurrence, though at relatively low amounts, of C22:6n-3c in all S. latissima tissues cultivated at sea in vertical longlines off Póvoa de Varzim (Table 10). This important n-3 PUFA is generally absent or exists in very small amounts in different brown macroalgal species (417,418). Fatty acids, such as C18:1n-9c and C20:4n-6c, were found at higher levels in stipes of S. latissima (Table 10), the more rigid parts of the algae, pointing to a strong structural role of these compounds (76). An increase in C20:4n-6c levels has also been reported when algae are submitted to stressful conditions to produce structurally unique oxylipins that may be involved in the defense and innate immunity of these photosynthetic marine organisms (73). As a free fatty acid, C18:1n-9c is suggested to be

98 RESULTS AND DISCUSSION

involved in the pathogen defense of land plants (419,420). Assuming a similar function in macroalgae, higher levels of this and other compounds would suggest that the ability of macroalgae to cope with specific or multiple environmental pressures relies on a combination of physiological strategies, including the accumulation of specific fatty acids. Similar trends of fatty acid distribution within algal organs were already described for some temperate species of Fucales and Laminariales, including S. latissima (76), that are closely related to the morphological, functional, and physiological differentiation of algal parts required for growth, photosynthesis, and energy storage (405).

3000 300

272.2 272.2 d,e,l 2500 250 c,d,t

b,h b,c,h 2000 a,b,h 200

f a,f d,f

1500 d,e 150 137.8 137.8 b,e

b,c mg/Kg dry algae dry mg/Kg 1000 a,i,k,m,n 100

16.3 16.3 66.5 66.5 15.2 15.2 d,p 500 a,d,p,n 50 b,c,v a,d,f,q,t b,e,f,v,w a,e,l a,b,n d 14.7 14.7 a,b,j e,h b,c,q g,p 0 0

∑ FA ∑ SFA ∑ MUFA ∑ PUFA

Light intensity (µmol/m2/s) SST (ºC)

Figure 26. Sum of saturated (ΣSFA), monounsaturated (ΣMUFA), polyunsaturated (ΣPUFA) and total fatty acid content (ΣFA) of S. latissima tissues cultivated at 5, 10, and 15 m deep subjected to different light intensity and sea surface temperatures (SST). Results are expressed as mean (standard deviation) of three determinations. Different letters denote statistically significant differences (p < 0.05) between samples (One-way ANOVA, Tukey's multiple comparison test). Identity of samples according to Table 5.

99 RESULTS AND DISCUSSION

4.1.2. Pigments

4.1.2.1. General overview

The analytical methodology employed in this work allowed the determination of six carotenoids, comprising five xanthophylls and β-carotene, as well as of chlorophyll a and of its demetalated derivative, pheophytin a (Figure 27). Compounds with chlorophyll c- like UV-vis spectrum were also detected in some samples (Figure 27). The pigment composition varied considerably amongst the analyzed kelps (Table 11). However, as expected, fucoxanthin was found in all samples, along with its cis isomers. Besides playing a central role in macroalgae as a component of the light harvesting complex for photosynthesis and photoprotection, this xanthophyll also displays promising effects in human health (as reviewed in (28,210). Among the carotenoids detected in this work, fucoxanthin was the dominant one, its relative content ranging from 4.3 up to 82.4 % of all quantified pigments. Total pigment concentrations ranged between 34.99 and 689.21 mg/kg of dry algae (Table 11). The lowest pigment levels were presented by whole- specimen samples of S. latissima from Amorosa beach, collected in January 2013 (Sl_W_Am_Jan13), whereas samples of whole specimens of S. polyschides grown in the IMTA system, and collected also in January 2013 (Sp_W_IMTA_Jan13), exhibited the highest amounts of photosynthetic pigments.

100 RESULTS AND DISCUSSION

20002000 1 L. ochroleuca

15001500

10001000 mAU 2 3 500 500 7 8 9' 9'' 4 6 00 00 1010 2020 3030 4040 5050 6060 Time (min.)

12001,2 1 S. latissima

8000,8 mAU

4000,4 2 3 7 8 9'' 4 6 0 0 10 20 30 40 50 60 Time (min.)

1200 120012001200 1200 1 1200 S. polyschides

800

800800800 800 mAU

400400400400 2 400 3 7

8 4 5 9'' 0 0 0 0 0 0 0 0 0 10 101010 20 2020200 30 3030 301040 4040 40 5020 5050 5050 6030 6060 6060 40 50 60 Time (min.)

Figure 27. HPLC-DAD carotenoid and chlorophyll profiles of acetone extracts from L. ochroleuca (Lo_W_IMTA_Jan13), S. latissima (Sl_W_IMTA_Apr13) and S. polyschides (Sp_W_N_Jan13). Detection at 450 nm. Fucoxanthin (1); fucoxanthin cis isomer 1 (2); fucoxanthin cis isomer 2 (3); violaxanthin (4); zeaxanthin (5); chlorophyll a (6); pheophytin a (7); β-carotene (8); chlorophyll c derivatives (9' and 9'').

101 RESULTS AND DISCUSSION

Table 11. Carotenoids and chlorophylls content in L. ochroleuca, S. latissima and S. polyschides extracts (mg/kg dry algae)1.

Carotenoids Chlorophylls Sample2 Fucox cis Fucox cis β- Chlor Chlor c Chlor c Fucox Violax Zeax Pheo a TOTAL isomer 1 isomer 2 Carot a deriv 1 deriv 2

57.87 4.12 3.35 1.76 13.70 0.91 nda 12.13 60.81 154.65 Lo_W_N_Oct12 nda (0.89)a (0.16)a,b,w (0.05)a (0.07)a,g,i (0.29)a (0.00)a (1.89)a,d,i,j,k,l,n,s,v,z (1.29)a (4.64) 55.99 3.41 3.05 4.00 15.99 82.44 Lo_B_N_Dec12 ndb ndb nda nda ndb (0.53)a,b (0.18)b (0.21)a,b,c (0.04)b (0.63)a,b,k,s,v,z (1.59) 4.39 0.42 0.38 2.71 36.06 57.87 101.83 Lo_S_N_Dec12 ndc ndb ndb nda (0.04)c (0.02)c (0.06)d (0.14)a,c (1.45)b (4.01)a,c (5.72) 51.80 2.96 3.25 3.49 22.14 83.64 Lo_W_N_Dec12 ndb ndb nda nqa nqb (1.09)a,d (0.07)a,b,d,e,f (0.52)a (0.01)d (0.54)b,d,k,r,s (2.23) 34.01 2.07 2.36 2.07 6.46 62.12 102.99 212.08 Lo_B_SBM_Jan13 ndb ndb nda (0.21)f (0.02)d,g,h,i,j (0.06)b,f,g,h,i (0.03)a,f,i,p (0.55)a,g,t,z (0.09)c (0.86)d (1.82) 12.12 0.95 0.71 0.89 3.67 43.34 35.70 97.38 Lo_S_SBM_Jan13 ndb ndb nda (1.76)c,e (0.04)c,g (0.15)e,d (0.09)e (0.52)a,f,t (0.04)d (0.12)e (2.72) 49.36 3.63 3.08 1.03 15.62 73.89 96.29 242.90 Lo_W_SBM_Jan13 ndb ndb nda (0.31)b,d (0.09)a,b,w (0.24)a,g,j (0.14)e,h (0.40)a,d,h (1.16)e (0.91)d (3.25) 163.65 7.97 7.41 2.16 61.26 4.56 84.11 109.46 440.58 Lo_B_SBM_Jun13 nda ndc,f,g,i (0.98)g (0.00)k,u (0.11)k,y (0.08)i,p (0.81)c (0.30)c (3.63)f (3.63)d (9.54) 161.71 9.56 8.03 1.72 15.73 62.58 111.78 371.11 Lo_B_Am_Apr13 ndb nda nqc,f,g,j (4.61)g,h (0.02)l (0.15)k (0.08)a,i,j (0.06)d (1.03)c (2.17)d (8.12) 87.20 6.92 6.69 2.80 1.25 18.23 21.34 84.94 102.94 332.31 Lo_W_IMTA_Jan13 ndb (0.54)i,k (0.11)k,m (0.42)l,m,y (0.02)k (0.04)d (0.65)b (4.65)e,h,k,m,r (5.18)f (3.52)d (15.13) 153.89 9.60 9.70 5.69 0.90 132.73 144.44 456.95 Lo_B_IMTA_Apr13 ndb ndc,f,g,l nda (0.28)h (0.17)l (0.13)n (0.31)l (0.01)a (6.00)c (16.34)f (23.24) 35.43 2.16 1.93 1.71 6.90 15.22 11.66 75.01 Sl_B(5m)_PV_Jul12 ndb ndb nda (0.16)f (0.08)e,j,n,o,p (0.05)i,o,p,q (0.02)a,m (0.78)a,i,j,l,m (0.15)g (0.07)b (1.31) 33.70 3.00 2.38 1.81 40.89 Sl_B(10m)_PV_Jul12 ndb ndb nda nqc,f,gl,n nda ndb (0.11)f (0.18)a,b,i,o,q (0.03)c,h,j,p,r (0.06)a,i,n (0.38) 20.32 1.87 1.44 1.71 11.00 36.34 Sl_B(15m)_PV_Jul12 ndb ndb nda nda ndb (0.33)e,j (0.07)f,g,p,q,r (0.12)e,q,s (0.03)a,o (1.24)a,d,i,j,k,l,n,o (1.79) 21.31 1.50 1.96 1.97 8.25 34.99 Sl_W_Am_Jan13 ndb ndb nda nda ndb (0.18)j (0.08)c,h,n,r (0.22)f,o,r,s (0.01)a,i,p (0.41)a,d,i,j,k,l,n,p (0.90) 89.56 5.94 5.32 3.14 41.31 145.27 Sl_W_IMTA_Jan13 ndb ndb nda nda ndb (4.39)i (0.02)m,s (0.08)t,u (0.39)d,k (3.89)q (8.77)

102 RESULTS AND DISCUSSION

Table 11. Cont.

Carotenoids Chlorophylls Sample2 Fucox cis Fucox cis β- Chlor Chlor c Chlor c Fucox Violax Zeax Pheo a TOTAL isomer 1 isomer 2 Carot a deriv 1 deriv 2

126.01 8.27 6.62 2.93 1.44 32.59 66.64 244.50 Sl_B_IMTA_Apr13 ndb nda nda (0.51)l (0.26)u,t (0.47)l,v (0.18)k (0.03)d (0.71)q,r (1.61)a (3.77) 87.56 5.96 6.05 2.32 0.60 17.62 120.11 Sl_W_IMTA_Apr13 ndb nda nda ndb (2.01)i (1.21)m,v (0.37)m,u,v (0.32)f,p (0.11)e (1.96)e,h,m,o,p,s,t,u (5.98) 79.11 4.80 4.68 1.44 1.66 3.32 25.93 49.03 169.97 Sp_W_N_Jan13 nda nda (0.59)k (0.02)s,v,w (0.01)t (0.01)g,h,j,m,n,o (0.05)e (0.08)f (6.95)e,h,r,v (0.58)a,e (8.29) 235.95 14.33 11.36 2.66 179.81 245.10 689.21 Sp_W_IMTA_Jan13 ndc ndb nda nda (9.95)m (1.23)x (0.07)w (0.02)g (19.80)w (22.49)g (53.56) 58.61 4.08 3.38 0.65 9.53 40.13 116.38 Sp_B_IMTA_Apr13 ndb ndb nda nda (0.17)a (0.12)a,b,w (0.13)a (0.03)e (1.96)a,d,i,j,k,l,n,s,x (0.26)c,e (2.67) 92.14 6.43 5.20 4.03 27.30 64.26 199.36 Sp_S_IMTA_Apr13 ndb ndb nda nda (2.82)i (0.29)m (0.45)t (0.04)b (0.55)b,h,q,r,u (0.96)a (5.11) 166.37 9.36 8.87 2.82 2.31 19.79 209.52 Sp_W_IMTA_Apr13 ndb nda nda ndb (2.41)g (0.03)l,t (0.05)x (0.18)k (0.02)h (0.25)e,h,r,m,o,p,x,z (2.94)

1 Results are expressed as the mean (standard deviation) of three determinations; nq, not quantified; nd, not detected. Fucox, fucoxanthin; Violax, violaxanthin; Zeax, zeaxanthin; β-Carot, β-carotene; Chlor a, chlorophyll a, Pheo a, pheophythin a; Chlor c deriv, chlorophyll c derivative. Different superscript letters denote statistically significant differences (p < 0.05) in the same column (One-way ANOVA, Tukey's multiple comparison test). 2 Identity of samples according to Table 5.

103 RESULTS AND DISCUSSION

4.1.2.2. Harvesting period and origin vs pigment profile

In their natural habitats, seaweeds grow in exceptionally diverse and dynamic light climate, which is the most important and one of the most complex abiotic factors affecting these organisms (196). Factors other than light, such as SST to which macroalgae are subjected in the normal course of the seasons, can also influence their metabolism and, consequently, their chemical composition (5,86,414,421). The ability to acclimate and adjust photosynthesis and growth to the rapid changes in light and temperature regimes may rely on different strategies (e.g., pigment composition plasticity), as a prerequisite for macroalgal life under seasonal changes (5,414). One of the many outcomes of this study was the identification of significant seasonal variations in the photosynthetic pigment composition of the analyzed kelps. L. ochroleuca, collected at Praia Norte in October 2012 (Lo_W_N_Oct12) and December 2012 (Lo_W_N_Dec12) exhibited considerable differences in both qualitative and quantitative pigment profiles: zeaxanthin and β- carotene were found only in the whole-specimen samples collected in October (Lo_W_N_Oct12), which also presented significantly higher levels of chlorophyll c derivative (Table 11). Still, L. ochroleuca blades from São Bartolomeu do Mar, harvested in January 2013 (Lo_B_SBM_Jan13) and June 2013 (Lo_B_SBM_Jun13), showed even greater differences: the last contained nearly twice the levels of pigments, amongst which carotenoids corresponded to 56.1% of the total quantified ones (Table 11). In fact, the specimens collected in June were exposed to higher light intensity and longer photoperiod, along with increasing seawater temperatures, than the ones collected in January, as in this region June coincides with the end of spring and the beginning of summer.

Overall, these results point to the occurrence of photoprotective mechanisms in the algae that deflects energetic resources to pigment biosynthesis, ensuring the ecological success of the species (422). Regarding kelps cultivated in IMTA, total pigment contents were generally higher (Table 11). Some qualitative differences were also noticed, although with less significance. Comparing the whole individuals of each studied macroalgae species from wild natural stocks and IMTA, with identical sampling period, some observations can be highlighted. For L. ochroleuca, the cultivated macroalgae (Lo_W_IMTA_Jan13) exhibited almost 1.4 times more pigments than the wild macroalgae from São Bartolomeu do Mar (Lo_W_SBM_Jan13). In S. latissima, the differences were even higher. Cultivated macroalgae (Sl_W_IMTA_Jan13) displayed almost 4.2 times more pigments than wild S. latissima collected at Amorosa (Sl_W_Am_Jan13). Significantly higher pigment contents were also detected in S. polyschides from the IMTA system (Sp_W_IMTA_Jan13): almost 4.1 times more than in

104 RESULTS AND DISCUSSION

the macroalgae collected at Praia Norte (Sp_W_N_Jan13). Although these results could immediately imply that algae cultivation in a pilot-scale land-based system provides higher levels of photosynthetic pigments than those from wild natural stocks, such assumption would prove to be tendentious. As for fatty acid composition (subsection 4.1.1.3.), the differences found for pigment profiles are most likely the result of the combined influence of several ecological conditions under which seaweeds persists in their natural habitat, as well as of the small-scale conditions within the IMTA system (37,51).

4.1.2.3. Pigment distribution within thalli

Many macroalgae, particularly the kelps, display strong morphological thallus differentiation, which has been shown to translate into biochemical gradients (76–79). The kelps under study are economically important and edible macroalgae species, with potential to be produced sustainably through commercial aquaculture, namely S. latissima (132). Therefore, the selection of thallus parts rich in valuable bioactives, such as carotenoids, may improve their nutritional value. Pigment composition of different algal thallus sections, when available, was assessed. Although it has been previously reported that pigment concentrations are commonly lower in the meristematic areas of algal thalli (76,77), such a trend was not observed in the total pigment contents for most of the analyzed algal tissues (Table 11). These contrasting results can be possibly attributed to the high turnover rate of basal structures, such as stipes, which may compensate better for the detrimental effects of combined environmental factors (221). The stipes analyzed herein exhibited generally higher chlorophyll relative content than blades, pointing to a high metabolic activity of this algal tissue. On the other hand, a dominance of carotenoids was found in blades. Carotenoids play an important role in the function and structural integrity of the chloroplast thylakoid membrane, and blades are structurally defined by a much greater density of thylakoid and mitochondrial cristae per unit volume than stipes (423,424). Thallus age, metabolic activity, and physiological function, but also location of specific thallus parts in the water column, support the dynamic in pigment profiles (224,225).

4.1.2.4. Pigment composition of S. latissima tissues vs cultivation depth

As referred above, because environmental parameters change with depth, it is possible to predict that physiological responses of macroalgae, at a given moment, should vary along the gradient, resulting in significant metabolic changes. Previous reports have shown that the perennial kelp S. latissima can acclimate to different light and temperature

105 RESULTS AND DISCUSSION

conditions, within its limits of tolerance, by relying on different mechanisms, such as regulation of pigment levels (41,414,425,426). Samples of S. latissima collected in the longlines cultivated at sea, in July, were subjected to decreasing light intensities and temperatures with increasing depth. The total pigment amounts of S. latissima blade samples (samples Sl_B(5m)_PV_Jul12, Sl_B(10m)_PV_Jul12 and Sl_B(15m)_PV_Jul12) decreased along depth (5–15 m) (Table 11). Overall, the carotenoid contents were not statistically different (p > 0.05), except for fucoxanthin and its cis isomer 2 in blades collected at 15 m deep (Sl_B(15m)_PV_Jul12). Nevertheless, the largest variability was observed within chlorophyll pigments: blades harvested at 5 m deep (Sl_B(5m)_PV_Jul12) showed higher chlorophyll amounts than blades from deeper locations. Studies on photosynthesis and pigment composition under different temperature and light conditions revealed that algal acclimation patterns depend on the position of the photosynthetic tissue in the water column (223,427). Therefore, it seems that the ability of macroalgae to cope with specific or multiple environmental pressures depends on a combination of local adaptations.

106 RESULTS AND DISCUSSION

4.2. Non-enzymatic α‑linolenic acid derivatives from the sea: Macroalgae as novel sources of phytoprostanes

This work aimed at assessing the presence of different naturally occurring classes of free phytoprostanes, readily bioavailable and absorbed by the human body, in twenty-four macroalgae species belonging to Chlorophyta, Ochrophyta, and Rhodophyta collected along the Western coast of Portugal and from IMTA systems (Table 7).

4.2.1. Occurrence of α‑linolenic acid in macroalgae

The n-3 PUFA α-linolenic acid, precursor of phytoprostanes, was identified and quantified in the twenty-four selected macroalgae species (Table 12). The content of this compound ranged between ca. 0.7 and 5.6 g/kg of dry algae, C. tamariscifolia and U. lactuca presenting, respectively, the lowest and the highest amounts (Table 12). Indeed, previous studies have reported that members of the order Ulvales presented α-linolenic acid as the characteristic PUFA (130,418). The presence of α-linolenic acid in all the analyzed species enables and emphasizes the importance of assessing the occurrence of its autoxidation products, the phytoprostanes.

4.2.2. Occurrence of free phytoprostanes in macroalgae

Among the ten phytoprostane standards available, only three were detected in the analyzed algal samples (Table 12). Neither 16-series of F1t-phytoprostanes, nor 9-series of

D1t-phytoprostanes, were detected in the studied species. Phytoprostane identity was confirmed according to their molecular masses, the precursor ions (m/z 327.2 and 307.2), the characteristic MS/MS fragmentation product ions, and the corresponding retention times. The mass spectrometric information on the phytoprostanes detected in one macroalgae sample (C. tomentosum) is summarized in Figure 28. Both 9-F1t- phytoprostane and 9-epi-9-F1t-phytoprostane (Figure 28A) showed the same transition from the precursor ion at m/z [M-H]- 327.2 to the product ion at m/z 171.2 (Figure 28B and C). Thus, their identification was only possible by the comparison of their retention times: 9-F1t-phytoprostane eluted at 1.75 min, whereas 9-epi-9-F1t-phytoprostane eluted at 1.93 min (Figure 28B and C). Contrary to prostaglandins, phytoprostanes are non- enzymatically formed as regio- and stereoisomeric mixtures (161). The analytical conditions employed in this study did not allow enantiomer separation. Therefore, 16-B1- phytoprostane and 9-L1-phytoprostane (Figure 28A) were identified and quantified according to their specific transitions from the precursor ion m/z [M-H]- 307.2 to the product ions m/z [M-H]- 235.2 and m/z [M-H]- 185.2, respectively (Figure 28D and E).

107 RESULTS AND DISCUSSION

The qualitative profile of phytoprostanes found in the analyzed samples showed great variability among Chlorophyta, Ochrophyta, and Rhodophyta phyla, and even between close relatives within single genera, that is, 9-F1t-phytoprostane and 9-epi-9-F1t- phytoprostane were detected in F. spiralis, but in F. serratus and F. guiryi none of the studied phytoprostanes was identified.

In the three green macroalgae studied (C. tomentosum, U. lactuca, and Ulva sp.), as well as in two of the red (S. coronopifolius and G. vermiculophylla) and eight of the brown species (C. spongiosus, F. spiralis, L. ochroleuca, P. pavonica, S. latissima, S. polyschides,

S. vulgare, and S. scoparium), both 9-F1t-phytoprostane and 9-epi-9-F1t-phytoprostane were identified. Also, 16-B1-phytoprostane was detected in four of the brown species (P. pavonica, S. latissima, S. vulgare, and S. scoparium). The brown macroalgae B. bifurcata contained only 16-B1-phytoprostane. None of the screened phytoprostanes were detected in ten seaweed samples (A. armata, C. tamariscifolia, C. usneoides, F. guiryi, F. serratus, Gigartina sp., H. filicina, P. canaliculata, P. cartilagineum, and S. muticum). The species C. spongiosus and C. tomentosum exhibited higher diversity in phytoprostanes.

The amount of phytoprostanes found in the analyzed samples is shown in Table 12.

F1t-phytoprostanes were the dominant class determined in this study, whereas L1- phytoprostanes were detected in only two algae species, and at very low levels. Concerning each phytoprostane class, both 9-F1t-phytoprostane and 9-epi-9-F1t-phytoprostane were found in higher concentration in the brown seaweed S. latissima (700.94 and 667.62 ng/100 g of dry algae, respectively); F. spiralis exhibited the lowest amount of these phytoprostanes (18.61 and 17.27 ng/100 g of dry algae, respectively). The sample with the lowest content of 16-B1-phytoprostane was S. vulgare (4.00 ng/100 g of dry algae), whereas C. tomentosum was found to have the highest amount (14.42 ng/100 g of dry algae). C. spongiosus and C. tomentosum were the only species that presented 9-L1- phytoprostane (4.67 and 6.36 ng/100 g of dry algae, respectively).

As observed with the qualitative profile, the quantification of total phytoprostanes in the studied macroalgae samples revealed significant variability. The total phytoprostane content ranged between ca. 6 and 1381 ng/100 g of dry algae. The macroalgae species showing the highest amount of phytoprostanes (S. latissima) was from IMTA systems. However, no conclusions can be drawn regarding the advantages of IMTA for obtaining higher yields of phytoprostanes, as no marine counterpart of this species was analyzed.

108 RESULTS AND DISCUSSION

Table 12. α-Linolenic acid (g/Kg dry algae) and phytoprostane (ng/100 g dry algae) content in the analyzed macroalgae species1.

Phytoprostanes Species α- 9-epi-9-F1t- Linolenic 9-F1t-PP 16-B1-PP 9-L1-PP TOTAL PP acid 0.96 A. armata nd nd nd nd – (0.00)d,e 3.57 5.68 5.68 B. bifurcata nd nd nd (0.04)e,f (1.09)d (1.09) 4.09 75.25 38.05 10.53 4.67 128.49 C. spongiosus (2.77)i (3.71)d,e (6.57)d (2.32)b,c (1.17)a (6.78) 3.77 22.67 32.49 14.42 6.36 75.94 C. tomentosum (0.28)i (3.38)e (4.45)d (2.10)a (0.25)a (9.22) 0.69 C. tamariscifolia nd nd nd nd – (0.01)b 2.62 C. usneoides nd nd nd nd – (0.05)f 5.08 F. guiryi nd nd nd nd – (0.06)b 4.23 F. serratus nd nd nd nd – (0.06)c,d 3.98 18.61 17.27 35.87 F. spiralis nd nd (0.14)d,e (3.04)e (1.32)d (2.18) 1.66 Gigartina sp. nd nd nd nd – (0.02)h G. 1.79 54.46 43.49 97.96 nd nd vermiculophylla (0.03)h (6.31)d,e (2.14)d (4.91) 3.43 H. filicina nd nd nd nd – (0.08)f 3.90 36.42 21.07 57.48 L. ochroleuca nd nd (0.13)d,e (6.23)d,e (5.54)d (11.69) 5.03 26.33 28.91 6.39 61.36 P. pavonica nd (0.08)a (4.15)d,e (3.60)d (0.52)d (7.03) 4.40 P. canaliculata nd nd nd nd – (0.10)c 2.63 P. cartilagineum nd nd nd nd – (0.01)g 2.01 700.94 667.62 12.33 1380.90 S. latissima nd (0.07)h (53.14)a (51.34)a (0.55)a,b (103.83) 1.10 413.35 305.97 719.62 S. polyschides nd nd (0.03)i (50.88)b (39.56)b (24.22) 3.94 S. muticum nd nd nd nd – (0.22)d,e 3.62 99.94 39.54 4.00 143.48 S. vulgare nd (0.12)e,f (17.63)d (9.62)d (0.86)d (26.92) S. 0.78 38.10 36.80 74.90 nd nd coronopifolius (0.01)i (4.91)d,e (2.44)d (4.93) 0.80 334.28 170.23 7.43 511.94 S. scoparium nd (0.01)i (44.67)c (15.97)c (0.37)c,d (52.69) 5.11 74.95 55.17 130.13 U. lactuca nd nd (0.01)b (6.10)d,e (4.04)d (2.14) 3.49 88.28 64.98 153.25 Ulva sp. nd nd (0.30)f (23.82)d,e (6.72)d (25.44)

1 Results are expressed as mean (standard deviation) of three determinations; PP, phytoprostane; nd, not detected (nd). Different superscript letters denote statistically significant differences (p <0.05) in the same column (One-way ANOVA, Tukey's multiple comparison test).

109 RESULTS AND DISCUSSION

To the best of our knowledge, no information was ever published on the content of naturally occurring free phytoprostanes in macroalgae. Therefore, the results obtained can be compared only with those of previous work performed on plant material and other foodstuffs. Karg et al. (169) found that both D1- and F1-phytoprostanes were the dominant classes in vegetable oils, whereas B1- and L1-phytoprostanes, including their enantiomers, were minor components (169). In more recent years, studies have found that F1t- phytoprostanes were generally the most abundant class in several other natural matrices, including in the green table olive ‘Manzanilla de Sevilla’ (188), in different almond cultivars (189), in red wine and must (192), and in gulupa shell (190). Likewise, in macroalgae, F1t-phytoprostanes were the main phytoprostanes detected. This class of compounds was also found to occur in the leaves, flowers, and roots of taxonomically distinct plant species, ranging from 43 to 1380 ng/g of dry weight (187). Although structural differentiation in macroalgae is likely to be accompanied by variation in chemical composition, the present work used whole algae individuals in the same stage of development, rendering impossible to conclude about the presence and distribution of phytoprostanes within thallus parts of the selected species. Although it was theoretically expected to find both 9- and 16-series of F1-phytoprostanes in equal amounts, we were not able to ensure this hypothetical relation in the analyzed algae material. In fact, previous studies reported only that 9- and 16-series (previously referred to as types II and I, respectively) of A1-, B1-, and E1-phytoprostanes were of equal abundance in different plant tissues (169). Moreover, Imbusch et al. (186) stated that due to the isomeric complexity of

F1-phytoprostanes, identification and quantification of this class of compounds are technically difficult. Studies reporting the presence of F1-phytoprostanes have therefore focused on the differentiation between free and esterified isomers (169,186,187,428). Still, it is important to highlight that the analytical methodology employed in this work was capable of differentiating 9-F1-phytoprostane from 16-F1-phytoprostane (188–190,192); however, the occurrence of these regio-isomers was not of equal proportions. For instance, in refined sunflower oil the 9-F1-phytoprostane was found in 44.48 ng/mL, whereas the

16-F1-phytoprostane was found in 24.40 ng/mL; in 0.8° and 0.4° extra virgin oil 9-F1- phytoprostane were not detected, whereas 16-F1-phytoprostane was found in 2.13 and 3.70 ng/mL, respectively (188). The reason behind these differences is still unclear, and more studies are needed to clarify the underlying response mechanisms of individual free phytoprostanes in natural products.

110 RESULTS AND DISCUSSION

1.75 327.2 → 171.2

171.2

1.93 327.2 → 171.2

171.2

2.85 307.2 → 235.2

235.2

3.01 307.2 → 185.2

185.2

Figure 28. Representative UHPLC-QqQ-MS/MS chromatogram of detected phytoprostanes (C. tomentosum) (A), presumed fragmentation and MRM transitions for quantification of 9-F1t- phytoprostane (B), 9-epi-9-F1t-phytoprostane (C), 16-B1-phytoprostane (D), and 9-L1- phytoprostane (E).

111 RESULTS AND DISCUSSION

PCA was performed to detect possible distribution patterns of the determined phytoprostanes (Figure 29).

Variables: PC1 and PC2 (92.9%) Observations: PC1 and PC2 (92.9%)

A G1 B

G5 G4

G3 PC2 (28.9%) PC2 PC2 (28.9%) PC2 G2

PC1 (64.0%) PC1 (64.0%)

Figure 29. Projection of macroalgae (A) [variables: A. armata (AA), B. bifurcata (BB), C. spongiosus (CS), C. tomentosum (Ctom), C. tamariscifolia (CT), C. usneoides (CU), F. guiryi (FG), F. serratus (Fser), F. spiralis (Fspi), Gigartina sp. (Gsp), G. vermiculophylla (GV), H. filicina (HF), L. ochroleuca (LO), P. pavonica (PP), P. canaliculata (PC), P. cartilagineum (Pcart), S. latissima (SL), S. polyschides (SP), S. muticum (SM), S. vulgare (SV), S. coronopifolius (SC), S. scoparium (SS), U. lactuca (UL), and Ulva sp. (Usp)] and loadings (B) by phytoprostane composition

[variables: 9-F1t-phytoprostane (9-F1t-PP), 9-epi-9-F1t-phytoprostane (9-epi-9-F1t-PP), 16-B1- phytoprostane (16-B1-PP), and 9-L1-phytoprostane(9-L1-PP)] into the plane composed by principal components PC1 and PC2 containing 92.9% of the total variance.

PCA of the normalized phytoprostane data set explained 92.9% of total variations, PC1 accounting for 64.0% of the variance and PC2 for 28.9%. Five groups were distinguished (Figure 29A). One group (G1) includes C. spongiosus and C. tomentosum, the only two macroalgae species in which 9-L1-phytoprostane was identified. G2 contains G. vermiculophylla, S. coronopifolius, U. lactuca, and Ulva sp., which presented similar amounts of both 9-F1t-phytoprostane and 9-epi-9-F1t-phytoprostane, whereas G3 comprises the three macroalgae species that clearly stood out for their high levels in these exact phytoprostanes (S. latissima, S. scoparium, and S. polyschides). The low amounts of

9-F1t-phytoprostane and 9-epi-9-F1t-phytoprostane in F. spiralis and L. ochroleuca led to the inclusion of these two species in another group (G4), together with A. armata, C. tamariscifolia, C. usneoides, F. guiryi, F. serratus, Gigartina sp., H. filicina, P. canaliculata, P. cartilagineum, and S. muticum, in which none of the available phytoprostanes was identified. Finally, B. bifurcata, P. pavonica, and S. vulgare were

112 RESULTS AND DISCUSSION

grouped together (G5) due to their similar amounts of 16-B1-phytoprostane (Figure 29).

The total amount of phytoprostanes is visibly influenced by the levels of both 9-F1t- phytoprostane and 9-epi-9-F1t-phytoprostane, confirming that F1t-phytoprostane is the dominant phytoprostane class in the analyzed samples (Figure 29B).

Altogether, these observations suggest that the large variations observed in phytoprostane composition can be partially explained by intrinsic factors and/or extrinsic factors.

Phytoprostanes have been recent targets of lipid research, which has generally highlighted their importance not only as excellent biomarkers of the oxidative degradation of plant-derived foodstuffs, but also as biologically active molecules with potential benefits in human health [reviewed in (159)]. Studies have been shown that certain phytoprostane classes were active in various experimental models (166,169,170,429). However, further research on bioavailability and bioactivity of such compounds is required to assess the real effect of phytoprostanes in human health.

4.2.3. Free phytoprostanes vs α-linolenic acid

Pearson correlations were calculated to establish a potential relationship between the presence of α-linolenic acid in algae material and phytoprostane composition. Our results showed a lack of correlation between the amount of α-linolenic acid and total phytoprostane content (r = −0.318). However, the possibility of enzymatic oxidation of α- linolenic acid cannot be ignored. Although this compound is prone to undergo autoxidation reactions, it can also be released from membrane lipids and metabolized by the enzymatic action of LOX (430). In fact, several studies have already reported the presence of diverse structurally unique oxylipins from enzymatic routes in macroalgae (431–436). Furthermore, Barden et al. (185) conducted a clinical trial with healthy volunteers whose diet was supplemented with linseed oil. They detected high levels of phytoprostanes, but they were not able to assess whether phytoprostanes increased due to enhanced concentration of α-linolenic acid or by direct intake from linseed oil (185).

113 RESULTS AND DISCUSSION

4.3. Phlorotannins

The aim of this work was to establish the phlorotannin composition of Fucus spp. widely represented in the Northern Portuguese coastline (Figure 14, Table 8). HPLC- DAD-ESI/MSn and UPLC-ESI-QTOF/MS analyses were employed to provide evidence of the presence of phlorotannins, their varying degree of polymerization and tentative identification in purified extracts from F. guiryi (Fg), F. serratus (Fser), F. spiralis (Fspi), and from wild-sourced and aquaculture-grown F. vesiculosus (Fves-w and Fves-a, respectively).

4.3.1. Phlorotannin profile of Fucus spp.

The procedure employed herein for obtaining phlorotannin-rich fractions from the different Fucus species benefited from the purification of the extract by using a C18 Sep- Pak cartridge (Figure 15). This step allowed the retention of phlorotannins and removal of other co-extracted compounds, such as polysaccharides, with which phlorotannins are usually associated (289). In fact, preliminary MS analyses were conducted in the SPE washing fractions and no phlorotannins were detected. These fractions were then discarded, and methanol eluates were considered of suitable purity for MS profiling analysis.

The total phlorotannin content in the five samples of Fucus species under study was determined by the specific DMBA colorimetric method, using their monomeric unit (phloroglucinol) as standard. Total phlorotannin amount (mg PGE/Kg dry algae) was ordered as follows: Fves-a (9.55 ± 0.20) < Fspi (16.72 ± 0.22) < Fves-w (32.76 ± 0.72) < Fg (181.70 ± 1.46) < Fser (264.73 ± 2.78). All samples were screened for their phlorotannin composition by HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS. The peaks were not abundant in the UV chromatograms recorded at 280 nm (data not shown) and could not be related to the MS ions corresponding to phlorotannins. Thus, the Extracted Ion Chromatogram (EIC), together with the analysis of the MS fragmentation (Ion Trap) of the deprotonated molecular ions ([M-H]-), as well as their exact molecular mass (QTOF), were used as an attempt to identify simple phloroglucinol polymers (fucols/phlorethols/fucophlorethols), polymers with additional OH groups (fuhalols/hydroxyfuhalols) and derivatives presenting dibenzodioxin moieties (eckols/carmalols). The tentative identification of phlorotannin structures was based on the loss of phloroglucinol units (126/125/124 amu) or their derivatives (Table 13 footnote) and considering that the C-C linkages between phloroglucinol units, characteristic of fucols, are of difficult fragmentation, when compared to C-O-C bonds,

114 RESULTS AND DISCUSSION

characteristic of phlorethols. Apart from the loss of neutral fragments, fragments corresponding to the loss of neutral fragments with one or two protons less were also observed. For instance, fragmentation of phloroglucinol may present a deprotonated molecular ion with less 1 amu or 2 amu than the parent compound (126 amu), which corresponds to the loss of one or two hydrogen atoms (125 amu and 124 amu, respectively).

The twenty-two phlorotannins detected in this work were then tentatively identified using mass spectrometry, as well as literature data (57,245,282,437–440). The analytical methodology employed herein had already been successfully used (57), allowing an adequate resolution of phlorotannins with between 3 and 8 phloroglucinol units. Furthermore, any polymer with mass lower than 1200 Da could be detected and the gradient used allows the elution of compounds with several degrees of polymerization. Preliminary experiences were performed with softer gradients at the end to search for other polymers; nevertheless, as these were not observed, a gradient with a fast increase of the organic phase at the end for cleaning was used.

The identified polyphenols exhibited distinct molecular weights (370–746 Da), and relatively low degree of polymerization (3–6 phloroglucinol units) (Table 13). Though there are seaweed species with monomeric and dimeric phlorotannins (441), it seems they are not commonly found in Fucus spp. extracts. The variability observed for the overall phlorotannin composition amongst the analyzed algal extracts (Figure 30) points also to the influence of species-specific factors and/or extrinsic factors. For instance, in a previous work undertaken by our group (57), phlorotannins of higher degree of polymerization were tentatively identified in a Fucus species. This can be explained, at least in part, by the geographical location of the F. spiralis used in that work: a southern location, with generally higher temperatures and light exposure, can be determinant for the profile of secondary metabolites. Besides phlorotannin amount, these abiotic factors can also be responsible for the production of phlorotannins with higher degree of polymerization, explaining the differences observed between seaweed species from southern and northern origin (57). Of the Fucus spp. studied so far, a study of Heffernan and co-workers (290) with F. serratus and F. vesiculosus harvested off the Irish coast described the presence of phlorotannins with 3 and up to 12 phloroglucinol units. However, most of the polyphenols found in Fucus spp. were of low molecular weight, which is in accordance with our findings for species of this genus. Other studies (246,248,282,289,303,304) are also in agreement with this and it seems that the abundance of low molecular weight phlorotannins can be characteristic of Fucus spp.

115 RESULTS AND DISCUSSION

To the best of our knowledge, this was the first work confirming the presence and degree of polymerization of phlorotannins in Portuguese-sourced brown macroalgae, specifically in F. guiryi and F. serratus, as well as in aquaculture-grown F. vesiculosus. Furthermore, the information gained in this work ascertains the role of advanced analytical tools in facilitating the use of this valuable natural resource for the development of macroalgal-based products, opening doors for future application of phlorotannin-rich extracts in commercial areas related to nutraceuticals, pharmaceuticals, and cosmetics.

The main features of the different phlorotannin classes identified in the extracts from Fucus spp. will be discussed below.

116 RESULTS AND DISCUSSION

Intensity 106 A: EIC 373 4 Fves-w 1 3 2 1 0 107 B: EIC 497 3 7 2 1 0 107 18 C: EIC 621 0.8 16 19 0.4 14 15 17 0.0 107 21 D: EIC 7 45 1.0 22 0.5 0.0 0 2 4 6 8 10 12 14 16 18 Time [min] Intensity 106 Fves-a E: EIC 497 5 4 7 2 0 5 F: EIC 621 10 13 4 3 2 1 0 5 10 20 G: EIC 7 45 4 22 2 0 0 2 4 6 8 10 12 14 16 18 Time [min] Intensity 106 Fg H: EIC 369 6 4 4 2 0 6 I: EIC 497 10 5 10 1.5 6 8 1.0 11 0.5 0.0 106 12 J: EIC 493 1.00 0.75 0.50 0.25 0.00 0 2 4 6 8 10 12 14 16 18 Time [min] Intensity 106 Fser 2 K: EIC 373 1.0 0.5 0.0 106 9 L: EIC 497 2.0 1.5 11 1.0 5 0.5 0.0 0 2 4 6 8 10 12 14 16 18 Time [min] Intensity 105 M: EIC 373 5 Fspi 2 4 3 2 1 0 106 3 N: EIC 369 2 1 0 6 10 10 O: EIC 497 2.0 1.5 1.0 7 0.5 0.0 0 2 4 6 8 10 12 14 16 18 Time [min]

Figure 30. Extracted Ion Chromatograms (EIC) obtained from HPLC-DAD-ESI/MSn of purified phlorotannin extracts from wild-sourced and aquaculture-grown F. vesiculosus (Fves-w and Fves- a, respectively), F. guiryi (Fg), F. serratus (Fser), and F. spiralis (Fspi). [M-H]- (m/z): 1–2, 373; 3–4, 369; 5–11, 497; 12, 493; 13–19, 621; 20–22, 745.

117 RESULTS AND DISCUSSION

Table 13 Retention times (Rt), molecular formula and mass spectrometric data of molecular ions and main observed fragments of phlorotannins in the extracts of wild-sourced F. vesiculosus (1, 7, 14–19, 21, 22), aquaculture-grown F. vesiculosus (5, 7, 13, 20, 22), F. guiryi (4–6, 8, 10–12), F. serratus (2, 5, 9, 11) and F. spiralis (2, 3, 7, 10).a

Compoundb Phlorotannin Rt Molecular MS [M-H]- MS2 [M-H]-, m/z (%, -lossc) MS3 [(M-H)→base peak]-, m/z (%, -lossc) oligomer (min) formula m/z

1 trimer 3.9 C18H14O9 373.0567 355(100, -18), 329 (40, -44), 247(30, -126), 229 (30, -126), 215(100, -140) 231(60, -142)

2 trimer 6.6 C18H14O9 373.0560 247(100, -126), 233(70, -140) 229(100, -18)

3 trimer 14.5 C18H10O9 369.0249 238(100) 195(100, -43), 167(60), 112(50, -126)

4 trimer 18.6 C18H10O9 369.0246 351(50, -18), 325(100, -44), 307(40, -62) 307(100, -18), 297(60, -28), 281(45, -44)

5 tetramer 3.2 C24H18O12 497.0715 479(100, -18), 353(10, -144) 331(70) 461(100, -18), 435(40, -44), 353(20, -126)

6 tetramer 4.6 C24H18O12 497.0719 479(100, -18), 461(35, -36), 355 (50, -142) 420(35), 353(80, -126), 337(100, -142)

7 tetramer 5.2 C24H18O12 497.0717 479(100, -18), 371(5, -126), 353(25, -144), 339(100, -140), 229(5, -250) 339(30, -158)d

8 tetramer 6.7 C24H18O12 497.0713 479(10, -18), 355(100, -142), 311(50) 311(100, -44), 229(10, -126)

9 tetramer 8.2 C24H18O12 497.0715 355(100, -142), 235(40, -262) 269(40), 229(100, -126)

10 tetramer 8.5 C24H18O12 497.0716 235(100, -262) 207(75, -28), 191(100, -44)

11 tetramer 9.4 C24H18O12 497.0715 479(25, -18), 373(100, -124), 265(50) 233(50, -140), 139(100)

12 tetramer 17.4 C24H14O12 493.0405 475(100, -18), 367(15, -126) 431(60, -44), 405(100)

13 pentamer 4.2 C30H22O15 621.0899 603(100, -18), 577(35, -44), 559(40), 497(5, - 585(100, -18), 559(50), 477(25, -126) 124), 477(5, -144)

14 pentamer 4.7 C30H22O15 621.0883 603(100, -18), 495(10, -126) 585(100, -18), 463(35, -140), 477(25, -126), 459(60, -144)

e 15 pentamer 6.7 C30H22O15 621.0902 603(100, -18), 495(5, -126), 461(39, -160), 461(60, -142), 355(100, -248) 355(45, -266)

f 16 pentamer 7.4 C30H22O15 621.0887 603(100, -18), 479(10, -142), 461(5, -160), 479(70, -124) , 335(100, -268) 353(20, -268)

g 17 pentamer 7.8 C30H22O15 621.0885 603(100, -18), 479(50, -142), 461(60, - 461(60, -142) , 353(100, -250), 335(25, -268) 160)g, 353(30, -268)

h h 18 pentamer 8.2 C30H22O15 621.0901 603(100, -18), 463(5, -158), 339(10, -282) 477(40, -126), 463(50, -140), 339(100, -264)

118 RESULTS AND DISCUSSION

Table 13. Cont.

Compoundb Phlorotannin Rt Molecular MS [M-H]- MS2 [M-H]-, m/z (%, -lossc) MS3 [(M-H)→base peak]-, m/z (%, -lossc) oligomer (min) formula m/z

i 19 pentamer 8.5 C30H22O15 621.0900 603(100, -18) , 479(5, -142), 337(25, -284), 479(50, -124), 339(100, -264), 229(60) 229(10)

20 hexamer 6.4 C36H26O18 745.1040 727(100, -18), 601(10, -144) 709(100, -18), 602(20, -125), 585(20, -142)

21 hexamer 9.9 C36H26O18 745.1050 727(100, -18), 601(35, -144), 461(5, -284), 601(35, -126), 583(90, -144), 479(50, -248), 335(12, -410), 229(5) 353(60, -374), 229(20)

22 hexamer 10.3 C36H26O18 745.1048 727(100, -18), 601(25, -144), 479(10, -286), 601(45, -126), 583(60, -144), 461(35, -266), 353(12, -392), 229(5) 335(100, -392), 229(45) a Ions in bold comprise the loss of phloroglucinol moieties. b Peak identity as in Figure 30. c 62: 44+18; 140: 124+16; 142: 126+16/124+18; 140: 124+16; 142: 124 + 18; 144: 126+18; 158: 124+16+18; 160: 126+16+18/124+18+18; 170: 126+44; 248: 124+124; 250: 126+124; 262: 124+124+14; 264: 124+124+16; 266: 124+124+18; 268: 126+126+16/124+126+18; 282: 124+124+18+16; 284: 124+124+18+18; 286: 124+126+18+18; 374: 124+124+126; 376: 126+126+124; 392: 124+124+126+18; 410: 126+126+126+16+16. d7 MS3(497→339): 230; MS4(497→479→229): 229(100); 201(25); e15 MS4(355): 229(100, -126); f16 MS4(479): 339(100, -140), 230(40); g17 MS3(461): 230; MS4(461→230): 229; h18 MS3(339): 230; MS4(621→603→463): 339(100); MS4(621→603→339): 230(100); i19 MS3(621→603): 479(50, -124), 337(100, -266), 229(65); MS4(621→603→337): 229(100).

119 RESULTS AND DISCUSSION

4.3.1.1. Phlorotannin trimers

Apart from Fves-a, trimers of phloroglucinol (1–4) were found in all purified extracts (Figure 30). Compounds 1 and 2 have the same [M-H]- at m/z 373, but slightly different fragmentation patterns, suggesting that they would be isomers (Table 13). The MS2 fragmentation of compound 1, detected only in Fves-w (Figure 30A), exhibited the loss of a single unit of phloroglucinol (–126 amu) and of a derivative with water (124+18 amu) to give rise to the ions at m/z 247 and 231, respectively. Important losses of 18 and 44 amu were also detected, the latter probably generated by the combined elimination of an ethylene group and water, a consequence of the internal cleavage of benzene ring structures (282). Likewise, in the MS2 fragmentation of compound 2, detected in both Fser and Fspi extracts (Figure 30K and M), the loss of only one phloroglucinol unit was observed (Table 13). In MS3 and MS4 (data not shown) of compounds 1 and 2 no additional losses of phloroglucinol or derivative were observed, the ion at m/z 229 in MS3 does not undergo further fragmentation into phloroglucinol, suggesting the presence of a fucol moiety (two units of phloroglucinol linked by an aryl-aryl bond). Compounds 1 and 2 were then tentatively identified as fucophlorethol isomers, as they are formed by phloroglucinol units linked through both ether and phenyl bonds. Either one of them may, in fact, correspond to fucophlorethol A (Figure 31), previously isolated from F. vesiculosus by Parys et al. (437).

The trimers 3 and 4, detected in the purified extracts of Fspi and Fg, respectively (Figure 30N and H), presented deprotonated molecular ions with less 4 amu than the others (m/z 369), consistent with three-ringed phloroglucinol containing dibenzo[1,4]- dioxin structural elements, such as dioxinodehydroeckol derivatives (Figure 31). Dioxinodehydroeckol, also known as eckostolonol, is a common phlorotannin reported in the composition of several seaweed species belonging to the orders Fucales and Laminariales (57).

120 RESULTS AND DISCUSSION

TRIMERS 142 PENTAMERS 126 160 248 266 142 126

(4) 44 62 44 124 144

(1)

TETRAMERS (13) (15)

229 262 142 HEXAMERS

(6) (9) 392 142 126 126 286 266

44 262

144 142/144 (12) 126 125 44 (22) (10) (20)

Figure 31. Proposed fragmentation patterns of the structures tentatively identified in phlorotannin purified extracts from Fucus spp.: (1) m/z 373; (4), m/z 369; (6, 9 and 10), m/z 497; (12), m/z 493; (13 and 15), m/z 621; (20 and 22), m/z 745.

121 RESULTS AND DISCUSSION

4.3.1.2. Phlorotannin tetramers

Phlorotannins composed of four phloroglucinol units (5–12) were generally the most representative of the analyzed extracts (Figure 30, Table 13). Contrary to Fves-w, in which only one tetramer (7) was detected, more than two of these oligomers were found in the remaining extracts (Figure 30). Excepting compound 12, the remaining tetramers (5–11) presented deprotonated molecular ions at m/z 497 (Table 13). In the MS2 and MS3 fragmentation of compounds 5 and 6 the loss of a single phloroglucinol unit indicates the presence of only one aryl-ether bond, likely corresponding to difucophlorethol isomers (Table 13). As far as we are aware, to date these phlorotannin oligomers have not yet been described in any Fucus species; however, the difucophlorethol A isomer had already been isolated from other brown seaweeds of the order Fucales (Himanthalia elongata (Linnaeus) S.F. Gray and Cystophora retroflexa (Labillardière) J. Agardh) (438,439). In their MS fragmentation (MS2 and MS3), compounds 7–9 exhibited losses of one and two phloroglucinol units, the ion characterizing the fucol moiety (m/z 229 in MS3) being observed, differing from each other by their base peaks and the relative abundance of their product ions (Table 13). Based on the MS data collected (Table 13), two phloroglucinol units linked by an aryl-ether bond (diphlorethol) may be present, leading to the tentative identification of compounds 7–9 as fucodiphlorethol isomers. As it happened with 7–9, the loss of a phloroglucinol unit (–124 amu) was detected in the MS2 of compound 11, originating the base peak at m/z 373 that is fragmented in MS3 with loss of 140 amu (124+16). No other loss of phloroglucinol was noticed, leading to the putative identification of compound 11 as a fucodiphlorethol isomer. These tetramers of phloroglucinol isomers have already been identified by our group (57) in purified extracts of F. spiralis and Cystoseira tamariscifolia (Hudson) Papenfuss, as well as by Wang et al. (282) in Sephadex subfractions of the ethyl acetate fraction from F. vesiculosus ethanol:water (80:20, v/v) extract. This particular phlorotannin assemblage has been associated to a number of interesting properties (e.g., free radical scavenging (442) and anti-allergic (278)), making their presence in these extracts of great relevance for further biological studies.

Despite sharing the same molecular ion of the previously identified tetramers (m/z 497), the main MS2 fragments observed for compound 10 result from the loss of two phloroglucinol molecules and a methyl group (262 amu = 124+124+14), to give rise to the base peak, which did not originate other phloroglucinol units in MS3. As so, compound 10 should correspond to two fucol moieties linked by an aryl-ether bond, tentatively labelled as bisfucophlorethol (Figure 31). Bisfucophlorethols, together with several other

122 RESULTS AND DISCUSSION

phlorotannin oligomers, were found by Glombitza and Hauperich (440) in Cystophora torulosa (R. Brown ex Turner) J. Agardh (Fucales).

The tentative identification of the tetramer 12, detected only in the purified extract from Fg (Figure 30J), proved to be a challenging task mainly because of its atypical molecular formula (C24H14O12). Its deprotonated molecular ion (m/z 493) is 4 amu lower than those of the previous tetramers. Based on the MS data and published literature (245), it is possible that the chemical structure of compound 12 resembles that of a fucofuroeckol containing an additional OH group in its backbone (Figure 31).

4.3.1.3. Phlorotannin pentamers

Among the analyzed phlorotannin purified extracts, the ones from both wild-sourced and aquaculture-grown F. vesiculosus (Fves-w and Fves-a, respectively) were the only exhibiting five-ringed phloroglucinol oligomers (Figure 30C and F). In all the pentamers (13–19), as well as in most of the phlorotannins detected in this work, the base peak in MS2 results from the loss of water from the deprotonated molecular ion (Table 13). Both MS2 and MS3 fragmentation of compounds 13 and 14 are similar, with losses of a single phloroglucinol unit (Table 13) being observed, with no further loss of phloroglucinol or phloroglucinol derivative in MS4 (data not shown). The chemical features of compounds 13 and 14 are therefore consistent with the ones of trifucophlorethol isomers (Figure 31). As far as we know, this was the first tentative identification of this fucophlorethol-type phlorotannins in Fucales.

All the remaining pentamers (15–19), detected only in Fves-w (Figure 30C), had similar MS2 fragmentation patterns, characterized by losses of phloroglucinol (–126 amu) or a phloroglucinol derivative (–142/146/158/160 amu), as well as by the simultaneous loss of two units of phloroglucinol combined with oxygen and/or water (– 266/268/282/284 amu) (Table 13). The same losses were detected in the MS3 fragmentation. In the MS2 of compound 19, in the MS3 of compounds 17 and 18, as well as in the MS4 of 15–19, an ion at m/z 229/230 was found (Table 13). As for several other phlorotannins detected herein, this ion may be due to the loss of three phloroglucinol units linked to each other through aryl-ether bonds [229 = 621–392 (124+124+126+18 amu)]. Additionally, this ion does not undergo further fragmentation in MS4, pointing to a fucol moiety (two units of phloroglucinol linked by an aryl-aryl bond), as observed in the fragmentation of other compounds described above. In some cases, like that of compound 18, it was possible to observe the sequential loss of phloroglucinol units: MS4 (621 → 603 → 463): 339; MS4 (621 → 603 → 339): 230 (Figure 32). Pentamers 15–19 were then

123 RESULTS AND DISCUSSION

tentatively identified as isomers of fucotriphlorethol (Figure 31). In the work previously conducted by our group (57) it was already identified a fucotriphlorethol derivative in the seaweed species Cystoseira usneoides (Linnaeus) M. Roberts, belonging to Fucales. Still, of relevance, was the isolation of fucotriphlorethol A from F. vesiculosus extracts (437), suggesting that one of the 15–19 pentamers detected herein, in the same seaweed species, may, in fact, correspond to that isomer.

Intensity 106 -MS2 (621.1) 463.1 (-158:124+18+16) 1.5 477.2 (-144:126+18) 603.1 (-18) 1.0 244.9 (-376: 126+126+124) 339.0 (-282:124+124+18+16) 585.1 0.5 0.0 105 -MS3 (621.5 → 603.4) 4 339.0 (264:124+124+16) 463.1 (-158:124+18+16) 3 477.0 (-126) 585.0 2 228.9 244.8 353.0 (-250:126+124) 559.0 1 0 3 104 -MS (621.5 → 339.2) 3 229.9 (-110: 126-16) 2 213.9 1 322.2 337.0 0 4 104 -MS (621.5 → 603.4 → 463.2) 338.8 (-124) 2.0 319.9 1.5 229.8 1.0 201.8 264.9 0.5 292.8 0.0 105 4 1.00 -MS (621.5 → 603.4 → 338.9) 0.75 0.50 229.9 (-110: 126-16) 0.25 0.00 200 300 400 500 600 m/z

Figure 32. Mass spectra analysis of the pentamer 18 detected in wild-sourced F. vesiculosus (Fves-w).

4.3.1.4. Phlorotannin hexamers

The extracts of F. vesiculosus (Fves-w and Fves-a) were the only ones exhibiting phlorotannin hexamers (20–22) (Figure 30D and G). The three hexamers had the same deprotonated molecular ion at m/z 745, but different fragmentation patterns (Table 13). In the MS3 of compound 20 detected in Fves-a (Figure 30G), the loss of water and the fragmentation of a phloroglucinol unit (–125 amu) indicates the presence of a single ether bond in its structure (Figure 31). The loss of 125 amu was observed only for this compound (Table 13). A similar fragmentation pattern was recently reported for phorethols and fucophlorethols extracted from Sargassum fusiforme (Harvey) Setchell (Fucales) (285). Although the MS fragmentations provided raw data to ensure the identity of compound 20, it can be included within the fucophlorethol-type, and possibly labelled as tetrafucophlorethol.

Compounds 21 and 22 showed a MS fragmentation similar to that of the pentamers referred above (15–19), though with an additional phloroglucinol unit. Besides the ion

124 RESULTS AND DISCUSSION

[(M-H)–18]- as base peak, their MS2 fragmentation exhibited the ions at m/z 601 (–144), 461/479 (–284/286), 335/353 (–410/392) and the radical fucol at m/z 229, the corresponding ions being also found in the MS3. These data suggest the presence of four phloroglucinol units linked by aryl-ether bonds, and of a fucol moiety (ion observed at m/z 229), leading to the tentative identification of compounds 21 and 22 as fucotetraphorethol isomers (Figure 31), already isolated from H. elongata and C. retroflexa (Fucales) (438,439).

4.3.2. Phlorotannin extracts from Fucales as bioregulators engaged in inflammation-related mediators and enzymes

Among the most promising effects already described for phlorotannins, the anti- inflammatory potential has attracted particular attention (54,275,302,342,344,347,352,356). In this work, the anti-inflammatory capacity of phlorotannin-targeted extracts from different species of Fucales, widely represented on the Portuguese coastline and still underexplored in this field, was examined through a multiple-method approach of well-documented in vitro cell and cell-free assays.

4.3.2.1. Quantitative overview

The methodology employed herein for obtaining high-purity phlorotannin extracts (Figure 16) was already successfully applied for biological assessment (54,56), and with which we were able to extract almost all free phlorotannins, the so-called extractable polyphenols (249). The extraction yields were of around 20% of dry algal weight, which were in agreement with other reports for some species of Fucales (282,443). The amount of phlorotannins in each purified extract was determined through the DMBA spectrophotometric assay, by interpolation on the phloroglucinol calibration curve (y= 0.0213x + 0.0131; r2 = 0.9997) and expressed as PGE. Significantly higher levels of phlorotannins were found in the extracts of Fg and Fser (288.36 ± 23.44 and 269.20 ± 47.45 μg PGE/100 mg DE, respectively), followed by Fspi (165.95 ± 12.83 μg PGE/100 mg DE), and by the extracts of both Fves-w and Fves-a (144.51 ± 12.91 and 110.28 ± 3.36 μg PGE/100 mg DE, respectively) (Figure 33).

125 RESULTS AND DISCUSSION

F v e s- a c

F v e s- w b,c

F sp i b Samples F se r a

F g a

50 100 150 200 250 300

Concentration (µg PGE/100 mg DE)

Figure 33. Phlorotannin content in the purified extracts from Fucus spp. Fg, F. guiryi; Fser, F. serratus; Fves-w, F. vesiculosus-wild; Fves-a, F. vesiculosus-aquaculture; PGE, phloroglucinol equivalents; DE, dry extract. Results represent the mean ± standard deviation of five determinations performed in duplicate. Different letters denote statistically significant differences (p < 0.05) between samples (ANOVA, Tukey's multiple comparison test).

Despite belonging to the same genus, the macroalgae samples under investigation showed clearly different phlorotannin content. This variability can be attributed, in part, to species-specific factors, along with the broad fluctuations of environmental surrounding conditions that characterize the marine ecosystem. For instance, F. vesiculosus collected along the wild natural stocks (Fves-w) presented higher levels of phlorotannins than its counterpart cultivated in the pilot land-based installation (Fves-a). Regardless of the ecological and socio-economic advantages of IMTA, the complexity of environmental changes under which a species persists within its natural beds is more likely to provide chemically richer profiles. Indeed, phlorotannin composition in Phaeophyceae shows plasticity face to different abiotic factors, and biotic interactions (254). In the present study, the phlorotannin content of F. vesiculosus from both wild (144.51 µg PGE/100 mg DE = 324.20 mg PGE/kg dry algae) and IMTA system (110.28 µg PGE/100 mg DE = 163.36 mg PGE/kg dry algae) was higher than that of F. vesiculosus from Iceland (79.39 mg PGE/kg dry algae), previously documented by Wang and colleagues (282). On the other hand, F. spiralis analyzed herein exhibited much less phlorotannins (165.92 µg PGE/100 mg DE = 362.94 mg PGE/kg dry algae) than the same species from a southern location of the Portuguese coast (54). This variation confirms again the influence of abiotic and/or biotic factors in phlorotannin levels, suggesting that higher phlorotannin content seems to be positively correlated with generally higher temperatures and light exposure.

126 RESULTS AND DISCUSSION

4.3.2.2. Anti-inflammatory activity

4.3.2.2.1. Lipoxygenase inhibitory potential

Among the most relevant enzymes involved in the biosynthesis of mediators closely related to the pathogenesis of allergy- and inflammatory-related diseases, LOX is highlighted (444). Therefore, LOX inhibitors are expected to suppress the exacerbation of inflammatory states.

The capacity of phlorotannin purified extracts to inhibit LOX was evaluated in a cell- free system. All the analyzed extracts inhibited LOX in a dose-dependent manner (Figure

34). Fg and Fser extracts were the most potent (IC50 = 82.10 and 110.16 µg/mL, respectively; p > 0.05), whereas those obtained from Fspi and Fves-w displayed IC50 values higher than 350 µg/mL (p > 0.05) (Table 14). Under the assayed concentrations, it was only possible to calculate IC25 for Fves-a (348.68 ± 17.90 µg/mL) (data not shown), once maximum LOX inhibition was of around 35% for the highest concentration tested (500 µg/mL) (Figure 34, Table 14). To the best of our knowledge, the inhibitory activity of phlorotannin purified extracts from F. guiryi, F. serratus, F. spiralis, and F. vesiculosus towards LOX was assessed herein for the first time.

100 F g

n F se r

o 75

i t

i F spi

i h

n F v e s -w

i 50

X F v e s -a

O L

% LOX %LOX inhibition %

10 100 1000

ConcentrationC o n c e n tr a t io n (µg ( g DE/mL) D E /m L ) (Log ( L o g scale) s c a le )

Figure 34. Lipoxygenase (LOX) inhibition of phlorotannin purified extracts in cell-free assay. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis; Fves-w, F. vesiculosus-wild; Fves-a, F. vesiculosus- aquaculture; DE, dry extract. Results represent the pooled mean ± standard deviation of three independent experiments performed in triplicate.

As reviewed by Barbosa and collaborators (22), although many studies have focused on LOX substrates (highly unsaturated fatty acids), products (leukotrienes), and LOX isoforms isolated from macroalgae, only a few have documented the LOX inhibitory

127 RESULTS AND DISCUSSION

potential of brown algal phlorotannins (355,357). Shibata and collaborators (355) showed that oligomers of phloroglucinol (eckol, phlorofucofuroeckol A, dieckol, and 8,8'-bieckol) isolated from Eisenia bicyclis (Kjellman) Setchell had pronounced inhibitory effects on LOX. Years later, Kurihara et al. (357) examined the LOX inhibitory potential of fucophlorethol C isolated from the brown seaweed Colpomenia bullosa (D.A. Saunders) Yamada. The authors found that this phloroglucinol oligomer effectively inhibited LOX

(IC50 = 215 µM) by a mixed type inhibition (competitive and non-competitive) and concluded that the free phenolic hydroxyl groups in the molecular structure were important for the observed activity (357).

In this work, a strong negative correlation between total phlorotannins and IC50 values was found (Pearson correlation; r= –0.9118; p < 0.0001), suggesting that the observed activity might be proportional to phlorotannin concentration, probably as a result of synergism phenomena between the overall constituents. The Fucus genus, rich in phlorotannins belonging to the class of fucophloretols (57,286), is a good example of this.

For instance, Fg, the most effective against LOX, presented an IC50 of 82.10 µg/mL, which corresponds to 0.24 µg PGE/mL (Table 14). This value is significantly lower than that found by Kurihara and co-workers (357) for the isolated fucophlorethol C (IC50 of 215 µM = 80.47 µg/mL), proving that the synergism between fucophlorethols and other phlorotannins present in Fucales extracts can overcome the activity of the isolated compounds themselves. The IC50 values obtained herein indicated also that the use of purified phlorotannin extracts can be advantageous over that of isolated compounds. For instance, Shibata et al. (355) found that 8,8'-bieckol was the most effective compound regarding LOX inhibition (IC50 = 24 µM = 17.80 µg/mL). Despite being more potent than fucophlorethol C (IC50 = 80.47 µg/mL) (357), the IC50 of 8,8'-bieckol is suggested to be clearly higher than those obtained herein with purified phlorotannin extracts (ranging between 0.24 and 0.60 µg PGE/mL) (Table 14). In addition to phlorotannin-rich extracts, this survey highlights the capacity of fucophlorethols, previously characterized in Fucales (57,286), to inhibit LOX in a dose-dependent manner (Figure 34).

128 RESULTS AND DISCUSSION

Table 14. IC50 values found for the phlorotannin purified extracts on LOX inhibition.

LOX inhibition2 Sample1 µg DE/mL µg PGE/mL Fg 82.10a ± 9.60 0.24a ± 0.03 Fser 110.16a ± 2.47 0.30a ± 0.01 Fspi 362.42b ± 8.13 0.60c ± 0.01 Fves-w 364.84b ± 23.19 0.53b ± 0.03 Fves-a > 500 > 0.55

1 Identity as in Table 8. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis; Fves-w, F. vesiculosus-wild; Fves-a, F. vesiculosus-aquaculture; LOX, lipoxygenase; PGE, phloroglucinol equivalents; DE, dry extract. 2 Values correspond to the pooled mean ± standard deviation of three independent experiments, performed in triplicate. Different superscript letters denote statistically significant differences (p < 0.05) in the same column (One-way ANOVA, Tukey's multiple comparison test).

4.3.2.2.2. Effect on inflammatory mediators

Phlorotannins have already demonstrated capacity to modulate NO levels, either by direct scavenging or by decreasing NO production, through the action in inflammatory signaling cascades and inhibition of enzymes involved in its production (340,342).

The ability of the phlorotannin purified extracts to decrease NO levels in culture medium of LPS-exposed macrophages was then evaluated. Preliminary experiments (MTT reduction assay) were conducted to assess the range of non-cytotoxic concentrations for which the exposure to the purified extracts did not significantly affect cell viability. The evaluation of the phlorotannin-treated RAW 264.7 cells viability indicated that, even at the highest concentration, phlorotannin purified extracts did not affect mitochondrial activity (Figure 35). At non-cytotoxic concentrations, Fves-w purified extract was the most effective in NO reduction (IC25 = 56.52 µg/mL), while Fves-a extract displayed the lowest capacity to decrease NO levels (IC25 = 317.41 µg/mL; p < 0.05) (Table 15). Broad outline, the overproduction of NO induced by LPS was efficiently surmounted when RAW 264.7 cells were treated with the purified phlorotannin extracts (Figure 35). At the highest concentration tested (500 µg/mL), Fves-a, Fser, Fspi, Fg and Fves-w extracts reduced NO levels by 50.3, 49.8, 47.7, 40.9 and 38.5%, respectively, when compared to the corresponding control (Figure 35).

129 RESULTS AND DISCUSSION

Fg Fser Fspi 125 125 125 C ell viability C ell viability C ell viability

100 100 N O 100 N O N O

* * * * * 75 * * * * 75 75 * * * * * * * * * * *

* * * * * * *

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0 0 0 C ontrol 31.25 62.5 125 250 500 C ontrol 31.25 62.5 125 250 500 C ontrol 31.25 62.5 125 250 500

C o n c e n tr a tio n ( g D E /m L ) CConcentrationo n c e n tr a tio n ( (µg g DE/mL)D E /m L ) Concentration (µg DE/mL) CConcentrationo n c e n tr a tio n ( (µg g DE/mL)D E /m L )

Fves-w Fves-a 125 125 C ell viability C e ll v ia b ility 1 2 5 C e ll v ia b ilit y 100 100 N O N O N O 1 0 0

75 * * * * 75

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50 50 % **** 25 5 025

0 2 5 0 C ontrol 31.25 62.5 125 250 500 C ontrol 31.25 62.5 125 250 500

CConcentrationo n c e n tr a tio n ( (µg g DE/mL)D E /m L ) 0 CConcentrationo n c e n tr a tio n (µg( g DE/mL)D E /m L ) C o n t ro l 3 1 .2 5 6 2 .5 1 2 5 2 5 0 5 0 0

Figure 35. Effect of phlorotannin purified extracts on the viability and NOC levelso n c e n tofr a RAWt io n ( 264.7g /m L )cells pre-treated for 2 h with the extracts, followed by 22 h co- treatment with LPS (1 μg/mL) or vehicle (culture medium). Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis; Fves-w, F. vesiculosus-wild; Fves-a, F. vesiculosus-aquaculture; DE, dry extract. Results represent the pooled mean ± standard deviation of four independent experiments, performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 face to the respective control.

130 RESULTS AND DISCUSSION

Although no correlation was found between the amount of phlorotannins and the IC25 values obtained for cellular NO levels (Pearson correlation; r = –0.4994; p = 0.0580), the results presented herein are in agreement with previous published data (54,341), highlighting the potential of Fucales extracts to reduce this pivotal mediator involved in the signaling and pathogenesis of inflammation. Nevertheless, the qualitative composition of the extracts cannot be excluded. For instance, Fg is significantly richer than Fspi regarding total phlorotannin amounts (288.36 and 165.92 µg PGE/100 mg DE, respectively), but the two species displayed similar capacity to reduce NO in cell culture medium (IC25 values of 97.73 and 95.86 µg/mL, respectively) (Table 15). Additionally, for the highest concentration tested (500 µg/mL), the NO reduction in cells supernatant was not significantly different (40.9 and 47.7%, p > 0.05, respectively) (Figure 35).

Table 15. IC50 values found for the phlorotannin purified extracts on NO levels in RAW 264.7 cell culture medium.

NO reduction2 Sample1 µg DE/mL µg PGE/mL Fg 97.73a ± 25.65 0.28a,c ± 0.07 Fser 77.04a ± 15.55 0.21a,b ± 0.04 Fspi 95.86a ± 18.64 0.16b,d ± 0.03 Fves-w 56.52a ± 2.58 0.08d ± 0.00 Fves-a 317.41b ± 31.61 0.35c ± 0.03

1 Identity as in Table 8. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis; Fves-w, F. vesiculosus-wild; Fves-a, F. vesiculosus-aquaculture; NO, nitric oxide; PGE, phloroglucinol equivalents; DE, dry extract. 2 Values correspond to the pooled mean ± standard deviation of four independent experiments, performed in triplicate. Different superscript letters denote statistically significant differences (p < 0.05) in the same column.

To our knowledge, this was the first report regarding the effect of phlorotannin extracts from Fg, Fser and Fves-a on NO in LPS-stimulated RAW 264.7 cells. In fact, few surveys have addressed the anti-inflammatory activity of phlorotannin extracts from Fucales (54,91,108,341), the great majority of them focusing seaweeds from Eisenia, Ecklonia and Ascophyllum genera (274,275,302,340,342,347,348,351). Zaragozá and collaborators (341) had conducted a survey with F. vesiculosus extracts rich in polyphenols and obtained an IC50 of ca. 95 µg/mL for NO reduction in the cell culture supernatant. Despite being the same species, the authors found a much higher phlorotannin amount, which can be explained by seaweed geographic location and time of harvest. In addition, the kind of extract itself (hydroethanolic), containing several other classes of bioactive compounds, can be the reason for the higher inhibitory activity observed by those authors (341). Considering Fucus spp., a purified phlorotannin extract

131 RESULTS AND DISCUSSION

from F. spiralis was already chemically and biologically explored (54,57). However, the capacity of this kind of extract to reduce NO in LPS-stimulated RAW 264.7 cell culture medium was not reported. As far as we know, to date there is only another work focusing the effect of phlorotannins from Fucales in RAW 264.7 cells (91). The authors confirmed the richness in fucophloretols of Fucus distichus Linnaeus, also reported by Ferreres et al. (57) in seaweeds for this genus and demonstrated its capacity to decrease mRNA expression of acute and chronic inflammatory biomarkers (91).

As far as we are aware, none of the studies conducted with isolated phlorotannins addressed the capacity of fucols and fucophlorethols, characteristic of Fucales, to reduce the NO levels in RAW 264.7 cell culture medium. The existing studies are mainly devoted to compounds of eckols class (274,275,342,347). However, purified phlorotannin extracts of the seaweed species evaluated herein seem, again, to be advantageous face to isolated compounds. For instance, of the isolated compounds studied so far, phlorofucofuroeckol A was one of those presenting lower IC50 in this inflammation model (6.95 µM = 4.19 µg/mL) (347). Even so, phlorotannin extracts seem to be more promising: the highest concentration tested herein was lower than that of phlorofucofuroeckol A, and still leads to a NO reduction between 38.5 and 50.3% (achieved with 500 µg/mL, which corresponds to a variation between 0.55 and 1.44 µg PGE/mL) (Figure 35).

According to the discussed above, the anti-inflammatory effects of the purified phlorotannin extracts can occur by different mechanisms, including the well-documented antioxidant activity of phlorotannins (282). In fact, the decrease of NO concentration can be due to the interference with NO production in LPS-stimulated macrophages, but also to the direct scavenging of the NO produced (54). The anti-radical potential of the phlorotannin purified extracts was determined using a cell-free assay with a NO donor. All extracts tested showed a clear concentration-dependent scavenging activity against •NO (Figure 36).

132 RESULTS AND DISCUSSION

100 F g

g F se r n

i 75 g

n F spi

e v

a F v e s -w

c 50

O F v e s -a

NO scavenging NO

 •

% % %

50 500 5000

ConcentrationC o n c e n tr a t io n (µg( g DE/mL) D E /m L ) (Log ( L o g scale) s c a le )

Figure 36. Nitric oxide radical (•NO) scavenging of phlorotannin purified extracts in cell-free assay. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis; Fves-w, F. vesiculosus-wild; Fves-a, F. vesiculosus-aquaculture. Results represent the pooled mean ± standard deviation of three independent experiments performed in triplicate.

Under the tested concentrations, Fg exhibited the highest scavenging potential (IC50 =

451.91 µg/mL), Fves-a being the least active (IC50 = 2072.32 µg/mL; p < 0.05) (Table 16). A negative correlation was observed between the total phlorotannin levels in the purified extracts and the IC50 values for NO scavenging (Pearson correlation; r = –0.6363; p = 0.0108). However, once the concentration ranges required for the cell-free assay were significantly higher (78–1250 µg/mL for Fg and Fspi and 156–2500 µg/mL for Fser, Fves- w and Fves-a, of DE) (Figure 36) than those employed in cell bioassays (Figure 35), it seems that the decrease in NO observed in the cell system may be due to the modulation of specific inflammatory cascade steps, rather than to direct radical scavenging activity.

Likewise, Lopes et al. (54) assumed that the anti-inflammatory potential of phlorotannins could result from the combination of several factors, including the scavenging of inflammatory mediators and the modulation of their production. In fact, previous works with phlorotannins from different species of Fucus have demonstrated the ability of these polyphenols to modulate the expression of key mediators involved in inflammatory responses (91,108).

133 RESULTS AND DISCUSSION

• Table 16. IC50 values found for the phlorotannin purified extracts on NO scavenging.

•NO scavenging2 Sample1 µg DE/mL µg PGE/mL Fg 451.91a ± 130.13 1.30a ± 0.38 Fser 1214.73b ± 269.47 3.27b ± 0.73 Fspi 801.97a,b ± 166.24 1.33a ± 0.28 Fves-w 1330.61b ± 271.63 1.92a ± 0.39 Fves-a 2072.32c ± 73.81 2.29a,b ± 0.08

1 Identity as in Table 8. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis; Fves-w, F. vesiculosus-wild; Fves-a, F. vesiculosus-aquaculture; NO, nitric oxide; PGE, phloroglucinol equivalents; DE, dry extract. 2 Values correspond to the pooled mean ± SD of 3 independent experiments, performed in triplicate. Different superscript letters denote statistically significant differences (p < 0.05) in the same column.

4.3.3. Phlorotannin extracts from Fucales in allergy network

Despite representing a major opportunity for drug discovery, anti-allergenicity studies focused on seaweed-derived compounds, including polyphenols, are still scarce.

This work aimed at evaluating, for the first time, the anti-allergic effects of phlorotannin purified extracts obtained from different Fucus species. Among the samples available (Table 8), F. guiryi (Fg), F. serratus (Fser), F. spiralis (Fspi) and the wild- sourced F. vesiculosus (Fves-w) were selected for this study.

The anti-allergic potential of the phlorotannin extracts was assessed on RBL-2H3 cell degranulation experimentally induced by two different stimuli: the calcium ionophore A23187 and the IgE/antigen complex. Cell degranulation was then determined by monitoring the concomitant release of histamine and β-hexosaminidase into cell supernatant. In addition, the capacity of the phlorotannin purified extracts to inhibit HAase and to directly alter β-hexosaminidase activity in the supernatant of degranulated RBL-2H3 cells were also screened.

4.3.3.1. Effect of phlorotannin purified extracts on cell degranulation

Acknowledging that the responsiveness of RBL-2H3 cells to different stimuli is highly dependent on experimental conditions (445), concentrations of A23187 and IgE/antigen used in this study to elicit a significant RBL-2H3 cell degranulation were chosen based on dose-response assays, considering i) cell viability, and ii) significant differences in β- hexosaminidase and histamine release, when compared to basal levels (Figure 37). RBL- 2H3 cells sensitized with increasing concentrations of anti-DNP IgE (12.5–1000 ng/mL),

134 RESULTS AND DISCUSSION

and triggered with the same concentration range of allergen, exhibited a bell-shaped dose- response curve, with maximum release of both β-hexosaminidase and histamine at 50 ng/mL (p < 0.001; Figure 37A). With IgE/antigen, β-hexosaminidase increased by approximately 3-fold above basal levels (p < 0.0001; Figure 37A) and histamine increased from the basal value of 0.22 ± 0.05 µM towards 0.69 ± 0.23 µM (p < 0.001). With A23187, a dose-dependent increase of β-hexosaminidase and histamine release was noticed; however, ionophore concentrations higher than 150 ng/mL led to a significant reduction of cell viability (p < 0.05; Figure 37B) and, thus, were not considered. At 150 ng/mL, ionophore stimulation caused a 3-fold increase (p < 0.0001; Figure 37B) of β- hexosaminidase levels, and histamine levels increased towards 0.89 ± 0.03 µM (p < 0.01), similarly to that observed with IgE/antigen complex at 50 ng/mL (Figure 37).

Prior to testing phlorotannin purified extracts for their anti-allergic potential in stimulated-RBL-2H3 cells, their cytotoxicity levels were assessed through the MTT assay (Figure 38). Either with or without degranulation stimuli, a significant increase in the ability of RBL-2H3 cells to reduce MTT was observed for some of the analyzed extracts (p < 0.05; Figure 38). No differences were observed between treated and untreated RBL- 2H3 cells in CVS assay (data not shown), suggesting that the increase in MTT mitochondrial reduction was due to an increase in cell metabolic activity, rather than to cell proliferation. Thus, all extract concentrations (125–500 µg DE/mL) were considered to proceed with the anti-allergic experiments.

135 RESULTS AND DISCUSSION

* * * * 125 * * * e * * * * * s 4

a 1.5

e n

100 e

)

i a

* *

e t

* * * e

s c

3 l

r d

r 1.0 * * *

increase) increase)

* * * n

T 2

T l

50 l

MTT MTT reduction

(fold (fold

o * *

( (

* * i 0.5

% % Histamine Histamine release

% H e

Hexosaminidase Hexosaminidase release 1

25 -

-  0 0 0.0 C ontrol 12.5 25 50 500 1000 C ontrol 12.5 25 50 500 1000 C ontrol 12.5 25 50 500 1000

CConcentrationo n c e n tr a tio n (ng/(n g /mLm L) ) C oConcentrationn c e n tr a tio n (n(ng/g /mmLL ) CConcentrationo n c e n tr a tio n (ng/(n g /mLm L) )

125

e 6 * * * * *

s 1.0 a

e * * * *

e n

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)

r i

* a

t e

* * e

e * * * *

c l

* * * s * * * *

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a 4

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* * * * e

increase) increase)

T 0.5

T l

50 l

MTT MTT reduction

(fold (fold

o 2

( (

* * * i

% % Histamine Histamine release

% H

e Hexosaminidase Hexosaminidase release

25 -

-  0 0 0.0 C ontrol 100 150 200 300 400 C ontrol 100 150 200 300 400 C ontrol 100 150 200 300 400

CConcentrationo n c e n tr a tio n (ng/(n g /mLm L) ) CConcentrationo n c e n tr a tio n(ng/ (n gmL/m )L ) CConcentrationo n c e n tr a tio n (ng/(n g /mLm L) )

Figure 37. Effect of IgE/antigen (A) and calcium ionophore A23187 (B) on the cell viability (MTT reduction), and on β-hexosaminidase and histamine released from RBL-2H3 cells. Results are expressed as the mean ± standard deviation of at least four independent experiments, performed in duplicate. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 face to the respective control.

136 RESULTS AND DISCUSSION

A23187 IgE/antigen

* # # 150 # # # 175 # *

125 # # 150

o i

t 100 125

u d

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0 0 C ontrol 125 250 500 C ontrol 125 250 500 [Fg] (µg DE/mL)

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0 0 C ontrol 125 250 500 C ontrol 125 250 500 [Fser] (µg DE/mL)

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0 0 C ontrol 125 250 500 C ontrol 125 250 500 [Fspi] (µg DE/mL)

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t 100

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e 100 r

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T 75 M

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0 0 C ontrol 125 250 500 C ontrol 125 250 500 [Fves-w] (µg DE/mL) 150 Withoutw /o s t istimulusm u lu s

Withw s t stimulusim u lu s

o i

t 100 c

Figure 38. Effectu of phlorotannin purified extracts on the viability of RBL-2H3 cells with and

d e

without stimulationr by the calcium ionophore A23187 or by IgE/antigen. Fg, F. guiryi; Fser, F.

serratus; Fspi, F. T spiralis, Fves-w, F. vesiculosus-wild; DE, dry extract. Results are expressed as the

M 50 *, # ## mean ± SD of at least four independent experiments, performed in duplicate. p < 0.05; p <

0.01; ###p < 0.001;% face to the respective control.

137 0 C o n t ro l 125 250 500

C o n c e n tr a t io n ( g d r y p u r if ie d e x t r a c t/m L ) RESULTS AND DISCUSSION

4.3.3.1.1. A23187-mediated cell degranulation

All phlorotannin purified extracts were able to decrease the degranulation induced by the non-immunological A23187 stimulus, in a dose-dependent manner (Figure 39). At the highest tested concentration of Fg, Fser, Fspi and Fves-w extracts, the levels of released β-hexosaminidase decreased by 77.5 ± 6.8%, 78.8 ± 7.8%, 31.3 ± 7.7% and 33.2 ± 12.2%, respectively, when compared to the corresponding control (Figure 39). As it can be seen in Figure 39, with the exception of Fves-w extract, which effect was lower than 50%, all the other Fucus spp. purified extracts were able to significantly reduce histamine levels (p < 0.05) (Figure 39, Table 17). With 500 µg DE/mL of Fg, Fser and Fspi, the levels of histamine decreased by 67.4 ± 12.7%, 67.1 ± 14.7% and 55.1 ± 11.9%, respectively, when compared to control with A23187 (Figure 39). The extract from Fves-w only promoted a reduction of 27.9 ± 14.8% of the histamine released (Figure 39).

Fg Fser

125 125

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# 75 75 *

* *

% %

50 # # # 50 # # #

* * * * * * 25 25

0 0 C ontrol 125 250 500 C ontrol 125 250 500

C o n c e n tr a t io n ( g D E /m L ) CConcentrationo n c e n tr a t io n ( (µg g DDE/E /mL)m L ) Concentration (µg DE/mL)

Fspi Fves-w

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% % % % # # 50 50

25 25

0 0 C ontrol 125 250 500 C ontrol 125 250 500

C o n c e n tr a t io n ( g D E /m L ) C o n c e n tr a t io n ( g D E /m L ) Concentration (µg DE/mL) Concentration (µg DE/mL)

Figure 39. Effect of phlorotannin purified extracts on β-hexosaminidase and histamine released from RBL-2H3 cells when stimulated with the calcium ionophore A23187. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis, Fves-w, F. vesiculosus-wild; DE, dry extract. Results are expressed as the mean ± standard deviation of at least four independent experiments, performed in duplicate. *, #p < 0.05; **, ##p < 0.01; ***, ###p < 0.001 face to the respective control.

138 RESULTS AND DISCUSSION

Table 17. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase and histamine released by A23187-stimulated RBL-2H3 cells.1

A23187-mediated degranulation Species β-Hexosaminidase Histamine µg DE/mL µg PGE/mL µg DE /mL µg PGE/mL

Fg 335.76a ± 20.65 0.86a ± 0.05 357.74a ± 114.69 0.92a ± 0.29

Fser 356.75a ± 24.02 1.01b ± 0.07 395.95a ± 87.92 1.12a ± 0.25

Fspi > 500 > 0.93 312.94a ± 105.91 0.58a ± 0.20

Fves-w > 500 > 0.86 > 500 > 0.86

1 Values correspond to mean ± standard deviation of at least three independent experiments. Different superscript letters denote statistically significant differences (p < 0.05) in the same column. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis; Fves-w, F. vesiculosus-wild; PGE, phloroglucinol equivalents; DE, dry extract.

4.3.3.1.2. IgE/antigen-mediated cell degranulation

Although to a different extent, all phlorotannin purified extracts were also able to decrease the degranulation induced by the immunological stimulus (Figure 40, Table 18). At the highest concentration tested, the extracts from Fg, Fser, Fspi and Fves-w induced a maximum β-hexosaminidase decrease of 79.4 ± 10.3%, 61.7 ± 4.6%, 43.7 ± 9.0% and 34.3 ± 13.2%, respectively, when compared to control (Figure 40). The purified extracts were also capable of reducing histamine release by 49.1 ± 7.6%, 54.7 ± 14.7%, 44.1 ± 9.8% and 38.5 ± 4.6%, respectively, face to the correspondent control (Figure 40).

Table 18. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase and histamine released by IgE/antigen-challenged RBL-2H3 cells.1

IgE/antigen-mediated degranulation Species β-Hexosaminidase Histamine µg DE/mL µg PGE/mL µg DE/mL µg PGE/mL

Fg 339.44a ± 39.20 0.87a ± 0.10 478.40a ± 12.42 1.23a ± 0.03

Fser 376.32a ± 54.72 1.06a ± 0.15 434.49a ± 35.29 1.23a ± 0.10

Fspi > 500 > 0.93 > 500 > 0.93

Fves-w > 500 > 0.86 > 500 > 0.86

139 RESULTS AND DISCUSSION

Fg Fser

125 125

100 100 * * * 75 * * * 75

* * *

% % % % # # # * * * 50 50

* * * * 25 25

0 0 C ontrol 125 250 500 C ontrol 125 250 500

C o n c e n tr a t io n ( g D E /m L ) C o n c e n tr a t io n ( g D E /m L ) Concentration (µg DE/mL) Concentration (µg DE/mL)

Fspi Fves-w

125 125

100 100

# # *

75 * # # # 75 # %

% *

% %

50 50

25 25

0 0 C ontrol 125 250 500 C ontrol 125 250 500

C o n c e n tr a t io n ( g D E /m L ) C o n c e n tr a t io n ( g D E /m L ) Concentration (µg DE/mL) Concentration (µg DE/mL)

β-Hexosaminidase Histamine

Figure 40. Effect of phlorotannin purified extracts on β-hexosaminidase and histamine released from RBL-2H3 cells when stimulated with IgE/antigen. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis, Fves-w, F. vesiculosus-wild; DE, dry extract. Results are expressed as the mean ± standard deviation of at least four independent experiments, performed in duplicate. *, #p < 0.05; **, ##p < 0.01; ***, ###p < 0.001; ****p < 0.0001 face to the respective control.

Even though all phlorotannin-targeted Fucus spp. extracts were able to act upon cellular events triggered by the immunological reaction, and on cellular events downstream the Ca2+ influx caused by the chemical stimulus (Figures 39 and 40; Tables 17 and 18), the ones obtained from the species Fg and Fser were, in general, the most potent in reducing RBL-2H3 cell degranulation elicited either by A23187 or by IgE/antigen (Figures 39 and 40; Tables 17 and 18).

The higher anti-degranulation effects of Fg and Fser extracts can be partly explained by their total amount of phlorotannins (Figure 33). In fact, positive correlations were found between the phlorotannin content and the maximum reduction of β- hexosaminidase released by RBL-2H3 cells stimulated with A23187 (Pearson correlation; r = 0.9115, p < 0.0001) and IgE/antigen (Pearson correlation; r = 0.8755, p = 0.0002), as well as between the amount of phlorotannins and the maximum reduction of histamine in

140 RESULTS AND DISCUSSION

IgE/antigen-stimulated RBL-2H3 cells (Pearson correlation; r = 0.8943, p < 0.0001). The results obtained so far suggest that, besides phlorotannin content, the anti-allergic effects can also be explained by species-specific variations regarding their qualitative profile. Although the studies devoted to the evaluation of the anti-allergic properties of phlorotannins are still scarce and confined to few seaweed species, particularly those from Eisenia and Ecklonia genus, some of them have reported the anti-degranulation capacity of phlorotannins. Sugiura et al. (381) reported a dose-dependent decrease of histamine released from RBL-2H3 and KU812 cells stimulated with A23187, when pre-treated with a methanol:chloroform (1:2, v/v) extract of Eisenia arborea Areschoug. In a similar survey performed with Ecklonia stolonifera Okamura, the authors found that the methanol:chloroform (1:2, v/v) extract was also capable of inhibiting RBL-2H3 cell degranulation in a dose-dependent manner (382). The removal of phenolic compounds by treating the crude extract with polyvinylpolypyrrolidone, and the decline in the activity of the resulting fraction, led the authors to conclude that the compounds responsible for degranulation inhibition were of phenolic nature, most likely phlorotannins (382). The same research group had previously demonstrated that methanol extracts from seven species of brown seaweeds inhibited histamine release from IgE/antigen-stimulated-RBL- 2H3 cells, and also attributed this effect to phlorotannins (377).

Beyond non-targeted brown seaweed extracts, some studies have been conducted with isolated phlorotannins, and have proved their ability to suppress cell degranulation through multifunctional inhibitory effects (276,278,372,373,375,384). Some phlorotannins isolated from Ecklonia cava Kjellman have demonstrated anti-allergic potential by inhibiting the degranulation of KU812 and RBL-2H3 cells stimulated with A23187 and sensitized with IgE (278,373). Regarding the effect of isolated compounds on IgE-sensitized RBL-2H3 cells, eckol, phlorofucofuroeckol A, 6,6'-bieckol and dieckol led to a dose-dependent inhibition of β-hexosaminidase release, IC50 values ranging between 38 and 98 µM (corresponding to ca. 23–32 µg/mL) (278,373). Fucodiphlorethol G was the most effective at inhibiting cell degranulation (IC50 value of 31.65 µM ≈ 15.77 µg/mL), while phloroglucinol did not reach 50% of inhibition (278,373). Regarding β- hexosaminidase release from IgE/antigen-stimulated RBL-2H3 cells, Fg and Fser extracts exhibited much lower IC50 values (0.87 and 1.06 µg PGE/mL; Table 18) than those of the isolated compounds, emphasizing the interest in exploring phlorotannin-targeted extracts from Fucus spp. for their anti-allergic activities.

When triggered with A23187, eckol, phlorofucofuroeckol A, and fucodiphlorethol G were previously able to reduce histamine levels by over 50%, the latter showing the strongest effect (IC50 value of 55.12 µM ≈ 27.47 µg/mL) (278). As for the immunological

141 RESULTS AND DISCUSSION

stimulus, the phlorotannin purified extracts from Fg, Fser and Fspi presented lower IC50 values (0.92, 1.12 and 0.58 µg PGE/mL, respectively; Table 17), reinforcing the potential of Fucus genus towards allergy-related conditions.

The anti-allergic activity of phlorotannins isolated from E. arborea was also explored in RBL-2H3 cells, by evaluating the capacity to inhibit histamine and β-hexosaminidase release (276,372). All the isolated phlorotannins inhibited basophil degranulation; of them, phlorofucofuroeckol B (IC50 value of 7.8 µM ~ 4.70 µg/mL) presented an inhibitory activity 3–6 times greater than that of the natural inhibitor epigallocatechingallate and of the commercial anti-allergic drug Tranilast (372). These authors reported that the anti- allergic activity of the isolated compounds could be positively influenced by their molecular weight and number of free OH groups (372).

The Fucus species evaluated in this survey have been previously characterized for their qualitative phlorotannin profile in the most recent work conducted by our group (286). Contrary to Sugiura and co-workers (372), the best anti-allergic effects were displayed by the extracts obtained from Fg and Fser, which were mainly composed by phlorotannins of lower molecular weights (286). On the other hand, Fves-w, richer in phloroglucinol pentamers and hexamers (286), was the one with the lowest anti-allergic potential. Among other factors, this can result from differences in phlorotannin interactions when isolated or when present in an extract. Moreover, face to the IC50 values reported so far for isolated phlorotannins, it seems advantageous to use purified extracts: they were effective at lower doses, probably as a result of synergism phenomena between the overall constituents.

The anti-allergic effects of phlorotannin purified extracts reported herein can be explained by a possible binding modulation between IgE and FсεRI, as result of the formation of insoluble complexes between phlorotannins and potential allergens, rendering Fucus spp. extracts to be hypoallergenic. Additionally, a reduction in the increase of cytoplasmic Ca2+, immediately preceding cell degranulation, should not be set aside. In fact, it has been reported that these marine polyphenols are responsible for cell membrane stabilization, alleviating the increase of intracellular Ca2+ levels (373). Some authors have attributed the anti-allergic effects of phlorotannins to i) their capacity to alleviate the increase of intracellular Ca2+, correlated with the decrease of histamine release, ii) the suppression of the binding between IgE and FсεRI receptor, and iii) the suppression of FсεRI expression (278,373–375).

Phlorotannins, such as dieckol, have also proved their anti-allergic activity in in vivo models, which was mainly linked to anti-inflammatory effects (375). The complex

142 RESULTS AND DISCUSSION

crosstalk between allergy and inflammation has indeed been widely recognized (363,446). The phlorotannin purified extracts of Fucus spp. analyzed herein have also demonstrated their anti-inflammatory effects (subsection 4.3.2.2.1), among which their capacity to inhibit LOX activity, an enzymatic system with key roles in both allergic and inflammatory reactions (444), is highlighted (53). Thus, phlorotannin purified extracts can contribute to ameliorate the symptoms of allergy, through mechanisms also involving LOX inhibition.

4.3.3.2. Allergy-related enzymes

4.3.3.2.1. β-Hexosaminidase inhibition

Among the enzymatic systems, granule-associated β-hexosaminidase is an important exoglycosidase, released in parallel with histamine, and thus experimentally used as a degranulation marker (385). Although no direct association between this enzyme and the allergic process was found (447), studies have demonstrated that β-hexosaminidase represents the dominant glycosaminoglycan-degrading glycosidase released by chondrocytes into the extracellular compartment, its activity being elevated in synovial fluid of patients with osteoarthritis (448) and rheumatoid arthritis (449). Inhibition of this enzyme may then arise as a potential strategy to prevent cartilage matrix glycosaminoglycan degradation triggered by inflammatory and autoimmune states (448,449).

All phlorotannin purified extracts from Fucus spp. analyzed herein were able to directly interact with the β-hexosaminidase present in the supernatant of degranulated RBL-2H3 cells, inhibiting this enzyme in a clear dose-dependent manner (Figure 41). At the highest concentration tested (500 µg DE/mL), Fg, Fser, Fspi and Fves-w purified extracts inhibited β-hexosaminidase by 88.1 ± 3.2%, 85.3 ± 3.0%, 71.4 ± 8.7% and 71.2 ± 3.1%, respectively (Figure 41). Extracts from both Fg and Fser revealed the most promising inhibitory activity, with IC50 values of 60.15 and 80.84 μg/mL, respectively (Table 19).

143 RESULTS AND DISCUSSION

n 100

o t

i F g

b i

h F se r n

i 75

s F spi

a d

i F v e s -w n

i 50

inhibition

Hexosaminidase Hexosaminidase

β x

e 25

% %

- 

% 0

10 100 1000

CConcentrationo n c e n tr a t io n (µg( g DE/mL)D E /m L ) (Log( L o g scale) s c a le )

Figure 41. β-Hexosaminidase inhibition of phlorotannin purified extracts in cell-free systems. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis, Fves-w, F. vesiculosus-wild; DE, dry extract. Results are expressed as the mean ± standard deviation of at least three independent experiments, performed in duplicate.

Beyond avoiding β-hexosaminidase release (subsections 4.3.3.1.1. and 4.3.3.1.2.), phlorotannin purified extracts directly inhibited β-hexosaminidase activity, also with a strong negative correlation between the IC50 values and the total phlorotannin content (Pearson correlation; r = –0.8141, p = 0.0013).

As far as we are aware, this was the first report on β-hexosaminidase enzyme inhibition of phlorotannin-rich extracts, opening doors for their exploitation as potential chondroprotective agents.

Table 19. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase inhibition.1

β-Hexosaminidase inhibition Species µg DE/mL µg PGE/mL

Fg 60.15a ± 8.44 0.15a ± 0.02

Fser 80.84a ± 17.18 0.23a,b ± 0.05

Fspi 249.87b ± 55.72 0.47c ± 0.10

Fves-w 200.78b ± 54.33 0.35b,c ± 0.09

1 Values correspond to mean ± standard deviation of at least three independent experiments. Different superscript letters denote statistically significant differences (p < 0.05) in the same column. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis, Fves-w, F. vesiculosus-wild; PGE, phloroglucinol equivalents; DE, dry extract.

144 RESULTS AND DISCUSSION

4.3.3.2.2. Hyaluronidase inhibition

Other enzymatic systems are also involved in allergy, often participating in the inflammatory circuits. HAase, for instance, acts in coordination with other separate enzymes (e.g., β-glucuronidase and β-N-acetyl hexosaminidase) and mediates the degradation of HA, a polysaccharide of high molecular weight, especially found in the extracellular matrix of connective tissues (e.g., skin, umbilical cord, synovial fluid and vitreous humor), and with key-roles in several physiological and pathological events (450). HAase-mediated depolymerization of HA has been known to be involved in inflammatory and allergic states, as well as in migration of cancer cells (450). Therefore, the modulation of HAase by suitable inhibitors seems to be useful to control certain pathological processes.

The capacity of phlorotannin purified extracts to prevent HA degradation via HAase inhibition was screened using a cell-free system and DSCG as positive control. A dose- dependent behavior towards HAase inhibition was observed for all the analyzed phlorotannin purified extracts (Figure 42). At the highest concentrations tested, Fg, Fser, Fspi and Fves-w purified extracts inhibited HAase in 74.7 ± 8.1%, 61.3 ± 9.7%, 52.9 ± 7.0% and 86.6 ± 9.4%, respectively (Figure 42). The best inhibitory activities were found for Fg and Fser extracts (IC50 = 690.80 and 817.54 μg/mL) (Table 20), both being much more efficient in inhibiting this enzyme than the reference drug DSCG (IC50 = 1104.84 ± 30.23 μg/mL, p < 0.0001).

100

F g n

o F se r i

t 75 i

b F spi

h n

i F v e s -w

HAase HAase inhibition H

% % %

100 1000 10000

C o n c e n t r a t io n (  g D E /m L ) (L o g s c a le ) Concentration (µg DE/mL) (Log scale)

Figure 42. Hyaluronidase (HAase) inhibition of phlorotannin purified extracts in cell-free systems. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis, Fves-w, F. vesiculosus-wild; DE, dry extract. Results are expressed as the mean ± SD of at least three independent experiments, performed in duplicate.

145 RESULTS AND DISCUSSION

As it was observed for the other biological activities explored in this work, a strong correlation was found between the IC50 HAase inhibition values and phlorotannin content (Pearson correlation; r = –0.9177, p < 0.0001). To the best of our knowledge, this was the first report on the HAase inhibitory potential of phlorotannin-rich extracts from Fg, Fser and Fves-w. Among the species studied herein, the extracts from Fspi were significantly more active (IC50 value of 1675.79 µg dry extract/mL = 7.04 µg dry algae/mL) than that from the same species harvested at a southern location of the Portuguese coast (IC50 value of 0.79 mg dry algae/mL) (57). Indeed, the F. spiralis material analyzed before contained much more phlorotannins (54) than the one studied herein, suggesting that the activity not only reflects the quantity of phlorotannins, but also their individual contribution and interactions. HAase inhibitory activity of phlorotannin-rich extracts was compared with that of DSCG, Fg and Fser extracts showing stronger inhibitory activity (Table 20). In fact, studies have already documented the higher HAase inhibitory capacity of brown seaweed extracts face to DSCG, for which phlorotannins were the main contributors (380,382,383). Isolated phlorotannins of E. bicyclis were indeed found to act as competitive inhibitors of HAase, the higher molecular weight compounds being the most active (376). Although displaying IC50 values between 40 and greater than 800 µM (~ 30 – > 300 µg/mL), the isolated compounds were generally less effective in preventing HA degradation via HAase inhibition than the phlorotannin purified extracts under investigation (IC50 values lower than 5 µg PGE/mL; Table 20).

Table 20. IC50 values found for the of phlorotannin purified extracts on hyaluronidase (HAase) inhibition.1

HAase inhibition Species µg DE/mL µg PGE/mL

Fg 690.80a ± 86.83 1.77a ± 0.22

Fser 817.54b ± 44.97 2.32b ± 0.13

Fspi 1675.79c ± 98.53 3.13c ± 0.18

Fves-w 1673.35c ± 125.63 2.88c ± 0.22

1 Values correspond to mean ± standard deviation of at least three independent experiments. Different superscript letters denote statistically significant differences (p < 0.05) in the same column. Fg, F. guiryi; Fser, F. serratus; Fspi, F. spiralis, Fves-w, F. vesiculosus-wild; PGE, phloroglucinol equivalents; DE, dry extract.

146

CHAPTER IV

CONCLUSIONS

147

CONCLUSIONS

5. Conclusions

The work conducted under the scope of this thesis allowed to draw the following conclusions:

 A clear seasonal variation in both fatty acid and pigment profiles of the kelps L. ochroleuca, S. latissima and S. polyschides was found, for which environmental parameters, such as temperature and luminosity, as well as algal reproductive periods were thought to be determinant.

 While intra-thallus variability was mainly attributed to physiological functions of the respective thallus section, the overall differences observed in fatty acids and pigments of S. latissima tissues cultivated at different depths at sea were linked to temperature and luminosity changes along the water column.

 The first approach to the occurrence of free phytoprostanes in macroalgae revealed

that F1t-phytoprostanes and L1-phytoprostanes were, respectively, the dominant and minor classes in all analyzed samples. However, both the occurrence and distribution of phytoprostanes are highly unpredictable, not being correlated with the amounts of α-linolenic, differing even among close relatives within a single genus and as a consequence of the surrounding conditions.

 Low molecular weight phlorotannins (370–746 Da) were proved to be present in purified extracts from different Fucus species. The dominant category of phlorotannins was that of fucophlorethols, suggesting a dependence between taxon and the structural type of phlorotannins.

 Different phlorotannin profiles of wild-sourced and IMTA cultivated species confirmed the influence of external factors on the polyphenolic composition.

 Phlorotannin-targeted extracts from Fucus spp. showed a marked potential to act upon different mediators important in the pathophysiology of inflammatory-related conditions. Despite modulating free radicals associated with the exacerbation of inflammation, phlorotannin extracts demonstrated to be effective in inhibiting LOX.

 Phlorotannin-targeted extracts from Fucus spp. acted as multi-target agents in the multifactorial etiology of allergic diseases, modulating critical steps of the allergic response.

149 CONCLUSIONS

 It was demonstrated that the unique phlorotannin profile of the selected Fucus species, in terms of both qualitative and quantitative composition, is behind the observed bioactivities.

 The many outcomes support the potential of polyphenol components as valuable naturally occurring pharmacological alternatives with a large spectrum of activity, also contributing to the valorisation of Fucus genus both as food and for nutraceutical applications.

 The possibility of designing new functional foods and pharmaceuticals is challenging, and the incorporation of phlorotannin-derived products needs further insights to ensure the relation between efficacy and safety, and in vivo studies for a more detailed understanding of the mechanisms beyond the documented biological activities.

150

CHAPTER V

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