Mutton, Robbie John

UHI Thesis - pdf download summary

The Bioactivity and Natural Products of Scottish Seaweeds

Mutton, Robbie John

DOCTOR OF PHILOSOPHY (AWARDED BY OU/ABERDEEN)

Award date: 2012

Awarding institution: The University of Edinburgh

Link URL to thesis in UHI Research Database

General rights and useage policy Copyright,IP and moral rights for the publications made accessible in the UHI Research Database are retained by the author, users must recognise and abide by the legal requirements associated with these rights. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement, or without prior permission from the author.

Users may download and print one copy of any thesis from the UHI Research Database for the not-for-profit purpose of private study or research on the condition that:

1) The full text is not changed in any way 2) If citing, a bibliographic link is made to the metadata record on the the UHI Research Database 3) You may not further distribute the material or use it for any profit-making activity or commercial gain 4) You may freely distribute the URL identifying the publication in the UHI Research Database Take down policy If you believe that any data within this document represents a breach of copyright, confidence or data protection please contact us at [email protected] providing details; we will remove access to the work immediately and investigate your claim.

Download date: 02. Oct. 2021

‘The Bioactivity and Natural Products of Scottish Seaweeds’ ©

A thesis presented for the degree of Doctor of Philosophy (Ph.D) at the University of Aberdeen

Robbie John Mutton

B.Sc. University of Newcastle upon Tyne. United Kingdom M.Sc. University of Newcastle upon Tyne. United Kingdom

May 2012

Environmental Research University of the Highlands and University of Aberdeen Institute Islands King’s College North Highland College Executive Office Aberdeen Castle Street Ness Walk AB24 3FX Thurso Inverness KW14 7JD IV3 5SQ

©This copy of this thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotations from the dissertation, nor any information derived there from, may be published without the author’s prior, written consent.

Declaration

I, Robbie John Mutton, confirm that I composed this thesis, that it has not been accepted in any previous applications for a degree, that the work is my own, and that all quotations have been distinguished by quotation marks and the sources of information specifically acknowledged.

Acknowledgements

Acknowledgments

I would like to start by thanking my Director of Studies, Kenny Boyd for his guidance, time, patients and friendship over the last few years. It has been greatly appreciated.

My thanks are also due to my other supervisors, Alex Ford and Sandy Gray.

I would also like to acknowledge the Scottish Funding Council, Highlands and Islands Enterprise and the European Regional Development Fund for funding this project.

I also wish to thank Professor Alan Harvey and his staff at the Strathclyde Innovation in Drug Research unit at Strathclyde University and Dr Charlie Bavington and his staff at Glycomar for carry out a number of assays.

Thank you to Ian Megson and Sherine Deakin for helping me with the EPR study

Thanks also to Jon Igoli for help with NMR analysis of my sample.

Thanks to Stuart Gibb and past and present colleagues at the ERI. I would however like to thank Angela Squier, Irina Foss, Mona Larson, Silvia Batchelli and Sabine Freitag for their friendship and support.

Thanks to Holly Owen and Joanne Ryrie for their contribution to this project.

To Mum and Dad, thank you for always believing in me.

Finally, a big thank you to Kathleen. Your encouragement and support means the world to me.

i Abstract

Abstract

Seaweed has traditionally been used in Scotland and other countries both as food and for medicinal purposes, which has led to seaweed being investigated for their natural product content. Despite over 30 years of research, the majority of species found in Scotland have yet to have their chemistry examined.

Extracts of seaweed were tested for antimicrobial activity against marine bacteria. Extracts of Palmaria palmata , Ulva linza , Chondrus crispus and Pelvetia canaliculata showed no detectable activity, while ethyl acetate extracts of Fucus serratus , Halidrys siliquosa, Osmundea pinnatifida and Polysiphonia fucoides showed activity against at least six of the seven strains. Extracts were screened for radical scavenging activity against ABTS, DPPH and superoxide radicals. At least one extract from each brown seaweed showed radical · scavenging of at least 80 % towards ABTS + with an ethyl acetate extract of P. fucoides · and H. siliquosa quenching DPPH by at least 90%. Radical scavenging activity appears to be dependent total phenolic content of extract. Extracts were subjected to a series of assays relevant to human health. Ethyl acetate extracts showed high antiparasitic activity against Trypanosoma brucei with a P. fucoides extract showing antibacterial activity toward Staphylococcus aureus . Extracts of H. siliquosa and F. serratus showed cytotoxicity to Hela cells with extracts of H. siliquosa showing cytotoxicity to LN CAP AS and PC 3 cell lines.

An extract of H. siliquosa underwent chromatography and by applying assay guided fractionation, several active fractions were identified. These were analysed using NMR and LC/MS and four compounds identified: (2 E. 6 E. 14 E)-l-(l’-hydroxy-4’-methoxy-6’- methyl-phenyl)-5, 13-dihydroxy-12-one-3, 7, 11, 15-tetramethylhexadeca-2, 6, 14-triene, a known antibacterial compound, previously identified in H. siliquosa ; (2E, 6 E, 10 E, l4 E)-1- (1’-hydroxy-4’-methoxy-6’-methyl phenyl)-5, 12 dihydroxy-3.7, 11, 15-tetramethyl hexadeca-2.6, 10, l4-tetraene, previously identified in Cystoseira elegans , and now in H. siliquosa ; and two compounds that have not been reported before.

Keywords: Seaweed, Algae, Natural Products, Bioactivity, Scotland

ii Content

Contents

Acknowledgements i Abstract ii Contents iii Abbreviations ix

Chapter 1 : Introduction 1 1.1. A Brief History of Natural Products 1 1.1.1. Before the Common Era 1 1.1.2. The Middle Ages 3 1.1.3. The Modern Era 3 1.2 Marine Natural Products 6 1.2.1 Background 6 1.2.2 Seaweed as a Source of Natural Products 9 1.2.3 The Highland and Islands of Scotland and Seaweed 10

Chapter 2 : Natural Products from Seaweed found in Caithness Waters and their 13 Bioactivity 2.1 Introduction 13 2.1.1 Methodology 14 2.1.2 Caithness Coastal Geography 18 2.1.3 The Review 18 2.2 Chlorophyceae (Green Seaweed) 18 2.3 Phaeophyceae (Brown Seaweed) 21 2.4 Rhodophyceae (Red Seaweed) 41 2.5 Conclusion 55

Chapter 3 : Antifouling Activities of Scottish Seaweed 59 3.1 Introduction 59 3.1.1 What is Biofouling? 59 3.1.2 Effects of Biofouling on Marine Surfaces 59

iii Content

3.2 Materials and Method 62 3.2.1 Materials 62 3.2.2 Bacterial Strains 63 3.2.3 Preparation of Extracts 63 3.2.4 Antimicrobial Assay 66 3.3 Results 68 3.4 Discussion 71 3.4.1 General Discussion 71 3.4.2 Correlation of Antibacterial activity and Shore Position of Seaweed 73 3.5 Conclusion 75

Chapter 4 : Antioxidants from Seaweed 77 4.1 Introduction 77 4.2 Materials and Method 80 4.2.1 Materials 80 4.2.2 Preparation of Extracts 80 4.2.3 2,2-Diphenyl-2-Picrylhydrazyl Hydrate Radical Scavenging Assay 81 4.2.4 2,2-Azinobis (3-Ethlybenzothiazoline-6-Sulphonic Acid) 83 Diammonium Salt Radical Cation Discolourisation Assay 4.2.5 Total Phenolic Content (TPC) 83 4.2.6 Ability of Seaweed Extracts to Quench Superoxide Radicals 84 4.2.7 Ability of Seaweed Extracts to Quench Hydroxyl Radicals 84 4.3 Results 85 4.3.1 DPPH Assay 85 4.3.2 ABTS Assay 87 4.3.3 Total Phenolic Content 91 4.3.4 Quenching Ability of Seaweed Extracts against Superoxide using 93 Electron Paramagnet Resonance 4.3.5 Quenching Ability of Seaweed Extracts against Hydroxyl Radicals 95 using Electron Paramagnet Resonance 4.4 Discussion 95 4.4.1 Radical Scavenging Activities of Different Seaweed Classes 95 4.4.2 Radical Scavenging Activity of Different Solvent Fractions 96

iv Content

4.4.3 Correlation of Total Phenolic Content and Radical Scavenging 96 Activity 4.4.4 Radical Scavenging Activity and Structure of Radicals 103 4.4.5 Electron Paramagnetic Resonance 104 4.5 Conclusion 107

Chapter 5 : Assessment of the Bioactivity of Scottish Seaweed Extracts 109 5.1 Introduction 109 5.1.1 Identification of Leads 109 5.1.2 The Screening of Seaweed Extracts 111 5.1.2.1 Antimicrobial Activity 111 5.1.2.2 Cytotoxic Activity 112 5.1.2.3. Miscellaneous 113 5.1.2.4 Screening Seaweed Species from the United Kingdom 114 5.1.3 Developing a Screening Programme 114 5.2 Materials and Method 115 5.2.1 Materials 115 5.2.2 Preparation of Extract 115 5.2.3 Assays 118 5.2.3.1 Alamar Blue Antibacterial Assay using S. aureus and E. coli 118 5.2.3.2 Alamar Blue Antibacterial Assays using N. farcinica 118 5.2.3.3 Alamar Blue Assay to Determine Drug Sensitivity of African 119 Trypanosomes in vitro (Trypanosoma brucei brucei S427) 5.2.3.4 Alamar Blue Cytotoxicity Assays 120 5.2.3.5 XTT Cytotoxic Assay against HeLa cells 120 5.3 Results 120 5.3.1 Antibacterial Screening 120 5.3.2 Antiparasitic Assay 124 5.3.3 Cytotoxicity Screening 126 5.4 Discussion 133 5.4.1 Development of a Screening Network 133 5.4.2 Antibacterial Screening 134 5.4.3 Antiparasitic Screening 135

v Content

5.4.4 Cytotoxic Screening 135 5.5 Conclusion 137

Chapter 6 : Column Chromatography and Assay Guided Fractionation of an Ethyl 139 Acetate Extract of the Brown Seaweed Halidrys Siliquosa 6.1 Introduction 139 6.1.1 Criteria for Choosing Species for Further Study 139 6.1.2 Halidrys siliquosa 140 6.1.3 Strategy 141 6.2 Material and Methods 142 6.2.1 Materials 142 6.2.2 Extraction of H. siliquosa 143 6.2.3 Column Chromatography of an Ethyl Acetate Extract of H. 145 Siliquosa 6.2.4 Chromatography of Fraction E 145 6.2.5 Chromatography of Fraction A 146 6.2.6 Chromatography of Fraction B 146 6.2.7 Chromatography of Fraction C 146 6.2.8 Chromatography of Fraction D 147 6.2.9 DPPH Assay Radical Scavenging Method 147 6.2.10 ABTS Assay Radical Scavenging Method 147 6.2.11 Disk Diffusion Assay Method 148 6.2.12 Analysis of Fractions by RP-HPLC/PDA 148 6.3 Results 149 6.3.1 Column Chromatography 149 6.3.2 Radical Scavenging Assays 151 6.3.3 Disk Diffusion Assay 153 6.3.4 RP-HPLC/PDA Analysis of H. siliquosa Sub-factions 154 6.4 Discussion 163 6.4.1 Radical Scavenging Activity of H. siliquosa Sub-fractions 163 6.4.2 Antibacterial Activity of H. siliquosa Sub-Fractions 164 6.4.3 RP-HPLC/PDA Analysis of H. siliquosa Sub-factions 165

vi Content

6.4.4 Can Observed Bioactivity be Assigned to Signals in the RP- 167 HPLC/DA Chromatograms? 6.5 Conclusion 168

Chapter 7 : Identification of Compounds from Halidrys siliquosa using Mass 170 Spectrometry and Nuclear Magnetic Resonance 7.1 Introduction 170 7.2 Materials and Methods 172 7.2.1 Materials 172 7.2.2 Extraction and Purification of H. siliquosa 172 7.2.3 Minimum Inhibition Concentration (MIC) of Fraction Cb 173 against the ABTS Radical 7.2.4 Identification of Compounds Responsible for Observed ABTS 173 Radical Activity from Fraction Cb 7.2.5 Semi-preparative HPLC of Sub-fraction Ca 174 7.2.6 RP-HPLC/DA Analysis of Ca α, Ca β, Ca γ and Ca δ 175 7.2.7 Semi-preparative HPLC of Ca α and Ca δ 175 7.2.8 Semi-preparative HPLC of Sub-fraction Ab 176 7.2.9 RP-HPLC/DA Analysis of Ab fractions 178 7.2.10 LC/MS Analysis of Selected Sub-factions 178 7.2.11 NMR Analysis of Selected Sub-fractions 178 7.3 Results 179 7.3.1 Minimum Inhibition Concentration (MIC) of Fraction Cb 179 Against the ABTS Radical 7.3.2 Identification of Compounds Responsible for Observed ABTS 179 Radical Activity from Fraction Cb 7.3.3 Semi-preparative HPLC of Sub-fraction Ca 181 7.3.4 Semi-preparative HPLC of Sub-fraction Ab 181 7.3.5 HPLC/MS of Cb, Ca and fractionated samples of Ca 183 7.3.6 HPLC/MS of Fractionated Samples of Ab 185 7.3.7 Nuclear Magnetic Resonance Analysis of Selected Fractions 187 7.4 Discussion 187 7.4.1 Identification of Compounds Responsible for Observed ABTS 187

vii Content

Radical Activity from Fraction Cb 7.4.2 Identification of Compounds from H. siliquosa Fractions 192 7.4.3 Use of Semi-preparative HPLC on Ab and Ca Sub-fractions 208 7.5 Conclusion 208

Chapter 8 : Conclusion and Future Work 210 8.1 Conclusion 210 8.2 Future Work 213 8.2.1 Screening of Seaweed Species 214 8.2.2 Isolation and Analysis of Pure Compounds 214 8.2.3 Ecological Functions of Natural Products from Seaweeds 215 8.2.3.1 Antimicrobial Activity 215 8.2.3.2 Antifeedant Activity 216 8.2 Limitations of this Study 217

References 220 Appendix I 241

viii Abbreeviations

Abbreviations

ABTS 2-Azinobis (3-Ethlybenzothiazoline-6-sulphonic Acid) Diammonium Salt

AD Anno Domini

ASE Accelerated Solvent Extraction

BC Before Christ

DCM Dichloromethane

DNA Deoxyribonucleic Acid

DPPH 2,2-Diphenyl-2-picrylhydrazyl Hydrate

EDTA Ethylenediaminetetraacetic Acid

EPR Electron Paramagnetic Resonance

ESI Electrospray Ionisation

EtOAc Ethyl Acetate

GC Gas Chromatography

HBSS Hank’s Buffered Saline Solution

HMBC Heteronuclear Multiple Bond Coherence

HPLC High Performance Liquid Chromatography

HSQC Heteronuclear Single Quantum Coherence

HTS High Throughput Screening

IP Intellectual Property

MA Marine Agar

MALDI Matrix Assisted Laser Desorption Ionisation

MB Marine Broth

ix Abbreeviations

MeOH Methanol

MIC Minimum Inhibitory Concentration

MRSA Methicillin Resistant Staphylococcus aureus

MS Mass Spectromatry m/z Mass to Charge Ratio

NCI National Cancer Institute

NMR Nuclear Magnetic Resonance

PBS Phosphate Buffer Solution

PDA Photodiode Array

ROS Reactive Oxygen Species

RP Reverse Phase

SIDR Strathclyde Innovations in Drug Research

TB Tuberculosis

TBT Tertiary Butyl Tin

TLC Thin Layer Chromatography

TOF Time of Flight

TPC Total Phenolic Content

UV Ultraviolet

VRS Vancomycin-Resistant Enterococci

x Chapter 1

1. Introduction

In the marine environment, submerged surfaces undergo a stepwise accumulation of unwanted marine life called biofouling. This has a range of disadvantages especially to manmade structures and to living hosts. It can be noted that seaweed do not accumulate this layer and therefore they must somehow regulate the biofouling process. Many theories exist as to how this regulation may occur with one proposal being that seaweed produce compounds that inhibited the biofouling process at some stage. In addition seaweed has been used effectively in the Highlands and Islands of Scotland as a traditional medicine for many years to treat various conditions. Indeed growing research has shown that natural products from seaweed are active against a number of medically related targets and it is possible that pharmaceuticals derived from seaweed natural products could be a reality. Despite these observations, seaweeds sampled in the North of Scotland have yet to be evaluated for their natural product content. Here we can ask several research questions relating to these observations. Are seaweeds from the Caithness coast able to produce a compound or a series of compounds that could potentially regulate the accumulation of biofouling on its surface? Do seaweeds from Caithness produce compounds that could be of benefit to human health by showing activity to relevant bioassays? If the answer to either of these two questions is yes then what are the compound that are responsible the observed activity, can they be isolated and could they be used to our advantage?

1.1. A Brief History of Natural Products

1.1.1. Before the Common Era

Humanity’s relationship with its natural surroundings has been inevitably close. At some point during our brief period on this planet, humans began to exploit (although unbeknown to then at the time) the quite remarkable chemistry of nature by using various plants for their medicinal properties.

1 Chapter 1

Exactly when humans began to utilise natural remedies is something that we are unlikely to discover. There is of course little physical evidence of the use of plants as medicines or indeed other uses before written records began to emerge 5000 years ago. However some evidence is available that has literally been frozen in time. Otzi the Iceman is a 5300 year old naturally preserved mummy found in 1991 in the Alps on the Austrian/Italian border, was found carrying with him two fungi that are now known to have medicinal properties. One of these fungi Piptoporus betulinus , is known to be an effective treatment for an intestinal parasite that Otzi was infected with (Otzi Museum, 2008).

The first written record of plants being used as medication was penned by the Sumerians in the Middle East over 5000 years ago. The relationship the Sumerians had with plants and spiritualism is highlighted in the poem ‘ Epic of Galgamesh ’ (Swerdlow, 2000a). Around the same time as the Sumerians began documenting the use of plants as medication, Shen Nong (a Chinese Emperor) is widely credited with starting the culture of traditional Chinese medicine and his work is documented in the Shennong Bencao Jing, which is loosely translated as ‘The Divine Farmer’s Materia Medica’. It is however highly likely that this document is not actually from the time of Shen Nong, but was created around 2000 years ago from various older texts of unknown authorship. During his life, Shen Nong was said to have tasted several hundred plants and observed the effects both positive and negative. He later described methods of preparation and dosage information for treatments (Swerdlow, 2000b). Legend has it that Shen Nong was able to do all of this with the aid of a transparent body, where he was able to observe the effects of the plants on his organs.

Other ancient cultures such as the Greeks, Romans and Egyptians have all embraced the use of plants as medicinal agents. It was the ancient Greeks who popularising the more dangerous and sinister side of natural products. Socrates, the ancient Greek philosopher, was famously sentence to death for ‘corrupting the youth and impiety’. He was required to carry out his own death sentence by consuming a hemlock based drink; a punishment that would have taken sometime to complete and been considerably painful. Plato, a student of Socrates documents the death of his mentor at the end of his final dialogue, Phaedo (Plato, ca. 360BC)

2 Chapter 1

The Bible also makes several references to plants being used as medicines. The book of Ezekiel describes different types of trees lining the banks of a river with fruit being provided throughout the year. In addition, the use of the leaves as medicine is mentioned specifically (Ezekiel 47:12, New King James Version). However, there is no mention of the types of trees used or indeed the specific medical conditions the leaves were intended to treat. An additional reference to natural products can be found in the New Testament during the nativity. Here the well known Wise Men from The East presented gifts of gold, frankincense and myrrh to Jesus at his birth (Matthew 2:11, New King James Version). Christian traditions tell us that these gifts have symbolic meaning, with frankincense being used as an incense and myrrh as an anointing oil. However it is worth bearing in mind that the origin of the Wise Men who presented these gifts was The East. During this time and in the Middle East, both frankincense and myrrh were used as traditional medicines, and it may have been that these travellers were bringing medication to help a newborn baby.

1.1.2. The Middle Ages

During this time, there was very little significant advancement in the exploration of new plants as potential treatments. In fact, a comprehensive book on the history of natural medicines glosses over nearly 800 years (600AD – 1400AD) of history in just two pages (Swerdlow, 2000). There may be several reasons for this, for instance in Western Europe there may well have been some reluctance in using natural products for medicinal purposes as an individual making up or distributing such concoctions could very well have been accused of witchcraft, which in many cases resulted in a death sentence. However, at this time in the Middle East the first pharmacies opened and trade in medicinal plants became more prominent.

1.1.3. The Modern Era

During the 16 th century, Jesuits isolated quinine from the bark of the cinchona tree in South America where it was used as a treatment for malaria and fevers (Claydon, et al., 2001). This advancement ultimately helped with the colonisation of many tropical areas by Europeans. The consumption of quinine as a tonic became a daily routine for many

3 Chapter 1 colonists in order to gain some protection from the disease. This practice of consuming the tonic still continues today however, the function of its consumption has changed from medicinal to a more social and leisurely aspect usually with a good glug of gin and slice of lemon and lime. Quinine, is still used as treatment for malaria in many countries today, however the World Health Organisation (WHO) recommends that it should only be used when all other potential treatments have been ruled out (WHO, 2001).

William Shakespeare describes the effects of several medicines from plants in a number of his plays. In ‘ Othello ’, (act III, scene 3) the character Lago makes reference to the drowsy effects of poppies, which would be due to the opiate content (Shakespeare, 1977). In ‘Much Ado About Nothing ’, (act III, scene 4) a sick Beatrice who appears to have a cold, is advised by Margaret to take the essential oil of Carduus benedictus which is known to have appetite stimulation properties. However this plant appears to have been chosen out of jest, as a play on words between the name of the plant and with reference to Beatrice’s love interest, Benedick (Shakespeare, 1977).

In more recent history the study of the natural product content of microbes such as bacteria and fungi has become a popular avenue of research after what could arguably be the most important event in natural product chemistry, when bacterial growth was shown to be inhibited by what was found to be the Penicillium mould. This observation was first made by John Sanderson in the 1870’s and subsequent work carried out by Sir Alexander Fleming, Howard Flory and Sir Ernst Chain lead to the isolation of penicillin, with the three being jointly awarded the 1945 Nobel Prize for Medicine (Nobel Foundation, 2010).

In the last 100 years, the discovery and development of chromatography has allowed for an explosion in the amount of natural products being isolated. Column chromatography is still the traditional method used by natural product chemists in the isolation process. Here development in packing materials has led to a range of stationary phases being made available with silica gel still being used most widely. Other developments include bonded silica gel allows for reverse phase chromatography and polyacrylamide beads are suited to the isolation of peptides and carbohydrates. Gas chromatography (GC) is one of the more modern methods of chromatography originating around 60 years ago. An inert gas, usually helium is used as a mobile phase and stationary phase is made up of liquid. GC is often used in the separation of volatile mixtures such as petrol or essential oils. A few years

4 Chapter 1 after the discovery of GC, high performance liquid chromatography (HPLC) was developed. Here solvents under high pressure are used as a mobile phase to elute a sample through tightly packed stationary phases allows compounds to be eluted at a higher resolution than with traditional column chromatography. In addition, HPLC benefits from being able to change the mobile phase composition to a high degree of accuracy during a run which aids in the repeatable separation of compounds (Cannell, 1998).

Both GC and HPLC are useful techniques on their own, however they are usually coupled to a detector which allows them to become powerful analytical tools. Although a range of detectors are available, mass spectrometry is perhaps the most recognised of these. Mass spectrometry has been around in some form since the end of the 19 th century. Mass spectrometers became commercially available during the 1940’s and were used mainly for quantitative analysis. Since then, the development of electrospray ionisation (ESI), matrix assisted laser desorption ionisation (MALDI) and other ionisation sources have allowed for larger and more complex compounds such as proteins to be analysed by MS. In addition, mass spectrometers such as ion traps, time of flight (TOF) and orbitraps have been developed. Ion traps allow for the trapping of ions which can then be released according to the mass to charge ratio ( m/z ) by altering the potential of the electrodes. This gives a high resolution spectrum and allows for MS n experiments which are useful in structural elucidation. TOF measure the m/z by accelerating ions with a known energy and recording the time they take over a known distance. This allows for high accuracy of mass to be established. Orbitraps also trap ions allowing for the benefits of an ion trap, but they are also able to provide a high accurate mass (Griffith, 2008).

Nuclear magnetic resonance (NMR) has been widely available since the middle of the 20 th century. Development of NMR has been rapid with ever increasing field strengths which have now reached above 1000 MHz allowing for higher resolution. In addition, the introduction of 2D and now 3D experiments means that more complex structures and compounds in mixtures can be identified relatively quickly.

It is of course without surprise that with the aid of modern isolation methods and drug development practices, along with thousands of year worth of traditional medicine, many of today’s modern chemotherapeutic agents have their origins from natural products. A recent review of drugs from natural products has highlighted 661 different compounds that

5 Chapter 1 were introduced onto the market between 1981 and 2006 that are natural products, natural product derivatives, mimics and synthetic compounds with a natural product pharmacophore with only 310 compounds being totally synthetic (Newman and Cragg, 2007).

1.2 Marine Natural Products

1.2.1 Background

Water covers approximately two thirds of the Earths surface, yet much is still unknown about what lies beneath the waves, including natural products from marine plants and animals. The study of marine natural products is a relatively new area of natural product research. 35 years ago there was relatively little in the way of marine natural products research with relatively few compounds having been isolated. Consequently it would have been relatively easy to perform a comprehensive review of the literature relating to isolated natural products (Faulkner, 1977). A combination of more sensitive spectroscopic instruments and the increased likelihood of finding potentially high value compounds have led to rapid growth within the sector.

Research into marine natural products produces its own challenges over its terrestrial counterpart by its very nature. The simplest sampling method of going to the seashore and collecting intertidal plants and animals is not only dependant on the weather, as with terrestrial sampling, but also by tides where there may only be a window of 30 minutes to find and collect sufficient amounts of sample for further investigation. Any other sampling venture out with the intertidal zone will of course require specialised equipment and training. For example, to study the natural products of a sponge from waters of a depth of about 10 m, may require, a boat, diving equipment (along with the necessary training) and staff to conduct the sampling activity. However, the vast majority of the ocean is far from land and of considerable depth and therefore the likelihood is that residential voyages on large research ships with other specialised equipment such as submersible vessels may be the reality. With the demand on research vessels and costs being high it may prove to be difficult to fund such ventures.

6 Chapter 1

It is not just the logistics of obtaining a sample that are a challenge to marine natural product chemists. Many of the diverse species that live in the oceans around the world thrive in distinct regions due to the conditions of the local environment: should the local environment change, either naturally or as a consequence of human presence, the organisms may very well produce a stress response. This response is ultimately a change in the chemical makeup of the organism, for example it is know that some seaweed produce a chemical response due to grazers feeding on it, producing chemicals that prevents the grazers from feeding on the seaweed further (Kurata, et al., 1997). Therefore, even sampling may cause some form of response, but this can be limited by looking after samples in an appropriate manner (Wright, 1998). Many researches around the world are interested in isolating bioactive natural products from marine bacteria, with some of the more exotic species investigated being found in environments such as the sediment and mud from the ocean floor (Bull and Stach, 2007). At the more extreme end of the spectrum, bacteria from the bottom of the Marianas Trench are being investigated for their natural product content. In a recent study the bacteria Dermacoccus abyssi , from Mariana Trench sediment, produces a class of compounds called dermacozines which have been shown to have radical scavenging, anti-tumour and anti-bacterial properties (Abdul- Mageed, et al., 2010).

It is with the advancements over the last 100 years in chromatography and analytical devices and with the growth in marine natural products that have led to our ability to isolate and characterise an array of compounds from marine sources. Despite these advancements in technology allowing for quicker and easier structural elucidation of natural products, some compounds still prove to be a challenge. Palytoxin (fig 1.1) is a highly toxic compound from a Hawaiian sample of “Limu-make-o-Hana”, the deadly seaweed of Hana, which incidentally is not a seaweed but actually a zoanthids. The compounds was isolated in 1971, however it took nearly 11 years for the structure to be correctly determined (Ciminiello, et al., 2010).

7 Chapter 1

OH O

O O OH HO OH H2N

OH OH HO

O OH OH

HO OH

OH OH OH

OH OH

HO OH O O O OH OH OH

OH OH HO N N O H H

OH OH HO OH HO

OH O OH O HO O OH OH OH O OH

OH OH OH HO OH

OH Figure 1.1. Palytoxin

Other triumphs in marine natural product chemistry are the characterisation of the brevetoxins backbone (fig 1.2). Brevetoxins are compounds originating from blooms of the algae Kerenia brevis which can be eaten by shellfish when used as foods, they cause neurotoxic shellfish poisoning, which can be fatal. There are nine toxins made up of two differenet cyclic backbones, brevetoxin A and B (Wang, 2008).

8 Chapter 1

R1O Brevetoxin A

O OR2

O O O O O

O O O O

R1O

OR2

O Brevetoxin B O

O O O O O

O O

O O O

Figure 1.2. The Brevetoxins

Nowadays, marine natural product science has flourished into a well respected branch of chemistry with research being carried out on a diverse array of marine plants and animal life from tropical waters all the way to Arctic and Antarctic waters. From this global research, a number of compounds originating from marine sources are currently used as licenced pharmaceuticals. Ziconotide was the first compound to be approved and is currently used to in pain management. It was isolated from the marine snail Conus magus (DailyMed, 2008). Trabectedin originally isolated from the sea squirts Ecteinascidia turbinata and is currently licenced in the UK in the treatment of soft tissue sarcoma (Rinehart, 2000; Nice, 2010). Eribulin has been approved for use in metastatic breast cancer and was first isolated from the marine sponge Halichondria okadai (Adams, 2011).

1.2.2 Seaweed as a Source of Natural Products

Since seaweed were first investigated for their natural product content around 40 to 50 years ago, about 3000 compounds have been identified. This is around one fifth of all

9 Chapter 1 marine natural products. With interest growing rapidly in compounds from marine bacteria and microalgae in recent years, macroalgae or seaweed has taken a back seat and account for only around 10% of natural products being isolated since 2005.

There is a stark contrast between the different classes of seaweed when it comes to the type and number of natural products isolated from them. Chlorophyta (green seaweed), tend not to contain large compounds or varied natural products from a range of different structural classes. Only around 300 compounds have been isolated from green seaweed since the 1960’s with the most recent review of marine natural products by Blunt, et al. (2010), suggesting that only around 10 new compounds are found each year. Phaeophyta (brown seaweed) have yielded a larger number of compounds than the green seaweed, including diterpenes, polyphenols and phlorotannins with the majority of compounds being isolated from only just a few genera. Rhodophyta (red seaweed) have yielded over 1500 new compounds, many of which are chlorinated and or brominated, with the majority of compounds isolated originating from Osmundea spp (Maschek and Baker, 2008).

1.2.3 The Highland and Islands of Scotland and Seaweed

The Highlands and Islands of Scotland have a strong heritage of using seaweeds in a medicinal capacity. Macroalgae from all three classes have been used in traditional medicine across the region. An unspecified green seaweed, probably Ulva lactuca , applied to the temples and forehead of an individual is said to stop a bleeding nose. The brown seaweed Pelvetia canaliculata has been used to treat rheumatism of the knee by applying the seaweed boiled in seawater, to the knee (Beith, 1995).

Several species have been traditionally used in cooking and to some extent are still used today. The red seaweed Palmaria palmata and Chondrus crispus are two examples of marine algae that may be consumed as part of rural Highland diet. P. palmata would traditionally be made into a soup which itself could be used to relieve stomach upsets and indigestion and also helped restore the condition of damaged skin. In some parts of the Islands eating raw P. palmata would be used to improve ones eyesight. Boiled, P. palmata and the liquor could be used as a laxative, with other preparations being used to treat migraines, kidney stones and remove worms as well as helping to remove the placenta

10 Chapter 1 after birth. C. crispus has mainly been used in cooking where it is used to thicken various dishes and provide nourishment (Beith, 1995).

The above examples seem to have a firm basis in delivering extracts of seaweed or the seaweed itself to the patient in a way that allows natural products to elicit some active effect. There is however cases where the role of the plant seems more obscure. A condition known as ‘falling of the uvula’ appeared to be a feared in the Highlands and Islands at some point in time. However it is with some luck that seaweed offers good protection from this awful ailment. An individual must collect a piece of red seaweed at low tide from a rock pool, tie a cord round it and present it to the patient and say the words ‘Ann an ainm an Athar, a’ Mhic, agus an Spioraid Naoimh, air cioch-shlugain (patient name)’. This translates as ‘In the name of the Father, Son and Holy Ghost, for the uvula of (patient name)’ (Beith, 1995). It is however unlikely that such an approach could have been based on the natural product content of the seaweed.

Despite seaweed being used extensively for their health benefits in the Highlands and Islands for many years, there is precious little peer reviewed literature available on the matter. It is striking that within a region with such a rich heritage of using natural resources more research has not been carried out. This project aims to address this issue.

It is very easy to use a database such as ‘Web of Knowledge’, and using certain key words, such as natural products, activity, Scotland, UK, algae and seaweed to find no articles relating to natural products from Scottish seaweed species. In fact, there is little evidence of investigations of natural products and bioactivity from seaweeds around the rest of the United Kingdom. Of course seaweeds found in Scottish waters are also found in many other parts of the world, a minority of which have previously been investigated with a small proportion of these being extensively examined for their natural product content.

To address the research questions set out at the beginning of this chapter, this project has two distinct aims: 1. To assess the bioactivity of extracts from a range of local seaweed species and in doing so, develop access to a range of bioassays suitable for the task 2. To identify and isolate from the most promising species indicated above, natural products responsible for the observed bioactivity

11 Chapter 1

Firstly it is important to identify the species of seaweed found in Caithness. Continuing on from this using the peer reviewed literature, compounds previously isolated from these species will be identified along with any bioactivity described.

The results from this literature review will allow the selection of a number of species for further study. Extracts from the seaweed samples will undergo a series of assays that will effectively evaluate their bioactivity against as wide a range of targets as possible. From the results of the bioassays and the information gathered from the literature review, it will be possible to select suitable seaweed species for a further chemical analysis in order to identify bioactive compounds.

12 Chapter 2

2. Natural Products from Seaweed found in Caithness Waters and their Bioactivity

2.1 Introduction

The study of seaweed as a source of natural products began to gain momentum in the 1970’s. However at that time, the vast majority of research focused almost solely on the isolation and characterisation of natural products, with many new compounds and classes of compounds being discovered. After the initial interest in seaweed as a source of natural products the focus of marine natural products research turned toward more exotic species such as sponges, tunicates, micro-organisms, coelenterates and molluscs (nudibranchs) to the exclusion of seaweeds (Faulkner, 2000a). Natural products from seaweed account for 13.6% of all marine natural products highlighted in the annual review of “Marine Natural Products” published in Natural Product Reports. Green seaweeds accounts for 1.2% of natural products with brown and red seaweeds being more fruitful account for 5.3% and 7.1% respectively (fig.2.1). In contrast natural products form sponges, coelenterates and micor-organism compise of almost two thirds of the total amount of natural products with 34.4%, 20.0% and 11.8% respectively (Faulkner, 1984a, 1984b, 1986, 1987, 1988, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000b, 2001 and 2002, Blunt, et al., 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010 and 2011).

Figure 2.1 Distrabution of natural products per group as discribed by Faulkner, (1984a, 1984b)

13 Chapter 2

In more recent times, interest in establishing the bioactivity of these natural products both in terms of exploring their ecological function and pharmaceutical potential has grown. A range of properties are now being reported for seaweed natural products including antibiotic, antifungal, anticancer and antiviral activities (Amico, et al., 1980; Bouaicha, et al., 1993a; Piattelli, et al., 1995). The ecological function of a number of compounds has also been explored, with anti-bacterial (anti-biofouling), sex hormones and anti-feedants being identified (Culioli, et al., 2008; Kusumi, et al., 1986a; Stratmann, et al., 1992).

However, in the case of most species, the study of seaweed natural products has been on the whole limited to cataloguing their chemistry. Therefore with the advancement of biological assays, both in terms of sophistication and diversity, and the advances in analytical technology the re-examination of previously studied seaweed and those that remain unstudied would seem to be justified (Faulkner, 2000a).

2.1.1 Methodology

This chapter concentrates on exploring to what extent the natural products from seaweed species found on the Caithness coastline, in northern Scotland, have been investigated. The first major task is to identify the species of seaweed that are present on the Caithness coastline. Fortunately, an extensive study of the seaweed species on the local shores has already been carried in 2004 (McDougall, 2004). Here 18 coastal sites, consisting of both sheltered and exposed locations, were surveyed for their seaweed population diversity and are shown on Figure 2.2.

14 Chapter 2

3 17 16 No. Location 1 Ackergill 2 9 18 2 Brims Ness 11 12 3 Brough 14 4 Castlehill 5 8 4 5 Crosskirk 10 13 6 Forse, Lybster 7 North Head 1 8 Point of Ness 9 Port of Brims 15 10 Sandside Bay 7 11 Sandside Head 12 Scotland’s Haven 13 Sgarbach 14 Skirza 15 Staxigoe 6 Harbour 16 St John’s Point 17 The Haven 18 Thurso

Figure 2.2. Sites surveyed for seaweed population diversity by McDougall, (2004)

In addition to the survey conducted by McDougall, (2004), a seashore guide (Gibson, et al., 2001) was used along with the Marine Life, Information Network (MarLIN) website to identify seaweed that were not previously highlighted.

Utilising these three sources, it is possible to identify 129 different species of seaweed native to Caithness, 21 green seaweed (table 2.1), 42 brown seaweed (table 2.2) and 65 red seaweed (table 2.3).

15 Chapter 2

Species Species Blidingia marginata Rosenvigella tortuosa Blidingia minima Spongomorpha arcta Chaetomorpha ligustica Ulothrix flacca Chaetomorpha linum Ulothrix speciosa Cladophora rupestris Ulva intestinalis Cladophora sericea Ulva lactuca Codium fragile Ulva linza Monostroma grevillei Ulva prolifera Prasiola stipitata Ulvella lens Pringsheimiella scutata Urospora penicilliformis Rhizoclonium tortuosum Table 2.1. Green seaweed native to the Caithness coast

Species Species Alaria esculenta Halidrys siliquosa Ascophyllum nodosum Herponema velutinum Asperococcus fistulosus Himanthalia elongata Chorda filum Hincksia hincksiae Cladostephus spongiosus Hincksia sandriana Colpomenia peregrina Laminaria digitata Desmarestia aculeata Laminaria hyperborea Dictyosiphon chordaria Leathesia marina Dictyosiphon foeniculaceus Litosiphon laminariae Dictyota dichotoma Myriotrichia clavaeformis Ectocarpus fasciculatus Petalonia fascia Ectocarpus siliculosus Pelvetia canaliculata Elachista fucicola Pilayella littoralis Elachista scutulata Ralfsia verrucosa Elachista stellaris Saccharina latissima Fucus ceranoides Saccorhiza polyschides Fucus distichus Sauvageaugloia chordariiformis Fucus serratus Scytosiphon lomentaria Fucus spiralis Sphacelaria cirrosa Fucus vesiculosus Sphacelaria rigidula Fucus vesiculosus var linearis Spongonema tomentosum Table 2.2. Brown seaweed native to the Caithness coast

16 Chapter 2

Species Species Acrochaetium alariae Lomentaria articulata Acrochaetium microscopicum Lomentaria clavellosa Acrochaetium parvulum Mastocarpus stellatus Acrochaetium secundatum Membranoptera alata Acrochaetium sparsum Mesophyllum lichenoides Aglaothamnion hookeri Nemalion helminthoides Aglaothamnion sepositum Odonthalia dentate Ahnfeltia plicata Osmundea hybrid Bonnemaisonia hamifera Osmundea pinnatifida Callophyllis laciniata Palmaria palmata Catenella caespitosa Phycodrys rubens Ceramium nodulosum Phyllophora crispa Ceramium pallidum Phymatolithon calcareum Ceramium shuttleworthianum Phymatolithon purpureum Ceramium virgatum Plocamium cartilagineum Chondrus crispus Plumaria plumose Chondrus verrucosus Polyides rotundus Conferva atropurpurea Polysiphonia atlantica Corallina officinalis Polysiphonia brodiei Cryptopleura ramosa Polysiphonia elongate Cystoclonium purpureum Polysiphonia fucoides Delesseria sanguinea Polysiphonia nigra Dilsea carnosa Polysiphonia stricta Dumontia contorta Porphyra dioica Erythrotrichia carnea Porphyra purpurea Furcellaria lumbricalis Porphyra umbilicalis Gastroclonium ovatum Ptilota gunneri Gelidium pusillum Rhodochorton purpureum Gelidium spinosum Rhodomela confervoides Heterosiphonia plumosa Rhodothamniella floridula Lithophyllum crouanii Scinaia furcellata Lithophyllum incrustans Vertebrata lanosa Lithothamnion glaciale Table 2.3. Red seaweed native to the Caithness coast

This study used the annual review of “Marine Natural Products” published in Natural Product Reports, previously compiled by the late John Faulkner (Faulkner, 1984a, 1986, 1987, 1988, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000a, 2000b, 2001 and 2002) and now compiled by John Blunt, Brent Copp, Murray Munro, Peter Northcote and Michèle Prinsep (Blunt, et al., 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010 and 2011) as a guide to identify the natural products produced by the seaweed species found on the Caithness coast.

17 Chapter 2

2.1.2 Caithness Costal Geography

Caithness is a county in the far north of the Highland region of Scotland. Its coastline totals 353.2 km (high water), with the north coast starting around 4 km east of Melvich and the east coast staring around 5 km north of Helmsdale with both coastlines meeting at Duncansby Head, at the far north easterly point of the mainland UK. The coastline features a mixture of exposed areas as well as many sheltered bays of varying size.

2.1.3 The Review

The aim of this review is to gain an understanding of how extensively seaweed species that are found on the Caithness coastline have been studied. Documented here, is a comprehensive analysis of available literature, highlighting studies that have led to identification and isolation of novel natural products and describing any associated bioactivity.

2.2 Chlorophyceae (Green Seaweed)

To date little research into the natural products from green seaweed has been carried out. This is the case for not only Scottish species but throughout the rest of world as well. One possible reason for this void is that previous research into green seaweed has produced largely disappointing results, with very few natural products being isolated.

21 different species of green seaweed have been identified around the Caithness coastline (McDougall, 2004). However, only six of these species have been investigated for their natural product content.

U. prolifera is an edible seaweed which is primarily consumed in Japan and other far- eastern countries. It has been suggested that wide consumption of this seaweed is responsible for some degree of protection from developing certain types of cancer. Studies have shown that extracts of U. prolifera do indeed have anti-carcinogenic properties both in-vivo and in-vitro . In-vivo studies have used chemically induced tumours on mice to study the anticancer activity of the seaweed extract. The chlorophyll derived compound

18 Chapter 2 pheophytin-a 1, found in U. prolifera, has previously been thought to be the molecule responsible for the observed activity and while it is clear from in-vivo studies that pheophytin-a exhibits anti-carcinogenic properties, it is not clear if it is solely responsible (Hiqashi-Okaj, et al., 1999).

O O

N O O H N N O H N

Pheophytin-a has also been shown to have potent anti-inflammatory activity. Two in-vitro studies have been conducted; one monitoring the production of superoxide anions in mouse macrophage and the other was using the migration of human polymorphonuclear leukocytes (PMN) as a measurement of their chemotactic activity. These showed that - pheophytin-a greatly reduced the O 2 production and PMN migration. In an in-vivo assay, using a chemically induced inflammation on the ears of mice, the addition of pheophytin-a greatly reduced this inflammation (Okai and Higashi-Okai, 1997).

Methanol extracts from two members of the Ulva genus, U. intestinalis and U. lactuca , have been tested for antibacterial, antifungal, phytotoxicity and insecticidal activity in an extensive study with a further 31 seaweed species. The U. intestinalis extract showed only a mild antibacterial effect, where as methanol extracts from U. lactuca showed significant phytotoxic activity and a moderate antifungal activity across a wide range of fungi. U. lactuca also showed a small positive result in the insecticidal assay. In these cases the natural products responsible were not identified (Rizvi and Shameel, 2005).

The steroid 3-O-β-D glucopyranosyl clerosterol 2 has been isolated from an extract of U. lactuca . The steroid also exhibits potent anti-inflammatory activity in chemically induced oedema in mice ears. It also has been shown to have antibacterial, antifungal and anti- yeast activity, in some cases proving to be more effective than commercial counterparts (Awad, 2000).

19 Chapter 2

Glu O 2

U. lactuca has also been shown to contain a large number of relatively simple bromophenols, 2-bromophenol 3, 4-bromophenol 4, 2,4-dibromophenol 5, 2,6- dibromophenol 6 and 2,4,6-tribromophenol 7. Additionally it was shown that the concentration of these compounds varies throughout the year. All five compounds have been shown to disrupt levels of calcium ions in endocrine cells. An endocrine cells ability + to release hormones into the body is controlled by the influx of Ca into the cell, therefore a hormonal imbalance will occur where levels cannot be controlled. 5 and 7 were shown to be the most potent of the five bromophenols (Hassenklöver, et al., 2006).

OH OH OH OH OH Br Br Br Br Br Br

Br Br Br 3 4 5 6 7

Further studies have identified a possible biosynthetic route to 2,4,6-tribromophenol derived from of L-tyrosine 8. The following intermediate compounds have been isolated, 4-hydroxyphenyllactic acid 9, 4-hydroxyphenylacetic acid 10 , 4-hydroxymandelic acid 11 , 4-hydroxybenzaldehyde 12 , 4-hydroxybenzoic acid 13 and 3,5-dibromo-4-hydroxybenzoic acid 14 . The bromination step is thought to occur using the enzyme bromoeperoxidas (BPO) (Flodin and Whitfield, 1999).

20 Chapter 2

O OH O OH OH OH OH H2N HO O O

OH OH OH OH 8 9 10 11

O HO O HO O

Br Br OH OH OH 12 13 14

BPO has also been found in U. lens , a green seaweed, where it is involved in the production of brominated methane (Ohshiro, et al., 1999).

2.3 Phaeophyceae (Brown Seaweed)

The brown seaweed have been studied much more extensively than their green counterparts. 43 different species of brown seaweed were identified on the Caithness coast with 12 of them having been investigated for their natural product content. Brown seaweed is far more abundant by mass than both green and red species, making it much easier to harvest and extract large quantities for natural products research.

A. nodosum is a brown seaweed common around most of the British coastline. A series of sulphated L-dihydroxyphenylalanine (L-DOPA) 15 derivatives have been identified from this species. These were identified as DOPA-3-SO 4 16 , DOPA-4-SO 4 17 and DOPA-3,6- diSO 4 18 (Laycock and Ragan, 1984). L-DOPA is more commonly associated as a treatment for Parkinson disease. L-DOPA is used due to its high affinity for crossing the blood brain barrier where it is metabolised by aromatic L-amino acid decarboxylase along with vitamin B6 to dopamine (Voet and Voet, 1995).

21 Chapter 2

O O O HO O OH S OH O NH O NH HO 2 HO 2 15 16

O O O HO O OH S OH O O NH O NH O O 2 HO O 2 S O S O O O 17 18

A sex hormone, identified as 1(3 E,5 Z,8 Z)-undecatetrane 19, with the trivial name finavarrene after Finavarra; a location in Galway Bay, Ireland where the work was carried out, has been identified in A. nodosum . The hormone is produced by the eggs of the female plant and has been shown to strongly attract the male sperm. Its activity was demonstrated by collecting the product directly from a GC column and embedding the compound into small droplets of Vaseline. Fresh spermatozoids from A. nodosum were shown to be strongly attracted to the droplets (Muller, et al., 1982a). Fucoserraten 20 , a C8 unsaturated hydrocarbon with the double bonds at the 1, 3 and 5 position, was isolated from the brown seaweed F. serratus and shown to be of biological significance as a female sex attractant (Muller and Aenicke, 1973). Two new C21 lipids, heneicosa-1,6,9,12,15,18- hexaene, 21 and heneicosa-1,6,9,12,15-pentaene, 22 , have been isolated from a sample of F. vesiculosus . Despite similar compounds being sex attractants, no similar biological activity for these compounds has been determined (Halsall and Hills, 1971).

A sex pheromone from the seaweed E. siliculosus was isolated and identified using various spectrochemical data and later named ectocarpene 23 (Muller, et al., 1971). The biosynthesis of ectocarpene has been studied by following the incorporation of tritium and deuterium from unsaturated medium chained fatty acids (Boland and Mertes, 1985). Further research in to the biosynthesis of ectocarpene has revealed that two enzymes; 9- lipoxygenase and hydroperoxide-lyase may have an important role in forming a precursor, from an unsaturated fatty acid molecule, which then undergoes a Cope rearrangement forming ectocarpene (Pohnert and Boland, 1997).

22 Chapter 2

Desmarestene 24 , which has shown to be the major signalling compound in the release and attraction of sperm in D. Aculeata along with two other sex pheromones, ectocarpene 23 and viridiene 25 . (Muller and Peters, 1982) C. spongiosus , was also shown to contain desmarestene (Muller, 1986). A sex pheromone that is released from the eggs of D. dichotoma to attract sperm has been isolated and identified as n-butyl-cyclohepta-2,5-diene or dictyotene 26 . In addition to ectocarpene, dictyotene and finavarrene produced by E. siliculosus , a further sex hormone, hormosirene 27, has been identified (Stratmann, et al., 1992).

19 20

23 24 25 H H

26 27 H

A total of 11 diterpenes with a nine membered carbon ring and unsaturated side chains 28 - 38 have been isolated from Dictyota dichotoma . Acetyldictyolal 28 , hydroxyacetyldictyolal 29 , isodictyohemiacetal 30 and dictyodiacetal 31 were isolated from a specimen from Oshor Bay, Japan using a methanolic extract over silica gel chromatography (Enoki, et al., 1982a). Hydroxyacetyldictyolal 29 has also been named fukurinolal (Ochi, et al., 1982). Compounds 28 , 29 , 30 have been tested for anti viral activity against Poliomyelitis virus I and Herpes simplex virus I , however the results showed no significant activity (Siamopoulou, et al., 2004). Dictyolactone 32 , which has previously been isolated from the sea hare, Aplysia depilans (Finer, et al., 1979) has now been found in D. dichotoma from an unspecified location. The compound showed promising algaecidal activity towards Heterosigma akashiwo (Kim, et al., 2006). Two further compounds containing a furan ring (dictyofuran T 33 and dictyofuran C 34 ) have been isolated from D. dichotoma sampled in Japan, however no biological activity has been reported for these two compounds (Enoki, et al., 1983a). 4-Acetoxydictyolacetone 35 , dictyotalide A 36 and B 37 and finally nordictyotalide 38, isolated from a sample of D.

23 Chapter 2 dichotoma from Yagachi, Japan have all been shown to exhibit cytotoxicity against B16 mouse melanoma cells (Ishitsuka, et al., 1988). Compounds 28 , 32 and 37 have been tested for their antifungal activity against Aspergillus fumigatus , Microsporum canis and Trichophyton mentagrophytes . MIC values were in the range of 180-200 µg ml -1 while the positive control, ketoconazole showed activity between the range of 2-5 µg ml -1. 28 and 32 showed significant activity against human nasoparynx carcinoma and also murine leukaemia. 32 showed stronger activity than the positive control mercaptopurine against human non-small cell lung carcinoma. 37 was also tested against the three cancer cell line but only showed weak activity (Bouaicha, et al., 1993a). In a similar study, 29 was also tested against the same cancer cell lines and showed significant activity against human nasoparynx carcinoma and murine leukaemia. It was weakly active against human non- small cell lung carcinoma (Bouaicha, et al., 1993b).

R 2 O AcO O R1 R1 O O R3

28 R1 = H 30 R1 = H, R2 = OH, R3 = CH2 32 29 R1 = OH 31 R1 = OAc, R2 = OMe, R3 = CHOMe

H H OAc O O H H

O O

33 34 35

O O O

OHC O O OAc O O

36 37 38

Diterpenes from the hydroazulenoid family have also been isolated form D. dichotoma . They are patchydictyol A 39 , dictytriene A 40 , dictytriene B 41 , dictyone 42 , dictyriol 43 (Oshoro Bay, Japan) (Enoki, et al., 1982b) and isodictytriol 44 (Yagachi, Japan) (Kusumi, et al., 1986b). The cytotoxicity of 39 and 42 has been examined with both compounds showing good activity against two mouse cell lines, NIH3T3 and KA3IT (Gedara, et al.,

24 Chapter 2

2003). 39 has since undergone further testing against cervical and colon cancer and leukaemia cell lines showing moderate activity against each (Kolesnikova, et al., 2006). 39 has also under gone antiviral screening against Poliomyelitis virus I and Herpes simplex virus I , and results showed no significant antiviral activity (Siamopoulou, et al., 2004).

Dictyol A 45 and B 46 (Fattorusso, et al., 1976) are two diterpenes isolated form a sample of D. dichotoma . These compounds have undergone antiviral screening against Poliomyelitis virus I and Herpes simplex virus I , and results showed no significant antiviral activity (Siamopoulou, et al., 2004). The dictyol family has been expanded by the isolation of dictyol C 47, from a sample from Sicily, Italy, dictyotadiol 48 and dictyol B acetate 49 (Amico, et al., 1980; Faulkner, et al., 1977). 47 and 49 have under gone antiviral screening against Poliomyelitis virus I and Herpes simplex virus I , but showed no significant antiviral activity (Siamopoulou, et al., 2004).

A monoacetylated version of dictyol D, dictyol-D-2β-acetate 50 has been identified along with dictyoxide 51 (Palermo, et al., 1994). Both compounds have undergone antiviral screening against Poliomyelitis virus I and Herpes simplex virus I , with results again showing no significant antiviral activity (Siamopoulou, et al., 2004).

Dictyone acetate 52 exhibited good activity against to two mouse cell lines, NIH3T3 and KA3IT where as dictyol E 53 (Amico, et al., 1980) performed better against KA3IT than NIH3T3. A further diterpene, 3,4-epoxy 13-hydroxypachydictyol A 54 showed no activity in the same assays (Gedara, et al., 2003).

Dictyol I acetate 55 was isolated from a sample of D. dichotoma from the north Adriatic (de Rosa, et al., 1986). A new diterpene; isopachydictyolal 56 has also been isolated from D. dichotoma . (Siamopoulou, et al., 2004). Isopachydictyol A 57 and dictyotatriol A 58 was isolated from a sample of D. dichotoma from Cadiz, Spain (Duran, et al., 1997). The anti viral activity of isopachydictyol A and 56 was investigated with results showing that it had no significant antiviral activity against Poliomyelitis virus I and Herpes simplex virus I (Siamopoulou, et al.,2004). A further dictyol compound was isolated from D. dichotoma . Dictyol J 59 was tested for algicidal activity, showing high activity towards H. akashiwo (Kim, et al., 2006).

25 Chapter 2

A cyclic diterpene 60 and its corresponding acetate 61 have been isolated from D. dichotoma . No biological activity was identified for these compounds (Blount, et al., 1982). Patchytriol 62 is a diterpene found in D. dichotoma , driving its name from the acetylated version, acetoxypachydiol, originally isolated from Pachydictyon coriaceum (Gonzalesz, et al., 1987). Again no biological activity is reported for this compound.

H H

H H H H H H OH 39 40 41

H H H H O H OH H OH OH OH H H H OH OH OH 42 43 44

OH OH O H H H H H

OH OH HO 45 46 47

OAc H H AcO H

HO H HO HO OH 48 49 50

H H O O H AcO HO H OH 51 52 53

H H H AcO O H H O H HO HO OH OH 54 55 56

26 Chapter 2

H H H

HO OH H HO H H HO HO OH Cl 57 58 59

OH OAc

HO HO OH O O O O 60 61 62

Dilophol 63 and 3-acetoxyacetyldilophol 64 were isolated from a sample of D. dichotoma from Oshoro Bay, Japan. No biological activity has been reported for these compounds (Enoki, et al., 1984).

H H AcO OH OAc 63 64

The dicyclic diterpene, dictymal 65 was isolated from a Japanese sample of D. dichotoma . No biological data for dictymal has been reported (Segawa, et al., 1987). α-Dictalediol monoacetate 66 is a major metabolite found in D. dichotoma. No bioactivity has been reported (Gonzalesz, et al., 1987).

HO H H

H CHO AcO H 65 66

Four new diterpenes from D. dichotoma , 14-oxo-3,7,18-dolabellatriene 67 , 3,4-epoxy- 7,18-dolabelladiene 68 , 3,4-epoxy-14-oxo-7,18-dolabelladiene 69 and 3,4-oroxy-7,18- dolabelladiene 70 were all isolated from a sample colleted from Sicily, Italy. All exhibit

27 Chapter 2 antibacterial properties against a number of gram positive and negative bacterial strains (Amico, et al., 1980). In later studies on these dolabellanes, structural corrections were made, reversing the stereochemistry of the epoxide. Compound 69 has been shown to possess antiviral, antibacterial and cytotoxicity properties (Piattelli, et al., 1995). 69 has been tested for its antifungal activity against A. fumigatus , M. canis and T. mentagrophytes . MIC values were in the range of 130-160 µg ml -1 while the positive control, ketoconazole showed activity between the range of 2-5 µg ml -1. 69 was also tested for its cytotoxic properties against three mammalian cancer cell lines. No significant activity was observed against human nasopharynx carcinoma and human non-small lung carcinoma. However the strong activity observed against murine leukaemia suggests that this compound may act specifically against this cell line (Bouaicha, et al., 1993a). 13 new dolabellanes 71 -83 and a new dolastane 84 has been isolated from samples of D. dichotoma from Japan, Australia, the North Atlantic and the Mediterranean (Sullivan, et al., 1986). A further 13 dolabellanes 85 -97 from D. dichotoma have also been isolated from a sample collected in Cadiz, Spain (Duran, et al., 1997).

10-acetoxy-18-hydroxy-2,7-dolabelladiene 98 has been isolated from two samples of D. dichotoma , one from Sicily, Italy (Amico, et al., 1980) and the other from Chubut, Argentina (Palermo, et al., 1994). 98, isolated from a sample collected in the Aegean Sea, Greece, has been tested against the Poliomyelitis virus I and Herpes simplex virus I , and results showed no significant antiviral activity (Siamopoulou, et al., 2004).

28 Chapter 2

O O O O H H H H H

67 68 69 OH HO H O HO OH H OH H O O 70 71 72 HO R

OH O O O OH 73 R = H 75 76 74 R = OAc

HO O O O OH O OH 77 78 79

O AcO OH OAc OH OAc

80 R = CHO 82 83 81 R = COOH

29 Chapter 2

OAc H H OH AcO OAc AcO HO HO O 84 85 86

OAc OAc H H H HO AcO HO OAc OAc HO AcO 87 88 89

H H H HO HO OH HO HO HO 90 91 92

OAc H H HO O HO HO H 93 94 95

HO CHO OAc CHO OAc H H AcO H H

96 97 98

Epoxydictymene 99 a tricyclic diterpene has been isolated from a D. dichotoma sample from Oshoro Bay, Japan (Enoki, et al., 1983b).

H H H O H 99

30 Chapter 2

Two new dolastanes were isolated, dichotone 100 and dichotodione 101 from D. dichotoma (Ali and Pervez, 2003a). Dichotenol A 102 , B 103 and C 104 are three similar diterpenes which have all been isolated from a sample of D. dichotoma from the Arabian Sea (Ali, et al., 2004). No biological activity has been identified for these five compounds. O O H O R R2 O OH O O OH O OH OH H OAc H R1 100 R = H, R = OH 1 2 102 R = H 104 101 R1 = ketone, R2 = H 103 R = OH

The first in a series of new tricarbocyclic diterpenes has been identified as dictyoxetane 105 from the seaweed D. dichotoma . No biological activity has been reported (Welch and Samartino, 1985).

H O OH O

105

Four compounds from the dolastane family isolated from a sample of D. dichotoma from the Karachi, Pakistan, have been identified as dichototetraol 106 , dichotopentaol 107 , (Ali and Pervez, 2003b) dichotenone A 108 and B 109 (Ali, et al., 2003). No biological activity has been described.

OH OH O OH HO OH OH OH OH OH OH OH OH

106 107 108

OH O

OH OH OAc

109

31 Chapter 2

Crenulacetal A 110 is an unusual compound from D. dichotoma , as it has a cyclooctene ring fused to a cyclopropane. Similar compounds have been found to exhibit pesticidal activity as well as functioning as fish antifeedants, although this has not been confirmed for this compound. Two related compounds, crenulacetal C 111 and D 112 , have been isolated from an unidentified sample of seaweed from the Dictyota family (and should be emphasised that this specie may not be found in the waters of Caithness) (Kusumi, et al., 1986a). Acetoxycrenulide 113 isolated from a Greek sample of D. dichotoma was tested against Poliomyelitis virus I and Herpes simplex virus I , with results showing no significant antiviral activity (Siamopoulou, et al., 2004). 113 was also tested against a number of different cancer cell lines and showed weak activity against human nasoparynx carcinoma, murine leukaemia and human non-small cell lung carcinoma (Bouaicha, et al., 1993b).

H H OH OAc

H H H OMe H O MeO O MeO OMe H H 110 111

H OAc H OAc

H H H MeO O O H OMe O 112 113

A cyclic diterpene and its corresponding acetate have been isolated from D. dichotoma 114 and 115 . (Blount, et al., 1982) No biological activity has been reported on the two compounds so far.

OHC OHC OHC OHC

HO AcO

114 115

32 Chapter 2

Fucosterol, 116 from D. dichotoma , collected from Hurgada, Egypt, displays potent activity towards mouse P388 leulemia cells. (Gedara, et al., 2003) It has also been isolated from a Greek sample and tested for its anti-viral activity against Poliomyelitis virus I and Herpes simplex virus I with results showing no significant antiviral activity (Siamopoulou, et al., 2004). 116 has been tested for it antiviral activity against HIV-1 reverse transcriptase but has been found to be inactive (Ahn, et al., 2006). Fucosterol has also been reported to decreases the blood glucose and liver glycogen levels in rats. (Lee, et al., 2004) Fucosterol has also been found in a sample of the brown seaweed F. vesiculosus from an unspecified location (Halsall and Hills, 1971).

HO 116

Three tricyclic diterpenes tricyclodictyofuran A 117 , B 118 and C 119 have been isolated from D. dichotoma , collected from Oshoro Bay, Japan. (Enoki, et al., 1985)

H OH H H H H H H H H H H H H O O O OMe

117 118 119

Sanadaol 120 which have previously been isolated from P. coriaceum (Ishitsuka, et al., 1982) has now been found in a sample of D. dichotoma . The compound has been tested for algicidal activity which showed high activity towards H. akashiwo (Kim, et al., 2006).

H OH

OHC H

120

33 Chapter 2

Two new diterpenes, ent-erogorgiaene 121 and (+)-1,5-cyclo-5,8,9,10- tetrahydroerogorgiaene 122 were isolated from D. dichotoma collected from Troitsa Bay, Russia (Kolesnikova, et al., 2006). The biological activity of these compounds has not been described.

H H H H

121 122

A Greek sample of D. dichotoma has given rise to the following compounds; bicyclosesquiphellandrene 123 , germacrene D 124 and axenol 125 . These compounds have undergone antiviral screening against Poliomyelitis virus I and Herpes simplex virus I, with results showing no significant antiviral activity (Siamopoulou, et al., 2004). Germacrene D is a compound that is found in large proportions in plant essential oils, especially in oil from leaves. Many of these essential oil mixtures have been tested for their bioactivity and have shown antimicrobial and cytotoxic properties. Axenol 125 showed moderated activity towards human cancer cell lines of the cervix and colon as well as leukaemia (Kolesnikova, et al., 2006).

123 124 125

Three bromophenols have been isolated in a sample of F. vesiculosus , lanosol 126 , lanosol methyl ester 127 and lanosol ethyl ester 128 . These compounds were later found to exhibit anti-feeding activity against young abalone, species Haliotis discus hannai (Kurata, et al., 1997). All three compounds have been tested for their cytotoxic activity against DLD-1 and HCT-116, human colon cancer lines. Lanosol has an IC 50 value of around 20 µmol for both cell lines. This value decreases with an increase in length of the ether chain. (Shoeib,

34 Chapter 2 et al., 2004) Lanosol ethyl ester has been tested against a series of terrestrial and marine bacteria and fungi and showed good activity (Barreto and Meyer, 2006). These three compounds have also been found in the red seaweed, Vertebrata lanosa . (Shoeib, et al., 2004)

HO O O

Br Br Br

HO Br HO Br HO Br OH OH OH 126 127 128

Examination of the brown seaweed H. siliquosa collected from Cumbrae, UK, has led to the identification of six unusual molecules 129-134. These molecules are classed a polyprenyl hydroquinol mono-methyl esters, which are essentially a hydroquinol methyl ester with varying types of unsaturated sidechain. No biological activity was described in this study (Higgs and Mulheirn, 1981a). In addition to these compounds, further research identified eight similar compounds 135-142, also from H. siliquosa , from the Brittany coast. Compounds 133, 135-140 and 142 have all been tested for their antifouling properties against a series of marine bacteria. Compounds 133, 135 and 139 each showed strong antibiotic properties towards the tested bacteria. It should be noted that enatiomeric differences play a crucial role in the biological activity of these compounds. The difference in activity between 137 and 138 is striking, with one of the compounds showing activity at 5 µg ml -1 and the other compounds with activity above 100 µg ml -1. It is unfortunate that the authers do not make clear which isomer is more active (Culioli, et al., 2008).

35 Chapter 2

OH O O

129 OCH3 OH O

O 130 OCH 3 O

OH OH O

131

OCH3 OH O O

OH 132

OCH3 OH O O

OH 133 OCH3 OH OH

OCH3 134

36 Chapter 2

OH O O

135 OCH3 O

OH OH O 136

OCH3

OH O OH

137 O

OCH3

OH O OH

138 O

OCH3 OH OH OH

O 139

OCH3 OH O O

OH 140 OCH3

OH 141 OH O

O O H3CO 142

37 Chapter 2

A series of five acetylated polyhydoxyphenyl ethers have been isolated from the acetylated extracts of Halidrys siliquosa collected in Brittany, France: tetrafuhalol-A-undecacetate 143 , tetrafuhalol-B-undecacetate 144 , tetrafuhalol-C-undecacetate 145 , pentafuhaloltridecacetate 146 and desacetoxyheptafuhalolheptadecacetate 147 . These consist of multiple configurations of ether linked acetylated phenol subunits. These compounds are considerably larger than other natural products found in seaweed. No biological activity has been reported (Glombitza, et al., 1980). Diphlorethol pentaacetate 148 was isolated form a French sample of H. elongata (Glombitza, et al., 1977) and was then isolated from a sample of Chorda filum with further isofuhalols 149-160 . The isofuhalols are a series of two to five, benzene rings, joined together through ether bonds. A crude extract sample was acetylated leading to full or partial acetylation of the isofuhalols (Grosse-Damhues and Glombitza, 1984). It should be made clear that in order to isolated these compounds the extracts were acetylated, therefore the natural products from these seaweed samples are the non-acetylated phenol equivalents.

Further examination of the brown seaweed H. elongate from Brittany, France, showed the presence of an eight-ringed phlorotannin 161 . This phlorotannin is a series of ether linked methylated benzene rings (Grosse-Damhues, et al., 1983). No bioactivity has been reported for this compound. Phloroglucinol triacetate 162 , difucol hexaacetate 163 , trifucol nonaacetate 164 , tetrafucoldodecaacetate A 165 and B 166 were isolated from a sample of F. vesiculosus (Glombitza, et al., 1975). These compounds were later found in H. elongata . No biological activity has been reported (Glombitza, et al., 1977).

38 Chapter 2

AcO AcO AcO AcO OAc OAc OAc

AcO O O OAc AcO O O OAc

AcO O AcO AcO AcO O OAc OAc

AcO OAc

AcO OAc AcO 143 144

AcO AcO AcO OAc OAc AcO O O OAc AcO O OAc AcO O AcO AcO OAc O AcO OAc OAc O OAc AcO O OAc AcO OAc OAc AcO OAc

145 146

H OAc AcO AcO AcO AcO AcO O O OAc O OAc AcO O AcO AcO AcO AcO O O OAc

AcO

AcO OAc

147

39 Chapter 2

OAc OAc OR OR

AcO O R1O O OR

OAc OAc OR2 OR3

148 149: R, R1, R2, R3 = Ac 150: R, R1, R3 = Ac, R2 = H 151: R, R1, R2 = Ac, R3 = H 152: R, R2, R3 = Ac, R1 = H

OR OR OR AcO AcO

RO O O OR O OAc

AcO O OAc AcO OR1 OR OR2 OAc 153: R, R1, R2 = Ac 154: R, R = Ac, R = H 2 1 AcO 156 155: R, R1 = Ac, R2 = H

RO RO R O RO RO R1O 1

O O OR O O OR

RO O OR RO RO RO O OR RO RO

OR OR

RO RO O 159: R, R1 = Ac 157: R, R1 = Ac 160: R = Ac, R1 =H 158: R = Ac, R1 = H OR

O O O O O

161

40 Chapter 2

OAc OAc AcO OAc OAc AcO OAc OAc AcO OAc OAc OAc AcO OAc OAc OAc AcO

162 163 164 OAc

OAc OAc OAc AcO OAc OAc AcO OAc OAc OAc OAc AcO OAc OAc OAc OAc AcO OAc OAc OAc AcO OAc OAc OAc 165 166

Fucoxanthin 167 , a common seaweed pigment, has been isolated from a cultivated sample of S. lomentaria grown in deep seawater. 167 has been shown to cell death of the HL-60 (leukaemia) cell line (Kim, et al., 2010). Other properties include antioxidant and also activity against GOTO cells and colon cancer cells (Mori, et al., 2004).

HO OAc

O O HO 167

2.4 Rhodophyceae (Red Seaweed)

Red seaweed, are often found in rock pools or attached to sheltered rock faces. Many red seaweed are of a symbiotic nature and can be found either near, attached to or even partially imbedded in other species of seaweed, mainly large brown species. Any mutual benefit gained has yet to be established. The natural products isolated from red seaweed

41 Chapter 2 tend to be halogentated compounds, with a tendency for bromides to be more prevalent than the other halides.

The species B. hamifera has been shown to contain a series of polybrominated heptane derived compounds, 168-180 from a sample taken from the Gulf of California, USA (Oliver, et al., 1980). Cartilagineal 181 is a monoterpene aldehyde isolated from a sample of P. cartilagineum from an unidentified location (Crews and Kho, 1974). A further monoterpene 182 has been isolated from a Chilean sample of P. cartilagineum . This compound is one of a growing number of unusual compounds isolated from this seaweed as the vast majority of monoterpenes isolated contain both bromine and chlorine atoms where as cartilagineal contains only chlorine (Diaz-Marrero, et al., 2002a). The bioactivity of 168 -182 has yet to be assessed. O O O Br Br Br

Br Br Br H Br H H Br Br 168 169 170 O O H O H I Br Br Br Br Br Br H Br Br H 171 172 173

OH OH COOH Br Br Br Br Br Br Br Br Br 174 175 176 OH COOH CO2Et Br Br Br Br 177 178 179

Cl Cl Cl OH Br OH Br Cl Br Br OH Cl O Br 180 181 182

42 Chapter 2

Two halogentated acyclic monoterpenes, (3R,4R)-l-bromo-7-chloromethyl-3,4-dichloro-3- methyl-lE,5E,7-octatriene, 183 , and oregonene A, 184 , have been isolated from P. cartilagineum collected in northern Spain. The ecological functions of these compounds have not been determined; however both antibiotic and antifungal activities have been assessed. A significant level of antifungal activity was observed for both compounds, but this observation was not mirrored with the antibiotic properties (Konig, et al., 1990). In addition to these two compounds, a further 21 polyhalogenated momoterpenes have been isolated from P. cartilagineum through a number of studies. 185-205 (Jongaramruong and Blackman, 2000). Plocamenol A 188 , B 189 and C 190 from a Chillean sample (Diaz- Marrero, et al., 2002a), 191 (Crews, et al., 1984), 192-202 (Mynderse and Faulkner, 1975), 203 all from a Californian sample (Mynderse and Faulkner, 1978) and finally 204 , 205 from an Antarctic sample have been isolated (Stierle and Sims, 1979). Despite a crude hexane extract of P. cartilagineum showing a strong anti-fungal activity, bioactivities of the 21 terpenes have yet to be established (Stierle and Sims, 1979).

43 Chapter 2

Cl Cl Cl Cl Cl Cl Cl O Br Br Br Cl H Br Cl H Cl 183 184 185 Cl Br Cl Cl Br Br Cl Cl Br Br Br Cl OH OH 186 187 188 Br Cl Cl Cl Cl Br Br Br Cl OH OH OH O Cl Br

189 190 191

CH2Cl Cl CH2Cl Cl CH2Cl Cl Br Cl Cl Cl Cl Cl

192 193 194

CH2Cl Cl CH2Cl Cl CH2Cl Cl Cl Cl Cl Br Cl Cl Cl 195 196 197 CH2Cl Cl Cl Cl Cl Br Br Br Br Cl Br Cl Br Cl 198 199 200 Cl Cl Cl Cl Br Br Br Br Br Br Cl Br Cl Br Cl 201 202 203 Cl Cl Cl Cl Br Cl Br Cl Br Cl

204 205

Four polyhalogenated fatty acid compounds 206-209 have been discovered from P. cartilagineum . Compounds 206 and 207 were isolated from a sample of seaweed from Maltese waters and compounds 208 and 209 were isolated from samples from around

44 Chapter 2

Corsica (Rezanka and Dembitsky, 2001). These compounds have yet to be tested for any potential bioactivity.

Cl Cl HOOC HOOC Br Br O Cl O Cl 206 207 Cl Cl HOOC HOOC Br OH Cl OH Cl 208 209

Three linear polyhalogenated monoterpenes 210 -212 have been isolated from a Portuguese sample of P. cartilagineum (Abreu and Galindro, 1996). No information on the activity of these compounds was described.

Cl Cl Cl Br Cl Cl H Br Br Cl Br Cl Cl Cl O 210 211 212

The first in a new series of tetrahydrofuran derivatives have been identified from Chilean samples of P. cartilagineum . They are a group of furan containing polyhalogentated monoterpenes, furoplocamioid A 213 , B 214 , C 215 (Darias, et al., 2001), 216 and 217 (Diaz-Marrero, et al., 2002b). No bioactivity for these five compounds has been identified. OH OH OH

Cl Cl Cl Br O Br O Br O Cl Cl Cl Br Br Br 213 214 215 Cl Br

Br Br Br Br O O Cl Cl 216 217

45 Chapter 2

Two, halogenated monoterpenes cyclised to form a six membered oxygen hetrocycles have been isolated from a Chilean sample of P. cartilagineum , the first being plocamiopyranoid 218 and the other pirene 219 (Cueto, et al., 1998). The bioactivity of these compounds has not been assessed.

Br HO Br O O Cl Br Br Cl Cl 218 219

Two polyhalogenated cyclic monoterpenes from P. cartilagineum 220 and 221 have been isolated from a Spanish sample of the seaweed (Gonzalez, et al., 1978). An additional four new cyclic polyhalogenated monoterpenes 222 -225 have been isolated from the same seaweed species. Three of these compounds 222 , 223 and 224 have been tested for their insecticidal properties against 10 species of insect. Compound 222 performed best and was shown to kill 80% of tobacco budworms. The compounds tested tended to kill less than 20% of the target insects. 223 and 224 have also been tested for their anti-fungal properties with poor activity being demonstrated (San-Martin, et al., 1991). Four further cyclic polyhalogenated monoterpenes 226 -229 have been isolated from an Antarctic sample (Stierle and Sims, 1979), but not been tested for bioactivity.

Two halogentated cyclic monoterpenes, 230 and coccinene 231 have been isolated from a Spanish sample of P. cartilagineum . The ecological functions of these compounds have not been determined. The antibiotic and antifungal activity of these compounds has been assessed with significant levels of antifungal activity being observed (Konig, et al., 1990). A further three compounds 232 -234 have been isolated from a Portuguese sample of P. cartilagineum (Abreu and Galindro, 1996), with no bioactivity being reported.

46 Chapter 2

Cl Cl Br Br Cl Cl Cl Cl

Cl Br Cl Br Cl Cl Cl Cl

220 221 222 223 Cl Cl

Cl Cl Cl Cl

Cl Cl Br Cl Cl Cl Cl Cl 224 225 226 227

Cl Br Cl Cl Br Cl Cl Cl Cl Br Cl Br Cl Cl Br Cl

228 229 230 231

Br Br Cl Cl Br Cl Br Cl Cl Cl

232 233 234

Delesserine 235 is a secondary sugar metabolite that has been isolated from the seaweed D. sanguinea (Yvin, et al., 1982). The bioactivity of delesserine has yet to be assessed. However aqueous extract from the Delesseriaceae family have been shown to posses significant anticoagulant properties however it is not clear which chemical is responsible for this activity (Poss and Belter, 1988; Yvin, et al., 1982).

OH H3CO O O O H OH 235

47 Chapter 2

A new tetrahydrofuran containing compound, spiro-bis -pinnaketal 236, has been isolated from O. pinnatifida had its structure determined by x-ray crystallography (Wiedenfeld, et al., 1985). No bioactivity for this compound has been identified.

O O O O OH OH 236

A new compound was first isolated as an alcohol 237 from O. pinnatifida of Tenerife in origin, (Gonzalez, et al., 1982) and was later isolated as the corresponding acetate 238 . The E isomer of the C-15 acyclic trienyne 239 has been isolated along with the corresponding acetate 240 from a further Tenerife sample. A further four similar compounds have been isolated (Gonzalez, et al., 1984; Norte, et al., 1991). The bioactivity of these eight compounds is as yet to be investigated.

Cl OH Cl OAc

H H 237 238 H H

Cl OH Cl OAc

239 240

Cl OAc Cl OH

H H 241 242 H H

Cl OH Cl OAc

243 244

48 Chapter 2

The two isomers of pinnatifidenyne ( Z 245 and E 246) have been identified (Gonzalez, et al., 1982). In addition to these compounds, the Z 247 and E 248 isomers of dihydrorhodophytin have also been isolated (Norte, et al., 1989). No activity for these four compounds has been identified.

Cl Cl H

O O Br Br H 245 246

Cl Cl H H H O O Br H Br H H 247 248

Two isomers of a nine membered cyclic ether from O. pinnatifida have been isolated as the E 249 and Z 250 isomers (Norte, et al., 1991). The activity of the two isomers has yet to be assessed.

Cl Cl

Br O Br O

249 250

Two fatty acids, 11-formyl-undeca-5,8,10–trienonic acid 251 and 9-hydroxy-eicosa- 2,5,7,11,14-pentaenoic acid 252 , have been isolated from the seaweed O. hybrida . (Higgs and Mulheirn, 1981b). Extracts of this seaweed have showed positive antibacterial activity towards S. aureus , E. coli and B. subtilis (Higgs, 1981).

COOH OH COOH OHC 251 252

49 Chapter 2

Hybridalactone 253 has been isolated from the O. hybrid (Higgs and Mulheirn, 1981b).

O O

O H H 253

Two similar unnamed compounds 254 , 255 have been isolated from O. pinnatifida collected in Tenerife with one compound being the aromatic version of the other (Gonzalez, et al., 1984). The biological activity of the compounds has not been determined.

Br Br

254 255

Dehydrothyrsiferol 256 has been isolated from a sample of O. pinnatifida from Tenerife (Gonzalez, et al., 1984). Dehydrothyrsiferol has been shown to be active against breast, lung and colon cancer cell lines through a number of anti-tumour assays (Pec, et al., 2003; Norte, et al., 1997).

OH H O O

O OH O H H Br 256

Three compounds from the chamigrene class, pinnatazane 257 (Rahman, et al., 1988) pinnatifenol 258 and 259 have been isolated from O. pinnatifida . 259 has previously been isolated from an unidentified Osmundea sample. A derivative of the diterpene phytol has also been isolated 260 . This compound has been synthesised many times and has been isolated from jasmine oil (Ahmed and Ali, 1991). The bioactivity of these compounds

50 Chapter 2 have yet to be assessed, however it is known that a number of chamigrene compounds possess cytotoxic activity (Francisco and Erickson, 2001).

O HO O Br Br Br

O O Cl Cl HO Cl

257 258 259

260

An in-depth investigation of the red seaweed R. confervoides yielded 37 different polybrominated phenolic compounds. There are a large variety of compounds present with either one, two or three polybrominated phenolic units which contain combinations of ethers, esters, acids, aldehydes, ketones, amides and sulphoxides to name a few. Rhodomevoidin 261 has three polybrominated phenolic units and was the first of these to be isolated from the seaweed (Xu, et al., 2003a). The bioactivity of this compound has not been assessed.

A -CH 2OH, 262 and a -CH 2OC 2H5, 263 substituted bromophenol were isolated from R. confervoides (Fan, et al., 2003a). Both these compounds were tested against eight strains of bacteria; two Staphylococcus aureus , two Staphylococcus epidermidis , two Escherichia. coli and two Pseudomonas aeruginosa strains. 262 showing weak activity against three of the strains of bacteria and 263 showed weak activity against five strains (Xu, et al., 2003b).

A polybrominated phenylpropylaldehyde 264 and a dimethly ether 265 derivative have been isolated along with a monobromoated phenyl methyl ester 266 from R. confervoides . There is a suggestion that the dimethyl ether maybe formed as a by-product in the extraction of the original phenylproylaldehyde (Fan, et al., 2003b). A collection of polyhalogenated monophenols 267-271 and diphenols 272-274 have been isolated (Fan, et al., 2003c). 274 is unusual as it is the only polyphenolic of this type to have an ether bridge to another phenolic unit, where other compounds have a methylene bridge group. Compounds 264-266, isolated from a Chinese sample, were tested against eight strains of

51 Chapter 2 bacteria; two S. aureus , two S. epidermidis , two E. coli and two P. aeruginosa strains. 272 showed moderate activity against the four Gram positive strains with no activity aginst the Gram negative bacteria. 273 shows weak activity against seven of the strains. 274 showed moderate to strong activity against seven of the strains. One strain of S. epidermidis was not affected by any of the compounds. 262 , 263 and 272 showed selectivity to wards Gram positive bacteria where 273 and 274 showed broad spectrum antibiotic activity (Xu, et al., 2003b).

Nine additional brominated phenols 275-283 have been isolated from R. confervoides , sampled from Qingdao, China. Compounds 275 , 277 , 282 and 283 were tested for antimicrobial activities against two strains of bacteria; S. aureus and E. coli and the yeast, Candida albicans . All compounds failed to show any activity at a concentration of 100 µg ml -1. Compounds 275 -279 , 282 , 283 were tested for cyctoxicity against several cancer lines including lung, breast, colon, stomach and hepatoma (liver) and no activity was observed at 10 µg ml -1 (Zhuo, et al., 2004). A series of four compounds 284-287 are the first known examples of bromophenols coupled to derivatives of amino acids or nucleosides. The four compounds were tested against several cancer lines and microorganisms, however these compounds were found to be inactive at 10 µg ml -1 (Zhao, et al., 2005). Four bromophenols coupled with methyl-γ-ureidobutyrate 288 -291 have been isolated from a sample of R. confervoides . A bromophenol 292 and three bromophenol sulphates 293 -295 from the same sample were also isolated and showed moderate activity towards five cancer cell lines (Ma, et al., 2006).

Methylrhodomelol 296 and rhodomelol 297 are two bromophenol, γ-lactone containing derivatives from the red seaweed Vertebrata lanosa . The author of this work entitle the paper “Antibiotics from Algae”, however no data is provided to support this statement. However the presence of the γ-lactone ring may contribute to an antibiotic effect much as a β-lactam would do in the case of penicillin (Glombitza, et al., 1995).

52 Chapter 2

Br Br OH HO O Br Br Br OH Br Br Br Br HO Br OH HO Br OH HO OH OH OH OH OH OH OH 261 262 263

Br O Br OMe O Br Br Br OMe OMe

HO HO HO OH OH OH

264 265 266

Br Br Br O Br Br OH OMe HO HO HO OH OH OH

267 268 269

OMe Br Br Br Br Br O O Br

HO HO HO Br OH OH OH OH OH 270 271 272

Br Br Br Br Br Br Br Br O

HO OH HO OH OH OH OH OH

273 274

53 Chapter 2

Br O O HO Br S HO OH O HO Br HO HO Br Br OH Br Br OH OMe 275 276 277 OH Br O HO Br O Br O OMe Br OH Br OH HO Br HO OH Br OH OH OMe Br OH OMe 278 279 280

O Br O Br O Br OH OH OMe MeO MeO MeO OH OH OH 281 282 283 O MeO HO MeO Br O Br O Br Br N N N Br Br O HO O HO O OH OH HO Br OH OH OH 284 285 286

N Br N O Br N N N HO OMe H N N HO H H HO O O HO Br OH Br OHOH 287 288

54 Chapter 2

O O HO OMe HO OMe N N N N H H H O O HO HO Br Br Br Br Br OH

Br OH HO Br OH 289 290 O HO OMe N N H OH O OR2 HO Br R1 Br OH Br Br Br OH OH Br OH OH

292 R1 = Br, R2 = H 291 293 R1 = Br, R2 = SO3H 294 R1= H, R2 = SO3H

HO OSO3H

HO Br Br

HO Br OH

295

O Br O Br O Br O Br

HO OMe HO OH O OH OH O OH OH OH OH 296 297

2.5 Conclusion

It is evident from this review of the literature that seaweed as a source of bioactive natural products is still largely an untapped resource with many seaweed species still to have their chemistry explored. Just less than 1/5 of the seaweed found on the shores of Caithness have been had their natural products studied. In addition, 206 identified compounds: 21 ,

55 Chapter 2

22 , 31 , 33 , 34 , 43 , 44 , 48 , 50 , 55 , 57 , 58 , 60 -66 , 71 -97 , 99 -112 , 114 , 115 , 117 -119 , 121 , 122 , 129-134, 141, 143-166, 168-182, 185-221, 225-229, 232-255, 257-261 , 267-271 , 276, 279-281 and 288-291 have not been assessed for their bioactivity, which comprises of 69% of the natural products isolated.

The majority of the seaweed which has been studied only contributes to one or two publications each, yielding a small number of compounds (table 2.4). However, two species, P. cartilagineum and D. dichotoma , have been extensively studied and accounts for 14 and 38 publications respectively. Two further species R. confervoides and O. pinnatifida have also been studied more extensively than the majority of species with seven and eight publications respectively. These four seaweed species contribute to over half of all natural products isolated in this study and are still heavily investigated and continue to yield novel compounds.

Species No. of Species No. of Publications Publications Dictyota dichotoma 38 Ulva prolifera 2 Plocamium cartilagineum 14 Vertebrata lanosa 2 Osmundea pinnatifida 8 Bonnemaisonia hamifera 1 Rhodomela confervoides 7 Chorda filum 1 Ectocarpus siliculosus 4 Cladostephus spongiosus 1 Ulva lactuca 4 Fucus serratus 1 Delesseria sanguinea 3 Himanthalia elongate 1 Fucus vesiculosus 3 Scytosiphon lomentaria 1 Halidrys siliquosa 3 Ulva intestinalis 1 Ascophyllum nodosum 2 Ulvella lens 1 Osmundea hybrida 2 Table 2.4. Number of publications per species

Out of the 129 different species on coast line of the northern United Kingdom, 108 species (84 %) (Tables 2.5, 2.6 and 2.7) have not been studied for their natural product content. This is a large proportion of the species which have not examined but it brings with it great potential in discovering new natural products and any biological function. Many species of brown seaweed such as F. vesiculosus , P. canaliculata and L. digitata are generally available in large quantities which make them suitable for obtaining sufficient amounts of extracts for natural product isolation and bioassays. Red seaweed are generally less abundant than brown seaweed making it more challenging to collect sufficient quantities for extraction purposes and further study. There are some red seaweed species such as P.

56 Chapter 2 palmata , a common edible seaweed, are available in larger quantities and are therefore suitable in aquaculture.

Species Species Blidingia marginata Pringsheimiella scutata Blidingia minima Rhizoclonium tortuosum Chaetomorpha ligustica Rosenvigella tortuosa Chaetomorpha linum Spongomorpha arcta Cladophora rupestris Ulothrix flacca Cladophora sericea Ulothrix speciosa Codium fragile Ulva linza Monostroma grevillei Urospora penicilliformis Prasiola stipitata Table 2.5. Green seaweed species found in Caithness, for which no information on natural product content could be found

Species Species Alaria esculenta Herponema velutinum Asperococcus fistulosus Laminaria digitata Colpomenia peregrina Laminaria hyperborea Desmarestia aculeata Leathesia marina Dictyosiphon chordaria Litosiphon laminariae Dictyosiphon foeniculaceus Myriotrichia clavaeformis Ectocarpus fasciculatus Petalonia fascia Elachista fucicola Pelvetia canaliculata Elachista scutulata Pilayella littoralis Elachista stellaris Ralfsia verrucosa Fucus ceranoides Saccharina latissima Fucus distichus Saccorhiza polyschides Fucus spiralis Sauvageaugloia chordariiformis Fucus vesiculosus var linearis Sphacelaria cirrosa Herponema velutinum Sphacelaria rigidula Hincksia hincksiae Spongonema tomentosum Hincksia sandriana Table 2.6. Brown seaweed species found in Caithness, for which no information on natural product content could be found

57 Chapter 2

Species Species Acrochaetium alariae Lithophyllum incrustans Acrochaetium microscopicum Lithothamnion glaciale Acrochaetium parvulum Lomentaria articulate Acrochaetium secundatum Lomentaria clavellosa Acrochaetium sparsum Mastocarpus stellatus Aglaothamnion hookeri Membranoptera alata Aglaothamnion sepositum Mesophyllum lichenoides Ahnfeltia plicata Nemalion helminthoides Callophyllis laciniata Odonthalia dentate Catenella caespitose Palmaria palmata Ceramium nodulosum Phycodrys rubens Ceramium pallidum Phyllophora crispa Ceramium shuttleworthianum Phymatolithon calcareum Ceramium virgatum Phymatolithon purpureum Chondrus crispus Plumaria plumose Chondrus verrucosus Polyides rotundus Conferva atropurpurea Polysiphonia atlantica Corallina officinalis Polysiphonia brodiei Cryptopleura ramose Polysiphonia elongate Cystoclonium purpureum Polysiphonia fucoides Dilsea carnosa Polysiphonia nigra Dumontia contorta Polysiphonia stricta Erythrotrichia carnea Porphyra dioica Furcellaria lumbricalis Porphyra purpurea Gastroclonium ovatum Porphyra umbilicalis Gelidium pusillum Ptilota gunneri Gelidium spinosum Rhodochorton purpureum Heterosiphonia plumosa Rhodothamniella floridula Lithophyllum crouanii Scinaia furcellata Table 2.7. Red seaweed species found in Caithness, for which no information on natural product content could be found

With this study, it is now possible to identify seaweed species which have not been studied at all and those species with limited evaluation that could benefit with further examination to identify new compounds and bioactivity. In addition, with the exception of a few samples, almost all species were sampled outside of British water with none being from Caithness or surrounding areas. There is evidence that the secondary metabolite content of seaweeds such as D. dichotoma varies with location which gives good reason that well studied species may be suitable candidates for further study.

58 Chapter 3

3. Antifouling Activity of Seaweed Extracts

3.1 Introduction

3.1.1 What is Biofouling?

In the marine environment biofouling can be defined as the unwanted accumulation of plants, animals and micro-organisms on submerged surfaces such as those of seaweed, rock, hulls of ships, oil rigs etc. Biofouling is a stepwise process with four recognised phases (Wahl, 1989). Firstly, within a few minutes of a surface being submerged in seawater, a thin layer of organic matter consisting of proteins and polysaccharides becomes attached. Bacterial colonisation follows about an hour after submersion with a range of marine bacteria attracted to the nutrient rich biofilm. After the development of the bacterial biofilm over several days simple unicellular organisms such as diatoms and protozoa begin to colonise the surface. The final stage of biofouling development is initiated by the recruitment of the spores and larvae of multicellular organism which then develop in to mature plants and animals which constitute the very visible endpoint of the biofouling process.

3.1.2 Effects of Biofouling on Marine Surfaces

The economic and environmental consequences associated with biofouling are often overlooked. The rough surface on boat hulls that biofouling can cause can lead to a reduction in the hydrodynamic properties of the vessel, decreasing the fuel efficiency of the vessel thereby increasing fuel costs. It is estimated that an increase in this roughness of 10 µm will lead to a decrease in fuel efficiency of around 1 %. With barnacles etc, causing an increase in roughness of centimetre scale, then the decrease in fuel efficiency will be much greater (Cooney, 1995). Additionally vessel down time required to remove fouling organisms adds to running costs. As a significant proportion of marine traffic is associated with the transportation of commercial goods, the increases in cost is ultimately forwarded

59 Chapter 3 to the consumers. In the past compounds such as tertiary butyl tin (TBT) have been used to prevent biofouling. Although these compounds are very effective as antifouling agents, they are incredibly toxic and can affect a variety of marine life including seaweed, molluscs and marine mammals with a recent study suggesting that TBT may cause serious immune function failure in adult harbour seals. Like most toxins, they can become integrated into the food chain and there is now evidence to suggest TBT could lead to health problems in humans such as cardiovascular, respiratory, kidney, liver, gastrointestinal and carcinogenic problems (Frouin, et al., 2008; Antizar-Ladislao, 2008). These compounds have subsequently been banned in most countries and have been replaced by copper and zinc based compounds. These in themselves are far from ideal and are also toxic although significantly less so than TBT (Rotterdam Convention, 2009). There is therefore the need to identify more environmentally friendly antifouling agents for use as coatings for submerged man made surfaces.

While we are relative novices in tackling biofouling in the marine environment, many plants and animals which spend the majority of their lives submerged in the sea seem to be able to limit the development of biofilm communities on their surfaces. In the case of seaweeds colonisation of surfaces by epibionts (an organism living on the surface of another) has a number of disadvantages for the host plant. In general the effects of such fouling on marine life are to shade the host (basibiont) from required sunlight, impede gas and nutrient exchange thus decreasing growth rate, and to increase friction which can result in the dislocation of fronds or entire plants (Sand Jensen, 1977; Harlin, et al., 1985; Siberstein, et al., 1986). As a consequence macroalgae have evolved a number of physical and chemical mechanisms to combat the growth of epiphytic organisms on their surfaces (Boyd, et al., 1999a). Physical adaptations include the shedding of the outer layer of cells and mucilaginous covering and the continuous erosion of the distal ends of blades as in Laminaria species and seagrass (Mann, 1973; Moss, 1973; Ott, 1980; Filion-Mykelbust and Norton, 1981; Wahl, 1989).

Chemical antifouling mechanisms have however received little attention. Seaweed species, like many classes of marine organisms, are a rich source of novel bioactive secondary metabolites (Blunt, et al., 2010). Although the ecological role of these metabolites has received little attention, a small number of compounds have been shown to exhibit biological activities consistent with them playing a role in fouling control. Tannin

60 Chapter 3 like compounds isolated from Sargassum natans (Linnaeus) Gaillon and Sargassum fluitans (Børgesen) Børgesen have been shown to have antibacterial activity against marine bacteria (Conover and Sieburth, 1964; Sieberth and Conover, 1965). The red seaweed Delisea pulchra (Greville) Montagne produces a series of brominated furanones which exhibit broad spectrum antifouling activity, inhibiting the settlement of both barnacle cyprids and Ulva lactuca Linnaeus gametes (de Nys, et al., 1995). The metabolites also inhibit the growth of marine bacteria and have recently been shown to interfere with N-acyl homoserine lactone signalling pathways important in the swarming and attachment of marine bacteria to surfaces (Givskov, et al., 1996; Gram, et al., 1996; Satuito, et al., 1997; Culioli, et al., 2008).

As outlined above, biofilm development in the marine environment is characterised by a sequence of events where an initially formed macromolecular film is first colonised by marine bacteria, followed by the settlement and attachment of free swimming algal spores and invertebrate larvae (Wahl, 1989). The ubiquitous nature of bacteria in the marine environment and their prominence at the early stages of biofilm development may allow them to strongly influence the establishment of other surface colonisers (Satuito, et al., 1997). Microbial biofilms have been shown to act as cues for the settlement of many marine organisms including sponges, cnidarians, molluscs, tube-building worms, barnacles, bryozoans, ascidians and algae (Krug, 2006). Thus the ability of seaweeds to manipulate the bacterial communities associated with their surfaces may be crucial to their biofouling control. For most marine organisms very little is known of the structure and function of the microbial communities which colonise their surface. However a number of studies have now at least highlighted important roles for surface associated bacteria in the marine environment which include the ability of such communities to protect host organisms from infection and to inhibit the settlement of the larvae of fouling organisms (Gil-Turnes, et al., 1989; Gil-Turnes and Fenical, 1992; Lau, et al., 2003; Bryan, et al., 1997; Maki, et al., 1988; Avelin, et al., 1993; Goecke, et al., 2010; Wiese, et al., 2009).

In theory at least, there are a number of chemical mechanisms which could be employed by seaweeds to influence the composition of their epibiotic bacterial community. Seaweeds could inhibit the development of a bacterial biofilm through the production of broad spectrum antimicrobial compounds and thus prevent the settlement of further fouling organisms which require biofilm specific cues for settlement. The antimicrobial activity of

61 Chapter 3 many seaweed species is well documented, however the fact that bacteria seem to be ubiquitous on marine macroalgae seems to exclude this hypothesis (Armstrong, et al., 2000; Boyd, et al., 1999a; Boyd, et al., 1999b). Alternatively, but along the same lines, seaweeds could play host to specific bacterial populations which, through competition for nutrients and space ultimately lead to the formation of a mature biofilm quite different from that which form on inanimate surfaces. This modification of the biofilm could be attained either by recruiting a population of bacteria which do not enhance the settlement of the larvae of fouling organisms or actively recruiting strains which deter the settlement of fouling larvae. The recruitment of such populations by the seaweed could be attained through the production of metabolites which favour colonisation by such bacteria either through inhibition of other species or through active recruitment of beneficial species.

Despite the plethora of literature concerning the settlement and attachment of invertebrate larvae on inanimate and living surfaces in the marine environment there is very little information on factors determining the composition of microbial communities present on marine organisms. This is possibly due to the lack of relevant bioassays and the predominant use of medical isolates to screen for and study the antimicrobial activity of marine natural products (Clare, 1996). This study assesses the anti-microbial activity of extracts from a range of intertidal seaweed, at environmentally relevant concentrations, against a panel of Gram negative and Gram positive surface associated marine bacteria. This strategy allows us not only to identify extracts which inhibit bacterial growth that could potentially find uses as antifoulants but also to assess the ecological relevance of these antimicrobial compounds to the plants themselves.

3.2 Materials and Methods

3.2.1 Materials

Methanol (HPLC grade) and ethyl acetate, hexane and dichloromethane (glass-distilled grade) were purchased from Rathburn Chemicals Ltd. (Walkerburn, UK). Milli-Q water was used unless otherwise stated. Marine Broth (MB) (Difco, Detroit, USA) was prepared

62 Chapter 3 according to manufacturers instructions. Marine Agar (MA) was prepared using Marine Broth (37.5 g) and bacteriological agar (Oxoid, Basingstoke, UK) (10.0 g) in water (1000 ml). Media was autoclaved at 121 °C for 15 minutes prior to use. Antibiotic assay discs (Whatman No.1, 6mm) were sterilised for 15 minutes at 121 °C.

3.2.2 Bacterial Strains The bacterial strains used in this study consist of both Gram positive and Gram negative bacteria isolated from a range of surfaces in the marine environment.

Four Gram positive bacteria; Bacillus sp , Bacillus licheniformis and Planococcus citreus , isolated from submerged stones (Peppiatt et al. 2000), and the unidentified strain IV/3(1), isolated from submerged stones within the vicinity of Thurso beach (Ruchonnet. 2007) were use.

The Gram negative bacteria included two different isolates of Cytophaga fucicola , which were isolated from the surfaces of marine algae (Peppiatt et al. 2000) and Oceanospirillum linum isolated from decaying seaweed (Boyd, Personal Communication) .

All bacterial cultures were maintained on MA plates prior to use.

3.2.3 Preparation of extracts

Seaweed species were selected based of a range of parameters; including class (Chlorophyceae, Phaeophyceae and Rhodophyceae), location on shore and represent specimens from both rock pools and exposed locations; in order to gain samples from a broad spectrum of coastal seaweed. The species collected and information relating to the collection of samples is summarised in Table 3.1.

63 Chapter 3

Shoreline Location of Date Species Class Exposure Position Sample Collected Cladophora rupestris Chlorophyceae Thurso All Rock Pool 22/01/07 (Linnaeus) Kützing (green) East Chlorophyceae Exposed rock Ulva linza Linnaeus Upper Thurso 20/07/07 (green) surface Chorda filum (Linnaeus) Phaeophyceae Lower Rock Pool Thurso 20/07/07 Stackhouse (brown) Phaeophyceae Exposed rock Thurso Fucus serratus Linnaeus Lower 22/01/07 (brown) surface East Phaeophyceae Exposed rock Thurso Fucus vesiculosus Linnaeus Middle 22/01/07 (brown) surface East Halidrys siliquosa (Linnaeus) Phaeophyceae Thurso Middle Rock Pool 22/01/07 Lyngbye (brown) East Pelvetia canaliculata Phaeophyceae Exposed rock Thurso (Linnaeus) Decaisne & Upper 22/01/07 (brown) surface East Thuret Rhodophyceae Exposed rock Thurso Chondrus crispus Stackhouse Middle 22/01/07 (red) surface East Rhodophyceae Middle- Thurso Corallina officinalis Linnaeus Rock pool 22/01/07 (red) Lower East Osmundea pinnatifida Rhodophyceae Exposed rock Thurso Lower 22/01/07 (Hudson) Stackhouse (red) surface East Palmaria palmata (Linnaeus) Rhodophyceae Exposed rock Middle Thurso 20/07/07 Kuntze (red) surface Plocamium cartilagineum Rhodophyceae Middle Rock Pool Thurso 20/07/07 (Linnaeus) P.S. Dixon (red) Polysiphonia fucoides Rhodophyceae Thurso Middle Rock pool 22/01/07 (Hudson) Greville (red) East Table 3.1. Summary of seaweed species collected for extraction including location and habitats

A total of thirteen different species of seaweed, were collected from the intertidal zone at sites in and around the town of Thurso on the north coast of Scotland. After collection samples were immediately returned to the laboratory and identified using standard field guides. The samples were then cleaned of obvious epiphytes, weighed and a known a mass was cut into approximately 2-3 cm pieces which were then covered with methanol at room temperature for 24 hours. The methanol was then decanted and the extraction process repeated a further two times to give a total of three extracts which were combined. The combined extracts were concentrated under vacuum and the residue diluted with water (50 ml). The aqueous suspension was then extracted sequentially with hexane, ethyl acetate and dichloromethane (3 x 50 ml for each solvent). The combined extracts for each solvent

64 Chapter 3 were then dried over sodium sulphate and evaporated to a gum. This gives a total of 39 extracts (three for each species)(Scheme 3.1).

Algae

Mix MeOH

Repeat Twice Action Algae/Methanol Infusion Product

Solvent Decant

Methanolic Extract Algae Solution

Evaporate

Crude Extract

Organic/Aqueous H2O Partit ioning He xane Repeat Twice

Organic Layer Aqueous Layer

Organic/Aqueous Partit ioning EtOAc

Repeat Twice

Organic Layer Aqueous Layer

Organic/Aqueous Partit ioning DCM Repeat Twice

Organic Layer Aqueous Layer

Dried over Na 2SO 4

Filter

Evaporate Solvent

Final Extract Fractions Scheme 3.1. Flow chart detailing the steps involved in the preparation seaweed extracts

65 Chapter 3

3.2.4 Antimicrobial assay

Antibiotic activities were assessed using a disc diffusion assay. The fraction of extract loaded on each disc was calculated to be equivalent to that extracted from a plant sample of equal volume to the disc, based on the equation below. Where M D is the loading 3 concentration applied to the disc, V D is the volume of the disk in cm , M A is the total mass 3 of extract from the seaweed in mg and V A is the volume of seaweed in cm . The loading concentration of extracts applied to each disc is shown in Table 3.2.

= (VD *M A ) M D VA

The volume of each seaweed sample was determined after extraction by transferring the extracted seaweed to a graduated cylinder and adding water to a given volume. The volume of seaweed was then determined as the difference between the volume of seaweed and water and the volume of water decanted from the mixture. The volume of the antibiotic discs was determined by measuring the diameter and height of the disk then applying the standard formula for a cylinder.

66 Chapter 3

Species Extract Loading concentration (mg) Hexane 0.93 Cladophora rupestris Ethyl Acetate 0.10 Dichloromethane 0.06 Hexane 2.30 Ulva linza Ethyl Acetate 0.80 Dichloromethane 0.01 Hexane 2.52 Chorda filum Ethyl Acetate 1.93 Dichloromethane 0.29 Hexane 1.94 Fucus serratus Ethyl Acetate 0.53 Dichloromethane 0.06 Hexane 0.54 Fucus vesiculosus Ethyl Acetate 0.74 Dichloromethane 0.01 Hexane 0.96 Halidrys siliquosa Ethyl Acetate 0.54 Dichloromethane 0.01 Hexane 1.58 Pelvetia canaliculata Ethyl Acetate 0.10 Dichloromethane 0.001 Hexane 1.68 Chondrus crispus Ethyl Acetate 0.32 Dichloromethane 0.62 Hexane 0.40 Corallina officinalis Ethyl Acetate 0.07 Dichloromethane 0.03 Hexane 0.63 Osmundea Ethyl Acetate 0.59 pinnatifida Dichloromethane 0.12 Hexane 0.90 Palmaria palmata Ethyl Acetate 0.25 Dichloromethane 0.07 Hexane 11.59 Plocamium Ethyl Acetate 2.54 cartilagineum Dichloromethane 1.19 Hexane 1.01 Polysiphonia Ethyl Acetate 1.41 fucoides Dichloromethane 0.03 Table 3.2. Loading Concentration of Extract Used Per Disc (mg)

Suspension cultures of seven bacterial strains representing Gram positive and Gram negative strains isolated from a range of surfaces in the marine environment (Boyd, et al., 1999; Peppiatt, et al., 2000; and Ruchonnet. 2007) were prepared in Marine Broth. The maintained cultures were transferred to MB (25 ml) and incubated at 29 °C with shaking for 3 days.

67 Chapter 3

Solutions of each extract were prepared in methanol at a concentration which allowed each disc to be saturated three times (3 x 17.5 µl), with drying in between, to give the desired mass of extract on each disc (M D). For each extract, 21 discs were prepared. Three discs prepared with each extract were then transferred to the surface of each of seven MA plates which had been freshly swabbed with liquid cultures of one of the seven surface associated marine bacterial strains. Control discs, used on each plate, were saturated three times with methanol. The plates were then incubated at 29 °C for 72 hours and visible zones of inhibited bacterial growth were recorded as the diameter of the zone, including the diameter of the disc.

3.3 Results

Methanolic extracts of thirteen species of seaweed were partitioned against water into three different solvents of increasing polarity (hexane, ethyl acetate and dichloromethane) to provide 39 different extracts. These were assessed for antimicrobial activity against seven strains of surface associated marine bacteria using a modification of the antimicrobial disc assay, where the amount of material deposited on each disc reflected the amount of material in the same volume of the extracted plant (Jenson, et al., 1998). While this could lead to some constituents, those not exclusively found in a single solvent, being assessed at concentrations lower than in the plant tissue it should preclude the assessment of activity at higher than naturally occurring concentrations. The results of the antimicrobial assays are summarised graphically in Figure 3.2.

Of the 39 extracts tested, 17 (43.6%) showed activity against at least one of the test strains with nine of the 13 (69%) seaweed species producing at least one extract which displayed antimicrobial activity against at least one of the bacterial strains. The extracts of Palmaria palmata , Ulva linza , Chondrus crispus and Pelvetia canaliculata ; showed no detectable levels of antibiotic activity towards any of the bacterial strains.

Extracts of Corallina officinalis show poor activity with the hexane extract inhibiting the growth of only a single strain of Oceanospirrillum linum and both the ethyl acetate and DCM extracts showing no detected activity against any of the strains.

68 Chapter 3

Hexane, ethyl acetate and DCM extracts of Polysiphonia fucoides and Cladophora rupestris all displayed antimicrobial activity. Although all three extracts of C. rupestris displayed antimicrobial activity two of these extracts, ethyl acetate and dichloromethane, only displayed activity against Planococcus citreus with the hexane extract active against both P. citreus and Oceanospirillum linum . The ethyl extract of P. fucoides displayed activity against all the strains with the exception of IV/3(1) with the hexane extract being active against all but IV/3(1) and one of the Cytophaga fucicola species.

Only one seaweed species, Osmundea pinnatifida , produced extracts active against all seven bacterial target strains with activity ranging from weak to very strong. Extract extracts of Plocamium cartilagineum and Fucus vesiculosus also showed broad spectrum antimicrobial activity inhibiting five of the seven target strains. Extracts of P. fucoides , Fucus serrat us and Halidrys siliquosa inhibited the growth of six of the seven target strains.

69 Chapter 3

Figure 3.2. Antimicrobial activity of extracts of seaweeds species against surface associated bacterial strains

70 Chapter 3

3.4 Discussion

3.4.1 General Discussion

While the antimicrobial activity of seaweeds has been assessed in the past, these studies have largely focused the activity of extracts against either human or animal pathogens and have rarely used ecologically relevant stains or environmentally relevant concentrations of extracts (Hornsey and Hide, 1974, 1976a, 1976b; Bansemir, et al., 2006). As a result, to date, the possible ecological role of antimicrobial seaweed metabolites has not been adequately assessed. However, using the methodology applied here I am able to assess the antimicrobial activity of extracts from a range of intertidal seaweeds at environmentally relevant concentrations.

In earlier work on British seaweed species, the extracts of P. palmata , P. canaliculata and Ulva , showed no antimicrobial activity against a range of human pathogenic bacteria (Hornsey and Hide, 1974). Here, dichloromethane extracts of P. palmata , P. canaliculata and Ulva compressa (Linnaeus) similarly showed none, or very little, antibiotic activity against a range of fish pathogenic bacteria even at relatively high concentrations (Bansemir, et al., 2006). However excised tissues and the water soluble portion of acetone extracts from C. crispus have been shown to exhibit a strong antimicrobial effect against Staphylococcus aureus , Escherichia coli , Bacillus subtilus and Proteus morganii (Hornsey and Hide, 1974, 1976a, 1976b). In contrast a more recent study suggests that the antimicrobial activity of C. crispus extracts was limited to yeast and fungi (Hellio, 2000)

The poor antimicrobial activity observed with extracts of C. officinalis confirms the lack of activity reported for extracts and excised thalli of this species in previous studies (Hornsey and Hide, 1974, 1976a). The lack of activity of these extracts in this study and in others suggests that although the mediation of bacterial surface colonisation by antimicrobial seaweed metabolites may occur, it is not a prerequisite for maintaining the plants position within the ecosystem.

71 Chapter 3

The antimicrobial activity of excised tissue from P. fucoides has not previously been demonstrated however extracts from a similar species, Polysiphonia lanosa (Linnaeus) Tandy, has been shown to have some antimicrobial activity (Hornsey and Hide, 1974, 1976a). Although the study of Hornsby and Hide (1974) indicated that excised tissues of C. rupestris had no antibiotic activity, Hellio (2004) showed the dichloromethane extract of this species to be active against both Gram negative and Gram positive marine bacteria. Interestingly, the dichloromethane extract of P. fucoides only displayed activity against C. fucicola . This suggests that this plant contains metabolites capable of inhibiting the growth of a broad spectrum of surface associated marine bacteria and that the seaweeds posses a multi-component antibacterial defence system.

The high level of activity of O. pinnatifida is consistent with previous reports where both excised tissues and extracts of this species have been shown to have activity against both Gram negative and Gram positive human pathogens but interestingly only against Gram positive marine bacteria (Hornsey and Hide, 1974, 1976a; Hellio, et al., 2000). The broad spectrum antimicrobial activity of H. siliquosa extracts against both marine bacteria and human pathogenic bacteria has been demonstrated previously (Hornsey and Hide, 1974, 1976a; Ruchonnet, 2007; Culioli, et al., 2008). Interestingly, in previous studies excised tissues from F. vesiculosus, F. serratus and P. cartilagineum did not show any antimicrobial activity against a panel of human pathogens. In the same study Chorda filum also displayed no activity while the extracts tested here inhibited three of the test strains.

The use of an antimicrobial assay system employing ecologically relevant strains and employing environmentally relevant concentrations has therefore demonstrated some notable differences compared to assay systems using either human pathogens or arbitrary concentrations of extracts (Table 6.3). While the broad spectrum activity of H. siliquosa and O. pinnatifida are not surprising based on studies using human pathogens, those of P. cartilagineum, F. vesiculosus and F. serratus are contrary to these studies. Similarly while the lack of activity of U. linza, C. filum and P. Palmata are in agreement with the results using human pathogens, the lack of activity displayed by C. crispus here is in contrast with the activity described against human pathogens. The data available on the activity of seaweed extracts against marine bacteria is probably too limited to draw any definite conclusions but the results of assays here tend to agree with those obtained against other strains of marine bacteria.

72 Chapter 3

Activity against surface Activity reported Activity reported associated marine bacteria at Seaweed species against human against marine environmentally relevant pathogens 1 bacteria 2,3 concentrations Cladophora rupestris -VE +VE +VE

Ulva linza ND ND -VE

Chorda filum -VE ND +VE

Fucus serratus -VE ND +VE

Fucus vesiculosus -VE ND +VE

Halidrys siliquosa +VE +VE +VE

Pelvetia canaliculata -VE ND -VE

Chondrus crispus +VE +VE AND -VE -VE

Corallina officinalis -VE ND +VE

Osmundea pinnatifida +VE ND +VE

Palmaria palmata -VE ND -VE Plocamium -VE -VE +VE cartilagineum Polysiphonia fucoides ND ND +VE Table 3.3. Summary of antimicrobial activity of seaweed extracts against marine bacteria and human pathogen 1. Hornsey and Hide, (1974), 2. Hellio et al., (2004), 3. Culioli, et al., (2008)

3.4.2 Correlation of Antibacterial Activity with Shore Position

On examination of the activities associated with the individual species of seaweed little correlation between the type of seaweed and activity of the extracts could be found. However, when the results from seaweed found on exposed rock surfaces are compared to those sampled from rock pools some interesting observations can be made. Of the six species of seaweed sampled from rock pools all exhibited activity against at least one strain of the test bacteria with three of the six species inhibiting five or more of the test strains. A total of 66.7% of the extracts prepared from species sampled from rock pools were active against at least one bacterial strain (Fig. 3.3). Broad spectrum antibiotic activity was observed with H. siliquosa , P. cartilagineum and P. fucoides extracts, however the remaining three species sampled from rock pools showed less activity. Extracts of C. filum

73 Chapter 3 were active against three of the four Gram positive bacteria while C. officinalis and C. rupestris show activity against one and two strains respectively.

Seaweed in rock pools spend all of their time underwater with a large proportion of this time, when the tide is out, spent in a relatively small volume of water exposed to higher temperatures and increased sunlight compared to bulk seawater. Such pools may provide ideal environments for bacteria to thrive in and it therefore seems logical that seaweed colonising such environments would have evolved some defence against colonisation by marine bacteria.

Figure 3.3. Summary of Activity with Respect to Shore Position

In contrast to the seaweeds sampled from rock pools, extracts from the seaweed sampled from and commonly found on exposed rocky surfaces tend to be less active with only 23.8% of extracts showing activity. These species can be further divided in to three shore positions; upper, middle and lower. The extracts of two seaweeds that occupy the upper tidal zone, U. linza and P. canaliculata , show no activity against any of the seven bacterial strains. In the locations studied, P. canaliculata is only found on the extreme upper shore on exposed rocks and as such experiences extreme conditions from desiccation and exposure to fresh water at low tide to complete submersion in salt water at high tide. Similarly U. linza , which although often protected from desiccation in pools was in this case sampled from exposed rock, is often subjected to a range of salinities ranging from seawater to completely fresh water. The shore locations occupied by these two species are conspicuous with low biodiversity probably due to the extreme environments in which they

74 Chapter 3 thrive. In addition to this, the two species from the lower shore, F. serratus and O. pinnatifida both exhibit both potent and broad spectrum activity. The remaining three species occupy the middle shore zone. One extract from F. vesiculosus shows broad spectrum activity against five of the bacterial strains. The remaining two species showed no antibiotic properties. While it is difficult to identify a definite trend, the seaweeds sampled here from the lower end of the intertidal zone certainly show more antibacterial activity than those on the upper shore where environmental conditions are more extreme.

3.5 Conclusion

The use of an antimicrobial assay system employing ecologically relevant strains and employing environmentally relevant concentrations of extracts allows us to make a number of observations regarding the possible ecological role of seaweed metabolites in mediating bacterial colonisation. The results obtained show that extracts from the majority of the species tested showed activity against at least one strain of ecologically relevant surface associated bacteria, with six species of seaweed showing a broad spectrum of activity over a range of Gram positive and Gram negative bacteria. Since the antimicrobial assays were carried out at environmentally relevant concentrations, it is possible to conclude that these species are able to produce potent antibacterial agents in amounts that are capable of inhibiting bacterial growth, thus confirming that seaweed have the potential to protect themselves from bacterial colonisation using chemically mediated mechanisms. Using a range of extraction solvents to partition initial extracts also provides evidence to suggest that a number of the seaweeds produce more than one antibacterial compound that can act independently on different bacterial strains. As such, these seaweed species have the potential as a source of antifouling compounds.

The ecological niche occupied by the seaweed species also seems to influence antibiotic production. The division of species by location from rock pool to the exposed shore line positions and also the location on the intertidal zone of the seaweed showed clear differences in anti-bacterial activity. All species found in rock pools were shown to exhibit anti-bacterial activity at environmentally relevant concentrations. In the case of seaweed found on exposed rock surfaces, extract extracts of those occupying the lower intertidal zone exhibit more antimicrobial activity than those found in the middile and upper shore

75 Chapter 3 positions. In more stable environments it is possible that seaweed species have developed antimicrobial defence mechanisms whereas in more variable environments it may be simply the case that being adapted to life under the physical stress is enough to ensure a species position with in the ecological niche.

76 Chapter 4

4. Antioxidants from Seaweed

4.1 Introduction

Oxidative stress is a phenomenon that affects all living organisms. Here, highly reactive oxygen containing molecules called reactive oxygen species (ROS) are able to interact and react with cellular components such as proteins, lipids and DNA, which can prove to be harmful to the host organism. ROS are highly reactive due to an unpaired electron in the valance shell, which can occur by a variety of means. ROS are formed in tissues where ·- there is a high metabolic rate, such as mitochondria where the production of O 2 and H 2O2 are prevalent under a process known as oxidative phosphorylation (Perry, et al., 2002). In addition, the presence of trace elements, such as iron and mercury can lead to the 2+ 3+ formation of ROS. Iron when it exists as Fe can easily be oxidised by O2 to Fe producing a superoxide radical. The enzyme superoxide dismutase, uses superoxide to produce hydrogen peroxide which ultimately leads to the formation of the highly reactive and destructive •OH radical as a product of the Fenton reaction (fig. 4.1) (Markesbery, 1997). 2+ + → 3+ + −• Fe O2 Fe O2 −• + + → + 2O2 2H H O22 O2 2+ + → 3+ +• + − Fe H O22 Fe OH OH Figure 4.1. Fenton chemistry reaction

There are of course other ways in which ROS can be formed. For example it is well known that UV light, more specifically UV-B, is of sufficient energy to cause the formation of ROS (fig 4.2), which is the major factor in skin cancer. Fortunately the majority of UV-B and all of the UV-C light is filtered out by the ozone layer (Halliwell and Gutteridge, 2007). Other factors which enhance the production of ROS include alcohol, tobacco smoke, diet, viral infection and biocides (Mena, et al., 2009).

UV→ • H O22 2 OH Figure. 4.2. Formation of hydroxyl radicals by UV-B light from hydrogen peroxide

77 Chapter 4

In humans, ROS are a contributing factor to a number of medical conditions such as liver problems, cancer, atherosclerosis and other cardiovascular conditions, along with the general aging process (Yan, et al., 1998; Zubia, et al., 2009). There is also increasing evidence linking free radicals with the onset of neurological conditions such as Parkinson’s disease and Alzheimer’s disease (Markesbery, et al., 1997; Perry, et al., 2002). Oxidative stress as a result from ROS can eventually lead to cell death (Halliwell and Gutteridge, 2007b).

In order to combat the destructive nature of these ROS, antioxidants provide a degree of protection by reacting with the ROS to form stable adducts. Some of these antioxidants are produced within the body, where as those that are not are acquired through diet. The tripeptide glutathione is a common antioxidant found in the body that is able to quench hydroxyl radicals. Some antioxidants are not mammalian metabolites and are acquired as part of a balanced diet, such as ascorbic acid (vitamin C) and tocopherol (vitamin E), along with various flavanoids and carotenoids being most commonly found in fruit and vegetables (Kaneto, et al., 1999). Type-2 diabetes is a condition that is characterised by the degradation of β-cells by oxidative stress leading to deficiency in insulin production and resulting in hyperglycaemia. Recent research has shown that in type-2 diabetic mice, a treatment with a combination of N-acetyl-L-cysteine, ascorbic acid and tocopherol offers some protection against the oxidation of β-cells (Kaneto, et al., 1999).

Recently the health benefits of antioxidants in the diet have received much publicity. A plethora of research has also been undertaken to investigate the benefits of consuming foods high in antioxidants and to determine which foods contain high levels of antioxidants. Foods that contain high levels of antioxidants are often classed as ‘superfoods’ along with other nutritionally beneficial foodstuffs such as tomatoes, broccoli, berries and green tea. While these foodstuffs contain antioxidant compounds, generally the amounts used in assay studies are far greater that what would be found in even large portions. This would seem to suggest that any potential benefit from consuming the ‘superfoods’ are likely to be minimal (Cancer Research UK, 2010). Although eating these ‘superfoods’ may not be as productive as the media suggest, these foods along with a host of other plants still contain a diverse range of antioxidant compounds that, once purified and developed could provide products with the potential in therapeutic applications. It is

78 Chapter 4 therefore key that the role of antioxidants is further investigated as possible treatments and as preventative measures to control the progression of disease. However it should be noted that consumer preference is for medication to be from natural sources rather than from synthetic compounds, due to consumer fears of negative effects synthetic medication (Zubia, et al., 2009). It should also be noted that natural sources are not perfect either and can also have their own negative side effects.

Although the interest in looking, not only at antioxidants but a whole range of biologically active marine natural products is increasing, the investigation into natural products from British waters has remained in its infancy. Seaweed species that can be found in British waters have been shown to contain a wide range of compounds such as carotenoids, flavonoids and terpenes (Chapter 2). Studies on seaweed from French (Zubia, et al., 2009) and Icelandic (Wang, et al., 2009) waters have shown that extracts from green, brown and red seaweed exhibit radical scavenging properties. The French study investigating solely red seaweed showed that extracts from 24 seaweed species exhibit a wide range of antioxidant activity in the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging assay, which is shown by a colour change from purple to pale yellow by the ● addition of H . The Icelandic study investigated 13 seaweed species with results showing that extracts from brown seaweed are more effective at quenching the DPPH radical than extracts from red or green seaweed. It is also suggested that phenolic acid compounds from the green seaweed Halimeda monile may help to protect the liver from damage by stimulating superoxide dismutase and catalase production in the liver of rats that have sustained liver damage from carbon tetrachloride exposure (Mancini-Filho, et al., 2009).

In this study, the radical scavenging potential of seaweed extracts was measured using two distinct analytical techniques. Firstly using spectrophotometric methods, and secondly using electron paramagnetic resonance (EPR). In the spectrophotometric study, the radical scavenging potential of extracts of 13 intertidal seaweed species (two green, five brown and six red (Table 3.1)) found commonly on the shore of the north of Scotland, was studied. Fractionated extracts were subjected to two different radical scavenging assays: the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging assay and the 2,2- azinobis (3-ethlybenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical cation discolourisation assay. In addition, extracts were examined for their total phenolic

79 Chapter 4 content by a modification of the Folin-Ciocalteu method. In the electron paramagnetic resonance study, the potential of seaweed extracts to quench superoxide and hydroxyl radicals in competition with the Tempone-H spin trap was studied.

4.2 Materials and Method

4.2.1 Materials

Spectrophotometric Methods: Methanol (HPLC grade) and ethyl acetate, hexane and dichloromethane (glass-distilled grade) were purchased from Rathburn Chemicals Limited (Walkerburn, UK). Ethanol was purchased from VWR International (Lutterworth, UK). 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH), 2,2-azinobis (3-ethlybenzothiazoline-6- sulphonic acid) diammonium salt (ABTS), potassium persulphate, gallic acid and anhydrous sodium sulphate were purchased from Sigma-Aldrich (Poole, UK). Folin- Ciocalteu 2M reagent was purchased from Fluka (Germany). Anhydrous sodium carbonate was purchased from Riedel-de-Haen (Germany). Absorption measurements were performed on a CamSpec M350 Double Beam UV-Vis Spectrophotometer.

Electron Paramagnetic Resonance Methods: Tempone-H, ethylenediaminetetraacetic acid (EDTA) and methanol (HPLC grade) were purchased from Sigma Aldrich (Poole, UK). Pyrogallol was purchased from Fisher Scientific (Loughborough, UK) and phosphate buffer solution (PBS) was purchased from PAA (Yeovil. UK). EPR measurements were performed on a Magnettech MS200 spectrometer.

4.2.2 Preparation of Extracts

The extracts that were used in this study were those described as in Chapter 3, section 3.2.2. A few of the extracts were not available in sufficient quantities to carry out the various assays. In these cases individual extracts from the same species were combined. The ethyl acetate and dichloromethane extracts of Pelvetia canaliculata , Polysiphonia

80 Chapter 4 fucoides , Halidrys siliquosa and Fucus vesiculosus were combined. In the case of Corallina officinalis all extracts were combined.

4.2.3 2,2-Diphenyl-2-Picrylhydrazyl Hydrate Radical Scavenging Assay

This assay was carried out using a modified technique of Miliauskas (2004). The assay measures the colour change of the purple DPPH radical to the pale yellow DPPH-H adduct (Fig 4.3).

N N H N +H N NO2 NO2 O2N O2N

NO2 NO2 (Purple) (Pale yellow) Figure 4.3. Quenching of the DPPH radical

Solutions of extracts were made by dissolving the dried extract (25 mg) in methanol (10 ● ml). A methanolic solution of DPPH (6x10 -5 M) was freshly prepared each day by ● ● dissolving 6 mg of DPPH in 250 ml of methanol. 3 ml of the DPPH solution was combined with 77 µl of the methanolic extract in a disposable cuvette. The reaction mixture was kept in the dark for 15 minutes at room temperature after which the absorption ● was measured at 515 nm. A control reading was taken using of 3ml DPPH solution and 77 µl methanol. This assay was performed on three aliquots of each extract. The radical savaging activity is expressed as a percentage of inhibition using the following formula: %I = [( − /) *] 100 AB AE AB

Where %I is the percentage inhibition, AB is the absorbance of the blank sample and A E is the absorbance of the extract solution. The average from each group of three aliquots was taken with the standard deviation being calculated.

81 Chapter 4

4.2.4 2,2-Azinobis (3-Ethlybenzothiazoline-6-Sulphonic Acid) Diammonium Salt Radical Cation Discolourisation Assay

This assay was carried out using a modified metheod of Re (1999), and measured the absorbance change of the dark blue ABTS radical to the colourless ABTS (fig 4.4).

O O O O S S O O NH4 NH4

N S N S

N + K2S2O8 N N N Antioxidant S N S N

O O NH4 NH S 4 S O O O O

(Blue) (Colourless) Figure 4.4. Activation of ABTS by Potassium Persulphate and Radical Quenching

● ABTS (10 mg) was dissolved in water (2.6 ml) to form a 7 mM solution. The ABTS + was formed by activating ABTS (2.6 ml) with a 2.45 mM solution of potassium persulphate (2.6 ml). This solution was left in the dark for 12 hours in order to generate sufficient levels of the radical. The solution was then diluted using ethanol to give a value of 0.700 ● ±0.020 AU on the spectrophotometer at 734 nm. 3 ml of the diluted ABTS + solution was combined with 30 µl of the methanolic extract solution (2.5 mg ml -1) and the absorption recorded every 30 seconds for 6 minutes. This assay was performed on three aliquots of ● each extract. A negative control using 3 ml of the ABTS + solution and 30 µl of methanol was also analysed. The results are presented as a decrease in absorption over time as well as a percentage of inhibition using the following formula: %I = [( − /) *] 100 AB AE AB

82 Chapter 4

4.2.5 Total Phenolic Content (TPC)

The method used was a modification if the technique described by Miliauskas (2004).

A calibration curve was first prepared using gallic acid as a standard phenolic compound. Solutions of Folin-Ciocaleu reagent (diluted 10 fold) and sodium carbonate (75 g l -1) were prepared in advance. Solutions containing varying concentrations of gallic acid were prepared (0.3 mg ml -1, 0.105 mg ml -1, 0.075 mg ml -1and 0.024 mg ml -1). 1 ml of each gallic acid solution was mixed with the Folin-Ciocaleu reagent (5 ml) and sodium carbonate solution (4 ml). The resulting mixture was shaken and then left to stand for 30 minutes. 3 ml of the mixture was then transferred to a cuvette and the absorption of the mixture recorded at 765 nm. This assay was performed on three aliquots of each extract. An average of the three aliquots was taken and the data was plotted as a line graph as gallic acid concentration against absorption.

In order to determine the TPC of seaweed extracts, solutions of each were prepared in methanol to give a final concentration of 2.5 mg ml-1. These were added to the Folin- Ciocaleu reagent (5 ml) and the sodium carbonate solution (4 ml). The resulting mixtures were shaken and then left to stand for 1 hour to react at room temperature, after which the absorption was measured at 765 nm. This assay was performed on three aliquots of each extract. The absorption value obtained using the seaweed extract is used to determine the equivalent concentration of gallic acid needed to give the same absorption from the calibration graph. The following formula is used to calculate the total phenolic content in gallic acid equivalents (GAE): C = (Vc / m) Where c is the equivalent gallic acid concentration determined from the calibration curve (mg ml -1), V is the volume of extract used (ml) and m is the mass of extract used in the assay (g). The average from each group of three aliquots was taken with the standard deviation being calculated.

83 Chapter 4

4.2.6 Ability of Seaweed Extracts to Quench Superoxide Radicals

Solutions of 10 mM EDTA in methanol and 100 mM tempone-H in 10 nM EDTA were made up in advance. A 100 mM solution of pyrogallol in PBS was freshly prepared before each run.

In a 1 ml eppendorf tube, 10 µl of extract solution was mixed with 10 µl of the pyrogallol solution and 10 µl of the tempone-H solution. 970 µl of PBS solution was added to make the mixture up to 1 ml prior to vortexing. Intensity readings were taken on the spectrometer at 0, 15, 30, 45 and 60 mins, vortexing the samples before each reading. A negative control was established using 980 µl PBS, 10 µl tempone-H solution and 10 µl methanol and a positive control contained 970 µl PBS, 10 µl tempone-H, 10 µl methanol and 10 µl pyrogallol solution was used.

It was necessary to first ascertain what concentration of extract would be appropriate to use in this study. Concentrations of the hexane extract of F. serratus at 10 µl ml -1, 0.1 µl ml -1, 1.0 ng ml -1 and 0.01 ng ml -1 were prepared in advance and analysed as described above. On the basis if this, a concentration of 1.0 ng ml -1 was chosen as it showed good activity between both positive and negative controls without the need for further dilution.

4.2.7 Ability of Seaweed Extracts to Quench Hydroxyl Radicals

Method A: In a 1 ml eppendorf tube, 10 µl of extract solution was mixed with 10 µl of a

10 µM solution of H2O2 in water and 10 µl of the Tempone-H solution. 970 µl of PBS solution was added to make the mixture up to 1ml. Intensity readings were taken at 0, 15, 30, 45 and 60 mins. A negative control was established using 980 µl PBS, 10 µl Tempone- H solution and 10 µl methanol and a positive control was 970 µl PBS, 10 µl Tempone-H,

10 µl methanol and 10 µl H 2O2 solution.

84 Chapter 4

Method B: To increase production of hydroxyl radicals 10 µl of a 5 µM of iron (II) chloride solution was substituted for 10 µl of PBS. The Fe 2+ ions present initiate Fenton chemistry which ultimately leads to greater hydroxyl radical production.

4.3 Results

4.3.1 DPPH Assay

A total of 34 extracts from 13 species of seaweed found on the north coast of Scotland were analysed for their radical scavenging activity using the DPPH assay. 13 out of the 34 extracts tested did not reduce the DPPH radical (fig. 4.5). These comprised all three extracts from Cladophora rupestris and Osmundea pinnatifida , the ethyl acetate and dichloromethane extracts of Ulva linza , Palmaria palmata , Plocamium cartilagineum and the dichloromethane extract of Fucus serratus . As a general trend the greatest reducing power was observed with the ethyl acetate extracts, with the hexane extracts showing more reducing power than the dichloromethane extract. The combined EtOAc/DCM extracts of both H. siliquosa and P. fucoides showed the highest percentage inhibition, exhibiting 91.1% and 92.5% inhibition respectively. The hexane extracts of H. siliquosa and P. fucoides and the EtOAc extract of Chorda filum gave inhibition values of 55.2%, 69.2% and 60.7% respectively. The EtOAc extract of F. serratus and the EtOAc/DCM extracts of F. vesiculosus and P. canaliculata gave values of 46.0%, 43.0% and 48.5% respectively. All other extracts gave inhibition values of under 40%. From the results, it is clear that ● extracts from brown seaweed show greater radical scavenging activity against the DPPH radical, whereas extracts from both green and red seaweed have either little or no affinity ● towards DPPH with the exception of P. fucoides .

85 Chapter 4

Figure 4.5. Percentage inhibition of the DPPH radical when exposed to seaweed extracts with standard deviation of each extract

86 Chapter 4

4.3.2 ABTS Assay

The blank sample showed that there is some natural quenching of the ABTS •+ radical in solution with a reduction in absorption of 1.8% over 6 minutes. Taking into account the natural quenching of ABTS •+ , it is clear that all extracts show at least some radical scavenging activity towards ABTS •+ (fig. 4.6 to 4.8). The hexane extracts of the seaweed extracts showed varying amounts of antioxidant activity. The extract from H. siliquosa showed the strongest activity after six minutes with 94.1% inhibition of the radical (fig.6 and 9). Hexane extracts of F. vesiculosus , C. crispus and P. fucoides showed inhibition of 42.0%, 45.0% and 49.4% respectively. All other extracts displayed inhibition levels of less than 40%.

Figure 4.6. Radical quenching ability of hexane extracts against the ABTS radical over monitored over six minutes

The ethyl acetate extract (including the combined ethyl acetate and DCM extracts) of each of the five brown seaweed showed inhibition levels of at least 82.9%, (fig. 4.7 and 4.9) with the extract from H. siliquosa inhibiting 99.9% of ABTS •+ . One final extract showed

87 Chapter 4 activity above 50% and that was the ethyl acetate extract of P. fucoides that exhibited 67.9% inhibition. It is striking that ethyl acetate extracts from H. siliquosa , F. serratus and F. vesiculosus showed such high levels of inhibition.

Figure 4.7. Radical quenching ability of ethyl acetate and combined extracts (c) against the ABTS radical over monitored over six minutes

Dichloromethane extracts showed less radical scavenging activity that the hexane and ethyl acetate extracts, with six of the seven extracts showing less than 15% activity. Only the extract from C. rupestris showed activity above 15% (fig. 8 and 9).

88 Chapter 4

Figure 4.8. Radical quenching ability of dichloromethane extracts against the ABTS radical over monitored over six minutes

Extracts of brown seaweed again show more radical scavenging activity than both red and green seaweed but not to the same extent as with the DPPH assay. Both green and red ● seaweed are able to quench the ABTS •+ radical more effectively than the DPPH in all extracts except for extracts of P. fucoides .

89 Chapter 4

Figure 4.9. Percentage inhibition of the ABTS radical when exposed to seaweed extract with standard deviation of each extract

90 Chapter 4

4.3.3 Total Phenolic Content

In a number of cases, due to extracts apparently containing large amounts of phenolic compounds, the intense blue colour produced did not allow the passage of sufficient light through the sample to successfully carry out the assay. It was therefore necessary to dilute the combined ethyl acetate and DCM extracts of P. canaliculata , P. fucoides and H. siliquosa from a concentration of 2.5 mg ml -1 to 1 mg ml -1, and the combined ethyl acetate/DCM extract of Fucus vesiculosus to 0.5 mg ml -1 so that a reading on the spectrophotometer could be obtained. It was also necessary to dilute the ethyl acetate extract of Chondrus crispus and the dichloromethane extracts of F. serratus and C. rupestris to a concentration of 1 mg ml -1, due to only small amounts of material remaining from previous assays. The phenolic content of the dichloromethane extracts of U. linza , C. filum and P. cartilagineum were not assessed due to a lack of material.

The amount of phenolic compounds found in the case of hexane extracts ranged from 15.6 mg g -1, in P. cartilagineum , up to 133.2 mg g -1, in F. vesiculosus (fig. 4.10). It is also worth noting that 10 of the hexane extracts had very similar levels of phenolic compounds with values of between 29.0 to 49.5 mg g -1. H. siliquosa contained 72.5 mg g -1of phenolic compounds.

The ethyl acetate extracts as well as the combined ethyl acetate/DCM extracts, on the whole contained more phenolics than their hexane counterparts with the exception of U. linza , P. palmata and P. cartilagineum which were shown to have low levels of phenolic compounds in their EtOAc extract. Over half of the extract from the EtOAc extract of F. vesiculosus was phenolic compounds. With a value of 510.3 mg g -1of seaweed extract, this is significantly higher than other extracts assessed.

91 Chapter 4

Figure 4.10. Total phenolic content of seaweed extracts with standard deviation of each extract

92 Chapter 4

4.3.4 Quenching Ability of Seaweed Extracts against Superoxide using Electron Paramagnetic Resonance

Initially a concentration of 1.0 mg ml -1 of seaweed extract was used. However this proved to be too concentrated and after 15 minutes the intensity readings for all extracts were above that of the positive control (Auto). It was necessary therefore to ascertain what concentration of extract would be appropriate to use in this study. Solutions of the hexane extract of F. serratus at 10 µl ml -1, 0.1 µl ml -1, 1.0 ng ml -1 and 0.01 ng ml -1 were prepared. 10 µl of these solutions replaced the 10 µl of methanol in the control samples. A concentration of 1.0 ng ml -1 was chosen as it showed good activity between both positive and negative controls without the need for further dilution (fig. 4.11).

Figure. 4.11. Superoxide scavenging of different concentrations of the hexane extract from F. serratus as shown as a reduction in intensity of tempone.

The same extracts were performed again at the lower concentration of 1.0 ng ml -1. This time all extracts were within the positive and negative controls after 60 minutes (fig. 4.12). The hexane extract of F. vesiculosus showed a 43.9% reduction of tempone compared to the positive control, showing that this extract has a strong ability to quench superoxide

93 Chapter 4 radicals. In addition the EtOAc extracts of F. serratus and F. vesiculosus showed a 34.5% and 21.3 % reduction of tempone compared to the positive control. Finally the hexane extract of F. serratus and H. siliquosa showed a 26.0% and a 13.6% reduction of tempone compared to the positive control.

Figure 4.12. Superoxide scavenging of seaweed extracts as shown as a reduction in intensity of tempone using EPR

The hexane extract of O. pinnatifida and C. rupestris and EtOAc extracts from H. siliquosa , O. pinnatifida and C. rupestris were also assessed for their ability to quench superoxide radicals. However the EtOAc extract of H. siliquosa was the only extract to be observed within the two controls with the others show reading high than that of the positive control.

It was clear from the results so far, that problems were occurring by showing greater intensities of tempone than the positive control. Many of the extracts appeared to be behaving as pro-oxidants (above pyrogallol positive control line). One possibility was that the majority of the extracts just do not quench superoxide radicals. It was therefore decided to investigate the extracts activity against hydroxyl radicals.

94 Chapter 4

4.3.5 Quenching Ability of Seaweed Extracts against Hydroxyl Radicals using Electron Paramagnet Resonance

This assay was first run using method A with the hexane and EtOAc extract of O. pinnatifida and C. rupestris , together with the EtOAc extract of H. siliquosa . The extracts were tested for hydroxyl radical scavenging, but the generation of hydroxyl radicals proved to be difficult. The same extracts were then subjected to the assay using method B. This method employs FeCl 2 to aid the formation of hydroxyl radicals using the Fenton reaction. However, after 30 minutes it was clear that all samples were outside of the control parameters and the run was terminated. It was obvious that there was a significant problem in generating hydroxyl radicals to a sufficient level that would allow for an effective evaluation of the extracts quenching abilities. This is perhaps due to natural decomposition of H 2O2 that was used in this experiment to H2O and O 2. Due to this, any results that were obtained in this experiment were unfortunately unreliable and therefore not presented here.

4.4 Discussion

4.4.1 Radical Scavenging Activities of Different Seaweed Classes

It is clear that the extracts from brown seaweed show higher radical scavenging activities than extracts of both green and red seaweed. This is most notable with the DPPH assay where, with the exception of P. fucoides , green and red seaweed show no more than 8.4% inhibition. This is less noticeable with the ABTS assay, however again with the exception of P. fucoides , only brown seaweed show scavenging ability of above 50% against ABTS •+ . The absence of radical scavenging activity in green seaweed suggests that they contain small amounts of compounds that are capable of radical scavenging. This is reflected in Chapter 2.2 where no radical scavenging activity was identified in compounds isolated from green seaweed. Despite large amounts of research on metabolites of red seaweed and their associated bioactivity, there seems to be a distinct lack of radical scavenging activity associated with compounds (Blunt, et al., 2010). This is confirmed in the present study and in other studies that also show poor radical scavenging activity of extracts from red seaweed (Wang, et al., 2009).

95 Chapter 4

4.4.2 Radical Scavenging Activity of Different Solvent Extracts

Of the eight seaweed species producing enough DCM extract for analysis, only three species showed activity towards the DPPH radical and all showed less than 25% inhibition of the ABTS radical. This shows that the DCM extracts contain small amounts of compounds that are capable of antioxidant activity. In contrast, EtOAc extracts in almost all extracts tested showed the highest radical scavenging activity, confirming that these extracts are rich in compounds capable of radical scavenging activities.

4.4.3 Correlation of Total Phenolic Content and Radical Scavenging Activity

A plot of the TPC against radical scavenging activity for all of the extracts tested (fig 4.13) suggest that there is no direct correlation between the two (DPPH R 2 = 0.27, ABTS R 2 = 0.49). While this is the case, it is possible to conclude that those extracts containing more than 100 mg g -1 of phenolic compounds, as measured as TPC, tend to have significant radical scavenging activity. Additionally very little activity is observed below TPC concentration of 29 mg g -1.

Figure 4.13. Correlation between the TPC and % inhibition of the ABTS and DPPH radicals for seaweed extracts

96 Chapter 4

Several studies have shown a relationship between TPC and radical scavenging activity as measured by the DPPH assay for both seaweed (especially red seaweed) and terrestrial plants (Duan, et al., 2006; Manchini-Filho, et al., 2009; Miliauskas, et al., 2004; Yuan, et al., 2005; Zhang, et al., 2007; Zubia, et al., 2007, 2009). There is little work on linking TPC with the scavenging of the ABTS radical from green and brown seaweeds.

These reports somewhat contradict the current study where TPC does not seem to correlate to the radical scavenging activity of either hexane or the ethyl acetate/DCM extracts of red seaweed against the DPPH radical (fig. 4.14). With the exception of P. fucoides , extracts showed little or no ability (<8.4%) to quench the DPPH radical, despite the TPC concentration for red seaweed reaching to 86.6 mg g-1. Thus we can conclude that phenolic compounds from red seaweed tend not to contribute towards radical scavenging activity of the DPPH radical.

Extracts of red seaweed seem to have more of an affinity towards the ABTS radical (fig. 4.14). However four of the hexane extracts all show similar levels of TPC (between 39 and 48 mg g -1 of extract) but show antioxidant activity ranging from 5-50%, suggesting that the radical scavenging activity could be independent of TPC. Bearing this in mind there seems to be a strong link between TPC and the radical scavenging activity of ethyl acetate/DCM extracts from red seaweed against the ABTS radical (R 2=0.84).

97 Chapter 4

Figure 4.14. Correlation between the TPC and % inhibition of the ABTS and DPPH radicals for red seaweed extracts

There appears to be a trend between radical scavenging activity and TPC with extracts from brown seaweed, with an increase in TPC generally corresponding to an increase in radical scavenging activity (R 2=0.37 and R 2=0.19) (fig. 4.15). Indeed previous research has shown that for six Icelandic species, including both Fucus species examined in this study, that TPC relates to radical scavenging activity (Wang, et al., 2009), although an earlier study showed that TPC and radical scavenging maybe independent of each other (Chandini, et al., 2008).

98 Chapter 4

Figure 4.15. Correlation between the TPC and % inhibition of the ABTS and DPPH radicals for brown seaweed extracts

Both the hexane and the EtOAc/DCM extracts of F. vesiculosus show much higher levels of TPC than similar extracts from other brown seaweed. It is therefore not surprising that this extract resulted in 95% inhibition of the ABTS radical. However the same extract only inhibits the DPPH radical 43%. The hexane extract of F. vesiculosus shows activity of about 40% less than what would have been expected for the observed TPC (Fig. 4.16).

99 Chapter 4

Figure 4.16. Correlation between the TPC and % inhibition of the ABTS and DPPH radicals for different solvent extractions of brown seaweed

The extracts of F. vesiculosus show much lower activity towards DPPH radicals than would normally be expected and the hexane extracts show much lower activity against ABTS radical. Indeed these results significantly skew any obvious relationship between TPC and radical scavenging powers. What this suggests is that the phenolic constituent of F. vesiculosus contains large amounts of phenolic compounds that are poor at radical scavenging. Despite the values obtained for F. vesiculosus, the other four brown seaweed show a clear trend between TPC and radical scavenging activity, with a linear trend against inhibition of the DPPH radical (R2=0.81) and a logarithmic trend showing a curve of best fit, against the ABTS radical (R 2=0.83) (fig. 4.17).

100 Chapter 4

Figure 4.17. Relationship of Scavenging activity and TPC of brown seaweed except for F. vesiculosus

While little correlation between radical scavenging activity and TPC is evident when considering the brown seaweed extract as a whole, there does seem to be some tendency, in the plot of % inhibition against TPC, for the activity of similar extracts against the same radical to group (fig 4.18).

101 Chapter 4

Figure 4.18. Grouping of similar extracts with similar radical scavenging activity and TPC from brown seaweed

Previous work on the green seaweed Halimeda monile suggests that an increase in TPC corresponds to increased radical scavenging activity (Manchini-Filho, et al., 2009). From the small selection of green seaweed studied, there does not appear to be a relationship between TPC and inhibition of the DPPH radical, however there does seem to be a trend that increased phenolic content corresponds to an increase in radical scavenging power against the ABTS radical (fig. 4.19). Interestingly the hexane and DCM extracts, all having similar TPC’s but very different levels of antioxidant activity against the ABTS radical. This would seem to suggest that the radical scavenging activity of the extract is independent of the TPC for hexane and DCM extracts. The data points from the ethyl acetate/DCM extracts are interesting as both samples have very different TPC levels with neither sample quenching the DPPH radical and the sample with higher TPC also showed higher inhibition of the ABTS radical then the sample with little TPC. It is clear that the two ethyl acetate/DCM extracts heavily influence the overall observed trend of extract activity against the ABTS radicals.

102 Chapter 4

Figure 4.19. Correlation between the TPC and % inhibition of the ABTS and DPPH radicals for green seaweed extracts

4.4.4 Radical Scavenging Activity and Structure of Radicals

The striking difference between the results obtained from both radical scavenging assays for many of the extracts is interesting. The difference can potentially be explained by the structure of the two radicals. The ABTS radical is a planer molecule with relatively little steric hindrance, this allows for incoming radical scavenging compounds to easily approach and quench the radical. In contrast, the reaction site on the DPPH radical is significantly more hindered with two benzene groups and two nitro groups in close proximity. These factors may be unfavourable for potential radical scavenging compounds and may result in no quenching of the radical or for quenching to occur at a slower rate. Many phenolic compounds that are found in macroalgae (Chapter 2) are bulky and may find it difficult to approach the more hindered DPPH radical compared to the ABTS •+ . This suggests that extracts from P. fucoides and those of brown seaweed may contain a group of small, but effective radical scavenging compounds that are not present in green and other red seaweed, with these seaweeds perhaps containing more bulky phenolic

103 Chapter 4 compounds. Alternatively these small effective radical scavenging compounds may be present in significantly larger amounts than in the green and other red seaweeds.

An interesting observation that can be made with these results is that each seaweed extract, except for extracts of P. fucoides , shows more affinity in scavenging the ABTS radical than the DPPH radical. Extracts from P. fucoides are able to reduce the DPPH radical at least 20% more than that of ABST radical. It is clear from theses observations that P. fucoides is unique on two accounts. Firstly of the 13 seaweed species examined here it is the only one which produces an extract which showed greater activity in the DPPH assay than the ABTS assay. Secondly, among the red seaweed, it is the only species to show significant levels of activity in both the DPPH and ABTS radical scavenging assays. A species similar to P. fucoides , P. stricta has been shown to also have potent DPPH radical scavenging properties with the ethyl acetate extract showing 93.9% inhibition at a concentration of 50 µg ml -1. The TPC of the ethyl acetate extract was 73.7 mg g -1 of dried seaweed matter (Duan, et al., 2006).

The work described in Chapter 3 suggests that there is a trend between antimicrobial activity and the position of the seaweed on the shore. No such correlation between radical scavenging activity of the seaweed extract and the position of the seaweed on the shoreline was found (Chapter 3).

4.4.5 Electron Paramagnetic Resonance

Although the use of the DPPH and ABTS radical scavenging assays are common in natural product chemistry, the use of EPR to asses the radical scavenging activity is much less common. There have been recent publications using enzymatic extracts from brown seaweed species to effectively quench superoxide and hydroxyl radicals using a spin trapping method (Ahn, et al., 2004; Je, et al., 2009). However it seems that the study documented here is the first to assess the quenching of superoxide and hydroxyl radicals by extracts of F. serratus, F. vesiculosus and H. siliquosa .

The EPR method used here measures the amount of tempone in the reaction mixture which is formed by either superoxide, generated from pyrogallol, or the hydroxyl radical, generated from H 2O2 reacting with tempone-H (fig. 4.20). The presence of an antioxidant

104 Chapter 4 in the reaction mixture competes with the tempone-H for the ROS and thus, the less tempone observed the greater radical scavenging activity. O O ROS

N N O O H Tempone-H Tempone Figure 4.20. Oxidation of tempone-H to tempone

There are a number of ways in which tempone can be reduced back to tempone-H such as with enzymes, thiols and vitamin C. (Dikalov, et al., 1997a). Previous research on the amount of vitamin C within seaweed shows that species from all three phyla and from intertidal and subtidal zones contain varying amounts of vitamin C (Norris, et al., 1936). Although the seaweed in this study are different to those used by Norris (1936), it should be acknowledged that there is the possibility that samples may also contain varying amounts of vitamin C. As vitamin C is an antioxidant compound, it would also react with the radicals, which would then no longer be able to react with tempone-H. However, it is unclear as to whether vitamin C is more effective at scavenging the superoxide radical or tempone. Should vitamin C be more effective at scavenging tempone then this would lead to less tempone being identified from the EPR results which could be interpreted as greater radical scavenging activity. However what is observed in many cases is contrary to this with extracts showing results above the positive control, in other words more tempone-H is being oxidised to tempone than in the positive control. In these cases, this suggests that extracts of seaweed are in fact pro-oxidant rather than antioxidant in this case.

Polyphenolic compounds are known to readily oxidise to produce superoxide and hydrogen peroxide usually in the presence of transition metals (Halliwell, 2008). When higher concentrations of seaweed extracts were being used, this pro-oxidant effect was allowing the generation of superoxide and hydrogen peroxide which were then being reacted with tempone-H to give the very large signals for tempone that were originally observed. With lower concentrations of extract, the level of superoxide and hydrogen peroxide being produced from the oxidation of polyphenols can be minimised allowing for scavenging of superoxide to be evaluated.

105 Chapter 4

A relationship between TPC and superoxide scavenging properties is again compromised by the very high TPC of the ethyl acetate extract of F. vesiculosus and its relatively low radical scavenging activity. However, the hexane extract of F. vesiculosus fits within a relationship with extracts of F. serratus and the hexane extract of H. siliquosa . Excluding the ethyl acetate extract of F. vesiculosus from this relationship, a very strong correlation (R 2 = 0.94) is observed between TPC and superoxide scavenging of the four extracts (fig. 4.21). This suggests that the hexane extract of F. vesiculosus is better as scavenging superoxide than both the ABTS and DPPH radicals compared to the other tested extracts.

Figure 4.21 Correlation between the TPC and % inhibition of superoxide for hexane extracts of F. serratus, F. Vesiculosus and H. siliquosa and EtOAc extact of F. vesiculosus

On removal of the remaining ethyl acetate extract, an examination of the relationship between activity of the three hexane extracts of F. serratus, F. Vesiculosus and H. siliquosa against TPC suggest a very strong correlation (R 2=1.00)(fig 4. 22). This suggesting that superoxide scavenging activity is dependent on the TPC of the seaweed extracts.

106 Chapter 4

Figure 4.22. Correlation between the TPC and % inhibition of superoxide from hexane extracts of F. serratus, F. Vesiculosus and H. siliquosa

There are of course no results for activity against the hydroxyl radical due to no generation of the radical from the hydrogen peroxide. However a previous study has shown that hydroxyl radicals are scavenged effectively by enzymatic extracts of the brown seaweed Undaria pinnatifida (Je, et al., 2009).

4.5 Conclusion

This study shows that extracts from red, green and brown seaweed are able to quench a number of very different free radicals. Green and red seaweed extracts with the exception of P. fucoides showed poor radical scavenging activity against the DPPH radical with the same extracts showing marginally better activity against the ABTS radical. Extracts of brown seaweed showed significantly better inhibition of both radicals. Five extracts showed >90% inhibition of the ABTS radical with H. siliquosa inhibiting the DPPH radical by 91.1%.

The general consensus from the literature is that there is a relationship between TPC and radical scavenging activity and the results in this study tend to confirm this position.

107 Chapter 4

However the current study also shows that such a relationship is not as straight forward as previous literature suggests. Factors such as the class of seaweed, the type of extraction method or type of extract, and finally the radical the extract is tested against are all shown to have an effect on any observed relationship. The TPC of extract from brown seaweed seem to influence activity against both ABTS and DPPH radicals. Radical scavenging activity of red seaweed extracts seem to be dependent on TPC against the ABTS radical but not the DPPH radical. Green seaweed also tends to show this pattern, however the small sample set should be increased to reinforce this observation.

The tested extracts from brown seaweed have been shown to inhibit the superoxide radical with this activity being dependant on TPC. There is the potential that natural products from these seaweed could be isolated and studied in greater detail with a prospect of them being used as commercial products in the health care industry.

The pro-oxidation effect that is observed cannot be overlooked. Phenolic compounds can and will undergo oxidation producing superoxide and hydrogen peroxide. Consequently the more phenolic compounds present in the sample the more superoxide and hydrogen peroxide generated. This is seen with the extracts of F. vesiculosus that were heavily affected by pro-oxidation of the phenolic compounds. The pro-oxidation of phenolic compounds suggest that there may be an optimum level of the phenolic that can be present before the antioxidant nature of these compounds are out competed by the oxidation of the phenolics. With current health advice suggesting that consumption of polyphenolics, usually through fruit, vegetable and teas that are rich in phenolic compounds could be of benefit to health, the results of the EPR assay suggests that this could result in a pro- oxidanat effect. This highlights the importance not to rely on the results from using a single radical to draw conclutions on antioxidant activity and to employ the use of several radicals to evaluate extracts and compounds for their antioxidant activity.

108 Chapter 5

5. Establishing a Screening Network to Assess the Bioactivity of Seaweed Extracts

5.1 Introduction

5.1.1 Identification of Leads

The screening of extracts and purified compounds to identify their bioactivity has been at the very heart of drug discovery from natural sources. This strategy has employed crude extracts, fractionated extracts and isolated natural products from a range of plants, bacteria and fungi. Increasingly, strategies have been developed employing a vast array of ever evolving assays in order to identify bioactive lead molecules for drug discovery programmes. As a result of the completion of the human genome it has been suggested that the potential number of drug targets in humans could range from 2500 to 5000. This compares with approximately 500 targets currently identified (Rollinger, et al., 2008).

There are several strategies for screening natural products for bioactivity and these can broadly be separated into physical and virtual screening strategies. Virtual screening uses a combination of 3D structural data from natural products and biological receptors to identify lead compounds which best fit specific molecular targets. This therefore requires significant work to establish full structural data for both the target receptor and a range of natural products. However once a target is stored digitally it is available for future use with virtually no maintenance required unlike with physical assays. Natural products that have undergone structure elucidation can quickly be screened against virtual receptors to identify leads (Rollinger, et al., 2008). Virtual screening has also greatly benefited from the advancement in analytical technologies. Mass spectrometry and NMR sensitivity has greatly improved over the last two decades allowing structures to be fully characterised from as little as a few nanograms of material (Harvey, 2007).

Physical screening on the other hand attempts to establish the efficacy of extracts, fractionated extracts or pure natural products on biological systems, isolated enzymes or

109 Chapter 5 targets designed to mimic a biological system. Traditional approaches of screening, where single extracts or fractionated extracts are assayed against single targets are now considered to be a slow and relatively inefficient strategy in drug discovery (Harvey, 2007). It can be considered a somewhat of a serendipitous approach as it relies on the chance that the right targets be employed in the first place. High throughput screening (HTS) utilises robotic and automated technology which increases the rate of assessment of samples, decreasing the amount of time an assay is in operation and increases the diversity of assays employed (Cordell, 1995). HTS however requires access to extensive natural product libraries which are costly to establish and maintain both in terms of time and money (Koehn, 2008).

It is generally accepted that the chance of identifying a successful hit in a screening programme is can be summarised on the basis of equation 5.1 (Pieters and Vlietinck, 2004).

Probability of Finding an Active Compound = S x CD x A

Equation 5.1. Probability of finding an active compound

Where S is the number of samples (extracts, pure compounds) in your library, CD is the chemical diversity of the library and A is the number of different assays you employ in your screening programme. In an ideal situation, it is best to optimise all three factors in order to maximise the chance of success. As ideal situations rarely exist, an appropriate balance of these factors needs to be reached. Analysing this approach in respect to the work carried out here, the chemical diversity, assuming that chemical diversity is some function of biological diversity, is fixed by the fact that only seaweed species from the Caithness coast were selected for study. From the work of McDougall (McDougall, 2004) this limits the number of species available for study to 129 seaweed species. The number of samples, in the case of crude extracts prepared by a single extraction method, is also limited by the relatively small number of sample species. The number of samples that can be prepared is always influenced by time and resources, such as the availability or not of automated extraction technologies such as Accelerated Solvent Extraction (ASE). In this case the effort available for the preparation of samples is that of a single individual over a limited period of time. This is in contrast to larger efforts at library development such as

110 Chapter 5 that carried out by the Developmental Therapeutics Program of the National Cancer Institute (NCI) in the USA who have a repository of 400,000 chemicals (National Cancer Institute, 2006). While it would be ideal to study all species found on the coast, the time and resources required to collect and process the seaweed is prohibitive for this study and only a subset can be studied. Additionally, undertaking even just the one assay, on such a large sample set would take a significant effort to complete. Even in the case of the NCI above they have screened only 80,000 of their 400,000 compounds. Therefore in this case, using equation 5.1, accepting that chemical diversity is fixed by studing seaweed from Caithness and the number of samples that can be processed by an individual is fixed by time, the probability of finding an active compound is now directly proportional to the number of assays employed. As a result it is now logical to increase the number of different assays that can be performed. While there are only a finite number of in-house assays that can be performed, as described in previous chapters, it was obvious that efforts must be made to maximise the number of those that can be performed by employing assays carried out by external organisations (both academic and commercial).

5.1.2 The Screening of Seaweed Extracts

As mentioned before the screening of seaweed extracts for bioactivity is not without precedent. The most commonly applied assays are those developed to identify antimicrobial activity and anti-cancer activity, although a number of other assays have been applied on the less widespread basis.

5.1.2.1 Antimicrobial Activity

From the literature it is clear that a large number of extracts have been tested for activity against human pathogenic bacteria. Indeed 44 species of seaweed from the Canary Islands were screened for their antimicrobial activity against a battery of nine different bacterial, fugal and yeast strains. Most seaweed extracts showed strong activity towards Staphylococcus aureus and Bacillus subtilis , however the other microbes appeared to be more resilient to the extracts (Gonzalez de Val, et al., 2001). Species from India, Mexico

111 Chapter 5 and Spain have also shown promising activity against S. aureus , B. subtilis , B. cereus but little activity against E. coli (Freile-Pelegrin and Morales, 2004; Salvador, et al., 2007; Kandhasamy and Arunachalam, 2008). A selection of red, green and brown British seaweed species have been tested for activity against the organism that causes TB, Mycobacterium tuberculosis , with an MIC of above 256 µg ml -1 (Spavieri, et al., 2010; Allmendinger, et al., 2010)

One of the more pressing issues within health sector is the increased prevalence of infections caused by the so called ‘super bugs’ such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). In these cases the bacteria have developed a resistance to traditional therapeutic agents. This has led to increased interest in finding compounds that are sufficiently active against MRSA. A recent study from Korea has highlighted the potential of seaweed as source of compounds active against anti-MRSA, with 13 out of 32 species exhibiting some activity against one or more strains of MRSA (Kim and Lee, 2008).

Seasonality has also been highlighted as a factor when screening seaweed extracts for antimicrobial activity. Specimins of Ulva lactuca , U. compressa , Cystoseira mediterranea C. usneoides and Sacchoriza polyschides sampled in August showed high antimicrobial activity against E. coli , S. aureus and Enterococcus faecalis while samples collected in February which shown no activity towards the same strains (Ibtissam, et al., 2009). A study by Hornsey and Hide, where samples of eleven seaweed species were collected each month for a year and subjected to antimicrobial screening against S. aureus showed variable monthly activity during the study period for certain species. Acetone extracts of Chondrus crispus , Laminaria digitata and Osmundea pinnatifida showed stronger activity against S. aureus over the winter months whereas extracts of Codium fragile , Dilsea carnosa , Dictyota dichotoma and Ascophylym nodosum showed stronger activity over the summer months (Hornsey and Hide, 1976a).

5.1.2.2 Cytotoxic Activity

A large proportion of the compounds which have been isolated from seaweed have had their cytotoxic potential evaluated against an array of cell lines (Chapter 2). It is not only

112 Chapter 5 pure isolated compounds that are assessed for cytotoxicity, extracts of many plants including seaweed are routinely screened as a first step in natural product isolation. Extracts of Brazilian red, green and brown seaweed have been shown to be active against the C32 skin cancer cell line with the benefit that many of the extracts do not damage to normal healthy cells (Rocha, et al., 2007). Extracts from the green seaweed Ulva prolifera , commonly found on the shores of the UK, displayed anti-carcinogenic properties both in- vivo and in-vitro (Hiqashi-Okaj, et al., 1999).

5.1.2.3 Miscellaneous

Different allergies, with a variety of symptoms affect a significant proportion of the population. Extracts from 20 species of seaweed from Cheju Island, (South Korea), have been shown to reduce the release of the amino acid histamine from cells which cause many of the symptoms associated with allergies (Lee, et al., 2006). African sleeping sickness is caused by the protozoa Trypanosoma brucei and affects 500,000 people in sub-Saharan Africa with a significant mortality rate. Extracts from British seaweed have been shown to act effectively against the protozoa. (Spavieri, et al., 2010; Allmendinger, et al., 2010)

A hexane extract of Plocamium cartilagineum showing strong anti-fungal activity (Stierle and Sims, 1979) and aqueous extracts from the Delesseriaceae family have been shown to possess a significant anticoagulant effect. However it is not clear what chemical is responsible for these effects (Poss and Belter, 1988; Yvin, et al., 1982).

Methanol extracts Ulva intestinalis and U. lactuta , were both tested for, antifungal, phytotoxicity and insecticidal activity. The methanol extracts from U. lactuta showed significant phytotoxic activity and a moderate antifungal response across a wide range of fungi. U. lactuta also showed a small positive result in the insecticidal assay. The active natural products have not been identified (Rizvi and Shameel, 2005).

113 Chapter 5

5.1.2.4 Screening Seaweed Species from the United Kingdom

There is limited literature on potential bioactivity of seaweed from the waters around the British Isles with the studies of Hornsey and Hide (1974, 1976a and 1976b) being the most comprehensive to date, despite being 35 years old. More recent studies by Spavieri, et al. (2010) and Allmendinger, et al. (2010) have explored seaweed sampled from Finavarra, (Ireland), Seacombe, (England), and Kimmeridge Bay, (England) for cytotoxicity and antiparacytic activity. Samples showed some activity towards Trypanosoma brucei rhodesiense and Trypanosoma cruzi .

There is little evidence of any wide ranging screening program being undertaken on seaweed extracts. It is known that geographical location and consequently the local environment plays a large part in which chemicals are present in a sample, thus making seaweed from waters around Scotland good candidates for a screening exercise.

5.1.3 Developing a Screening Programme

As already described, to increase the potential for a “hit” from a screening programme, the number of assays undertaken should be increased. In common with many natural products labs the ability to perform in house assays in using medically relevant isolates such as with E. coli and cancer cell lines either in a traditional set-up or employing HTS is limited. In order for this goal to be achieved, a network of collaborators willing to perform a number of assays, ideally using HTS, on seaweed extracts needed to be established.

Several considerations need to be taken in to account when deciding where to send extracts for analysis. Cost, turn around time and how many assays can realistically be performed must all be considered. Initial discussions were carried out with four organisations (two academic and two commercial) regarding the prospects of them carrying out assays. Of these only two were taken forward for a variety of reasons. Strathclyde Innovations in Drug Research (SIDR), a branch of the Strathclyde Institute of Pharmacy and Biomedical Sciences of Strathclyde University, Scotland, and Glycomar, a marine biotechnology company based in Oban, Scotland, both offered to carry out assays with out charge. SIDR

114 Chapter 5 required entering into a co-operation agreement, meaning that should anything worthwhile being discovered then efforts should be made to exploit this. Glycomar on the other hand were prepared to assess the extract as a favour in kind. With both institutions employing HTS it was assured that assays would be processed as soon as possible. With these assays being performed free of charge, it was made clear by both institutions the they would considered these samples to be of a lower priority and consequently turn around times would be longer than may be expected from a commercial service.

SIDR run 45 assays as a commercial service, including cell based assays to test for cytotoxicity, bacterial infections, glucose up-take and anti-inflammatory properties. They deploy isolated enzyme assays for fungal targets, obesity and diabetes, Gram negative bacteria and Alzheimer’s disease. A range of radioligand binding assays are used to show activity against a number of neurological conditions such as schizophrenia, Parkinson’s and Alzheimer’s disease. Glycomar were prepared to undertake the XTT cytotoxic viability assay using HeLa cells.

5.2 Materials and Methods

5.2.1 Materials

Methanol (HPLC grade), ethyl acetate (glass-distilled grade) were purchased from Rathburn Chemicals Ltd. (Walkerburn, UK). Milli-Q water was used unless otherwise stated. Sodium sulphate was purchased from Sigma-Aldrich (Poole, UK).

5.2.2 Preparation of Extracts

In this study seaweed were processed differently to those described in chapters 3 and 4. All seaweed were collected locally. Table 5.1 summarises the seaweed collected along with metadata regarding their collection.

115 Chapter 5

Shoreline Location of Date Species Class Exposure Position Sample Collected Thurso Cladophora rupestris Chlorophyceae All Rock Pool 22/01/08 East Kiess Codium fragile Chlorophyceae Middle Rock Pool 10/04/08 Harbour Kiess Ascophyllum nodosum Phaeophyceae Middle Sheltered 10/04/08 Harbour Exposed rock Thurso Fucus serratus Phaeophyceae Lower 22/01/08 surface East Exposed rock Thurso Fucus vesiculosus Phaeophyceae Middle 22/01/08 surface East Thurso Halidrys siliquosa Phaeophyceae Middle Rock Pool 22/01/08 East Exposed rock Thurso Pelvetia canaliculata Phaeophyceae Upper 22/01/08 surface East Exposed rock Thurso Chondrus crispus Rhodophyceae Middle 22/01/08 surface East Middle- Thurso Corallina officinalis Rhodophyceae Rock pool 22/01/08 Lower East

Furcellaria lumbricalis Rhodophyceae Unknown Unknown Unknown 2/04/09

Thurso Polysiphonia fucoides Rhodophyceae Middle Rock pool 22/01/08 East

Table 5.1. Summary of seaweed collected

Upon collection the samples were returned to the laboratory, where they were cleaned and identified using standard field guides. The seaweed were then cut in to pieces approximately 2.5 cm long, placed in a conical flask and covered with methanol. After 24 hours the methanol was decanted and the extraction process repeated a further two times to give a total of three extracts which were combined. The combined extracts were concentrated under vacuum and the residue diluted with water (50 ml). The resulting aqueous suspension was then extracted with ethyl acetate (3 x 50 ml). The combined ethyl acetate extract was dried over sodium sulphate and the solvent removed under vacuum.

The resulting gum was reserved and stored at -20°C. Using liquid N 2, the aqueous extract was frozen, forming a thin layer round the inside of a round-bottom flask. This was then freeze dried before redissolving in a minimal amount of methanol. The resulting solution was filtered and dried under vacuum before storing at -20 °C. The remaining solid was dissolved in a minimal amount of water and filtered before being freeze dried and stored at

116 Chapter 5

-20°C. Any undissolved matter was discarded. An outline of the extraction process is shown in Scheme 5.1. Algae

Mix MeOH

Repeat Twice Action Algae/Methanol Infusion Product

Solvent Decant

Methanolic Extract Algae Solution

Evaporate

Crude Extract

Organic/Aqueous H2O Partitioning EtOAc Repeat Twice

Organic Layer Aqueous Layer

Dried over Na 2SO 4 Freeze Dried

Filtered Redisolved in MeOH

Evaporate EtOAc Filtered

EtOAc Extract Fraction Methanolic Solution Solid Residue

Evaporate Redisolved in H 2O

MeOH Extract Fraction Filtered

Freeze Dried

Aqueous Extract Fraction

Scheme 5.1. Flow chart detailing the steps involved in the preparation seaweed extracts

117 Chapter 5

Extracts were sent on ice by post to the two institutions. Glycomar located in Oban carried out the XTT cytotoxic viability assay. Strathclyde Innovations in Drug Research (SIDR), part of the University of Strathclyde, Glasgow used the Alamar Blue assay to screen extract for antibacterial, antiparasitic and cytotoxic potential.

5.2.3 Assays

5.2.3.1 Alamar Blue Antibacterial Assays using Staphylococcus aureus and Escherichia coli

The Alamar blue assay was used to evaluate the antibacterial activity of extracts against S. aureus and E. coli was conducted using a modification of the method described by Baker and Tenover, (1996).

Bacterial cultures were incubated at 37 °C for 20 hours after which the solution was adjusted to correspond to a 0.5 McFarland standard and then diluted to 1 in 1000. Extracts were diluted to a concentration of 200 µg ml -1 in DMSO for the assay against S. aureus and to 500 µg ml -1 DMSO for the assay against E. coli . Plates were incubated for 20 hours at 37 °C to allow for further bacterial growth and for any antibiotic compounds to take effect. The fluorescence was measured at an excitation of 560 nm and emission of 590 nm. Results are presented as percentage of growth compared to a control sample.

MIC’s were established via a 1:1 sequential dilution of the original concentration. The positive control used a solution of Gentamycin (0.2 mM). This underwent a 1:1 sequential dilution to establish a MIC.

5.2.3.2 Alamar Blue Antibacterial Assays using Nocardia farcinica

Extracts were tested using the Alamar blue assay for activity against N. farcinica using a modification of the methods of Collins and Franzblau, (1997) and Franzblau, et al. (1998).

118 Chapter 5

Cultures of N. farcinica were incubated at 31 °C for four days after which the solution was adjusted to correspond to a 0.5 McFarland standard. This solution was further diluted a hundred fold. 100 µl of a 100 µg ml -1 solution of the extract was added to 100 µl of the N. farcinica culture which was then incubated for a further four days at 31 °C. Alamar blue solution was then added and the samples were incubated for 20 hours at 31 °C where upon the fluorescence was measured at an excitation of 560 nm and emission of 590 nm. Results are presented as percentage of growth compared to a control sample.

5.2.3.3 Alamar Blue Assay to Determine Drug Sensitivity of African Trypanosomes in vitro (Trypanosoma brucei brucei S427)

This assay was conducted using a modified version of procedure set out by Räz, et al. (1997). Stock solutions of extracts were prepared at a concentration of 10 mg ml -1 in DMSO. 4 µl of the stock solutions were transferred in to separate wells of the second column of a 12 x 8 well plate. To these was added 196 µl of HMI-9 medium (Hirumi and Hirumi, 1989) giving a solution with a final concentration of 200 µg ml -1. 100 µl of HMI- 9 medium was transferred to wells in column 1 and 3 through to 12. 1:1 serial dilutions of the solution in column 2 were then prepared using columns 3 to 11.

A positive control was established using a 1 mM solution of suramin in water, which was subjected to the same serial dilutions as the extracts using HMI-9 medium.

T. brucei brucei (S427) cell suspensions were prepared to a concentration of 2 - 3 x10 4 trypanosomes ml -1 where upon 100 µl of this solution was added to each well. The plates were incubated at 37 ºC, 5 % CO 2 with a humidified atmosphere for 48 hours. 20µl of Alamar blue was then added to the mixture followed incubation for a further 24 hours under the same conditions.

Fluorescence was measured at an excitation of 530 nm and emission of 590 nm. Results are presented as percentage of growth compared to a control sample.

119 Chapter 5

5.2.3.4 Alamar Blue Cytotoxicity Assays

Five cell lines were used in this study. DU-145 and PC-3 are both epithelial cell lines from a prostate carcinoma. HS27 is a fibroblast cell line of normal human foreskin. LNCAP AS is human prostate adenocarcinoma cells and Hep G2 is human Caucasian hepatocyte carcinoma. DU-145, HS27 and Hep G2 cells were prepared to a concentration of 0.5 x 10 5 cells ml -1 of Dulbecco's Modified Eagle Medium (DMEM) solution and LNCAP AS and PC-3 cells to 1.0 x 10 5 cell ml -1 of DMEM.

These cytotoxic assays were carried out using a modified version of the protocol highlighted by O’Brien, et al. (2000). Samples were incubated for 48 hours in a -1 humidified 5% CO 2 atmosphere. The concentration of extracts was 100 µg ml .

5.2.3.5 XTT Cytotoxic Assay against HeLa cells

Samples were dissolved in DMSO to a concentration of 50 mg ml -1 and then diluted further with Hank’s buffered saline solution (HBSS) to 100 µg ml -1. Extracts were then added to

HeLa cells and incubated for 24 hrs at 37 °C in 5% CO 2. 50 µl of XTT reagent was then added to each sample incubation continued for a further 4 hrs before fluorescence was measured at an excitation of 530 nm and emission of 590 nm. Results are presented as percentage viability. Positive controls consisted of doxorubicin at 0.5 µg ml -1 and 1.0 µg ml -1.

5.3 Results

5.3.1Anti-bacterial Screening

Seven extracts showed no activity against S. aureus with a further 24 giving no more than a 20% decrease in the bacterial population at a concentration of 200 µg ml -1 compared to a control sample (fig. 5.2). Only one extract, the ethyl acetate extract of Polysiphonia fucoids , showed good activity (< 20%) at this concentration, showing bacterial growth of 9

120 Chapter 5

% with respect the control sample with no extract showing moderate activity (20-50%). The minimum inhibitory concentration (MIC) of this extract was 200 µg ml -1 via sequential dilution of the extract. The aqueous extract of Cladophora rupestris was the only other extract that showed bacterial growth of less than 80% of the control.

Figure 5.2. Percentage of S. aureus growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue antibacterial assay

121 Chapter 5

Extracts in general showed poor activity towards E. coli . While the majority of extracts (73%) resulted in bacterial growth of less than 80 % of the control, all extracts failed to inhibit growth by more than 50% at a concentration of 500 µg ml -1 (fig. 5.3). Extracts of P. fucoides , Fucus vesiculosus and Pelvetia canaliculata were particularly ineffective against E. coli , failing to reduce growth below 85% of the control.

Figure 5.3. Percentage of E. coli growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue antibacterial assay

122 Chapter 5

Activity against N. farcinica was also weak (>50%) (fig. 5.4). Extracts were tested at a concentration of 100 µg ml -1 , which was considerably lower than used against E. coli and S. aureus , however none of the extracts showed any noticeable inhibition of N. farcinica . Over the whole sample set the growth of N. farcinica , compared to the control, ranged from 89.4 % to 100.2 %.

Figure 5.4. Percentage of N. farcinica growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue antibacterial assay

123 Chapter 5

5.3.2 Antiparasitic Assay

T. brucei brucei was more susceptible to inhibition by the extracts than the bacterial strains (fig. 5.5). It is interesting to note that all the aqueous extracts and all but one ( H. siliquosa ) of the methanol extracts from the seaweed produced an increase in the T. bucei brucei population from the control levels, with as much as a 13.6% increase in one case ( C. rupestris ) at 100 µg ml -1. The exception here was the methanol extract of Halidrys siliquosa which showed growth of 62.6% of a control sample of T. brucei brucei .

The ethyl acetate extracts showed considerably better activity towards the plasmodium, with six extracts ( Fucus serratus, Furcellaria lumbricalis, Corallina officinalis, C. rupestris, H. siliquosa and P. fucoides ) showing good activity (<20%).

The C. fragile ethyl acetate extract showed moderate activity by reduced growth to 22% compared to a control with a MIC of 50 µg ml -1 calculated via sequential dilution of the extract. The ethyl acetate extract of F. serratus reduced growth of T. brucei to 2.4% compared of the control with extracts from F. lumbricalis , C. officinalis and C. rupestris showing slightly better activity reducing growth to 1.5%, 0.7% and 0.6% of the control respectively. A MIC of 25 µg ml -1 was determined for these four of the extracts. The ethyl acetate extract of P. fucoides did not show as good activity as those mentioned above 100 µg ml -1. The ethyl acetate of this extract resulted in only a reduction to 7.3% when compared to the control. However the MIC for this extract, 12.5 µg ml -1, was less than those extract above which showed greater reduction in growth compared to the control. Similarly, the ethyl acetate extract of H. siliquosa , which showed growth of 2.4% of a control, had the lowest MIC of 3.1 µg ml -1.

124 Chapter 5

Figure 5.5. Percentage of T. brucei growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue antiparasitic assay

125 Chapter 5

5.3.3 Cytotoxicity Screening

The results of the XTT assay are calculated as % viability compared with a standard control. Therefore a lower value is indicative of more cytotoxicity. Only two extracts resulted in a decrease in viability of more than 50% in the HeLa cell line at 100 µg ml -1 (fig. 5.6). The ethyl acetate extract of F. vesiculosus showed moderate activity by reducing viability to 41.6% of the control whereas the same extract of H. siliquosa showed good activity by reducing cell viability to 13.2% of the control. Both these extracts showed enhanced inhibition over the highest concentration of the positive control (doxorubicin at 1.0 µg ml -1). A further seven extracts ( A. nodosum (MeOH and Aqueous), F. serratus (EtOAc), H. siliquosa (MeOH), P. canaliculata (EtOAc), P. fucoides (EtOAc) and C. crispus (Aqueous)) showed greater activity than lower concentration of positive control (doxorubicin, at 0.5 µg ml -1). Standard deviations for the viability of cells treated C. fragile, A. nodosum and F. lumbricalis extracts was not provided.

126 Chapter 5

Figure 5.6. Percentage viability of Hela cells, when exposed to a range of seaweed extracts as determined by the XTT cyctotoxic assay

127 Chapter 5

Analysis of the activities of the extracts against the HS27 cell line showed that the ethyl acetate extract of H. siliquosa was the only one that exhibited good activity resulting in cell growth of 17% of the control (fig. 5.7). All other extracts showed poor activity towards the HS27 cell line.

Figure 5.7. Percentage of HS 27 cell growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue cyctotoxic assay

128 Chapter 5

The extracts showed little or no activity towards the HepG2 cell line at a concentration of 100 µg ml -1 with the ethyl acetate extract of H. siliquosa showing the best activity at 88% cell growth of the control (fig. 5.8). In fact it appears that several of the extracts are capable of promoting growth of Hep G2 cells most notably the EtOAc extract of C. rupestris which shows growth of 122.1 % of the control sample.

Figure 5.8. Percentage of Hep G2 cell growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue cyctotoxic assay

129 Chapter 5

Similarly extracts showed weak activity against the DU145 cell line, with extracts resulting cell growth of above 92.8% of a control sample (fig. 5.9).

Figure 5.9. Percentage of DU145 cell growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue cyctotoxic assay

130 Chapter 5

Again, there is little activity from the extracts at 100 µg ml -1 towards the PC3 cell line (fig 5.10). The ethyl acetate extract of P. fucoides shows very mild activity against PC3 at 86% of the control. In addition the ethyl acetate extract of H. siliquosa showed activity at 53% of the control against PC3.

Figure 5.10. Percentage of PC 3 cell growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue cyctotoxic assay

131 Chapter 5

The aqueous extract of Fucus vesiculosus shows very mild active against LN CAP AS cell line at 82% of the control (fig 5.11). Again the EtOAc extract from H. siliquosa showed activity high within the moderate range against LN CAP AS at 21% of the control sample.

Figure 5.11. Percentage of LN CAP AS cell growth, compared to a control, when exposed to a range of seaweed extracts as determined by the Alamar Blue cyctotoxic assay

132 Chapter 5

5.4 Discussion

5.4.1 Development of a Screening Network

Initial contacts with four organisations offering assays targeted towards early stage drug discovery it was clear that that purchasing such services on a commercial basis was out of the questions without substantial funds being made available. With prices for individual assays ranging from £11.80 to £21.19 per sample per assay the total cost could reach as much as £699.27 per assay and several thousands of pounds for a complete screening of extracts. A more realistic approach appeared to be to entering in to some sort of agreement with each of the organisations which involved sharing any intellectual property arising from the study or simply asking them to carry out the analysis for free due to the academic nature of the study. These in fact were two routes that were taken forward, the former with SIDR and the later with Glycomar. The third option explored in the absence of any payment was to carryout the work myself in the lab offering the service. Although this route was productive, and led to the work carried out on the antioxidant potential of extracts using the spin trap assay described in Chapter 4, the results from this work are not presented in this chapter. The fourth organisation contacted was positive about the work and offered to carry out assays on the basis of shared IP, however undue delays with personnel being out of the country led to further delays and the work was then not taken forward.

It was initially hoped that with number of assays carried out by other institutions that the, extracts would be subjected to significantly more assays than the ten presented here. Four out of the nine assays that were undertaken by SIDR were not on the original list of 45 assays they proposed resulting in extracts not being assessed against 40 of the possible assays. It was initially expected that at least 50% of the available assays would have been completed on the extracts provided. This would have allowed for significant advancement in understanding the phytochemsitry of Scottish seaweed whereby the activity of extracts would have been evaluated using a diverse range of assay types. Also, all the assays carried out in this study were cell based assays, where cultured cells are subjected to a quantity of seaweed extract and after a period of time the amount of cells remaining

133 Chapter 5 compared to a control. In doing so, no isolated enzyme or radioligand based assays were carried out. Glycomar carried out the cytotocity assay free of charge and offered to conduct three anti-inflammatory assays, oxidative burst, elastase release and Nf-kB for a fee.

Reviewing the weaknesses in the approach, the small number of assays performed by SIDR compared with their capability was largely due to major staffing issues over the period of this study. This resulted in a back log of samples from all their collaborations and customers. It was not until late on into the project that this problem emerged, which did not allow for any effective backup plan to be formed. This shows the need for effective and regular communication between all collaborators in order to have a clear understanding of problems that are arising. However, on a positive note this has initiated a collaboration and there is fortunately a possibility that further assays will be completed allowing publication of a full dataset at a later date.

Despite the small number of assays carried out, those that were performed provide a useful insight into the bioactivity of the selected seaweed species.

5.4.2 Antibacterial Screening

The vast majority of extracts that were tested showed little antibacterial activity. The ethyl acetate extract of P. fucoides is the only extract showing good activity towards any of the three bacterial strains. This extract was active against the Gram positive bacterium S. aureus which is in agreement with a study by Hornsey and Hide (Hornsey and Hide, 1974). This activity reflects the observations made during the antibacterial assays carried out in chapter 3 of this thesis, where extracts of P. fucoides were shown to exhibit very strong antibacterial activity towards a number of marine bacteria, especially Gram positive bacteria. It is worth noting that in this study more activity was observed against Gram positive bacteria ( S. aureus and N. farcinica ) than Gram negative bacteria ( E. coli ) which is a common feature of other studies examining the antimicrobial activity seaweed (Salvador, et al., 2007; Freile-Pelegrin and Morales, 2004; Kim and Lee, 2008).

134 Chapter 5

Extracts of A. nodosum , C. crispus , C. fragile, F. vesiculosus and H. siliquosa have previously been shown to have possess activity against S. aureus , (Hornsey and Hide, 1976a; Ibtissam, et al., 2009). However this was not observed in the samples studied here.

The research conducted in chapter 3 also suggested that anti-bacterial activity of a seaweed extract is dependent on the location of the seaweed, with those found in rockpools all showing activity to at least one strain of from a test battery of seven bacteria and seaweed found on exposed surfaces showing increasing activity further down the inter tidal zone. However with the poor activity exhibited by the seaweed towards the three human pathogens, it is not possible to draw similar conclusions. It is however interesting to note that the antibacterial activity of extracts against marine strains appeared much greater than against human pathogens, however it should be noted that very different assays were used in each case.

5.4.3 Antiparasitic Screening

Ignoring the ethyl acetate extracts the only other extract exhibiting activity against T. brucei brucei was the methanolic extract of H. siliquosa. It is clear that ethyl acetate extracts are significantly more active against the plasmodium T. brucei brucei than both methanol and aqueous extracts with six out of the 11 ethyl acetate extracts showing promising activity. The antiparasitic activity of C. rupestris , C. officinalis and F. lumbricalis found in this study is mirrored in another study that showed low MIC values for each of the species tested against Trypanosoma brucei rhodesiense (Spavieri, et al., 2010; Allmendinger, et al., 2010). No such activity has been reported for the other species showing activity in this study. Again, activity of the extracts does not appear to depend on the preferred shoreline location of the seaweed.

5.4.4 Cytotoxicity Screening

In the search for potential anti-cancer compounds activity against the HS 27 is undesirable. HS 27 is used as a standard normal cell and a compound or extract with ideal activity would not damage this cell line. Of the 33 seaweed extracts tested 23 extracts showed no

135 Chapter 5 adverse effects on this cell line with a further eight showing only very minor activity. The ethyl acetate extract of H. siliquosa however drastically effected the viability of HS27 cells. Additionally, the ethyl acetate extract of H. siliquosa shows good activity towards both PC 3 and LN CAP AS, both prostate cancer cell lines, which may suggest some general toxicity of the extract. However, the same extract showed no effect towards a further prostate cancer cell line, DU145. This shows that although the extract damages normal cells, it also shows some degree of selectivity towards prostate cancer cells lines. The extract also showed no active against Hep G2, a hepatic cancer cell line, confirming that the ethyl acetate extract of H. siliquosa shows some selectivity towards the variety of cell lines used.

The extracts show slightly more activity towards HeLa cells, (a cervical cancer cell line) in the XTT cytotoxic assay. Again the ethyl acetate extracts strongly inhibited HeLa cells. The most surprising result was that the ethyl acetate extract of F. serratus reduced the viability of HeLa cells to 41.6%. This coupled with the fact that it did not effect the viability of HS27 cells, and the other cell lines tested, indicates that a compound or compounds present in the extract are capable of selectively targeting HeLa cells without damaging healthy normal cells.

The cytotoxicity of C. rupestris , C. officinalis and F. lumbricalis has previously been assessed against the L6 cell line (mammalian skeletal myoblast cells) with little activity being observed, which is reflected in this study confirming that these species have little potential as a source of cytotoxic agents for therapy (Spavieri, et al., 2010; Allmendinger, et al., 2010).

Extracts from C. fragile showed little cytotoxic activity towards the cell lines used in this study. In previous research however, methanolic and aqueous extracts from C. fragile have shown potent cytotoxic activity toward a mouse colon cancer cell line and poorer activity toward melanoma and leukaemia cell lines with no activity towards HeLa cells (Kim, et al., 2008). This suggests a degree of selectivity with the active compounds found in C. fragile . This is an important feature within extract screening and more broadly, drug discovery. Although it may well be initially exciting to find an extract that is capable of broad cytotoxic activity, it is not necessary desired as the compound may very well interact with essential biological systems. H. siliquosa and F. serratus have both been tested for

136 Chapter 5 their activity towards three leukaemia cell lines. F. serratus extracts showed little activity with extracts from H. siliquosa showing good activity against all cell lines (Zubia, et al., 2009). However, it appears from the work carried out here and by others that the cytotoxic activity of extracts from H. siliquosa are unselective and, if attributed to a single compound, would be undesirable as a potential therapeutic agent.

5.5 Conclusion

It is somewhat difficult to conclude whether a functional network of partners was formed during this study. Indeed extracts sent to Glycomar were promptly processed and results returned in good time; however involved only a single assay. Additionally, had SIDR not had the staffing issues that they experienced this aspect of the network would have been much more rewarding. However, it is clear that, in the absence of substantial funding, a sustainable network requires much effort to both initiate and sustain but is not an impossible goal. Such a network would provide better collaboration arrangments between all parties and allow for expansion utilising contacts from partner collaborators. This would have the potential increase the number of assays available and therefore increase the probability of identifying a “hit”.

This study shows that seaweed species from the coast of Caithness produce a number of compounds that have the potential to be of benift to human health and could be a valuable source of pharmaceutical compounds in the future. Here, three species of seaweed show activity towards the targets in this study which suggests that further investigation could beneficial in identifying bioactive natural products. The EtOAc extract of F. serratus shows selective cytotoxic activity against HeLa cells with out showing an effect towards healthy HS27 cells, which suggests that this species could be a potential drug source for treating cervical cancer. In addition, extracts from the red seaweed P. fucoides , have clear antibacterial including S. aureus , radical scavenging, and antiparasitic activities. Finally, H. siliquosa like P. fucoides has shown antibacterial, radical scavenging, and antiparasitic activity. In addition to this, H. siliquosa also shows some cytotoxic activity towards LN CAP AS, PC3 and HeLa cell lines.

137 Chapter 5

The three species highlighted here have not been extensively investigated. From H. siliquosa only 19 compounds being identified from three publicationsm with single compound identified from F. serratus . P. fucoides has yet to have it chemistry studied at all. These factors make each of these species suitable candidates to have their chemisty studied in greater detail.

Cell line/ Bacteria/ Seaweed species Plasmodium F. serratus H. siliquosa P. fucoides S. aureus - - 200.0 E. coli - - - N. farcinica - - - T. brucei 25.0 3.1 12.5 HeLa 100.0 100.0 - HS27 - 100.0 - Hep G2 - - - DU 145 - - - PC 3 - 100.0 - LN CAP AS - 100.0 - Table 5.2. Summary of activity from highlighted seaweed species stated as MIC (µg ml -1)

138 Chapter 6

6. Column Chromatography and Assay Guided Fractionation of an Ethyl Acetate Extract of the Brown Seaweed Halidrys siliquosa

6.1. Introduction

From the data presented in previous chapters, it is clear that the seaweed species studied have a diverse range of bioactivities. While many studies on marine plants and animals have assessed only the activity of extracts it is important to identify the compounds responsible for the bioactivity and if possible isolate and characterise them. As previously demonstrated, bioactive natural products have the potential to be key stepping stones in the development of new medicines and can also be used to shed light on the ecology of the seaweed through experiments designed to evaluate their function in nature.

6.1.1 Criteria for Choosing Species for Further Study

A range of criteria were developed to aid in choosing a seaweed for further analysis of its chemistry. Firstly, it was key that the chosen seaweed performed well in both radical scavenging assays as well as the anti-bacterial disk diffusion assay. These assays can be routinely applied in-house and would be used to assess the activity of the fractions after chromatography and guide isolation. Secondly, it was important that enough of the seaweed could be routinely collected, if needed, without any long term damage to the immediate ecosystem. Collecting sufficient quantities of seaweed that are either seasonal or sparse, such as many of the red species, would have taken considerable time and there is the possibility that removing of large amounts of the seaweed may result damage to the immediate ecosystem. Finally, and probably most crucially, the species chosen must not have been previously studied to any great extent. Several of the species such as Osmundea pinnatifida and Chondrus crispus which were screened earlier in this thesis to assess the bioactivity of extracts, have had their chemistry extensively explored over the last 20 years. Despite the novel compounds and indeed new classes of compounds still emerging

139 Chapter 6 from these species, and others that fall into this category, it was felt that investigating these types of seaweed would not be as productive as focusing on species less extensively studied to date.

6.1.2 Halidrys siliquosa

Using these criteria, it was clear that brown seaweed tended to be favoured and it was decided that Halidrys siliquosa would be a suitable candidate for further study. H. siliquosa , also commonly known as Sea Oak, is found abundantly in rock pools through out much of the British Isles (fig 6.1.). Out with British waters it can be found in many North East Atlantic costal areas ranging from Norway down to Portugal. It can also be found within the sublittoral zone below the brown seaweed Laminaria digitata . During low tides it is easy to harvest from rock pools. The appearance of the seaweed during it juvenile stage, is of an olive green colour and during its development into adulthood it darkens to become a dark brown/yellow colour. The structure of H. siliquosa consists of a flattened main stem out of which branches grow on alternative sides giving a distinct zigzag appearance. Each of these branches then sub-divide with a similar pattern emerging. At the end of each frond is situated an air bladder, used for buoyancy, which is slightly wider than the branches and between 1-4 cm in length. Specimens of H. siliquosa have been reported to grow to 2 m in length, however in locally specimens typically grow to only 0.5 m in length.

Seaweeds in general provide a home to a vibrant collection of life. In this respect H. siliquosa is no exception and has been shown to host a range of epiphytes, such as bacteria, algal larvae and bryozoans . H. siliquosa has been shown to host a smaller epiphyte population compared to other seaweeds in similar locations. This has been attributed to its ability to constantly shed its outer layer of cells (Tyler-Walters and Pizzolla, 2008).

140 Chapter 6

Figure 6.1. UK Distribution of H. siliquosa ; figure courtesy of Tyler-Walters and Pizzolla, 2008

In the preliminary screening exercise, hexane and ethyl acetate liquid-partitioned extracts of H. siliquosa demonstrated a broad spectrum of antibacterial activity across a range of Gram positive and negative marine bacteria, with extracts shown to be active against six of the seven strains that were available. The same fractionated extracts showed strong radical scavenging activity towards the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging assay and the 2,2-azinobis (3-ethlybenzothiazoline-6-sulphonic acid) diammonium salt radical cation discolourisation (ABTS) assay. Fractionated extracts of H. siliquosa showed cytotoxicity towards HeLa, PC 3 and LN CAP AS cancer cell lines. In addition and probably most importantly, H. siliquosa has not been extensively investigated with only three papers (Glombitza, et al., 1980; Higgs and Mulheirn, 1981a; Culiolo, et al., 2008) concerning the chemistry of the species mentioned in the annual publication “Marine Natural Products” (Faulkner, 1984a and Blunt, et al., 2010) over the past 27 years. Finally, a number of meroterpenes from H. siliquosa have been shown to be active against a number of marine bacteria responsible for biofouling (Culiolo, et al., 2008).

6.1.3 Strategy

The ethyl acetate extract of H. siliquosa which showed strong activity to both radical scavenging assays and the anti-bacterial disk diffusion assay will be prepared and subjected to column chromatography. Utilising assay guided fractionation, and employing

141 Chapter 6 a number of in-house assays allows for quick and easy identification of active fractions. Antibacterial activity of fractionated samples will be conducted by employing the disk diffusion assay described in Chapter 3. Here fractions of H. siliquosa will be evaluated for antibacterial activity against one Gram positive ( Planococcus citreus ) and one Gram negative ( Oceanospirillum linum ) strain. Both these bacterial strains were chosen as they showed particularly susceptibility to H. siliquosa extracts in Chapter 3. Radical scavenging activity of fractions will be evaluated by both the 2,2-diphenyl-2- picrylhydrazyl hydrate (DPPH) radical scavenging assay and the 2,2-azinobis (3- ethlybenzothiazoline-6-sulphonic acid) diammonium salt radical cation discolourisation (ABTS) assay described in Chapter 4. Coupled with a photo-diode array detection RP- HPLC will be used to assess the separation by column chromatography. This approach allows for the potential to indentify the number of compounds with in a specific sub- fraction and to initiate discussions on how best to proceed in isolating natural products.

The results from the antibacterial assay and the two radical scavenging assays along with the RP-HPLC analysis will allow for decision on which sub-fraction or sub-fractions should be examined more closely. Sub-fractions that show particularly strong activity in at least one of the assays and can be shown in the RP-HPLC/PDA analysis to be relatively pure or could yield pure compounds with minimal further chromatography are likely to be selected for further investigation. Additional chromatography and semi-preparative RP- HPLC will be used to further purify fractions.

6.2 Materials and Methods

6.2.1 Materials

Methanol (HPLC grade), hexane and ethyl acetate (glass-distilled grade) were purchased from Rathburn Chemicals Ltd (Walkerburn, UK). Ethanol was purchased from VWR International (Lutterworth, UK). Silica gel 60 Å for column chromatography, 2,2- diphenyl-2-picrylhydrazyl hydrate (DPPH) and 2,2-azinobis (3-ethlybenzothiazoline-6- sulphonic acid) diammonium salt (ABTS) were purchased from Sigma-Aldrich (Poole,

142 Chapter 6

UK). Thin layer chromatography silica gel plates 60 F254 were purchased from Merck (Germany).

Reverse phase HPLC with photodiode array (PDA) detection was carried out on a Thermo Separation Products SpectraSystem comprising: SCM1000 vacuum degasser, P4000 quaternary pump, AS3000 auto-sampler and a UV6000LP photodiode array. Seperation was achieved using a Phenomenex Synergi 4u MAX-RP 80Å column (250.0 x 4.6 mm). System control and data collection were performed using Chromquest software.

Absorption measurements were performed on a CamSpec M350 Double Beam UV-Vis Spectrophotometer.

Sufficient H. siliquosa for this study was collected from near Keiss harbour on the east coast of Caithness approximately 8 miles north of Wick, (58°32’52N 3°07’04E) on 10/04/2008.

6.2.2 Extraction of H. siliquosa

The seaweed was transported back to the laboratory and cleaned of obvious epiphytes and other marine life before being chopped into 2-3 cm piece and then 1.0 kg was transferred in its fresh state to a 2.5 l Winchester bottle which was then topped up with methanol. The methanol extract solution was decanted off after 24 hours and reserved. The extraction was repeated a further two times with methanol. The combined methanol extracts were concentrated in vacuo to give a crude extract and a small amount of water. The water was made up to 100 ml and was portioned with 3x100 ml ethyl acetate. The aqueous fraction was reserved. Further additions of 100 ml water followed by partitioning with ethyl acetate were required until no crude extract remained. The combined ethyl acetate fractions were dried over sodium sulphate and evaporated to dryness to give a dark green viscous liquid. The extraction protocol and solvent partitioning are outlined in Scheme 6.1.

143 Chapter 6

Algae

Action MeOH Product Mix

Solvent Repeat Twice Algae/Methanol Infusion

Decant

Methanolic Extract Algae Solution

Evaporate

Crude Extract

Organic/Aqueous H O EtOAc 2 Partitioning Repeat Twice

Organic Layer Aqueous Layer

Dried over Na 2SO 4

Filter

Evaporate Solvent

Final Extract

Scheme 6.1. Flow chart detailing the steps involved in the preparation seaweed extract

144 Chapter 6

6.2.3 Column Chromatography of an Ethyl Acetate Extract of H. Siliquosa

The ethyl acetate extract from H. siliquosa was subjected to silica gel column chromatography. 300 ml of silica was made into a slurry with hexane and added to a column (30 mm diameter). 1 g of the crude extract was dissolved in a minimal amount of ethyl acetate. This solution was loaded carefully to the column and eluted with a gradient of ethyl acetate in hexane (100 % hexane to 100 % ethyl acetate in 10 % increments in 100 ml aliquots). A total of 70 fractions were collected and thin layer chromatography (TLC) was used to visually assess the separation of the extract. Fractions were analysed twice via TLC, firstly using an mobile phase of 20:80 ethyl acetate:hexane and again with 40:60 ethyl acetate:hexane. This allowed for appropriately pooling similar fractions. This initial column resuted to five fractions, which were coded as E, A, B, C and D where fraction E is the most non-polar and fraction D being the most polar fraction. The TLC showed only multicomponent mixtures with no pure compounds isolated. It was therefore necessary to further purify the 5 isolated fractions by further employing column chromatography (scheme 6.2).

6.2.4 Chromatography of fraction E

A similar process to above was adopted for the chromatography of fraction E. In this step, a slurry of 200 ml of silica and hexane was used. 54 mg of E was dissolved in minimal amount of a hexane:ethyl acetate mixture (3:1) and added to the column. Due to this fraction being of a non-polar nature, the composition of the mobile phase was adapted. The initial mobile phase consisted of 100% hexane and was decreased to 65% hexane in 5% increments in 100 ml aliquots by the addition of ethyl acetate. From this column 30 fraction were collected and analysed by TLC, 10% ethyl acetate in hexane. From the TLC, fractions were grouped according to their TLC profile into five sub-fractions: Ea, Eb, Ec, Ed and Ee.

145 Chapter 6

6.2.5 Chromatography of fraction A

Fraction A was subjected to a modified method that was employed for fraction E. Fraction A yielded 293 mg which allowed for some of the fraction to be reserved for further analysis at a later stayed if required. A slurry of 200 ml of silica and hexane was prepared and added to the column. 100 mg of fraction A was dissolved in minimal amount of a hexane:ethyl acetate mixture (3:1) and loaded to the column. The initial mobile phase consisted of 100% hexane and was decreased to 60% hexane in 5% increments in 100 ml aliquots by the addition of ethyl acetate. From this column 47 fraction were collected with fraction 1 to 41 analysed by TLC, 25 % ethyl acetate in hexane and fractions 27 to 47 analysed with 40 % ethyl acetate in hexane. From the TLC, fractions were pooled according to their TLC profile into five sub-fractions: Aa, Ab, Ac, Ad and Ae.

6.2.6 Chromatography of fraction B

Fraction B was subjected to a modified method as employed previously. A slurry of 200 ml of silica and hexane was prepared and added to the column. With over 500 mg of fraction B at our disposal, this allowed for more material to be loaded to the column and allowed for some to be resurved for future use. 200 mg of B was dissolved in minimal amount of a hexane:ethyl acetate mixture (1:1) and loaded to the column. The initial mobile phase consisted of 95% hexane and was decreased to 50% hexane in 5% increments in 100 ml aliquots by the addition of ethyl acetate. From this column 38 fraction were collected and analysed with 30 % ethyl acetate in hexane. From the TLC, fractions were pooled according to their TLC profile into five sub-fractions: Ba, Bb, Bc, Bd, Be and Bf.

6.2.7 Chromatography of fraction C

Fraction C was subjected to a modified method as employed previously. A slurry of 200 ml of silica and hexane was prepared and loaded to the column. 58 mg of C was dissolved in minimal amount of a hexane:ethyl acetate mixture (1:1) and added to the column. The

146 Chapter 6 initial mobile phase consisted of 90% hexane and was decreased to 30% hexane in 10% increments in 100 ml aliquots by the addition of ethyl acetate. From this column 30 fraction were collected and analysed with 40 % ethyl acetate in hexane. From the TLC, fractions were pooled according to their TLC profile into five sub-fractions: Cb and Ca.

6.2.8 Chromatography of fraction D

Fraction D was subjected to a modified method as employed previously. A slurry of 200 ml of silica and hexane was prepared and added to the column. All 61 mg of D was dissolved in minimal amount of a hexane:ethyl acetate mixture (1:3) and loaded to the column. The initial mobile phase consisted of 100% hexane and was decreased to 0% hexane in 10% increments in 100 ml aliquots by the addition of ethyl acetate. From this column 59 fractions were collected with fraction 1 to 59 analysed by TLC with 40 % ethyl acetate in hexane and fractions 23 to 52 analysed by TLC with 60 % ethyl acetate in hexane. From the TLC, fractions were pooled according to their TLC profile into five sub- fractions: Da, Db, Dc and Dd.

6.2.9 DPPH Radical Scavenging Assay Method

This assay was carried out as described in section 4.2.3.

6.2.10 ABTS Radical Scavenging Assay Method

With the ABTS radical appearing to be more readily scavenged than the DPPH radical, which has already been highlighted in chapter 4, it was decided to reduce the amount of the fractions to be used from that used in Chapter 4. This was done to ensure that the compounds in the fractions did not scavenge all the available radical during the 6 minute reaction time. This could cause problems when trying to identify the effectiveness of different sub-fractions as the majority of them would register activity of around 95-100 %.

147 Chapter 6

The modified method is as follows. ABTS (10 g) was dissolved in water (2.6 ml) to give a ● 7 mM solution. The ABTS + was formed by activating ABTS (2.6 ml) with a 2.45 mM solution of potassium persulphate (2.6 ml). This solution was left in the dark for 12 hours. The solution was then diluted using ethanol to give an absorbance of 0.700 ± 0.020 at 734 ● nm. 3 ml of the diluted ABTS + solution was combined with 10 µl of the methanolic fraction solution (2.5 mg ml -1) and the decrease in absorption was recorded every 30 seconds for 6 minutes at 734 nm. Each fraction was analysed in triplicate. A blank ● consisted of the ABTS + solution (3 ml) combined with methanol (10 µl). The data was present as a percentage of inhibition.

6.2.11 Disk Diffusion Assay

A disk diffusion assay was used to assess antibacterial activity of fractions. Liquid cultures of Oceanospirillum linum and Planococcus citreus were prepared as described in 3.2 and introduced to marine agar plates. Triplicate sterilised 6 mm paper disks were infused with 100 µg of each fraction and added to the agar plates. A control disk with the same volume of methanol was placed at the centre of each plate. Plates were then incubated for 72 hrs at 27 °C. After 72 hrs, the plates were inspected visually for areas of bacterial inhibition. The zone of inhibition was measured as a diameter across the centre of the disk in millimetres. Some of the fractions have been described as giving an inhibition of “slight activity”, this is where a fine area of inhibition can be seen around the disk. Fractions that showed no activity were given a value of zero.

6.2.12 Analysis of Fractions by RP-HPLC/PDA

Sub-fraction Bb was used as a test subject in developing a suitable method with which to carry out the chromatographic analysis of the fractions. A total of 14 different methods were tested in which the length of run, the composition of the mobile phase, the amount of sample injected and the flow rate were all varied.

Sub-fractions were prepared into vials at a concentration of 1 mg ml -1 in methanol. Sub- fractions were then subjected to reverse phase HPLC with a diode array detector (RP-

148 Chapter 6

HPLC/PDA). 50 µl of each sub-fraction was injected on to the column and subjected to a mobile phase gradient starting with 80% methanol. After 10 minutes the composition of the mobile phase increased to 90% methanol, over a period of 60 minutes and was then increased over a five minute period to 100% methanol and held for 30 minutes after which the composition returned to its original composition of 80% methanol, over 2 minutes where it was maintained for a further 13 minutes.

6.3 Results

6.3.1 Column Chromatography

1 g of the crude extract (total yield 6.43 g) was successfully separated into five fractions (E, A, B, C and D). Each of them in turn underwent further column chromatography which yielded 22 sub-fractions. This process is presented in Scheme 6.2 with yields from each fraction. Utilising TLC to monitor the progress of the separation it was clear that no pure compounds were isolated from the chromatography.

149 Chapter 6

Dd 16mg

Dc 5mg

D 61mg

Db 12mg

Da 8mg

Ca 40mg

C 58mg

Cb 10mg

2mg Bf

Be 10mg

Bd 10mg

B 518mg

Bc 141mg

EthylAcetate extract Bb 24mg IncreasePolarity

Ba 6mg

Ae 4mg

A 293mg Ad 9mg

Ac 9mg column chromatography yields with associated

Ab 71mg

Aa 5mg

Ee 2mg

54mg E Ed 4mg

Ec 2mg

Eb 12mg

Ea 21mg

1st Gravity Column 2nd Gravity Column Scheme 6.2 Breakdown Scheme extract after ethyl acetate of

150 Chapter 6

6.3.2 Radical Scavenging Assays

15 out of the 22 sub-fractions assayed showed activity of over 50% inhibition in the DPPH radical scavenging assay with eight of these showing inhibition above 80%. (fig 6.2) Sub- fractions Ee and Ae are the only two fractions to show activity less than 40% with percentage inhibitions of 13%. All sub-fractions resulted in at least 13% inhibition. As with in chapter 4 the control sample showed only a slight decrease in absorption that is consistent with the addition of methanol diluting the radical solution.

Figure 6.2. Percentage inhibition of the DPPH radical by H. siliquosa sub-fractions

151 Chapter 6

The ABTS assay showed that four of the sub-fractions (Bc, Cb, Ca and Db) inhibited at least 50% or the radical with Cb showing 99% inhibition (fig. 6.3). All other sub-fractions showed activity below 50%. All of the tested sub-fractions resulted in at least 10% inhibition of the ABTS radical.

Figure 6.3. Percentage inhibition of the ABTS radical by H. siliquosa sub-fractions

152 Chapter 6

6.3.3 Disk Diffusion Assay

Out of the 22 factions, 15 (Ab to Dd) displayed activity against Planococcus citreus , with only one, Cb, showing activity towards Oceanospirillum linum (Table 6.1). Only one of the disks from fraction Bc showed very slight activity with the other two disks showing no activity.

Sub-Fraction Planococcus citreus Oceanospirillum linum Ea - - Eb - - Ec - - Ed - - Ee - - Aa - - Ab 8.00 ± 0.00 - Ac Slight activity - Ad 8.00 ± 0.00 - Ae 7.67 ± 0.58 - Ba 7.67 ± 0.58 - Bb Slight activity - Bc Slight activity - Bd 7.00 ± 0.00 - Be 7.00 ± 0.00 - Cb 11.33 ± 0.58 Slight activity Ca 9.00 ± 1.00 - Da 8.00 ± 0.00 - Db 9.00 ± 0.00 - Dc 8.00 ± 0.00 - Dd Slight activity - Table 6.1. Diameter of zone of inhibition (mm) of bacterial growth by H. siliquosa Sub- fractions

153 Chapter 6

6.3.4 RP-HPLC/PDA Analysis of H. siliquosa Sub-factions

Absorption chromatograms were recorded at a wavelength of 280 nm. This wavelength was chosen as this is where phenolics generally absorb UV light. As the radical scavenging activity of H. Siliquosa is dependant on the total phenolic content of the sample, it was therefore appropriate to use λ 280 nm to help identify compounds responsible for the observed activity.

Fraction E, despite its ID tag, was the most non-polar fraction. Using RP-HPLC/PDA on sub-fraction Ea (fig 6.4) it is possible to see that the majority of compounds analysed are eluted between 80 and 100 minutes, which corresponds to the time when 100 % methanol is used as the mobile phase. As the polarity of the fractions increase, more compounds are eluted earlier in the chromatogram. The intensity of the signal at 95.1 minutes in Eb (fig 6.5) is significantly stronger than those in other E sub-fractions. There are still several signals around the 50 mAU intensity that are being eluted with 100% methanol but despite this, the chromatogram of Eb shows one major compound In Ec (fig 6.6) a lone signal at 10.3 minutes could be a possible target for isolation. There are also two small broad signals at 65.0 and 78.1 minutes with many strong signals eluting with 100% methanol. Sub-fraction Ed (fig 6.7) showed good separation of compounds between 50 and 70 minutes, with three compounds showing strong signals. Between 78 and 110 minutes there are several strong signals, however separation of these is poor. The majority of the compounds eluted from Ee (fig 6.8) show an intensity below 10 mAU with more intense signals from 85 to 95 minutes. The intensity of these signals is relatively low in comparison with other chromatograms.

154 Chapter 6

Detector 1-Scan-280 nm robbie h.sil Ea 30/10/2008 13:34:12 60

40 mAU 20

0 20 40 60 80 100 120 Minutes

Figure 6.4. Chromatogram of sub-fraction Ea at λ 280 nm

Detector 1-Scan-280 nm 1000 robbie h.sil Eb 30/10/2008 15:36:58

750

500 mAU

250

0 20 40 60 80 100 120 Minutes

Figure 6.5. Chromatogram of sub-fraction Eb at λ 280 nm

200 Detector 1-Scan-280 nm robbie h.sil Ec 30/10/2008 17:39:43

150

100 mAU

0 20 40 60 80 100 120 Minutes

Figure 6.6. Chromatogram of sub-fraction Ec at λ 280 nm

155 Chapter 6

Detector 1-Scan-280 nm robbie h.sil Ed 30/10/2008 19:42:22

200 mAU 100

0 20 40 60 80 100 120 Minutes

Figure 6.7. Chromatogram of sub-fraction Ed at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil Ee 30/10/2008 21:45:07

20 mAU

0 20 40 60 80 100 120 Minutes

Figure 6.8. Chromatogram of sub-fraction Ee at λ 280 nm

The chromatogram of Aa, like Ed shows good separation around 50 to 70 minutes with a similar pattern emerging during elution with 100% methanol (fig 6.9). The most intense signals are at 50.5 and 58.4 minutes. The chromatogram of Ab shows two compounds eluted at 32.8 and 36.0 minute with high intensity (fig 6.10). The remainder of the compounds eluted have intensities below 30 mAU. The compounds eluting with 100% methanol are relatively small in this case. All signals bar one in the chromatogram of Ac show and intensity of below 30 mAU (fig 6.11). The remaining signal at 37.3 minutes showed and intensity of 60 mAU. Again compounds eluting with 100% methanol are relatively small. There are two prominent signals in the chromatograph of Ad at 90.5 and 99.8 minutes (fig 6.12). Several compounds are observed in the earlier part of the chromatogram with many having intensities close to 100 mAU. There are two large signals shown in the chromatogram of Ae at 17.6 and 19.3 minutes at around 400 and 250 mAU respectively (fig 6.13).

156 Chapter 6

Detector 1-Scan-280 nm robbie h.sil Aa 29/10/2008 19:09:53

400

mAU 200

0 20 40 60 80 100 120 Minutes

Figure 6.9. Chromatogram of sub-fraction Aa at λ 280 nm

300 Detector 1-Scan-280 nm robbie h.sil Ab 29/10/2008 21:12:36

200 mAU

100

0 20 40 60 80 100 120 Minutes

Figure 6.10. Chromatogram of sub-fraction Ab at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil Ac 29/10/2008 23:15:25

40 mAU

0 20 40 60 80 100 120 Minutes

Figure 6.11. Chromatogram of sub-fraction Ac at λ 280 nm

157 Chapter 6

300 Detector 1-Scan-280 nm robbie h.sil Ad 30/10/2008 01:18:21

200 mAU 100

0 20 40 60 80 100 120 Minutes

Figure 6.12. Chromatogram of sub-fraction Ad at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil Ae 30/10/2008 03:20:59 400

300

200 mAU

100

0 20 40 60 80 100 120 Minutes

Figure 6.13. Chromatogram of sub-fraction Ae at λ 280 nm

The chromatogram of Ba shows two major compounds at 32.2 and 33.7 minutes that elute together (fig 6.14). Several weaker signals have a longer retention time. Bb, like Ad, also shows broad intense signals that elute with 100% methanol, this time at 84.2 and 90.4 minutes in comparison with 90.5 and 99.8 minutes (fig 6.15). The chromatograph of Bc shows the compounds which are also present in Ad and Bb (83.2 and 90.2 minutes) (fig 6.16). Several prominent signals can be observed at shorter retention times. Two signals dominated the Bd chromatogram at 17.0 and 18.1 minutes (fig 6.17). Lower intensity compounds eluting between 10 to 30 minutes are more difficult to differentiate with compounds at 24.6 and 27.2 minutes being the most significant other signals. The chromatogram of Be shows a large number of signals between four and 25 minutes that have not separated well. (fig 6.18). Within this group the most intense peak is eluted at 13.8 minutes. The chromatogram of Bf shows a large number of compounds between four and 28 minutes (fig 6.19) with two distinct isolated peaks at 54.4 and 63.6 minutes.

158 Chapter 6

Detector 1-Scan-280 nm 300 robbie h.sil A03 26/09/2008 21:36:46

200 mAU

100

0 20 40 60 80 100 Minutes

Figure 6.14. Chromatogram of sub-fraction Ba at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil A04 26/09/2008 23:39:24 300

200 mAU

100

0 20 40 60 80 100 Minutes

Figure 6.15. Chromatogram of sub-fraction Bb at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil A05 27/09/2008 01:42:03 800

600

400 mAU

200

0 20 40 60 80 100 Minutes

Figure 6.16. Chromatogram of sub-fraction Bc at λ 280 nm

159 Chapter 6

Detector 1-Scan-280 nm 300 robbie h.sil A06 27/09/2008 03:44:55

200 mAU

100

0 20 40 60 80 100 Minutes

Figure 6.17. Chromatogram of sub-fraction Bd at λ 280 nm

Detector 1-Scan-280 nm 300 robbie h.sil A07 27/09/2008 05:47:39

200 mAU

100

0 20 40 60 80 100 Minutes

Figure 6.18. Chromatogram of sub-fraction Be at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil A08 27/09/2008 07:50:18 150

100 mAU

0 20 40 60 80 100 Minutes

Figure 6.19. Chromatogram of sub-fraction Bf at λ 280 nm

Both sub-fractions from C show that no compounds detected by UV are eluted after 40 minutes. The chromatogram of Cb shows two separate groups each of three signals above 100 mAU (fig 6.20). The strongest intensity compound has a retention time of 19.5

160 Chapter 6 minutes. The major peaks in the chromatogram of Ca at 17.1 and 27.1 minutes are perhaps made up of more than one compound (fig 6.21). Alternatively this could be a single compound eluting but showing fronting which is usually caused by column overload.

Detector 1-Scan-280 nm robbie h.sil A01 26/09/2008 17:31:05

400 mAU 200

0 20 40 60 80 100 Minutes

Figure 6.20. Chromatogram of sub-fraction Cb at λ 280 nm

600 Detector 1-Scan-280 nm robbie h.sil A02 26/09/2008 19:33:54

400 mAU

200

0 20 40 60 80 100 Minutes

Figure 6.21. Chromatogram of sub-fraction Ca at λ 280 nm

The chromatogram of Da shows several prominent compounds between 20 and 41 minutes (fig 6.22). The strongest of these compounds are at 21.1 and 24.4 minutes. Db has all major compounds eluted before 40 minutes (fig 6.23). The signals with the strongest intensity is eluted at 19.7 minutes with the next strongest eluted at 32.7 minutes. Dc has as a large collection of compounds that are eluted between four and 20 minutes (fig 6.24) which proves to be difficult to extract satisfactory data. There are four other compounds which have a longer retention time and are eluted at 77.9, 83.8 and 84.3 minutes and the final peak at 86.5 minutes. The chromatogram of Dd shows a small collection of compounds between four and 20 minutes (fig 6.25). Three compounds of higher intensity are eluted at 76.8, 83.7 and 84.2 minutes.

161 Chapter 6

Detector 1-Scan-280 nm robbie h.sil Da 30/10/2008 05:23:38

200 mAU 100

0 20 40 60 80 100 120 Minutes

Figure 6.22. Chromatogram of sub-fraction Da at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil Db 30/10/2008 07:26:07 400

300

200 mAU

100

0 20 40 60 80 100 120 Minutes

Figure 6.23. Chromatogram of sub-fraction Db at λ 280 nm

Detector 1-Scan-280 nm robbie h.sil Dc 30/10/2008 09:28:54

300

200 mAU

100

0 20 40 60 80 100 120 Minutes

Figure 6.24. Chromatogram of sub-fraction Dc at λ 280 nm

162 Chapter 6

Detector 1-Scan-280 nm robbie h.sil Dd 30/10/2008 11:31:32 800

600

400 mAU

200

0 20 40 60 80 100 120 Minutes

Figure 6.25. Chromatogram of sub-fraction Dd at λ 280 nm

6.4 Discussion

6.4.1 Radical Scavenging Activity of H. siliquosa Sub-fractions

It is clear from both the DPPH and ABTS radical scavenging assays that sub-fractions of H. siliquosa show varying degrees of activity. This suggests that H. siliquosa contains a number of different compounds with differing polarities that are able to act as radical scavengers.

Sub-fractions Ee and Ae show similar but significantly lower levels of activity towards the DPPH radical than the other sub-fractions. Sub-fraction Ee also exhibited low levels of radical scavenging towards the ABTS radical, showing similar levels of activity of two other sub-fractions (Ea and Ac). In contrast fraction Ae shows higher levels of activity than Ee against the ABTS radical. As mentioned in chapter 4, interactions of large compounds with the DPPH radical may be hampered due to steric hindrance of the radial and that these compounds could interact with the ABTS radical due to its less hindered structure. This suggests that sub-fraction Ee contains low amounts of radical scavenging compounds, with Ae perhaps containing larger compounds that are able to quench the planer ABTS radical but not the more hindered DPPH radical.

163 Chapter 6

It is interesting to note that the radical scavenging of the ABTS and DPPH radicals appears to be more predominant in the more polar fractions i.e. those that were eluted later from the original column. Only four sub-fractions (Bc, Cb, Ca and Db) show inhibition of the ABTS radical above 50 % and 13 sub-fractions show inhibition of the DPPH radical above 70 %, with nine of these sub-fractions being from B, C and D fractions This is relatively unsurprising considering that radical scavenging activity of H. siliquosa may well be dependent on the total phenolic content of the extract with phenolic compounds, especially polyphenolics, being polar in nature.

6.4.2 Antibacterial Activity of H. siliquosa Sub-Fractions

The antibacterial assays show that even at low concentrations, fractions of H. siliquosa are able to inhibit the growth of the Gram positive bacteria tested. There is a very clear boundary between sub-fraction Aa and Ab where no activity and activity is observed against Planococcus citreus . From this it is clear that there is a difference in activity between the polar and the non-polar sub-fraction with the extremely non-polar sub- fractions (Ea to Aa) showing no activity. It is curious to note that generally the activity against P. citreus appears at more or less a constant with inhibition zones of between 6 and 9 mm. However generally speaking, the lower activity is concentrated between sub- fractions Ab and Be, with very slightly more activity being observed between Cb and Dc.

Sub-fractions of H. siliquosa do not seem to be active at this concentration towards Oceanospirillum linum with only sub-fraction Cb showing slight activity. However, a previous study has shown that some compounds isolated from H. siliquosa as well as a crude extract show strong activity towards a range of four gram negative marine bacteria (Cilioli, et al., 2008). It should be emphasised that this does not mean that the other sub- fractions contain no compounds with antibiotic properties, only that any effect is not observed at the concentrations used.

It is however a little surprising that the activity against O. linum is so low compared with the crude hexane and ethyl acetate fraction from Chapter 3 which showed that these fractions were significantly more active against O. linum compared to P. citreus . It is important to realise that the sample of H. siliquosa from this study and the sample from

164 Chapter 6

Chapters 3, 4, and 5 are different samples. These samples were collected in different locations and a different time of the year. These two factors may account for the lack of observed activity against O. linum as it is known that differences in season and location can influence the metabolic profile of certain seaweed species including H. siliquosa (Hornsey and Hide, 1974 and 1976a).

6.4.3 RP-HPLC/PDA Analysis of H. siliquosa Sub-factions

The results from RP-HPLC/PDA analysis show that H. siliquosa contains a large number of different compounds within an ethyl acetate extract and consequently within each of the fractions produced by column chromatography. By examination of the chromatograms it is also possible to narrow down the number of compounds that could be responsible for anti-bacterial and/or radical scavenging activity. For example, the chromatogram of sub- fraction Cb (fig 20.) allows for the identification of at least six clear signals that corresponds to six different compounds. In addition, this sub-fraction also shows 100 % inhibition of the ABTS radical it is therefore possible to suggest that at least one of these signals is responsible for the observed activity.

Using the absorption spectra of each signal in the chromatogram it is possible to identify compounds that are found in more that one sub-fraction. This approach can be effectively used in the analysis of the large distinct peaks that are detected in Ad and Bb and at a lesser extent in Bc, between 80 and 100 minutes. Due to the unique UV/Vis spectra of these signals it is suspected that these are most likely to be chlorophyll a and b (fig 6.26.).

165 Chapter 6

90.85 Min 84.18 Min robbie h.sil Ad 30/10/2008 01:18:21 robbie h.sil A04 26/09/2008 23:39:24 1500 Lambda Max Lambda Max

1500

1000

1000 213 mAU mAU

500 500 271 272

0 0 400 401

250 300 350 400 450 250 300 350 400 450 nm nm Ad at 90 mins 403 nm Bb at 84 mins 400 nm

100.03 Min 90.53 Min 600 robbie h.sil Ad 30/10/2008 01:18:21 robbie h.sil A04 26/09/2008 23:39:24 Lambda Max Lambda Max 405

400 405

400 mAU

mAU 200

200

0 0

250 300 350 400 450 250 300 350 400 450 nm nm Ad at 100 mins 409 nm Bb at 90 mins 407 nm Figure 6.26. Spectra of Chlorphyll a and b in Sub-fractions Ab and Bb

In continuing this analysis it is possible to identify a further 12 compounds that are found in multiple sub-fractions (Table 6.2.). All bar two of the compounds that are listed here are found in adjacent sub-fractions. Compound 2 is not found in Dc, however it does have a large grouping of signal during the point at which compound 2 would be eluted. Compound 11 bridges between Ed and Aa without being detected in Ee. Ee shows many low intensity signals around the 80 to 100 minute period and it is possible that some of compound 11 could be present.

Compound 1 is more of a special case. Although it is found in adjacent sub-fractions Dc and Dd and give similar UV spectra, the spectra of the compound at Ad at this point also appears similar to Dc and Dd at 15.9 mins and 15.4 mins respectively. This could well be a coincidence with two compounds being eluted at the same time with very similar UV/Vis spectra.

166 Chapter 6

Compound Retention Time (mins) Sub-fraction 15.4 Dd 1 15.5 Ad 15.9 Dc 16.8 Dd 2 16.5 Db 17.4 Ad 3 17.6 Ae 20.7 Be 4 20.8 Ca 21.1 Bf 21.1 Da 5 21.1 Db 24.2 Ae 6 24.7 Ad 63.2 Be 7 63.6 Bf 83.7 Dc 8 83.8 Dd 84.2 Dd 9 84.3 Dc 92.7 Ec 10 92.8 Eb 92.7 Aa 11 93.1 Ed 95.1 Eb 12 95.1 Ec Table 6.2. Compounds found in Multiply Sub-fractions

6.4.4 Can the observed bioactivity be assigned to signals in the RP-HPLC/DA chromatograms?

Three different analyses have been carried out on sub-fractions from H. siliquosa ; the radical scavenging activities against two radical species, antimicrobial activity against a Gram positive and a Gram negative bacteria and finally RP-HPLC/DA analysis. With the activity of each sub-fraction known and chromatograms available for each, an attempt was made to attribute bioactivity with compounds within the sub-fractions.

When dealing with mixtures of unknown compounds it is of course impossible to identify which one or ones are biologically active simply by comparing assay results with chromatograms. What can be done is to compare the activity of sub-fractions that have

167 Chapter 6 compounds which can be found in multiple sub-fractions. Although this approach will not be able to tell if a compound is active it may provide some indication what compounds are not active. By comparing the intensity of a signal found in adjacent sub-fractions with the bioactivity, should the intensity of the signal be greatest in the sub-fraction exhibiting less activity, then it may be possible to conclude that this compound does not contribute significantly towards observed bioactivity.

For example, compound 8 is found in two sub-fractions, Dc and Dd. Dc shows more radical scavenging activity towards both radicals and shows greater antimicrobial activity than Dd. The intensity of the signal of compound 8 is 331 mAU in Dc and 848 mAU in Dd which suggests that this compound does not contribute significantly towards the bioactivity of the sub-fractions. This pattern can be seen with a further six compounds found in multiple sub-fractions, they are 1, 2, 4, 6, 7 and 9. For the remaining five compounds, no conclusion as to whether they are bioactive can be determined via this approach.

6.5 Conclusion

From the experiments conducted within this chapter, it is possible to select Ab, Cb and Ca as sub-fractions for further investigation.

Sub-fraction Ab, showed no activity towards either bacterial strain and relatively average radical scavenging activity. Despite this the chromatogram showed two very prominent peaks with only small amounts of other compounds visible. 71 mg of Ab was eluted during column chromatography meaning that a larger quantity of this sub-fraction was available for further purification.

Sub-fraction Cb showed strong antibacterial activity towards the Gram positive bacteria P. citreus and was the only sub-fraction to show activity against O. linum. In addition this sub-fraction had the highest radical scavenging ability against two different radicals. These factors mean that Cb is an excellent candidate for further investigations.

168 Chapter 6

Ca although not showing similarly high level of radical scavenging and antibacterial activity as Cb, does however show good radical scavenging activity along with inhibiting the growth of P. citreus . The chromatogram of Ca shows that it does not contain compounds found in Cb and therefore should be looked at in further detail.

Here two of the signals highlighted in the diode array analysis can be attributed to chlorophyll a and b which can be observed in sub-fractions Ad and Bb with small amounts found in Bc. Also, using the diode array, seven compounds found in multiple sub-factions are suspected of not being directly involved in the observed bioactivity of the parent sub- fraction. To confirm such a proposal, isolation of the compounds would be required followed by reanalysis in the assays used.

169 Chapter 7

7. Identification of Compounds from Halidrys siliquosa using Mass Spectrometry and Nuclear Magnetic Resonance

7.1 Introduction

Halidrys siliquosa has received little attention by natural products scientists. Since 1980, only three publication (Glombitza, et al., 1980; Higgs and Mulheirn, 1981a; Culiolo, et al., 2008) detailing natural products from H. siliquosa have been published, despite its prominence on coastlines especially that of Great Britain.

Work described in earlier chapters has shown that extracts from H. siliquosa are active against a range of marine bacterial strains, some medically relevant bacterial strains, some cancer cell lines and can also act as radical scavengers. Leading on from this, several fractionated extracts have been identified as having strong radical scavenging activity.

Identifying compounds responsible for observed activities in crude extracts is one of the main challenges in natural products chemistry. Traditionally this is achieved by isolating pure compounds from often complex mixtures using a variety of chromatography methods ranging from standard column chromatography as used in 6.2.3 to the use of ion exchange chromatography, planer chromatography, solid phase extraction, supercritical fluids and semi-preparative HPLC (Cannell, 1998). This then allows for structure elucidation and evaluation of the biological activities of individual compounds. There are of course several challenges, some of which have been highlighted earlier. Marine natural products are known for their relative instability compared to their terrestrial counterparts. Some marine organisms are known to die upon contact with air followed by rapid decomposition and compounds are known to be degraded by oxidation, polymerisation and enzymes. To prevent the breakdown of compounds before samples even reaching the laboratory they are usually kept cool or better, frozen as soon as possible after collection. Many samples are dried, freeze dried or directly extracted to preserve sensitive compounds. Indeed after extraction, compounds within extracts my still degrade if stored in solvent and/or at room temperature. It is therefore essential that samples are kept dry and at the lowest temperature available. With marine samples containing significant levels of water and with extraction taking place with 100% methanol this can result in the presence of

170 Chapter 7 surfactants that cause foaming and bumping when the sample is being concentrated under vacuum which ultimately increase the time of sample preparation and could jeopardises the stability of sensitive natural product. To reduce this, the sample is dried either by air or lyophilisation to remove the water prior to solvent extraction. A high degree of purity of natural products is often required in further analysis. To undertake a full characterisation of a compound a typical purity of 95% is normally required with greater purity of at least 99% required for pharmacokinetic studies. X-ray crystallography usually requires purity of 99.9%, however there are a number of challenges that occur through purification via chromatography. With the complex nature of crude and fractionated extracts many compounds have similar polarities and are therefore difficult to separate via either column chromatograph, HPLC or other methods. Quite often the solution to this is a combination of multiply steps of chromatography using different solid phases with complex mixtures of mobile phases. To further complicate the matter some compounds are sensitive to even small changes in pH. The use of buffers and careful selection of separation media can help to minimise this, however the loss of a biologically active compound may only be discovered during routine assay guided fractionation, which may require several attempts to identify the best course of action for purification (Wright, 1998).

With the advent of hyphenated HPLC techniques such as HPLC/MS/MS and HPLC/NMR, it is now possible to characterise, although not rigorously, compounds without isolating them beforehand. HPLC/NMR has limited use in the identification of marine natural products, however it has been used successfully in the structural determination of pentaprenylated-p-quinol from an Australian sponge, Dactylospongia sp (Dias and Urban,

2009) . Additional benefits that hyphenated HPLC brings to analysis include greater automation and throughput, shorter analysis time and reduced risk of contamination which all contribute to the increased efficiency of natural product isolation (Unni, 2004; Ali, et al., 2008).

Semi-preparative HPLC was used to further purify fractions obtained from chapter 6 with mass spectrometry and NMR used to aid identification of compounds in both sub-fractions and semi-preparative HPLC fractions. Additionally a combination of the ABTS radical scavenging assay and mass spectrometry was used to identify compounds that quench the radical.

171 Chapter 7

7.2 Materials and Methods

7.2.1 Materials

Methanol (HPLC grade) was purchased from Rathburn Chemicals ltd (Walkerburn, UK). Ethanol was purchased from VWR International (Lutterworth, UK), 2,2-azinobis (3- ethlybenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), potassium persulphate and dueturated chloroform were purchased from Sigma-Aldrich (Poole, UK).

Semi-preparatory High Performance Liquid Chromatography was performed using a Thermo Separation Product SpectraSeries comprising: P100 pump and a UV100 UV detector. Data was recorded using an Alpkem 310 chart plotter. Separation was achieved using a Phenomonex Gemini C18 5 µ 110Å column (250 x 10 mm).

Mass spectral analysis was performed using a Bruker esquire HCT ion trap with an ESI interface. This was coupled to a Dionex UltiMate 3000 HPLC comprising degasser, pump, flow manager and auto-sampler units.

Initial 1H NMR was performed on a JEOL (JNM LA400) 400 MHz spectrometer, with more detailed 1H NMR, 13 C NMR and 2D NMR spectra being run on a Bruker AV 300

(400MHz) and DRX 500 spectrometers using CDCl 3 as solvents and TMS as internal standard.

7.2.2 Extraction and purification of H. siliquosa

All extracts and fractionated extracts of H. siliquosa used in this chapter are those described in chapter 6.

172 Chapter 7

7.2.3 Minimum Inhibition Concentration (MIC) of Fraction Cb against the ABTS Radical

The minimum inhibition concentration (MIC) of Cb against the ABTS radical was determined by using different concentrations of the sub-fraction against the radical. Methanolic solutions of Cb at concentrations of 1.25 mg ml -1, 1.50 mg ml -1 and 1.75 mg ml -1 were prepared and subjected to the ABTS radical scavenging assay as previously described in section 6.2.10.

7.2.4 Identification of Compounds Responsible for Observed ABTS Radical Activity from fraction Cb by LC-MS

A Stock solution of the ABTS radical was prepared as described in section 4.2.4. In addition a methanolic stock solution of Cb (1.75 mg ml -1) was also prepared in advance. From these stock solutions, three solutions were prepared for analysis by LC-MS.

1. 3 ml of ABTS radical solution was added to 10 µl of methanol 2. 3 ml of ABTS radical solution was added to 10 µl of methanolic Cb solution 3. 3 ml of ethanol was added to 10 µl of methanolic Cb solution

5 µl of the solution was applied to the column and eluted using a solvent gradient of methanol in water (0.1% formic acid) over the course of 120 minutes. Mobile phase was held at 80 % methanol for 10 minutes before being increased to a 90% methanol over 60 minutes. Over the next five minutes the mobile phase was increased to 100 % methanol and remained at this level for 30 minutes. The mobile phase was then reduced back to 80% methanol over two minutes where it remained for the last 20 mintes. Flow rate was set at 500 µl min -1.

After discussion with mass spectrometry staff, mass spectrometry settings were as follows. All ions were detected in the positive ion mode with a scan range of between m/z 320 and 1000. For ionisation via electrospray ionisation (ESI) nebuliser drying gas pressure, temperature and flow were set to 2.0 bar, 250 ºC and 8.0 l min -1 respectively. Optics were

173 Chapter 7 set as follows, skimmer was set to 40.0 V, capillary exit at 128.5 V, octopole 1 at 12.0 V, octopole 2 at 1.7 V, octopole RF at 187.1 Vppn lens 1 and 2 at 5 V and 60 V respectively.

7.2.5 Semi-preparative HPLC of Sub-fraction Ca

Sub-faction Ca was chosen to under-go semi-preparative HPLC based on its activity to the ABTS and DPPH radical scavenging assays.

A solution of 1 mg ml -1 of extract in methanol was prepared. This solution was introduced to the column (Phenomonex Gemini C18 5 µ 110Å column (250 x 10 mm)) at 250 µl portions. An eluant of 90% methanol in water at a flow rate of 3 ml min -1 was used and the UV detector was set at 280 nm. After seven runs (1.75 mg extract) a total of 68 fractions had been collected and it was apparent that at this concentration it would take too long to process sufficient extract and as a result a higher concentration, 10 mg ml -1, was used. However at this higher concentration the injection volume had to be reduced to 100 µl in order to prevent overloading on the column and causing fronting on the chromatogram. This allowed for 1 mg to be loaded to the column for each injection. Using this method, 11 injections (11 mg) were made and 65 fractions collected, giving a total of 133 from 18 injection.

The chromatogram showed three major compounds followed by the elution of several minor compounds. The first two major compounds eluted closely together followed by the third major compound. The remainder of the chromatogram consisted of minor compounds. Some of the compounds from Ca eluted closely with others that were originally separated into separate fractions. On closer examination of the chromatogram it appeared that these compounds would be in both fractions and it was therefore decided to combine these fractions. Using the chromatogram the 133 fractions were sorted into 4 groups Ca α, Ca β, Ca γ and Ca δ. The first and second compounds eluted very close together and it was therefore decided to group these together. The third major compound was grouped with a minor compound for a similar reason. A further two minor compounds eluted very close together and was group together with a further minor compound. The final group consisted of a number of minor compounds that showed very weak intensity. The methanol from each fraction was evaporated and the remaining aqueous solutions

174 Chapter 7 were partitioned against ethyl acetate (3 x 25 ml). The ethyl acetate fractions were dried over sodium sulphate, filtered and then solvent removed under evaporation. A small amount of NaCl was added when partitioning Ca γ and Ca δ to aid with the separation. These four fractions were analysed using HPLC/diode array. Scheme 7.1 shows the origin of each fraction.

Scheme 7.1. Origin of Ca sub-fractions from an ethyl acetate extract fraction of H. siliquosa via column chromatography and semi-preparative RP-HPLC

7.2.6 RP-HPLC/PDA Analysis of Ca α, Ca β, Ca γ and Ca δ

The method applied here was as described in section 6.2.12.

7.2.7 Semi-preparative HPLC of Ca α and Ca δ

From the RP-HPLC/PDA analysis of these four sub-fractions, it was decided that fractions Ca α and Ca δ should undergo a second round of semi-preparative HPLC as both these fractions have major peaks surrounded by several minor peaks and it was felt that it was important to isolate the major constituent. The method was modified slightly by changing

175 Chapter 7 the composition of the mobile phase from 90% to 75% methanol. It was hoped that this change would aid in the separation of the peaks in the chromatogram and allowing for the isolation of pure compounds. Separation was achieved using a Phenomonex Gemini C18 5µ 110Å column (250 x 10 mm). All other variables were the same.

Samples were dissolved in methanol to give a 1 mg ml -1 solution. The methanolic solutions were introduced to the column at 200 µl portions. A total of 11 fractions were obtained from Ca α and 13 from Ca δ.

7.2.8 Semi-preparative HPLC of Sub-fraction Ab

Ab was chosen to undergo semi-prep HPLC based on RP-HPLC/PDA data from chapter 6 and also due to the large quantity of this fraction available (71 mg).

59 mg of Ab was dissolved in 9 ml of methanol and 4 x 150 µl aliquots injected onto the column, allowing 1 mg of Ab to be processed per injection. Separation was achieved using a Phenomonex Gemini C18 5 µ 110Å column (250 x 10 mm). A mobile phase consisting of 80 % methanol and flow rate of 5 ml min -1 was used. In total 100 fractions were collected from the four injections and were pooled based on the HPLC profile to give 19 fractions. Scheme 7.2 shows the origin of each fraction.

176 Chapter 7 column sub-fraction Ab

fractions 7.2. Origin of byScheme HPLC produced of

177 Chapter 7

7.2.9 RP-HPLC/PDA Analysis of Ab fractions

The method applied here was as described in section 6.2.12.

7.2.10 HPLC/MS Analysis of Selected Sub-factions

Several sub-fractions were analysed by LC/MS. From the conclusions drawn up at the end of chapter 6, it was clear that three fractions in particular, Cb, Ca and Ab merited further analysis. They were analysed along with Ca α sub-fractions, Ca β, γ and δ. Here fractions from Ab acquired through semi-preparative HPLC were also analysed. Cb, Ca and Ca sub- fractions were analysed using HPLC method A. Ab samples were analysed via method B.

HPLC methods Method A: Samples were dissolved in methanol to give a 1 mg ml -1 solution. The HPLC method applied is as described in section 7.2.4.

Method B: Method B differed from method A in that the flow rate was reduced to 200 µl min -1.

Mass spectrometer settings were as described in 7.2.4.

7.2.11 NMR Analysis of Selected Sub-fractions

NMR spectroscopy was carried out at the Strathclyde Institute of Pharmacy and Biological Sciences, University of Strathclyde, Glasgow.

Samples were transported to the University of Strathclyde on ice, upon arrival they were transferred to a freezer at -20 °C where they were stored prior to use. Due to time constraints it was not possible to analyses every fraction. Sub-fractions Ab, Cb and Ca were analysed as a priority due to reasons state in 6.5. In addition to these, fractions B and D, along with Da, Db, Dc and Dd were analysed. In addition to the H. siliquosa extracts,

178 Chapter 7 the hexane and ethyl acetate extracts of Pelvetia canaliculata were also examined. There was not enough material to conduct NMR analysis on semi-preparative HPLC products of Ab and Ca.

Samples were dissolved in deuterated chloroform. Proton NMR spectra were recorded for each of the samples listed above. On the basis of 1D NMR, fraction D and sub-fraction Ca were further analysed by carbon-13 NMR, Heteronuclear Multiple Bond Coherence (HMBC) and Heteronuclear Single Quantum Coherence (HSQC).

7.3 Results

7.3.1 Minimum Inhibition Concentration (MIC) of Fraction Cb against the ABTS radical

Cb was clearly the most active sub-fraction with respect to its ability to quench ABTS and DPPH radicals. In order to study the interaction of compounds in this extract with the ABTS radical it was necessary to establish the minimum amount of Cb needed to inhibit 100% of the ABTS radical. In addition, only 10 mg of Cb was obtained from the column and it was felt that appropriate measures should be taken to preserve this stock for any further investigation.

Concentrations of 1.25 mg ml -1 and 1.50 mg ml -1 showed radical scavenging ability of 68.6% and 84.3% respectively with 1.75 mg ml -1 showing complete inhibition of the ABTS radical.

7.3.2 Identification of Compounds Responsible for Observed ABTS Radical Activity from Fraction Cb

As described before, three LC/MS runs were performed; the first the ABTS radical solution (fig 7.1), secondly, on the Cb extract sub-fraction (fig 7.2) and finally on a solution which was a mixture of the ABTS and Cb solutions (fig 7.3).

179 Chapter 7

Intens. x10 8

3.5

3.0

2.5

2.0

1.5

1.0 0 10 20 30 40 50 60 70 Time [min] Figure 7.1. Total ion chromatogram of ABTS solution

Intens. x10 8

1.75

1.50

1.25

1.00

0.75

0 10 20 30 40 50 60 70 Time [min] Figure 7.2. Total ion chromatogram of methanolic Cb solution

Intens. x10 8

3.5

3.0

2.5

2.0

1.5

1.0 0 10 20 30 40 50 60 70 Time [min] Figure 7.3. Total ion chromatogram of ABTS and Cb solution

It is clear that many of the dominant peaks in the Cb solution (fig 7.2) are no longer present in the ABTS/Cb solution (fig 7.3). It can also be noted that there are several peak in the ABTS/Cb solution that are not found in the original Cb sample. Thus suggests that compounds from Cb are involved in the scavenging of the ABTS radical.

It is difficult to carry out investigations using mass spectrometry on ABTS due to oxidation of the ABTS radical when using standard ESI configuration. This problem can be resolved by using nanoESI with coated nanoelectrospray emitters (Marjasvaara, et al., 2008), which

180 Chapter 7 unfortunately is not available throughout this study. However, in this experiment it is not crucial to study the ABTS radical throughout the scavenging process, but rather the radical’s interaction with compounds in the seaweed extract.

The peaks present in figure 7.1 and 7.3 at 8.1, 10.6 and 12.1 minutes are common to both samples (fig 7.1 and 7.3) and are therefore attributed solely to compounds present in the ABTS radical solution. The broader peak at 12.1 minutes, although present in both samples, only shows fragments of the radical in the chromatogram of the unreacted ABTS solution, as described by Marjasvaara, et al. (2008). It appears that the broadness of the peak at 12.1 minutes could be masking a further peak at 11.8 minutes. At 11.8 minutes with in this broad signal, the fragments of ABTS at m/z 486, 485, 458, 487, 429, 285 257 255 and 230 can be seen (fig 7.4.).

Intens. +MS2(514.0), 11.8min #1064 x10 4 485.9 1.0

506.5 0.8

229.8 480.6 0.6

352.8 0.4 265.1 281.9 324.3 421.9

236.0 292.3 492.2 254.8 276.1 451.8 0.2 385.4 406.0 428.8 437.2 306.5 338.4 372.2 446.0 458.1 467.8 415.1 0.0 250 300 350 400 450 500 m/z Figure 7.4. Mass spectrum of compound (m/z 514) at 11.8 minutes showing fragmentation of ABTS as described by Marjasvaara, et al. (2008)

7.3.3 Semi-preparative HPLC of Sub-fraction Ca

The original chromatogram of Ca contained two main groups of peaks, at 17.1 and 27.1 minutes. Semi-preparative HPLC produced four sub-fractions and was successful at separating these two groups (fig 7.5 and 7.6). However, no further separation could be achieved. Analytical HPLC of Ca α shows, in addition to the major peak at 14.5 minutes, a number of impurities at shorter and longer retention times. The use of a modified gradient shows that Ca β contains the compounds responsible for the second major group of peaks around 28 minutes. The use of a modified gradient does indeed show that there are at least two compounds present at 27.4 and 28.6 minutes. Again minor peaks not observed in Ca with shorter retention times are present as in Ca α. The major peaks in Ca γ (fig 7.7) and

181 Chapter 7

Ca δ (fig 7.8) are those at short retention times (six - 10 minutes), again these were not observed in the sample prior to semi-preparative HPLC and are not shown here.

Detector 1-Scan-279 nm 400 robbie h.sil Ca Alpha

300

200 mAU

100

0 20 40 60 80 100 120 Minutes

Figure 7.5. Chromatogram of sub-fraction Ca α at 280 nm

Detector 1-Scan-279 nm robbie h.sil Ca Beta

200 mAU 100

0 20 40 60 80 100 120 Minutes

Figure 7.6. Chromatogram of sub-fraction Ca β at 280 nm

7.3.4 Semi-preparative HPLC of Sub-fraction Ab

Semi-preparative HPLC of Ab produced 19 fractions of which nine were analysed using RP-HPLC/PDA. Only even fractions were analysed as these correspond to the crest of peaks in the semi-preparative HPLC chromatogram. Isolating and analysing the crest of each peak will give a better chance of isolating pure compounds. The odd fractions consisted of either groups of minor compounds or the lull between two major compounds which will mixtures. Analytical HPLC of Ab initially showed the presence of two major compounds with retention times of 32.8 and 36.0 minutes, as well as a number of minor

182 Chapter 7 compounds at both shorter and longer retention times. Seven of the fractions (Ab2-6 and 12-18) contain no notable signals, despite the semi-preparative HPLC chromatogram clearly showing 10 minor compounds with retention times between 10.0 and 20.0 minutes and 30.0 and 42.0 minutes. Chromatographs of Ab2 and Ab4 contained small peaks around 17 minutes and Ab6 a single compounds at 31.5 minutes. The signals in these chromatograms were small and are not shown here. The chromatograms of Ab8 and Ab10 (fig 7.7 and 7.8) shows prominent peaks at 34.7 and 38.0 minutes respectively, with only minor compounds with shorter retention times. These two prominent peaks are the major constituents of the original chromatogram of Ab.

Detector 1-Scan-280 nm 20 robbie h.sil Ab8

10 mAU

-10

0 20 40 60 80 100 120 Minutes Figure 7.7. Chromatogram of sub-fraction Ab8 at 280 nm

Detector 1-Scan-280 nm 20 robbie h.sil Ab10

10 mAU

-10

0 20 40 60 80 100 120 Minutes Figure 7.8. Chromatogram of sub-fraction Ab10 at 280 nm

7.3.5. HPLC/MS of Cb, Ca and Fractionated Samples of Ca

The total ion chromatogram (MS) of Cb (fig. 7.9) contains 10 peaks compared to the six that were observed in figure 6.20. The separation of peaks is more defined using a different HPLC system and certainly peaks eluted between 20 and 30 minutes benefit

183 Chapter 7 greatly from this. The first seven peaks have the same mass as the most abundant ion and with similar retention times they may well be isomers.

Intens. x10 8 479.3

6 391.3

4 407.3

2 449.3

0 0 10 20 30 40 50 60 70 Time [min] Figure 7.9. Total ion chromatogram of Cb

The total ion chromatogram for Ca (fig 7.10) again shows more peaks than the chromatogram from RP-HPLC/PDA (fig 6.21) with an increase in definition between peaks, thus showing improved separation. Five compounds are eluted between 13 and 18 minutes with a further three eluted between 23 and 28 minutes.

Intens. x10 8 423.3 6

407.3 4 481.3 481.3 479.2 2 449.3

0 0 10 20 30 40 50 60 70 Time [min] Figure 7.10. Total ion chromatogram of Ca

Semi-preparative HPLC of the fractionated samples of Ca were also analysed by LC/MS. The total ion chromatogram showed that neither Ca β, γ, δ nor any of Ca α fractions contained any of the signals highlighted in figure 7.10. This suggests that the compounds observed in the total ion chromatogram of Ca have degraded. Sub-fractions of Ca δ were not analysed due to TIC of Ca δ suggesting that compounds have degraded.

184 Chapter 7

7.3.6 HPLC/MS of Ab fractions

The samples analysed above using RP-HPLC/PDA were further analysed here by HPLC/MS. The chromatograms of Ab2, 6, 12 and18 shows no significant signals and their chromatograms are not presented here. Retention times are longer here due to the reduction in flow rate from 500 µl min -1 to 200 µl min -1, this was done to improve the separation. There is only one major signal present in the total ion chromatogram of Ab4 which has a retention time of 38.6 minutes. This major peak shows a mass of 479.3 and an extracted ion chromatogram of this mass is presented in figure 7.11.

Intens. x10 6

2.0

1.5

1.0

0.5

0.0 0 10 20 30 40 50 60 70 Time [min] Figure 7.11. Extracted ion chromatogram of mass 479.3 from Fraction Ab4

The total ion chromatogram of Ab8 (fig 7.12) shows two clear groups of peaks. The first set of peak (34 - 40 minutes) can be attributed to the minor peaks observed in figure 7.7 at around 16 minutes. The second set (62 – 68 minutes) can be attributed to the major peak at 34.7 minute. The reduced flow rate gives a longer retention time for these peaks but also improves the separation revealing several peaks from both groups that were not observed in the RP-HPLC/PDA analysis.

185 Chapter 7

Intens. 8 x10 441.2 1.0

0.8 495.3 439.3 0.6 463.3 479.3 0.4

0.2 495.3

0.0 0 10 20 30 40 50 60 70 Time [min] Figure 7.12. Total ion chromatogram of Ab8

The total ion chromatogram of Ab10 (fig. 7.13) again shows two sets of peaks. The two peaks in that make up the first set (39 – 40 minutes) are the same that make up two of the minor compounds seen in Ab8 (fig. 7.12) with the same retention time and mass of the parent ion. These peaks are also observed in the PDA chromatogram of Ab10 (fig 7.8) as minor peaks at 18 minutes. The second set of peaks (63 – 68 minutes) can be attributed to the peak at 38 minutes on the PDA chromatogram of Ab10. Here the reduced flow allows for improved separation showing a single major and two minor peaks.

Intens. x10 8 441.2

2.5

2.0

1.5 479.3 495.32 1.0 463.3

0.5

0.0 0 10 20 30 40 50 60 70 Time [min] Figure 7.13. Total ion chromatogram of Ab10

The total ion chromatograms for Ab14 and Ab16, both showed minor components with retention times of 48 and 50 minutes respectively. These compounds were not visible in the respective PDA chromatogram.

186 Chapter 7

7.3.7 Nuclear Magnetic Resonance Analysis of Selected Fractions

From preliminary proton NMR analysis of the various fractionated extracts, seven of the samples were deemed unsuitable for further detailed analysis. Fraction B and sub-fraction Ab were not pure enough to make sufficient progress in identifying compounds. The initial proton NMR for Cb concluded that the sample was not concentrated enough for suitable evaluation, however there was not enough material to concentrate the sample to a sufficient level.

Ca was the only sample that was deemed suitable for more in-depth NMR analysis and subjected to high resolution proton and 13 C NMR and 2D HMBC and HSQC NMR. Spectral data can be viewed in Appendix I. Two of the most defined elements that can be taken from the 1D spectroscopy are the presence of a ketone shown at 214 ppm in the 13 C NMR and the singlet at 3.7 on the proton NMR corresponds to a methoxy group.

7.4 Discussion

7.4.1 Identification of Compounds Responsible for Observed ABTS Radical Activity from Fraction Cb

Comparing the total ion chromatograms (MS 2) of both unreacted and reacted Cb solutions (fig 7.14 and 7.15) it is obvious that many of the compounds in the reacted Cb solution have a greater retention time. This suggests that these compounds have interacted with the radical to produce compounds that are less polar than the originals.

187 Chapter 7

Intens. x10 7

1.0

0.8

0.6

0.4

0.2

0.0 30 35 40 45 50 55 60 65 70 75 Time [min]

Figure 7.14. Total ion chromatogram (MS 2) of Cb

Intens. x10 7

1.5

1.0

0.5

0.0 30 35 40 45 50 55 60 65 70 75 Time [min]

Figure 7.15. Total ion chromatogram (MS 2) of ABTS radical and Cb solution

To confirm that a number of compounds from Cb have reacted with the ABTS radical, several extracted ion chromatograms were analysed. It is clear that a number of compounds in the unreacted Cb solution are not observed in the chromatogram of the quenched radical solution at m/z 465.2 (fig 7.16) and m/z 479.2 (fig 7.17).

Intens. x10 7 2.5

2.0 Extract Solution 1.5 Extract and ABTS •+ Solution 1.0

0.5

0.0 47.5 50.0 52.5 55.0 57.5 60.0 62.5 65.0 67.5 70.0 Time [min]

Figure 7.16. Extracted ion chromatogram of Cb and Cb + radical (m/z 465.2)

188 Chapter 7

Intens. x10 7

4 Extract Solution •+ 3 Extract and ABTS Solution

0 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 Time [min] Figure 7.17. Extracted ion chromatogram of Cb and Cb + radical (m/z 479.2)

In addition, to confirm that the compounds highlighted in figures 7.16 and 7.17 are involved in radical scavenging it was possible to look for the products of this reaction.

Again using the total ion chromatogram (MS 2) of the Cb/ABTS mixture (fig 7.15) to identify particular masses, extracted ion chromatograms can be used to distinguish peaks which are observed in the reacted solution that are not observed in the extract fraction at m/z 509.3 (fig 7.18) and m/z 523.3 (fig 7.19).

Intens. 7 x10 •+ 1.0 Extract and ABTS Solution

0.8 Extract Solution

0.6

0.4

0.2

0.0 62.5 65.0 67.5 70.0 72.5 75.0 77.5 80.0 82.5 Time [min] Figure 7.18. Extracted Ion Chromatogram of Cb and Cb + radical (m/z 509.3)

Intens. x10 7 Extract and ABTS •+ Solution 1.5 Extract Solution

1.0

0.5

0.0 47.5 50.0 52.5 55.0 57.5 60.0 62.5 65.0 67.5 70.0 Time [min] Figure 7.19. Extracted ion chromatogram of Cb and Cb + radical (m/z 523.3)

These new compounds have the same chromatographic profile as those in figure 7.16 and 7.17, also these compounds have a greater retention time suggesting that the resulting

189 Chapter 7 products are less polar. There is evidence to suggest that the radical scavenging process may be more complex for phenolic compounds than an electron transfer as described in figure 4.3. It is strongly suspected that the radical scavenging activity of H. siliquosa extracts is due to high levels of phenolic compounds. Two studies do agree that the quenching process described in figure 4.3 does occur; this is a first step that creates a phenoxyl radical. One study however suggests that this phenoxyl radical could react with a further ABTS radical in a similar process as before to produce a phenoxide anion. Alternatively, phenoxyl radicals could undergo dimerization (Pannala, et al., 2001). A second study suggests that a phenoxyl radical undergoes a rearrangement to form a semiquinone which then reacts with a further ABTS radical molecule to form an ABTS- phenolic adduct. This adduct is unstable and breaks down via one of two routes. The first route produces a stable phenolic and ABTS derived adduct and a free benzothiazolium ion; which unstable itself, undergoes hydrolysis followed by oxidation to give a benzothiazolone. The alternative route produces another phenolic/ABTS derived adduct and also produces a free imine (Walter and Everette, 2009). These pathways are summarised in figure 7.20 using phenol as a model for the antioxidant. In the study presented in this chapter, efforts were made to identify the presence of the benzothiazolium ion, the benzothiazolone and the free imine within the total ion chromatogram (MS) of ABTS/Cb solution (fig 7.3). However none of these could be identified from the sample, suggesting that scavenging of the ABTS radical by Cb may occur by an alternative.

190 Chapter 7

ABTS ABTS O OH O

O ABTS

N N O O S S NH O O 4 O O N NH 4 S S N O O NH N S 4 O O S N O S S O O NH NH4 N

NH4 N Hydrolysis N O O HO S S N O O O S S S S N O O O O N NH NH [O] 4 4

N O O S S O O NH4 Figure 7.20. Radical scavenging of the ABTS radical as proposed by Walter and Everette, (2009)

What can be noted from the new compounds identified in figures 7.18 and 7.19 is that they show an increase in mass of 45.1 amu over the original compounds highlighted in figures 7.16 and 7.17. A mass gain of 45.1 could correspond to carboxylic acid group or perhaps an ethoxy group. With all new compounds gaining the same mass, this suggests that whatever is quenching the semiquinones must be in excess. With ethanol present in excess, this may be the compound incorporated into the observed product. A simple way to test this is to would be to repeat the experiment but substitute ethanol for methanol. A

191 Chapter 7 gain in mass corresponding to a methoxy group (31.0 amu) would confirm an interaction with the solvent.

This experiment clearly identifies a number of compounds in the Cb fraction of H. siliquosa involved in the radical scavenging of the ABTS radical. Here, eight isomers with a relative atomic mass of m/z 479.2 quench the ABTS radical.

It is also possible to show that not all compounds found in sub-fraction Cb are involved in the reaction. Three isomers with a relative atomic mass of m/z 449.2 eluting between 60 and 65 minutes are clearly visible in both the chromatograms of the Cb and of Cb/ABTS solutions (fig 7.21). Either these compounds are not phenolic in nature or they may be sufficiently hindered not to react with the ABTS radical.

Intens. x10 7 •+ 1.25 Extract and ABTS Solution

1.00 Extract Solution

0.75

0.50

0.25

0.00 50 55 60 65 70 75 80 Time [min] Figure 7.21. Extracted ion chromatogram of Cb (m/z 449.2)

7.4.2 Identification of Compounds from H. siliquosa Fractions

From the results of both NMR and LC-MS analysis it is possible to identify four meroditerpenes. Three of the compounds are found in sub-fraction Ca with the final compound being identified from Cb. The proton NMR of fraction Ca shows a singlet at 7.30 which could be attributed to a phenolic proton. Two protons with a shift of 6.53 and 6.57 suggesting that these are aromatic protons and several signals are found with a chemical shift of between 5.00 and 5.50 which would suggest that these are vinylic protons. Signals at 4.50 can be attributed to protons attached to C-OH group. A shift of 3.75 in the proton NMR suggests protons from a methoxy group. A shift of 3.30 suggests a CH 2 group adjacent to an aromatic ring. There are many signals between 1.00 and 2.30 ppm which can be attributed to protons on

192 Chapter 7

13 CH 3 and CH 2 groups. The C NMR from Ca shows a signal at 214.6 ppm suggesting the presence of a ketone. Several signals can found in the aromatic and alkene regions between 110ppm and 160ppm. Using J-modulation theses signals can be differentiated between quaternary and tertiary carbons. Quaternary carbons can be found at 153.0, 146.0, 139.9, 134.2, 127.6 and 125.6 with those found at 153.0 and 146.0 being aromatic. The tertiary carbons are found with shifts of 127.8, 125.7, 121.0, 120.2 114.0 and 113.0. Several methyl groups can be identified between 15.0 ppm and 25.0 ppm with a number of secondry carbons between 25.0 and 40.0 ppm. There is a primary carbon with a shift of 56 ppm which suggests a methoxy group which is in agreement with the proton NMR. Two tertiary carbons at 65.5 at 74.0 suggest that these carbons are attached to alcohol groups.

Utilising 1H and 13 C NMR along with HMBC and HSQC experiments, a common fragment can be identified for the three compounds from Ca. HSQC shows two protons at 6.53 ppm and 6.57 ppm coupled with tertiary carbons at 113 ppm and 114 ppm respectively suggesting an aromatic ring system with four substituents. The hydrogen atoms from these two CH groups show an HMBC coupling of equal intensity to a quaternary carbon at 146 ppm (corresponding to an aromatic carbon coupled to an alcohol). This leads to the conclusion that these three carbon atoms are not adjacent to each other. The carbon at 114 ppm does show an HMBC coupling to hydrogen atoms of a methyl group at 2.2 ppm which in turn shows a coupling to a quaternary carbon at 125.6 ppm. The carbon atom of the CH group at 113 ppm shows a similar HMBC coupling however this time it is to hydrogen atoms of a CH 2 group at 3.3 ppm with has HSQC coupling to a carbon at 32.0 ppm. The protons on this group then show a HMBC coupling to a quaternary carbon at

127.6 ppm. Hydrogen atoms on both the methyl and CH 2 groups show a HMBC coupling to the quaternary carbon at 146.0 ppm linking together the quaternary carbons at 125.6 ppm and 127.6 ppm. The distinctive singlet in the 1H spectrum at 3.75 ppm couples to a carbon at 56 ppm in the HSQC experiment. This confirms the presence of a methoxy group. HMBC data shows that the protons on the methoxy group couple to an aromatic carbon at 153 ppm. This data allows for the identification of the part the structure shown in Figure 7.22. HSQC coupling is detailed in table 7.1 and HMBC coupling is illustrated in figure 7.23.

193 Chapter 7

OMe Figure 7.22. Partial structure of meroditerpenes as determined by 1H and 13 C NMR

δ1H δ13 C 3.3 32 3.75 55.6 6.53 153.0 6.57 114.0 Table 7.1. HSQC couplings uses to identify phenolic unit

Figure 7.23. HMBC couplings used to identify phenolic unit

Both the carbon and the protons on the CH 2 group highlighted above show an HMBC coupling to a proton at 5.35 ppm and a carbon at 125.7 ppm respectively. The three protons described here all show a further HMBC coupling to a quaternary carbon at 134.2 ppm. The CH proton at 5.35 ppm shows a HMBC coupling to a primary carbon atom at 16.4 ppm of a methyl group (HSQC coupling to protons at 1.70 ppm). These protons at 1.70 ppm show an HMBC coupling to a quaternary carbon at 134.2 ppm. The protons on this methyl show a further HMBC coupling to a secondary carbon at 47.9 ppm which in turn has an HSQC coupling to protons at 2.20 ppm. These two protons (2.20 ppm) show a

HMBC coupling to the quaternary carbon at 134.2 ppm. The CH 2 protons at 2.20 ppm show two further HMBC couplings both to two very different tertiary carbons. Firstly to a carbon at 65.5 ppm, this in turn has a HSQC to a single proton at 4.5 ppm suggesting the

194 Chapter 7 presence of an alcohol. Secondly, to a carbon at 127.8 ppm, this in turn shows HSQC to a proton at 5.15 ppm leading to conclusion that this CH is bonded to a quaternary carbon via a double bond. Both the carbon and proton show HMBC coupling to a methyl group with the carbon showing a shift of 16.0 and the three hydrogens a shift of 1.68 ppm. The hydrogens on this methyl group and the single hydrogen on the CH at 5.15 both show an HMBC coupling to a secondary carbon at 39.5 ppm, this in turn show HSQC link to two hydrogens at 1.95 ppm. These two hydrogen atoms show HMBC linkage to a secondary carbon at 25.1 ppm with an HSQC link to two protons at 2.11 ppm. The continuation of the chain can no longer be continued using HMBC as there is no coupling to neighbouring carbon atoms or protons. It is suspected that this is due to the ending of the common structure (fig 7.24.) between identified compounds thus any possible HMBC signal would be weak and therefore not show. HSQC and HMBC coupling is illustrated in figure 7.25. This data is in agreement with the study by Banaigs, et al. (1983).

Figure 7.24. Common structure as determined by 1H and 13 C NMR

195 Chapter 7

δ1H δ13 C 1.68 16.0 1.70 16.4 1.95 39.5 2.11 25.1 2.20 47.9 3.30 32.0 4.50 65.5 5.15 127.8 5.35 125.7 Table 7.2 HSQC coupling of partial chain structure

Figure 7.25. HMBC coupling of partial chain structure

Compound A

Using the data provided in the study by Banaigs, et al. (1983) and the 1H and 13 C NMR data with the aid of HSQC coupling of Ca to identify two methyl groups (carbon shift at 18.7 ppm and 26.0 ppm and proton shift 1.85 ppm and 1.80 ppm respectively) that showed an HMBC coupling to each other and to a quaternary carbon at 139.9 ppm suggesting a termination in the chain. Both sets of protons on each primary carbon have an HMBC coupling to a tertiary carbon at 121 ppm with an HSQC coupling (table 7.3) to a proton at 4.95 ppm. The tertiary carbon then shows a HMBC coupling to a proton at 4.80 ppm which in turn has an HMQC coupling to a tertiary carbon at 74 ppm indicating the presence of an alcohol group. This tertiary carbon at 74 ppm has a larger shift than would usually be expected thus suggesting that it is adjacent to a ketone (fig 7.26). A ketone is observed in the 13 C NMR spectrum at 214.6 however no HMBC coupling is seen and therefore unable to use HMBC to provide a link to this fragment with the part structure in figure 7.24. LC-MS provides further evidence of for the part structure in figure 7.24 showing characteristic signals at m/z 151.0, 191.0, 205.0, 215.1, 255.1 and 269.1 (fig 7.27.).

196 Chapter 7

δ1H δ13 C 1.80 26.0 1.85 18.7 4.80 74.0 4.95 121.0 Table 7.3 HSQC coupling of compound A

Figure 7.26. HMBC coupling of compound A

Intens. +MS2(439.7), 13.1min #1076 x10 6 191.0 1.25

1.00

0.75

0.50 421.2

205.0

0.25 215.1 233.1 151.0 173.1 255.1 269.1 295.2 355.3 135.1 243.1 337.2 383.2 397.3 323.3 0.00 100 150 200 250 300 350 400 450 m/z Figure 7.27. Mass spectrum of compound A

In the mass spectrum the compound undergoes dehydration at the alcohol at position 5 leaving a double bond between carbon 4 and 5. Therefore the molecular ion as identified via mass spectrometry is reported with a loss of H 2O. In addition MS provides evidence for the remainder of the structure as proposed above. This leads the identification of compound A (fig 7.28) which has previously been identified in H. siliquosa and shows potent antibacterial activity (Culioli, et al., 2008). Figure 7.28 shows the fragmentation pattern of compound A. This conclusion is in agreement with the data published by Banaigs, et al. (1983).

197 Chapter 7 . Mass fragmentation Compound of A

Figure 7.28

198 Chapter 7

Compound B

Compound B also shows characteristic mass fragmentation at /z 151.0, 191.0, 205.0, 215.1, 255.1 and 269.1, which confirms the presence of the partial structure in figure 7.24 (fig 7.29.).

Intens. +MS2(407.6), 24.1min #2394 x10 6 151.0 3

191.0 255.1

351.2 2

203.1

217.1 269.2 243.1 1 323.2 297.1 229.1 177.1 337.3 135.0 159.1 285.2 311.2 365.3 123.1

0 100 150 200 250 300 350 400 m/z Figure 7.29. Mass spectrum of compound B

Using HMBC and previously published data it was again possible to identify two methyl groups (carbon shifts at 18.0 ppm and 25.9 ppm and proton shifts 1.6 ppm and 1.7 ppm respectively) through a quaternary carbon at 136.9 ppm, signifying a termination in the chain. Both sets of protons show a further HMBC coupling to a tertiary carbon at 120.2 ppm which has an HSQC coupling to a proton at 5.1 ppm (table 7.4). The tertiary carbon also shows a HMBC coupling to a proton at 2.28 ppm which in turn has a coupling to a secondary carbon at 34 ppm. No further HMBC couplings can be identified. This gives the part structure in figure 7.30. δ1H δ13 C 1.60 18.0 1.70 25.9 2.28 34.0 5.10 120.2 Table 7.4. HSQC coupling of compound B

Figure 7.30. HMBC coupling of compound B

199 Chapter 7

The NMR values documented here are in agreement with data published by Banaigs, et al. (1983). Bearing this in mind, it is suspected that the carbon at position 12 is tertiary with an alcohol group attached. Both alcohol groups on the chain are not seen in mass spectrometry data as compound B loses two molecules of water. The data from the mass spectrum shows the sequential loss of fragments at a mass of either m/z 12 or 14, which can be interpreted as a carbon atom and a CH 2. By using this data it is possible to identify the structure of compound B (fig 7.31). Compound B is a known compound that has previously been identified from a sample of the marine algae Cystoseira elegans . The bioactivity of compound B has been assessed with it showing no activity towards Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa and Klebsiella pneumoniae . This is the first time that this compound has been identified in a sample of H. siliquosa .

200 Chapter 7 ass spec fragmentation spec ass compound B of

Figure 7.31. Structure andFigure 7.31. Structure m

201 Chapter 7

Compound C

The structure of compound C was primarily solved using mass spectrometry data. The mass spectrum shows the characteristic fragments consistent with the fragmentation of compounds A and B that allows for the partial structure in figure 7.24 to be identified (fig 7.32).

Intens. +MS2(423.6), 15.1min #1318 x10 6 405.3

6 217.1 191.1

4 257.1 151.0

273.2 349.2 2 205.1 367.3 229.1 379.2 173.1 297.1 335.2 285.1 309.2 325.2 121.2 133.1 0 100 150 200 250 300 350 400 m/z Figure 7.32. Mass spectrum of compound C

The two relatively strong signals for the fragments at 349.2 and 353.2 are unusual and it is highly unlikely that an ion should fragment in this way with the difference of 4 protons between two such prominent signals. This suggests that keto-enol tautomerisation may be occuring. As a result the mass spectrum contains signals from both the ketone and the enol resulting in fragments m/z 349.2 (enol form) and m/z 353.2 (keno form) (fig 7.33). Previous research by Culioli, et al. (2008) has shown that similar meroditerpenes may also undergo some form of tautomerisation.

Fig 7.33. Keto-enol tautomerisation of compound C

Figures 7.34 and 7.35 show the full fragmentation of both the ketone and the enol version of compound C. It is believed that this is the first report of compound C.

202 Chapter 7 form

Figure compound 7.34. C Fragmentation of in ketone

203 Chapter 7 . . ] ] ] ] 2 2 2 2 O O O O 29 37 29 25 H H H H 22 28 24 20 349.2[C 297.1[C 325.3 [C 405.3 [C O 2 ]-H ] 3 4 O O 38 40 H H OH 28 28 M+H 423.6 [C Mass 440.6[C OH ompound C in form enol ...... ] ] ] ] ] ] ] ] . 2 2 2 2 ] 2 2 2 2 2 O O O O O O O O O 25 21 17 15 15 17 18 23 11 H H H H H H H H H OH OMe 19 17 15 13 9 12 14 16 18 285.2[C 229.1[C 203.1[C 151.1[C 191.1[C 217.1[C 243.1[C 273.2[C 257.1[C

Figure c 7.35. Fragmentation of

204 Chapter 7

Compound D

Compound D was identified from sub-fraction Cb and the structure solved by mass spectrometry with the aid of 1H NMR. Mass spectrometry does not show the characteristic mass fragment that would allow for the assignment of the part structure in figure 7.24. Gerwick and Fenical 1981 identified a hydroquinine compound with an unsubstituted chain from which a mass spectrum of the hydroquinine showed masses at m/z 137.0, 175 and 189 which are what would be expected if the methoxy group at position 4’ had been substituted for an alcohol group. Indeed the mass spectrum of compound D shows fragmentation at m/z 137.1, 177.0 and 189.0 which suggest a similar type of compound (fig 7.36).

Intens. +MS2(391.5), 27.5min #2824 x10 6 201.1 2.5 255.1

2.0

137.1 1.5

215.1 177.0 1.0 189.1 159.1 335.2 145.1 241.1 321.2 227.1 267.2 349.2 0.5 169.1 279.1 293.2 307.2 363.3 131.1 121.1

0.0 100 150 200 250 300 350 m/z Figure 7.36. Mass spectrum of compound D

The side chain shows a sequential fragmentation of either m/z 12 or 14 which can be either a carbon atom or a carbon and two protons. What is most notable is that there is no fragmentation of either m/z 16 or 18 which would depict a loss of an oxygen atom or a water molecule leading that has been observed in compounds A,B and C. This leads to a suggestion that the side chain is hydrocarbon in nature. When comparing the mass of compound D with the previous compound identified by Gerwick and Fenical, (1981), and then later by Culioli, et al. (2008), D is found to have less mass than the identified hydroquinone. This difference in mass of m/z 6 would suggest the D is fully unsaturated (fig 7.37). The accompanying proton NMR is similar to that of Ca and shows aromatic protons at 6.1 ppm and also protons at 6.5 ppm signifying a tertiary carbon with a double bond. The NMR also shows phenolic proton shift at 7.3 ppm. Between shifts of 5.00 and 5.50 there are a number of signals which would suggest that these are vinylic protons.

Various signals between 1.00 ppm and 2.30 ppm suggest an assortment of CH 3 and CH 2

205 Chapter 7 groups. As with the Ca fraction the signal at 3.75 indicates the presence of a methoxy group however there does not appear to be the alcohol signal that were shown in Ca at 4.5 ppm. The protons from a CH 2 group can be seen with a shift of 3.20 ppm. It should be emphasised that the NMR experiment is of the whole fraction where as the mass spectrometry analysis has been coupled to HPLC and hence why shifts for methoxy groups and CH 2 groups can be seen in the NMR but not seen in the mass spectrometry data. This is the first time that D has been identified and it is thought that it is the precursor for the series of meroditermpene that have been identified here and in the highlighted publications. It is not known if D is involved in the scavenging of the ABTS radical highlighted in this chapter.

206 Chapter 7 • • • • • ] ] ] ] ] ] 2 2 2 2 2 2 O O O O O O 25 27 23 21 29 19 H H H H H H 24 25 23 22 26 21 335.2 [C 335.2 321.2 [C 321.2 [C 307.2 293.2 [C 293.2 [C 349.2 [C 279.1 ] 2 O 35 H 29 M+H 391.5 [C 391.5 M+H • • • • • ] ] ] ] ] ] ] ] 2 2 2 2 2 2 2 2 • ] O O O O O O O O 2 19 17 15 11 O 13 13 15 19 9 H OH OH H H H H H H H H 20 15 14 12 8 11 13 14 16 . Mass fragmentation compound D of 267.2 [C 267.2 201.1 [C 201.1 [C 227.1 241.1 [C 241.1 [C 215.1 [C 189.0 [C 137.1 [C 177.0 [C 255.1

Figure 7.37

207 Chapter 7

7.4.3 Use of Semi-preparative HPLC on Ab and Ca Sub-fractions

Significant time and effort was spent on purifying extract sub-fractions in order to isolate and identify pure compounds using semi-prep HPLC and subsequent analysis by LC/MS and NMR. Despite this effort, analysis of semi-prep HPLC fractions of Ca by mass spectrometry did not show any of the signals that were highlighted in the parent Ca sub- fraction (fig 7.10). This suggests that the compounds listed from figure 7.10 and therefore the identified compounds A, B and C have broken down. As the compounds were identified after undergoing RP-LC/MS, it is unlikely that the reverse phase chromatography process is the cause. What is more probable is oxidation of the compounds. There is insufficient data to be able to identify products from the oxidation of these compounds. It can therefore be concluded that semi-preparative HPLC has not been successful with respect to compounds from Ca. In order to prevent the breakdown of natural products, a possible step could be to acetylate the compounds thus providing them with a degree of stability that could allow for isolation and characterisation. However after acetylation it can be difficult to undo the derivatisation and recover the products for analysis of their bioactivity.

The compounds present in Ab may have undergone a similar fate to those in Ca. Several signals highlighted in semi-preparative HPLC traces were collected and upon analysis by both diode array and mass spectrometry, failed to yield any pure compounds. In contrast to this, Ab2, 8 and 10, show signals in both diode array and mass spectrometry analysis, which can be seen in the original Ab sub-fraction. It is not possible to identify the compounds that are in each of these fractions due lack of suitable NMR data.

7.5 Conclusion

Chapter 6 concluded that sub-fractions Ab, Cb and Ca merited further investigation with respect to their composition. With Cb showing greatest radical scavenging activity out of the all sub-fractions, it was used in conjunction with the ABTS radical scavenging assay and mass spectrometry to identify compounds involved in the quenching of the radical. Several compounds of different masses from this sub-fraction are involved in scavenging of the ABTS radical. In addition, there are compounds within Cb that have been shown

208 Chapter 7 not to be radical scavengers. It is unfortunate that there was a limited amount of Cb available that meant analysis by NMR and further MS studies could not take place. It is important to stress that this sub-fraction is highly active in all three assays from chapter 6 and effort should be made to determine the structures of the compounds that have been identified here.

The combination of LC-MS and NMR including 2D experiments have allowed for identification of two known meroditerpene and also for the proposal of two unknown compounds from sub-fraction Ca (fig 7.38). Although not described here, these types of compound are known to be active against some marine bacteria (Culioli, et al., 2008). It would be highly desirable to isolate these compounds that have not been previously described, in order to characterise them fully and also to establish any bioactivity they may have. OH OH OH

Compound A O

OMe OH OH

Compound B OH OMe OH OH

OH Compound C (Enol) OMe

OH OH

Compound C (Ketone) O OMe

Compound D OH Figure 7.38. Proposed structures of compounds from H. siliquosa extract fraction using LC/MS and NMR

209 Chapter 8

8. Conclusion and Future Work

8.1 Conclusion

One of the aims of this project was “to assess the bioactivity of extracts from a range of local seaweed species and in doing so, develop access to a range of bioassays suitable for the task.” The work undertaken in this study has successfully evaluated seaweed from Scotland for their bioactivity by subjecting extracts of a number of intertidal seaweed species to a range of assays, via a network of collaborators.

Several of the species investigated showed no detectable activity to any of the seven bacterial strains while extract fractions of four species, Halidrys siliquosa , Polysiphonia fucoides , Osmundea pinnatifida and Fucus serratus , each showed antibacterial activity to at least six of the seven strains. The hexane and ethyl acetate fractions of P. fucoides showed particularly strong antimicrobial activity against the bacterial strains and the ethyl acetate fraction of O. pinnatifida was the only fraction to show detectable antimicrobial activity towards all seven bacterial stains. While several species such as H. siliquosa , O. pinnatifida show broad spectrum antimicrobial activity in this study and against human pathogens, extracts of Fucus vesiculosus , F. serratus and Plocamium cartilagineum only show activity against the bacterial strains from this study. This suggests that some species of seaweed produce compounds that are selective to only marine relevant bacteria where as other seem to produce broad spectrum antibiotic compounds. The antimicrobial activity of extracts against marine bacteria appears to be dependent to at least some degree on the preferred shore location of the seaweed with greater activity associated with extracts from species collected from rockpools and further down the shoreline. The ecological significance of this is that such seaweed species may have adapted antimicrobial chemical defence mechanisms to survive colonisation in more competitive ecological niches. These results confirm the presence of compounds that can inhibit the grown of a range of marine bacteria and therefore answer the research question regarding limiting the development of biofouling. This study shows that seaweed species have the potential to chemically control the population of surface accumulating bacteria. The impact of this research has allowed

210 Chapter 8 for H. Siliquosa extracts to be incorporated into paints and is currently undergoing testing to evaluate the effectiveness as an antimicrobial paint. .

It is perhaps a little more difficult to ascertain whether the research question has been satisfied regarding the radical scavenging assays in chapter 4. The research question asked at the beginning of the thesis specifies whether seaweeds produce compounds that could be of benefit to human health. Indeed extracts were evaluated for their radical scavenging potential using three different radicals. However the DPPH and ABTS radicals are not radicals that exist within human physiology, so although seaweed extracts quench these radicals it would be incorrect to state that compounds within the extract are of benefit to human health based on these assays. They were employed as they are well established, quick and easy to perform assays and the results offer a guide as to potential beneficial antioxidants. To fully answer the question posed, some extracts of brown seaweed were shown to quench superoxide radicals using EPR which may contain compounds that could be of benefit to human health. Generally extracts demonstrated strong radical scavenging activity in the spectrophotometric assays, however this was not mirrored in the electron paramagnetic resonance assays. This highlights a problem within antioxidant research, whereby well established, simple to perform assays which are widely employed, are often unable to quantify the real antioxidant potential of extracts and compounds, as these assays appear unable to differentiate between antioxidants and pro-oxidants. It is possible that the structure of the radical may also hide the true potential of an extract or compound. The DPPH radical is more hindered than the ABTS radical, this may prevent larger compounds from scavenging DPPH which could scavenge the ABTS radical. This illustrates the need to employ several radical scavenging assays within a study, rather than to rely on the results from a single radical assay to base conclusions on.

Extract fractions were subjected to range of assays designed to evaluate their potential as possible drug candidates. Extracts on the whole showed poor antimicrobial activity towards Staphylococcus aureus , Escherichia coli and Nocardia farcinica with the exception of an ethyl acetate fraction of P. fucoides that showed good activity against S. aureus . This seems to confirm that antimicrobial compounds that are produced by seaweed appear to be selective towards marine bacterial strains rather than compounds that have broad spectrum antibiotic properties. The aqueous and methanolic fraction of extracts showed little activity against Trypanosoma brucei brucei however the ethyl

211 Chapter 8 acetate fraction of most species showed very good activity. Ethyl acetate fractions of Chondrus crispus, F. vesiculosus and Ascophyllum nodosum showed poor activity. Extracts again showed poor activity against a number of cancer cell lines. The ethyl acetate fraction of H. siliquosa was the only fraction that showed cytotoxic activity against more than one cell line. However this extract also showed activity against the HS 27 cell line which is used as a standard model for health normal cell, which may indicate that the compounds responsible for the cytotoxic activity are not suitable as drug candidate. This study from chapter 5 confirms that seaweeds do produce compounds that could be benifical to human health. Not only does it confirm activity and thus answer the relevant research question but it takes it a step further by highlighting that there could be some degree of selectivity which would be highly desirable for a drug molecule.

Extracts of seaweed from the north of Scotland do show activity against a range of chemical and biological targets. The results presented here show that high value natural products from seaweed native to Scotland could be utilised and developed in to a range of products to suite the pharmaceutical, nutraceutical and marine industry markets.

The second aim based on the research questions was “to identify and isolate from the most promising species, natural products responsible for the observed bioactivity.” This has been accomplished in part. The use of semi-prep HPLC to isolate natural products from an H. siliquosa extract was not a success. No pure compound was isolated and fractions that were collected from the column showed not similarities to the parent fraction upon LC- MS. This led to the conclusion that compound could be air sensitive, however without the facilities to perform semi-prep HPLC in an inert atmosphere, this can not be confirmed. While no compounds have been isolated, and therefore no bioactivity can be assigned to an individual compound with a proposed structure, an extract of Halidrys siliquosa was subjected to assay guided fractionation and RP-HPLC/PDA analysis. This highlighted three sub-fractions and from one it was possible to identify several compounds that are involved in the radical scavenging activity of the ABTS radical. It was disappointing that there was not enough of this sample to undergo detailed NMR analysis however due to radical scavenging activity of brown seaweed being dependent on the total phenolic content it is likely that these compounds are phenolics. Several fractions were analysed by LC/MS and 2D NMR, where the structure of four compounds are proposed. Two known compounds, (2E. 6E. 14E)-l-(l’-hydroxy-4’-methoxy-6’-methyl-phenyl)-5, 13-dihydroxy-

212 Chapter 8

12-one-3, 7, 11, 15-tetramethylhexadeca-2, 6, 14-triene (compound A) and (2E, 6E, 10E, l4E)-1-(1’-hydroxy-4’-methoxy-6’-methyl phenyl)-5, 12 dihydroxy-3.7, 11, 15-tetramethyl hexadeca-2.6, 10, l4-tetraene (compound B) were identified and this is the first report of the latter compound in H. siliquosa . The structure of two new compounds (C and D) was proposed on the basis of the NMR and MS analysis. Despite identifying four compounds, the research question set out has not entirely been answered. Although these compounds were identified from fractions that showed good activity towards the chosen assays, it is unknown whether these compounds are thoses that produced the observed response. The compound previously identified from H. siliquosa is a known antimicrobial compound that has shown activity against some marine bacterial strains .Due to structural similarities it is likely that the compounds proposed from Ca also show some antimicrobial activity and may be responsible for the observed activity.

One final research question remains unanswered; can they be used to our advantage? As no pure compounds were isolated it is impossible to conclude whether an individual compound could be used to our advantage. This study at least highlights that compounds from seaweed could have health benefits. Had a pure compound been isolated from an active fraction, then it could have been fully characterised and then have been tested for activity using the relevant assay. Should results be positive from this, the network of collaborators would have been able to provide guidance on how best to proceed which may well have been to sell the IP to biotechnology company for further development.

8.2 Future Work

From the results described in Chapter 3, it is clear that seaweed from Scotland contain compounds that could form part of a chemical defence to biofouling. Several areas of future work could be undertaken. The study that is presented in Chapter 3 focuses on only 10 % of seaweed species that have been identified on local coastlines by McDougall, (2004) and Gibson, et al. (2001). This leaves 116 species that have yet to be screened against the bacterial cultures used here.

213 Chapter 8

8.2.1 Screening of Seaweed Species

The employment of various animals, plants and microorganisms as sources of bioactive compounds that may eventually find applications in commercial pharmaceuticals is still a vibrant and proven method of drug discovery (Chapter 1). Over the last 30 years marine organisms, including seaweed, have been investigated for their natural product content (Chapters 2 and 5). These investigations usually begin with screening fractionated extracts of the chosen specimens via a series of assays. The work presented here, due to limited time and other factors already highlighted, focused on a relatively small number of seaweed species and utilised a limited number of assays.

It has already been stated that in order to increase the chance of discovering a bioactive compound, then increasing in the number of samples and assays would be beneficial. In chapter 5, one of the discussion points was that SIDR only carried out cell based assays leaving out assays such as radioligand binding assays. It would be useful if the remaining assays were to be carried out on the samples that have already been processed to provide a broader overview of the potential of seaweed as a source of bioactive compounds. With only a small proportion of seaweed species from Scotland being evaluated for their bioactivity (chapters 3, 4 and 5), over 100 species have yet to be evaluated including a number of prominent species such as Fucus spiralis , Laminaria digitata and Ulva species. It is not inconceivable that every species found on the Scottish coast line could be evaluated; indeed the studies by Hornsey and Hide (1974) looked at 32 species. Such a task would take significant time and may present problems when collecting species that are found sparsely in the assigned location. However the results presented here do suggest that this may well be a worthwhile task.

8.2.2 Isolation and Analysis of Pure Compounds

The isolation of pure compounds is a priority for advancing this research. In the work described here, two species in particular ( H. siliquosa and P. fucoides) were highlighted by the screening program detailed in chapters 3, 4 and 5. H. siliquosa was chosen for a more in-depth examination which resulted in several compounds being identified by NMR and mass spectrometry data. The next logical step would be to continue with the purification

214 Chapter 8 of extracts with the aim of isolating pure compounds which can then be subjected to a variety of assays to produce individual compounds with defined bioactivities. This would allow access into the next phase of drug development.

As previously described in section 6.1.1, a species would not be selected for further study if removing sufficient quantities for an extraction could result in damage to the immediate ecosystem. It was for this reason that the chemistry of the red seaweed P. fucoides was not investigated further. P. fucoides merits further chemical analysis as this species has shown good potential as a source of bioactive natural products. The screening programme showed that extract fractions of P. fucoides showed good antibacterial activity against a range of Gram positive and Gram negative bacterial. It also shows greater radical scavenging activity and around twice the amount of phenolic compounds than the other red seaweed species tested. Crucially, P. fucoides has not had its chemistry studied in detail and would therefore benefit from undergoing a similar process to that of H. siliquosa .

8.2.3 Ecological Functions of Natural Products from Seaweed

8.2.3.1 Antimicrobial Activity

One of the more interesting conclusions to emerge from the experiments carried out in Chapter 3 was linking antimicrobial activity and shore position, which showed greater activity in species found in rock pools and those in lower shore line positions. The upper and lower shore line positions in this study had a sample set of two each with the middle shore position represented by three species. Although it is possible to observe patterns and report on them, the number of species (seven species on the exposed rock and the six species from the rockpool) is too small a data set to obtain statistically relevant conclusions. By increasing the number of species especially within these exposed shoreline positions to between 10-15 species of each brown, green and red, would provide a greater sample set thus providing results that are statistically significant. Species such as those in the upper infralittoral zone should be included in a study. Should the location of the seaweed be crucial for antimicrobial activity, it would be expected that such species would show strong antimicrobial activity towards a broad range of marine Gram positive and Gram negative bacteria.

215 Chapter 8

So far only laboratory based tests have been carried out to ascertain the antimicrobial activity of seaweed extracts using the disk diffusion assay. The next logical step is to develop a method to test antimicrobial activity of extracts in the environment. For example, extract solution could be swabbed onto sterile glass plates that in turn would be submerged in a marine environment. Once bacterial colonies are attracted to the plates they can be swabbed and bacteria (if any present) transferred to nutrient rich agar plates and then incubated. Bacterial colonies could then be isolated, enumerated and identified. This would allow for a number of observations to be made. Firstly the antimicrobial activity of seaweed extracts can be examined in the marine environment. Secondly, the differences in bacterial populations between samples and finally this type of experiment could provide information as to whether seaweed extracts or a combination of extracts could be utilised in coating to provide a suitable and effective prevention of biofouling on man-made structures in the marine environment.

8.2.3.2 Antifeedants

The seaweed species of Scotland provide a suitable habitat for a range of marine life, including the crustacean Echinogammarus marinus . Some seaweed species, usually those with broad leaves, such as F. vesiculosus and F. serratus , appear to be more susceptible to being consumed as food by marine organisms, whereas those species that are strand based, such as Chorda filum and Corallina officinalis usually appear to be more protected, however this is not always the case. There has been limited research into the antifeedant potential of seaweed, however a number of studies have highlighted a link between polyphenolic and phlorotannin levels and grazing habits of a range of marine life such as fish, sea urchins and snails (Cacabelos, et al., 2010; Svensson, et al., 2007; Wessels, et al., 2006; Pavia and Toth, 2000). This is contrary with what is observed, where species that contain more phenolic compounds, such as the Fucoids appear to be more susceptible to grazers. Generally, an organism would initially begin grazing on a seaweed sample whereupon a series of compounds would be released. What is not clear at the current time is to what extent and which species of seaweed from Scotland have antifeeding properties. In addition, during sampling expeditions for the studies in this thesis, it was observed that E. marinus made up a significant proportion of the attached epiphytes. E. marinus ,

216 Chapter 8 however have not been utilised as a test organism to determine the antifeedant properties of compounds in seaweeds found in Scotland.

In order to evaluate the antifeeding properties of seaweeds from Scotland a simple test could be implemented. Here extracts from a range of seaweed could be suspended in nutrient rich agar blocks. E. marinus could then be placed with the blocks in sea water and monitored each day for a week. At the end of the experiment several measurements could be taken. It is hoped that some of the E. marinus would have been able feed on the agar blocks, thus growing while others would not and subsequently die. The mass of the agar block could be measured providing information as to how much feeding had taken place. Also E. marinus moult when they grow and therefore it would be possible to count how many casts have been shed as an indication of whether the crustaceans have been feeding with the number of E. marinus that have died during the experiment would also provide similar conclusions.

8.3 Limitations of this Study

This research is limited by a number of factors. Some of these limitations have been briefly highlighted in several chapters. In this section greater detail of the implications that the limitations have on this study and the impact they may have on future work is discussed.

There are 129 species of seaweed found around the shore of Caithness. In this study only 10% of species have been assayed which leaves a large proportion that have not. Although this study has tried to select species from as broad a range of shore positions as possible whilest being realisitic with restricitons on time, this has led to some shore position being under represented such as upper and lower exposed areas. While this may not have any direct impact with relation to the research questions and the aims of the study it does however limit the ability to draw a definitive conclution with respect to the observed pattern between antimicrobial activity and shore postion. In section 8.2.3.2 it is recommended that for future work on antimicrobial activity that the number of seeweed species studied is increased to give and equil weighting per each area when making a conclusion, however in the current location this would lead to an imbalance of the number

217 Chapter 8 of species between exposed and sheltered loactions due to the shallow gradiant of the intertidal zone.

In addition, the antibacterial study in chapter 3 uses seven strains of marine bacteria and therefore any observed activity and subsequent conclusions are limited to this set of bacteria. There are of course more than seven strains of bacteria and it is highly possible that other marine bacteria would behave differently when exposed to extracts of seaweed and this may lead to a different conclusion. Using different bacterial strains is a consideration but the results here satisfactaully answers the initial reaserch question and aims.

Although it is clear that extracts of seaweed contain compounds that show activity aginst medically relevant targets, the research findings are limited to the number of assays carried out and the reason for the degree of limitation is discussed in chapter 5. To overcome the limitations that were found in this study it is imperative that all parties involved are full committed, clear on the aims and understands the impact that the the study has. This however was not the only limitation in this chapter with the possibility of further assays being carried out on a commercial basis. With the absence of sufficient funds it is not possible to utalise the commercial services of our colaborators. It would be beneficial for future work to secure additional funding so that assays could be carried out in a commercial capacity. At the very least this would insure that samples are processed and results returned within an appropriate time scale.

The access to equipment is a limitation that many research project encounter. This is not usually a problem at well established campus universities where departments are normally well equipt, for example you would expect a chemistry department to have a extensive range of analytical equiptment including NMR, mass spectrometry, GC, HPLC, IR etc. Indeed interdepartment usage of equipment is common place in most universities and require relatively little organisation. Although in this study there was on-site access to HPLC and mass spectromity, access to NMR was in Glasgow which required careful preparation and planning, as a result only one trip to Glasgow was undertaken. Greater access to NMR would have allowed for all extracts and fraction to be analysed which may have changed the outcome of the study and allowed for more compounds to be isolated. A

218 Chapter 8 future study specifically aimed at identifying novel compounds would require frequent and regular access to an NMR suite to maximise the outputs of that study.

When sampling in this study, only one sample was collected for each species. This limits the findings in this thesis to only those particular samples and not to the broader population of the species. By only sampling a species once there is the possibility that any observed activity could be coincidence and be unique to that sample. In reviewing the literature on natural products from seaweed it seems that in the vast majority of studies progress down the single sample route thus relying on the data from a single sample. To rectify this, multiple specamins of each selected species at different locations could be samples. This has its own limitation as by increasing the the number of replicates to a standard three, would triple the time of laboratory based work. It was felt that it was more productive and would therefore increase the chance of finding an active compound to study one sample from many species rather than for three samples in fewer species. Any future work will also have address this dilema and it will be up to the specific research questions and aims of those studies that will direct the approach that is taken.

219 References

References

Abdel-Mageed, W. M., Milne, B. F., Wagner, M., Schumacher, M., Sandor, P., Pathom-aree, W., Goodfellow, M., Bull, A. T., Horikoshi, K., Ebel, R., Diederich, M., Fiedler, H-P., and Jaspars, M. (2010) Dermacozines, a new phenazine family from deep- sea dermacocci isolated from a Mariana Trench sediment. Organic and Biomolecular Chemistry , 8, pp. 2352-2362.

Abreu, P. M., and Galindro, J. M. (1996) Polyhalogenated Monoterpenes from Plocamium cartilagineum from the Portuguese Coast. Journal of Natural Products , 59 , pp. 1159-1162.

Adams, B. (2011). Eisai marine sponge derived cancer drug approved in Europe. [online] Available at http://www.inpharm.com/news/152234/eisai-breast-cancer-drug-halaven- approved-europe [accessed on 9 th August 2011].

Ahn, C.-B., Jeon, Y.-J., Kand, D.-S., Shin, T.-S., and Jung, B.-M. (2004) Free radical scavenging activity of enzymatic extracts from a brown seaweed Scytosiphon lomentaria by electron spin resonance spectrometry. Food Research International , 37 , pp. 253-258.

Ahn, M.-J., Yoon, K.-D., Kim, C. Y., Kim, J. H., Shin, C.-G., and Kim, J. (2006) Inhibitory Activity on HIV-1 Reverse Transcriptase and Integrase of a Carmalol Derivative from a Brown Alga, Ishige okamurae . Phytotherapy Research , 20 , pp. 711-713.

Ahmed, V. U., and Ali, M. S. (1991) Terpenoids form the marine alga Laurencia pinnatifida . Phytochemistry , 30 , pp. 4172-4174.

Ali, I., Gupta, V. K., Aboul-Enein, H. Y., and Hussain, A . (2008) Hyphenation in sample preparation: Advancement from the micro to the nano world. Journal of separation Science, 31 , pp. 2040-2053

Ali, M. S., and Pervez, M. K. (2003a) Seco -dolastanes from the marine brown alga Dictyota dichotoma (Huds.) Lamour. Naturforsch B , 58 , pp. 438-442.

Ali, M. S., and Pervez, M. K. (2003b) Ring-A hydroxylated dolastanes from the marine brown alga Dictyota dichotoma (Huds.) Lamour. Natural Product Research , 17 , pp. 281- 286.

Ali, M. S., Pervez, M. K., Saleem, M., and Ahmed. F. (2003) Dichotenone-A and -B: Two New Enones from the Marine Brown Alga Dictyota dichotoma (Hudson) Lamour. Natural Product Research , 17 , pp. 301-306.

Ali, M. S., Pervez, M. K., Ahmed, F., and Saleem, M. (2004) Dichotenol-A, B and C: the C-16 oxidized seco -dolastanes from the marine brown alga Dictyota dichotoma (HUDS.) lamour. Natural Product Research , 18 , pp. 543-549.

Allmendinger, A., Spiveri, J., Kaiser, M., Casey, R., Hingley-Wilson, S., Lalvani, A., Guiry, M., Blunden, G., and Tasdemir, D. (2010) Antiprotozoal, Antimycobacterial and

220 References

Cytotoxic Potential of Twenty-Three British and Irish Red Algae. Phytotherapy Research , 24 , pp. 1099-1103.0

Ambrosio, A. L., Sanz, L., Sanchez, E. I., Wolfenstein-Todel, C., and Calvete, J. J. (2003) Isolation of two novel mannan- and L-fucise-binding lectins from the green alga Enteromorpha profilera : biochemical characterization of EPL-2. Archives of Biochemistry and Biophysics , 415 : pp. 245-250.

Amico, V., Oriente, G., Piattelli, M., Tringali, C., Fattorusso, E., Magno, S., and Mayol, L. (1980) Diterpenes dased on the dolabellane skeleton form Dictyota dichotoma. Tetrahedron , 36 , pp. 1409-1414.

Antizar-Ladislao, B. (2008) Environmental levels, toxicity and human exposure to tributyltin (TBT)-contaminated marine environment. A review. Environment International , 34 , pp. 292–308.

Armstrong, E., Boyd, K. G., and J. G. Burgess, J. G. (2000) Prevention of marine biofouling using natural compounds from marine organisms. Biotechnology Annual Review , 6, pp. 221-241.

Avelin, M. S., Vitalina, S., Rittschoff, M. D., and Nagabhushanam, R. (1993) Bacterial/barnacle interaction: potential use of juncellins or antibiotics to alter structure of bacterial communities. Journal of Chemical Ecology , 19 , pp. 2155-2167.

Awad, N. E. (2000) Biologically Active Steroid from the Green Alga Ulva lactuca . Phytotheraphy Research , 14: 641-643

Baker, C.N., and Tenover, F.C. (1996) Evaluation of alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci. Journal of Clinical Microbiology , 34 , pp. 2654-2659.

Bansemir, A., Blume, M., Schroder, S., and Lindequist, U. (2006) Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria. Aquaculture , 252 , pp. 79-84.

Barreto, M., and Meyer, J. J. M. (2006) Isolation and antimicrobial activity of lanosol derivative from Osmundaria serrata (Rhodophyto) and a visual exploration of its biofilm covering. South African Journal of Botany , 72 , pp. 521-528.

Beith, M. (1995) Herbal. In: Healing Threads. Edinburgh: Brilinn Limited, pp. 239-242.

Beuttner, G. R. (1987) Spin Trapping ESP Parameters of Spin Adducts. Free Radical Biology and Medicine , 3, pp. 259-303.

Blount, J. F., Dunlop, R. W., Erickson, K. L., and Wells, R. J. (1982) 2 Diterpenes with New Carbocyclic Ring-System from an Australian Collection of the Brown Alga Dictyota dichotoma . Australian journal of chemistry , 35 , pp. 145-163.

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. (2003) Marine Natural Products (2001). Natural Product Report , 20 , pp. 1-48.

221 References

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. (2004) Marine Natural Products (2002). Natural Product Report , 21 , pp. 1-49.

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. (2005) Marine Natural Products (2003). Natural Product Report , 22 , pp. 15-61.

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. (2006) Marine Natural Products (2004). Natural Product Report , 23 , pp. 26-78.

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. (2007) Marine Natural Products (2005). Natural Product Report , 24 , pp. 31-76.

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. (2008) Marine Natural Products (2006). Natural Product Report , 25 , pp. 35-94.

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2009. Marine Natural Products (2007). Natural Product Report ,26: 170-244

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2010. Marine Natural Products (2008). Natural Product Report ,27: 165-237

Blunt, J.W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. 2011. Marine Natural Products (2009). Natural Product Report ,28: 196-268

Boland, W., and Mertes, K. (1985) Biosynthesis of algal pheromones, A model study with the composite Senecio isatideus . European Journal of Biochemistry , 147 , pp. 83-91.

Bouaicha, N., Tringali, C., Pesando, D., Mallea, M., Roussakis, C., and Verbist, J. F. (1993a) Bioactive Diterpenoids isolated from Dilophus ligulatus . Planta Medica , 59 , pp. 256-258.

Bouaicha, N., Pesando, D., and Puel, D. (1993b) Cytotoxic Diterpenoids from the Brown Alga Dilophus ligulatus . Journal of Natural Products , 56 , pp. 1747-1752.

Boyd, K. G., Adams, D. R., and. Burgess. J. G (1999a) Antibacterial and repellent activity of marine bacteria associated with algal surfaces. Biofouling , 14 , pp. 227-236.

Boyd, K. G., Mearns-Spragg, A., and Burgess. J. G. (1999b) Screening of marine bacteria for the production of microbial repellents using a spectrophotometric chemotaxis assay. Marine Biotechnology , 1, pp. 359-363.

Bryan, P.J., Rottschof, D., and Qian, P.Y. (1997) Settlement inhibition of bryozoan larvae by bacterial films and aqueous leachates. Bulletin of Marine Science , 61 , pp. 849- 857.

Bull, A. T., and Stach, J. E. M. (2007) Marine actinobacteria: new opportunities for matural product search and discovery. Trends in Microbiology , 15 , pp. 491-499.

222 References

Cacabelos, E., Olabarria, C. Incera, M., and Troncoso, J. S. (2010) Do grazers prefer invasive seaweeds? Journal of Experimental Marine Biology and Ecology , 393 , pp. 182- 187.

Cancer Research UK. (2010) ‘Superfoods’ and Cancer. [online] Avalible at: http://info.cancerresearchuk.org/healthyliving/cancercontroversies/superfoods/ [Accessed 28 th September 2010]

Cannell, R. J. P . (1998) Natural Product Isolation. Totowa. Humana Press Inc.

Chandini, S. K., Ganesan, P., and Bhaskar, N. (2008) In vitro antioxidant activities of three selected brown seaweeds of India. Food Chemistry , 107 , pp. 707-713.

Cho, J. Y., Kwon, E.-H., Choi, J.-S., Hong, S.-Y., Shin, H.-W., and Hong, Y.-K. (2001) Antifouling activity of seaweeds extracts on the green alga Entromorpha prolifera and the mussel Mytilus edulis . Journal of Applied Phycology , 13 , pp. 117-125.

Ciminiello, P., Dell’Aversano, C., Fattorusso, E., and Forino, M. (2010) Palytoxins: A still haunting Hawaiian curse. Phytochemistry Review , 9, pp. 491-500.

Clare, A. S. (1996) Marine natural product antifoulants: status and potential. Biofouling , 9, pp. 211-229.

Claydon, J., Greeves, N., Warren, S., and Wothers, P. (2001) Organic Compounds. In: Organic Chemistry. 2 nd ed. Oxford. Oxford University Press. pp 2

Collins, L.A. and Franzblau S. G. (1997) Microplate Alamar Blue Assay versus BACTEC 460 System for High-Throughput Screening of Compounds against Mycobacterium tuberculosis and Mycobacterium avium . Antimicrobial Agents and Chemotherapy , 45 , pp. 1004-1009.

Conover, J. T., and Sieburth, J. McN. (1964) Effect of Sargassum distribution on its epibiota and antibacterial activity. Botanica Marina , 6, pp. 147-157.

Cooney, J. J. (1995) Organotin compounds and aquatic bacteria: a review. Helgolhnder Meeresunters . 49 , pp. 663-677.

Crews, P., and Kho. E. (1974) Cartilagineal. Unusual Monoterpene Aldehyde from Marine Alga. Journal of Organic Chemistry , 39 , pp. 3303-3304.

Crews, P., Naylor, S., Hanke, F. J., Hogue, E. R., Kho, E., and Braslau, R. (1984) Halogen Regiochemistry and Substituent Stereochemistry Determination in Marine Monoterpenes by 13 C NMR. Journal of Organic Chemistry , 49 , pp. 1371-1377.

Cueto, M., Darias, J., Rovirosa, J., and San Martin, A. (1998) Tetrahydropyran Monoterpenes from Plocamium cartilagineum and Pantoneura plocamioides . Journal of Natural Products . 61 , pp. 1466-1468.

223 References

Culioli, G., Ortalo-Magne, A., Valls, R., Hellio, C., Clare, A. S., and Piovetti, L. (2008) Antifouling Activity of Meroditerpenoids from the Marine Brown Alga Halidrys siliquosa. Journal of Natural Products , 71, pp. 1121-1126

DailyMed. (2008) Prialt (ziconotide ) injection. [Online] Available at http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=db671a67-346d-4d55-bfe3- eeecf60da01f [Accessed 9 th august 2011]

Darias, J., Rovirosa, J., San Martin, A., Diaz, A.-R., Dorta, E., and Cueto, M. (2001) Furoplocamioids A-C, Novel Polyhalogenated Furanoid Monoterpenes from Plocamium cartilagineum . Journal of Natural Products , 64 , pp. 1383-1387. de Nys, R., Steinberg, P., Willemson, P., Dworjanyn, S. A., Gabelish, C. L., and King, R. J. (1995) Broad spectrum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays. Biofouling , 8, pp. 259-271. de Rosa, S., de Stefano, S., and Zacodnik, N. (1986) Hydroazulenoid Diterpenes from the Brown Alga Dictyota dichotoma var. Implexa . Phytochemistry , 25 , pp. 2179-2181.

Dias, D. A., and Urban, S. (2009) Application of HPLC-NMR for the Rapid Chemical Profiling of a Southern Australian Sponge, Dactylospongia sp . Journal of Separation Science , 32 , pp. 542-548.

Diaz-Marrero, A. R., Cueto, M., Dorta, E., Rovirosa, J., San-Martin, A., and Darias, J. (2002b) New halogenated monoterpenes from the red alga Plocamium cartilagineum . Tetrahedron , 58 , pp. 8539-8542.

Diaz-Marrero, A. R., Rovirosa, J., Darias, J., San-Martin, A., and Cueto, M. (2002a) Plocamenols A-C, Novel Linear Polyhalohydroxylated Monoterpenes from Plocamium cartilagineum . Journal of Natural Products , 65 , pp.585-588.

Dikalov, S., Skatchkov, M., and Bassenge, E. (1997b) Quantification of Peroxynitrate, Superoxide, and Peroxyl Radicals by a New Spin Trap Hydroxylamine 1-Hydoxy-2,2,6,6- tetramethyl-4-oxo-piperidine. Biochemical and Biophysical Research Communication , 230 , pp. 54-57. (not sure if in)

Dikalov, S., Skatchkov, M., and Bassenge, E. (1997a) Spin Trapping of Superoxide Radicals by 1-Hydroxy-3-carboxy-pyrrolidine and 1-Hydroxy-2,2,6,6-tetramethyl-4-oxo- piperidine and the Stability of Corresponding Nitroxyl Radicals Towards Biological Reductants. Biochemical and Biophysical Research Communication , 231 , pp. 701-704.

Duan, X.-J., Zhang, W.-W., Li, X.-M., and Wang, B.-G. (2006) Evaluation of antioxidant properties of extract and fractions obtained from a red alga, Polysiphonia urceolata . Food Chemistry , 95 , pp. 37-43.

Duran, R., Zubia, E., Ortega, M. J., and Salva, J. (1997) New diterpenoids from the alga Dictyota dichotoma . Tetrahedron , 53 , pp. 8675-8688.

224 References

Enoki, N., Ishida, R., and Matsumoto, T. (1982a) Structures and conformations of new nine-membered ring diterpenoids from the marine alga Dictyota dichotoma . Chemistry Letters , pp. 1749-1752.

Enoki, N., Ishida, R., Urano, S., Ochi, M., Tokoroyama, T., and Matsumoto, T. (1982b) New hydroazululenoid diterpenes from the marine Dictyota dichotoma. Chemistry Letters , pp. 1837-1840.

Enoki, N., Shirahama, H., Osawa, E., Urano, S., Ishida, R., and Matsumoto, T. (1983a) Structure and conformation of furanocyclononene diterpenoids form the seaweeds Dictyota dichotoma . Chemistry Letters , pp. 1399-1402.

Enoki, N., Furusaki, A., Suehiro, K., Ishida, R., and Matsumoto. T. (1983b) Epoxydictymene, a new diterpene from the brown alga Dictyota dichotoma. Tetrahedron Letters , 24 , pp. 4341-4342.

Enoki, N., Shirahama, H., Furusaki, A., Suehiro, K., Osawa, E., Ishida, R., and Matsumoto, T. (1984) Absolute configuration and conformation mobility of dilophol and 3-acetoxyacetyldilophol. Chemistry Letters , pp. 459-462.

Enoki, N., Ishida, R., Urano, S., and Matsumoto, T. (1985) New tricarbocyclic cycropropanoid diterpenes from the brown alga Dictyota dichotoma . Tetrahedron Letters , 24 , pp. 1731-1734.

Fan, X., Xu, N. J. and Shi, J. G. (2003a) Two New Bromophenols from Red Alga Rhodomela confervoides . Chinese Chemical Letters , 14 , pp. 939-941.

Fan, X., Xu, N. J. and Shi, J. G. (2003b) A New Bromophenol and its Dimethyl Acetal from Red Alga Rhodomela confervoides . Chinese Chemical Letters , 14 , pp. 1045-1047.

Fan, X., Xu, N. J. and Shi, J. G. (2003c) Bromophenols from the Red Alga Rhodomela confervoides . Journal of Natural Products , 66 , pp. 455-458.

Fattorusso, E., Magno, S., Mayol, L., Santacroce, C., Sica, D., Amico, V., Oriente, G., Piattelli, M., and Tringali, C. (1976) Dictyol A and B, Two Novel Diterpene Alcohols from the Brown Alga Dictyota dichotoma . Journal of the Chemical Society Chemical Communications , pp. 575–576.

Faulkner, D. J., Ravi, B. N., Finer, J., and Clardy, J . (1977) Diterpenes from Dictyota dichotoma. Phytochemistry , 16 , pp. 991-993.

Faulkner, D. J. (1977) Interesting Aspects of Marine Natural Products Chemistry. Tetrahedron , 33 , pp. 1421-1443.

Faulkner, D. J. (1984a) Marine Natural Products: Metabolites of Marine Algae and Herbivorous Marine Molluscs. Natural Product Reports , 1, pp. 251-280.

Faulkner, D. J. (1984b) Marine Natural Products: Metabolites of Marine Invertebrates. Natural Product Reports , 1, pp. 551-598.

225 References

Faulkner, D. J . (1986) Marine natural products (1983-85). Natural Product Reports , 3, pp. 1-33.

Faulkner, D. J. (1987) Marine natural products (1985/86). Natural Product Reports , 4, pp. 539-576.

Faulkner, D. J. (1988) Marine natural products (1986/87). Natural Product Reports , 5, pp. 613-663.

Faulkner, D. J. (1990) Marine natural products (1987/88). Natural Product Reports , 7, pp. 269-309.

Faulkner, D. J. (1991) Marine natural products (1989). Natural Product Reports , 8, pp. 97-147.

Faulkner, D. J. (1992) Marine natural products (1990). Natural Product Reports , 9, pp. 323-364.

Faulkner, D. J. (1993) Marine natural products (1991). Natural Product Reports , 10 , pp. 497-539.

Faulkner, D. J. (1994) Marine natural products (1992). Natural Product Reports , 11 , pp. 355-394.

Faulkner, D. J. (1995) Marine natural products (1993). Natural Product Reports , 12 , pp. 223-269.

Faulkner, D. J. (1996) Marine natural products (1994). Natural Product Reports , 13 , pp. 75-125.

Faulkner, D. J. (1997) Marine natural products (1995). Natural Product Reports , 14 , pp. 259-302.

Faulkner, D. J . (1998) Marine natural products (1996). Natural Product Reports , 15 , pp. 113-158.

Faulkner, D. J. (1999) Marine natural products (1997). Natural Product Reports , 16 , p. 155-198.

Faulkner, D. J. (2000a) Highlights of Marine Natural Products Chemistry (1972-1999). Natural Product Reports , 17 , pp. 1-6.

Faulkner, D. J. (2000b) Marine natural products (1998). Natural Product Reports , 17 , pp. 7-55.

Faulkner, D. J. (2001) Marine natural products (1999). Natural Product Reports , 18 , pp. 1-49.

Faulkner, D. J. (2002) Marine natural products (2000). Natural Product Reports , 19 , pp. 1-48.

226 References

Filion-Mykelbust, C., and Norton, T. A . (1981) Epidermis shedding in the brown seaweed Ascophyllum Nodosum (L.) Le Jolis, and its ecological significance. Marine Biology Letters , 2, pp. 45-51.

Finer, J., Clardy, J., Fenical, W., Minale, L., Rocco, R., Battaile, J., Kirkup, M., and Moore, R. E . (1979) Structures of Dictyodial and Dictyolactone, Unusual Marine Diterpenoids. Journal of Organic Chemistry , 44 , pp. 2044-2047.

Flodin, C., and Whitfield, F. B. (1999) 4-Hydroxybenzoic acid: a likely precursor of 2,4,6-tribromophenol in Ulva lactuca. Phytochemistry , 51 , pp. 249-255.

Francisco, M. E. Y., and Erickson, K. L. (2001) Ma’iliohydrin, a Cytotoxic Chamigrene Dibromohydrin from a Philippine Laurencia Species. Journal of Natural Products , 64 , pp. 790-791.

Franzblau S.G., Witzig, R. S., McLaughlin, J. C., Torres, P., Madico, G. Hernandez, A., Degnan, M. T., Cook, M. B., Quenzer, V.K., Ferguson, R. M., and Gilman, R. H. (1998) Rapid, Low-Technology MIC Determination with Clinical Mycobacterium tuberculosis Isolates by using the Microplate Alamar Blue Assay. Journal of Clinical Microbiology , 36 , pp. 362-366.

Freile-Pelegrin, Y., and Morales, J. L. (2004) Antibacterial activity in marine algae from the coast of Yucatan, Mexico. Botanica Marina , 47 , pp. 140-146.

Frouina, H., Lebeuf, M., Saint-Louis, R., Hammill, M., Pelletier, É., and Fournier, M. (2008) Toxic effects of tributyltin and its metabolites on harbour seal (Phoca vitulina) immune cells in vitro. Aquatic Toxicology . 90 , pp. 243-251.

Gaquerel, E., Hervé, C., Labrière, C., Boyen, C., Potin, P., and Salaün, J.-P. (2007) Evidence for oxylipin synthesis and induction of a new polyunsaturated fatty acid hydroxylase activity in Chondrus crispus in response to methyljasmonate. Biochimica et Biophysica Acta , 1771 , pp. 565-575.

Gedara, S. R., Abdel-Halim, O. B., El-Sharkawy, S. H., Salama, O. M., Shier, T. W., and Halim. A. F. (2003) Cytotoxic hydroazulene diterpenes from the brown alga Dictyota dichotoma. Z. Naturforsch , 58 , pp. 17-22.

Gil-Turnes, M.S., and Fenical, W. (1992) Embryos of Homarus americanus are protected by epibiotic bacteria. The Biological Bulletin , 182 , pp. 105-108.

Gibson, R., Hextall, B., and Rogers, A. (2001) Photographic Guide to Sea and Shore Life of Britain and North-west Europe. Oxford, Oxford University Press.

Gil-Turnes, M.S., Hay, M.E. and Fenical, W. (1989) Symbiotic marine bacteria chemically defend crustacean embryos from a pathogenic fungus. Science , 246 , pp. 116- 118.

227 References

Givskov, M. R., de Nys, R., Manefield, M., Gram, R., Maximilien, L., Eberl, S., Molin, P. D., Steinberg, P., and Kjelleberg, S. (1996) Eukaryotic interference with homoserine lactone-mediated prokaryotic signaling. Journal of Bacteriology . 178 , pp. 6618-6622.

Glombitza, K.-W., Rauwald, H.-W., and Eckhardt, G. (1975) Fucole, Polyhyroxyoligophenyle aus Fucus vesiculosus . Phytochemistry . 14 , pp. 1403-1405.

Glombitza, K.-W., Rauwald, H.-W., and Eckhardt, G. (1977) Phlorotannine aus Himanthalia elongate . Phytochemistry , 16 , pp. 1614.

Glombitza, K.-W., Geisler, C., and Eckhardt, G. (1980) Polyhydroxyphenyl Ethers from Phaeophycea Halidrys siliquosa (III). Botanica Marina , 23 , pp. 735-740.

Glombitza, K.-W., Sukopp, I., and Wiedenfeld, H. (1985). Antibiotics from Algae XXXCII. Rhodomelol and Methyrhodomelol from Polysiphonia lanosa . Planta Medica , pp. 437-440.

Goecke, F., Labes, A., Wiese, J., and Imhoff. J. F. (2010) Chemical interactions between marine macroalgae and bacteria. Marine Ecological Progress Series , 409 , pp. 267-300.

Gonzalez, A. G., Arteaga, J. M., Martin, J. D., Rodriguez, M. L., Fayos, J., and Martinez-Ripolls, M. (1978) Two New Polyhalogenated Monoterpenes from the Red Alga Plocamiem cartilagineum . Phytochemistry , 17 , pp. 947-948.

Gonzalez, A. G., Martin, J. D., Martin, V. S., Norte, M., Perez, R., and Ruano, J. Z. (1982) Non-terpenoid C-15 metabolites from the red seaweed Laurencia Pinnatifida . Tetrahedron , 38 , pp. 1009-1014.

Gonzalez, A. G., Martin, J. D., Norte, M., Rivera, P., Perales, A., and Fayos, J. (1983) Structure and absolute configurations of Dictyota Sp . diterpenes. Tetrahedron , 39 , pp. 3355-3357.

Gonzalez, A. G., Arteaga, J. M., Fernandes, J. J., Martin, J. D., Norte, M., and Ruano, J. Z. (1984) Terpenoids of the red alga Laurencia pinnatifida . Tetrahedron , 40 , pp. 2751-2755.

Gonzalez, A. Manta, G., E., Martin, J. D., and Perez, C. (1987) Isolation and structure of pachytriol. Journal of Natural Products , 50 , pp. 500-502.

Gonzalez de Val, A., Platas, G., Basilio, A., Cabello, A., Gorrochategui, J., Suay, I., Vicente, F., Portillo, E., del Rio, M. J., Reina, G. G., and Pelaez, F. (2001) Screening of antimicrobial activities in red, green and brown macroalgae from Gran Canaria (Canary Islands, Spain). International Microbiology , 4, pp. 35-40.

Gram, R., de Nys, R., Manefield, M., Givskov, M., Steinberg, P. and Kjelleberg, S . (1996) Inhibitroy effects of secondary metabolites from the red alga Delisea pulchra on swarming motility of Proteus mirabilis . Applied and Environmental Microbiology , 62 , pp. 4284-4287.

228 References

Griffiths, J. (2008) A Brief History of Mass Spectrometry. Analytical Chemistry , 80 , pp, 5678-5683

Grosse-Damhues, J., Glombitza, K.-W., and Schulten, H.-R. (1983) An eight-ring phlorotannin form the brown alga Himanthalia elongate . Phytochemistry , 22 , pp. 2043- 2046.

Grosse-Damhues, J., and Glombitza, K.-W. (1984) Isofualols, a type of phlorotannin from the brown alga Chorda filum . Phytochemistry , 23 , pp. 2639-2642.

Halliwell, B., and Gutteridge, J. M. C. (2007a) Problems of the Skin. In: Free Radicals in Biology and Medicine. 3 rd ed. Oxford. Oxford University Press. pp. 388-390

Halliwell, B., and Gutteridge, J. M. C. (2007b) Consiquences of Oxidative Stress: Cell Death. In: Free Radicals in Biology and Medicine. 3rd ed. Oxford. Oxford University Press. pp. 199

Halliwell, B. (2008) Are polyphenols antioxidants or pro-oxidants? What do we learn form cell culture and in-vivo studies ? Archives of Biochemistry and Biophysics , 476 , pp. 107- 112.

Halsall, T. G., and Hills, I. R. (1971) Isolation of Heneicosa-1,6,9,12,18-hexane and - 1,6,9,13,15-pentaene from the Alga Fucus vesiculosus . Chemical Communication , pp. 448.

Harlin, M. M., Woelkerling, W. J., and Walker, D. I. (1985) Effects of a hypersalinity gradient on epiphytic Corallinaceae (Rhodophyta) in Shark Bay, Western Australia. Phycologia , 24 , pp. 389-402.

Hassenklöver, T., Predehl, S., Pilli, J., Ledwolorz, J., Assmann, M., and Bickmeyer, U. (2006) Bromophenols, both present in Marine Organisms and in Industrial Flame Retardants, Disturb Cellular Ca 2+ signaling in neuroendocrine cells (PC12). Marine Toxicology , 76 , pp. 37-45.

Hellio, C., Marechal, J. P. , Veron, B., Bremer, G., Clare, A. S., and Le Gal, Y . (2004) Seasonal variation of antifouling activities of marine algae from the Brittany coast (France). Marine Biotechnology , 6, pp. 67-82.

Higgs, M. D. (1981) Antimicrobial Components of the Red Alga Laurnecia Hybrida (Rhodophyta, Rhodmelacaea). Tetrahedron , 37 , pp. 4255-4258.

Higgs, M. D., and Mulheirn, L. J. (1981a) Six new metabolights from the brown alga Halidrys siliquosa (phaeophyta, Fucales). Tetrahedron , 37 , pp. 3209-3213.

Higgs, M. D., and Mulheirn, L. J. (1981b) Hybridalactone, an unusual fatty acid metabolite from the red alga Laurencia hybrida (Rhodophyta, Rhodomelaceae). Tetrahedron , 37 , pp. 4259-4262.

Hiqashi-Okaj, K., Otani, S., and Okai, Y. (1999) Potent suppressive effect of a Japanese edible seaweed, Enteromorpha prolifera (Sujiao-nori) on initiation and promotion phase of chemically induced mouse skin tumorigenesis. Cancer Letters , 140 , pp. 21-25.

229 References

Hirumi, K., and Hirumi, H. (1989) Continuous Cultivation of Trypanosoma brucei Blood Stream Forms in a Medium Containing a Low Concentration of Serum Protein without Feeder Cell Layers. The Journal of Parasitology , 75 , pp. 985-989.

Hornsey, I. S and Hide, D. (1974) The Production of Antimicrobial Compounds by British Marine Algae I. Antibiotic Producing Marine Algae. British Phycological Journal , 9, pp. 353-361.

Hornsey, I. S and Hide, D. (1976a) The Production of Antimicrobial Compounds by British Marine Algae II. Seasonal Variation in Production of Antibiotics. British Phycological Journal, 11 , pp. 63-67.

Hornsey, I. S and Hide, D. (1976b) The Production of Antimicrobial Compounds by British Marine Algae III. Distribution of Antimicrobial Activity within the Algal Thallus. British Phycological Journal , 11 , pp. 175-181.

Ibtissam, C., Hassane, R., Jose, M.-L., Francisco, D. S. J., Antonio, G. V. J., Hassan, B., and Mohamed, K. (2009) Screening of antibacterial activity in marine green and brown macroalgae from the coast of Morocco. African Journal of Biotechnology , 8, pp. 1258-1262.

Ishitsuka, I., Takenori, K., and Kakisawa, H. (1982) Acetylsanadaol, a Diterpene having a Novel Skeleton, from the Brown Alga, Pachydictyon coriaceum . Tetrahedron Letters , 23 , pp. 3179-3180.

Ishitsuka, M., Kusumi, T., Kakisawa, H., Kawakami, Y., Nagai, Y. and Sato, T. (1986) Structural elucidation and conformational analysis of germacrane-type diterpenoids from the brown alga Pachydictyon coriaceum . Tetrahedron Letters , 27 , pp. 2639-2642.

Ishitsuka, M. O., Kusumi, T., and Kakisawa, H. (1988) Antitumor Xenicane and Norxenicane Lactone from the Brown Alga Dictyota dichotoma . Journal of Organic Chemistry , 53 , pp. 5010-5013.

Jia, Z., Zhu, H., Misra, B., Mahaney, J. E., Li, Y., and Misra, H. P . (2008) EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol. Molecular Cell Biochemistry , 313 , pp. 187-194.

Jenson, P.R., Jenkins, K. M., Porter, D., and Fenical, W . (1998) Evidence that a new antibiotic flavone glycoside chemically defend the sea grass Thalassia testudinum against Zoosporic fungi. Applied and Environmental Microbiology , 64 , pp. 1490-1496.

Jongaramruong J., and Blackman, A. J. (2000) Polyhalogenated Monoterpenes from a Tasmanian Collection of the Red Seaweed Plocamium cartilagineum . Journal of Natural Products , 63 , pp. 272-275.

Kandhasamy, M., and Arunachalam, K. D. (2008) Evaluation of in vitro antibacterial property of seaweeds of south east coast India. African Journal of Biotechnology , 7, pp. 1958-1961.

230 References

Kaneto, H., Kajimoto, Y., Miyagawa, J.-I., Matsuoka, T.-A., Fujitani, Y., Umayahara, Y., Hanafusa, T., Matsuzawa, Y., Yamasaki, Y., and Hori, M. (1999) Beneficial Effects of Antioxidants in Diabetes – Possible Protection of Pancreatic β-Cells Against Glucose Toxicity, Diabetes , 48 , pp. 2398-2406.

Kato, N., Tanaka, S., Kataoka, H., and Takeshita, H. (1987) Total Sythesis of dictymal, a B-seco-dictymenoid aldehyde from and algae, Dictyota dichotoma . Chemistry Letters , pp. 2295-2298.

Kim, I. H., and Lee, J.-H. (2008) Antimicrobial activities against methicillin-resistant Staphylococcus aureus from macroalgae. Journal of Inductrial and Engineering Chemistry , 14 , pp. 568-572.

Kim, J. Y., Hamada, M. A. A. A., Fujuta, Y., and Ishibashi, F. (2006) Algicidal diterpenes from the brown algae Dictyota dichotoma . Bioscience Biotechnology Biochemsitry , 70 , pp. 2571-2574.

Kim, K-N., Kim, S.-H., Kim, W.-S., Kang, S.-M., Lee, K.-W., Lee, W. J., Park, S.-Y., Kim, S.-W., and Jeon, Y.-J. (2008) Antitumor Activities of Sea Staghorn ( Codium fragile ) against CT-26 Cells. Food Science and Biotechnology , 17 , pp. 976-982.

Kim, K.-N., Heo, S.-J., Kang, S.-M., Ahn, G., and Jeon, Y.-J. (2010) Fucoxanthin induces apoptosis in human leukemia HL-60 cells through a ROS-mediated Bcl-xL pathway. Toxicology in Vitro , 24 , pp. 1648-4654

Kolesnikova, S. A., Kalinovsky, A. I., Fedorov, S. N., Shubina, L. K., and Stonik, V. A. (2006) Diterpenes from the Far-eastern brown alga Dictyota dichotoma . Phytochemistry , 67 , pp. 2115-2119.

Konig, G. M., Wright, A. D., and Sticher, O. (1990) A new polyhalogenated monoterpene from the red alga Plocamium cartilagineum . Journal of Natural Products , 53 , pp. 1615-1618.

Konig, G. M., Wright, A. D., and de Nys, R. (1999) Halogenated Monoterpenes from Plocamium costatum and Their Biological Activity. Journal of Natural Products , 62 , pp. 383-385.

Koreeda, M., Weiss, G., and Nakanishi, K. (1973) Absolute configuration of natural (+)- abscisic acid. Journal of the American Chemical Society , 95 , pp. 239-240.

Krug, P.J. (2006) Defense of benthic invertebrates against surface colonization by larvae: A chemical arms race. In: (Clare A. and Fusetani N, eds) Marine Molecular Biotechnology , 2, Springer, Berlin. pp. 1-53

Kurata, K., Tanigughii, K., Takashima, K., Hayashi, I. and Suzuki, M. (1997) Feeding-deterrents bromophenols from Odonthalia corymbifera . Phytochemistry , 45 , pp. 485-487.

231 References

Kusumi, T., Nkongolo, D. M., Goya, M., Ishitsuka, M., Iwashita, T., and Kakisawa, H. (1986a) Structures of Crenulacatals A, B, C and D. The new diterpenoids from the brown algae of Dictyotaceae. Journal of Organic Chemistry , 51 , pp. 384-387.

Kusumi, T., Nkongolo, D. M., Ishitsuka, M., Inouye, Y., and Kakisawa, H. (1986b) Structure and absolute configuration of isodictytriol, a new diterpene from the brown alga Dictyota dichotoma . Chemistry Letters , pp.1241-1242.

Lau, S.C.K., Thiyagarajan, V., and Qian, P.Y. (2003) The bioactivity of bacterial isolates in Hong Kong waters for the inhibition of barnacle ( Balanus amphitrite Darwin) settlement. Journal of Experimental Marine Biology and Ecology , 282 , pp. 43-60.

Laycock, M. V., and Ragan, M. A. (1984) L-3,4-dihydroxyphenylalanine-3-sulfate from the brown alga, Ascophyllum nodosum . Journal of Natural Products , 47 , pp. 1033-1035.

Lee, H. J., Kim, Y. A., Ahn, J.-W., Na, H.-J., Kim, H.-M., and Seo, Y. (2006) Screening of Korean Marine Plants for Their Inhibitory Effect of Histamine Release from RPMC in vitro . Biotechnology and Bioprocess Engineering , 11 , pp. 80-83.

Lee, Y. S., Shin, K. H., Kim, B.-K. and Lee, S. (2004) Anti-diabetic Activities of Fucosterol from Pelvetia siliquosa . Archives of Pharmacal Research , 27 , pp. 1120-1122.

Ma, M., Zhao, J., Wang, S., Li, S., Yang, Y., Shi, J., Fan, X., and He, L. (2006) Bromophenols Coupled with Methyl γ-Ureidobutyrate and Bromophenol Sulfates from the Red Alga Rhodomela conferoides . Journal of Natural Products , 69 , pp. 206-210.

Manchini-Filho, J., Novoa, A. V., González, A. E. B., de Andrade-Wartha, E. R. S., de O e Silva, A. M., Pinto, J. R., and P Mancini, D. A. (2009) Free Phenolic Acids from the Seaweed Halimeda monile with Antioxidant Effect Protecting against Liver Injury. Z. Naturforsch , 64c , pp. 657-663.

Mann, K. H. (1973) Seaweeds: their productivity and strategy for growth. Science , 182 , pp. 975-981.

Maki, J., Rittschof, D., Costlow, J., and Mitchel, R. (1988) Inhibition of attachement of larval barnacles, Balanus amphitrite , by bacterial biofilms. Marine Biology , 97 , pp. 199- 206.

Marjasvaara, A., Janis, J., and Vainiotalo, P. (2008) Oxidation of a laccase mediator ABTS as studied by ESI-FTICR mass spectrometry. Journal of Mass Spectrometry , 43 , pp. 470-477.

Markesbery, W. R., (1997) Oxidative Stress Hypothesis in Alzheimer’s Disease. Free Radical Biology and Medicine , 23 , pp. 134-147.

Maschek, J. A., and Baker, B. J. (2008) The Chemistry of Algal Secondary Metabolites In: C. D. Amsler, ed. 2008. Algal Chemical Ecology . Berlin: Springer-Verlag. Ch. 1.

Maximilien, R., de Nys, R., Holmstrom, C., Gram, L., Givskov, M., Crass, K., Kjelleberg, S., and Steinberg, P. (1998) Chemical mediation of bacterial surface

232 References colonisation by secondary metabolites from the red alga Delisea pulchra . Aquatic Microbial Ecology , 15 , pp. 233-246.

McConnell, O.J., and Fenical, W. (1980) Halogen chemistry of the red alga Bonnemaisonia . Phytochemistry , 19 , pp. 233-247.

McDougall, K. E., (2004) Intertidal seaweed species richness: a comparison of exposed and sheltered rocky shores of Caithness , Heriot-Watt University, Edinburgh, 2004

Mena, S., Ortega, A., and Estrela, J. M. (2009) Oxidative stress in environmental induced carcinogenesis. Mutation Research , 674 , pp. 36-44.

Miliauskas, G., Venskutonis, P. R., and van Beek, T. A. (2004) Screening of Radical Scavenging Activity of some medicinal and Aromatic Plant Extracts. Food Chemistry , 85 , pp. 231-237.

Mori, K., Ooi, T., Hiraoka, M. Oka, N., Hamada, H., Tamura, M., and Kusumi, T. (2004) Fucoxanthin and Its Metabolites in Edible Brown Algae Cultivated in Deep Seawater. Marine Drugs , 2, pp. 63-72.

Moss, B. L. (1973) The control of epiphytes by Halidrys siliquosa (L.) Lyngb. (Phaeophyta, Cystoseiraceae). Phycologia , 21 , pp. 185-191.

Muller, D. G., Jaenicke, L., Donike, M., and Akintobi, T. (1971) Sex attractant in a brown alga: Chemical Structure. Science , 171 , pp. 815-817.

Muller, D. G., and Aenicke, L. J. (1973) Fucoserraten, the female sex attractant of Fucus Serratus L. (phaeophyta). FEBS letters , 30 , pp. 137-139.

Muller, D. G., and Gassmann, G. (1980) Sexual hormone specificity in Ectocarpus and Laminaria (Phaeophyceae). Naturwissenschaften , 67 , pp. 462-463.

Müller, D. G., Gassmann, G., Boland, W., Marner, F., and Jaenicke, L. (1981) Dictyota dichotoma (Phaeophyceae):Identification of the Sperm Attractant. Science , 212 , pp. 1040-1041.

Muller, D. G., Gassmann, G., Marner, F-J., Boland, W., and Jaenicke, L. (1982) The Sperm Attractant of the Marine Brown Alga Ascophyllum nodosum (Phaeophyceae). Science , 218 , pp. 1119-1120.

Muller, D. G., and Peters, A. (1982) Idenification of a sexual hormone and related substances in the marine brown alga Desmarestia . Naturwissenschaften , 69 , pp. 290-291.

Muller, D. G., (1986) Sexual Pheromone in Cladostephus (Sphacelariales, Phaeophyceae). Naturwissenschaften , 73 , pp. 99-100.

Mynderse, J. S., and Faulkner, D. J. (1975) Polyhalogenated Monoterpenes from the Red Alga Plocamium cartilagineum . Tetrahedron , 31 , pp. 1963-1967.

233 References

Mynderse, J. S., and Faulkner, D. J. (1978) Variations in the Halogenated Monoterpene Metabolites of Plocamium cartilagineum and P. violaceum . Phytochemistry , 17 , pp. 237- 240.

National Cancer Institute. (2006) Drug Discovery at the National Cancer Institute: Fact Sheet. [online] Available at http://www.cancer.gov/cancertopics/factsheet/NCI/drugdiscovery [Accessed 19 th April 2011]

Newman, D. J., and Cragg, G. M. (2007) Natural Products as Sources of New Drugs over the Last 25 Years. Journal of Natural Products , 70 , pp. 461-477.

NICE . (2010) Soft cell sarcoma – Trabectedin. [Online] Available at http://guidance.nice.org.uk/TA185 [Accessed 9 th August 2011]

Nobel Foundation (2010). The Nobel Prize in Physiology or Medicine 1945. [online] Available at http://nobelprize.org/nobel_prizes/medicine/laureates/1945/ [Accessed 11 th November 2010]

Norris, E. B., Simeon, M. K., and Williams, H. B. (1936) The Vitamin B and Vitamin C Content of Marine Algae. Journal of Nutrition , 13 , pp. 425-433.

Norte, M., Fernandez, J. J., Cataldo, F., and Gonzalez, A. G. (1989) E- dihydrorhodophytin, a C-15 acetogenin form the red alga Laurencia pinnatifida . Phytochemistry , 28 , pp. 647-649.

Norte, M., Gonzalez, A. G., Cataldo, F., Rodriguez, M. L., and Brito, I . (1991) New examples of acyclic and cyclic C-15 acetogenins from Laurencia pinnatifida . Reassignment of the absolute configuration for E and Z pinnatfidienyne. Tetrahedron , 47 , pp. 9411-9418.

Norte, M., Fernandez, J. J., Souto, M. L., Gavin, J. A., and Garcia-Gravalos, M. D. (1997) Thyrsenols A and B, Two Unusual Polyether Squalene Derivatives. Tetrahedron , 53 , pp. 3173-3178.

O’Brien, J., Wilson, I., Orton, T., and Pognan, F. (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry , 267 , pp. 5421-5426.

Ochi, M., Masui, N., Kotsuki, H., Miura, I., and Trkoroyama, T. (1982) The structures of fukurinolal and fukurinal, two new diterpenoids from the brown seaweed Dilophus okamurai Dawson. Chemistry Letters , pp. 1927-1930.

Ohshiro, T., Nakano, S., Takahashi, Y., Suzuki, M., and Izumi, Y. (1999) Occurrence of bromoperoxidase in the marine green macro-alga, Ulvella lens , and emission of volatile brominated methane by the enzyme. Phytochemistry , 52 , pp. 1211-1215.

Okai, Y., and Higashi-Okai, K. (1997) Potant anti-Inflammatory Activity of Pheophytin A Derived from Edible Green Alga, Enteromorpha prolifera (Sujiao-Nori). International Journal of Immunopharmacology , 19 , pp. 355-358.

234 References

Osman, A. M., Wong, K. K. Y., and Fernyhough, A. (2006) ABTS radical-driven oxidation of polyphenols: Isolation and structural elucidation of covalent adducts. Biochemical and Biophysical Research Communications, 346 , pp 321-329.

Ott, J. A. (1980) Growth and production in Posidonia oceanica (L.) Delile P.S.Z.N.I. Marine Ecology , 1, pp. 47-64.

Otzi Museum. (2008) Otzi’s First-Aid Kit. [Online] Available at http://www.iceman.it/en/node/288 [Accessed 4th November 2010]

Palazon, J. M., and Martin, V. S. (1988) Enantioselective total synthesis of 6(R), 7(R)- 3-cis -9-cis -12-cis , 6-acetoxy-7-chloropentadeca-3,9,12-trien-1-yne and its 3-trans -isomers. Tetrahedron Letters , 29 , pp. 681-684.

Palermo, J. A., Bernardo, J. J., and Seldes, A. M. (1994) Dictyl-D-2β-Acetate and other Diterpenoids from the Brown Alga Dictyota dichotoma . Anales de la Asociación Química Argentina , 82 , pp. 355-358.

Paquette, L. A., Sun, L.-Q., Friedrich, D., and Savage, P.B. (1997) Highly enantioselective total synthesis of natural epoxydictymene. An alkoxy-directed cyclization route to highly strained trans -oxabicyclo(3.3.0)octanes. Tetrahedron Letters , 38 , pp. 195- 198.

Pavia, H., and Toth, G. B. (2000) Inducible Chemical Resistance to Herbivory in the Brown Seaweed Ascophyllum Nodosum . Ecology , 81 , pp. 3212-3225.

Pec, M. K., Aguirrea, A., Moser-Thier, K., Fernandez, J. J., Soutob, M. L., Dortab, J., Diaz-Gonzalez, F., and Villar, J. (2003) Induction of apoptosis in estrogen dependent and independent breast cancer cells by the marine terpenoid dehydrothyrsiferol. Biochemical Pharmacology , 65 , pp. 1451-1461.

Peppiatt, C.J., Armstrong, A., Pisacane, A., and Burgess, J. G. (2000) Antibacterial Activity of Resin Based Coatings Containing Marine Microbial Extracts. Biofouling , 15 , pp. 225-234.

Pereira, H. S., Leao-Ferreira, L. R., Moussatche, N., Teixeira, V. L., Cavalcanti, D. N., Costa, L. J., Diaz, R., and Frugulhetti, I. C. P. P. (2004) Antiviral activity of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis against human immunodeficiency virus type 1 (HIV-1). Antiviral Research , 64 , pp. 69-76.

Perry, G., Cash, A. D., and Smith, M. A. (2002) Alzheimer Disease and Oxidative Stress. Journal of Biomedicine and Biotechnology , 2, pp. 120-123.

Piattelli, M., Tringali, C., Neri, P., and Rocco, C. (1995) Stereochemistry and Conformation of Dolabellane Diterpenes: An NMR and Molecular Mechanics Study. Journal of Natural Products , 58 , pp. 697-704.

235 References

Pieters, L. and Vlietinck, A. J . (2004) Bioguided isolation of pharmacologically active plant components, still a valuable strategy for the finding of new lead compounds? Journal of Ethnopharmacology ; 100 , pp. 57-60.

Plato. (ca 360 BC) Phaedo. Translated by Banjamin Jowett., 1892. Oxford University Press [Online] Available from: http://oll.libertyfund.org/?option=com_staticxt&staticfile=show.php%3Ftitle=766&chapter =93700&layout=html&Itemid=27 [Accessed 9 th September 2010]

Pohnert, G., and Boland, W. (1997) Pericyclic reactions in nature: Synthesis and cope rearrangement of thermolabile bis-alkenylcyclopropanes from the female gametes of marine brown algae (Phaeophyceae). Tetrahedron , 53 , pp. 13681-13694.

Poss, A. J., and Belter, R. K. (1988) Vitamin C in Organic Synthesis: Reaction with p - Hydroxybenzyl Alcohol Derivatives. Journal of Organic Chemistry , 53 , pp. 1535-1540.

Rahman, A., Ahmed,, V. U., Bano, S., Abbas, S. A., Alvi, K. A., Shaiq, M., Lu, H. S. M., and Clardy, J. (1988) Pinnatazane, a bridged cyclic ether swsquiterpene from Laurencia pinnatifida . Phytochemistry , 27 , pp. 3879-3880.

Raz, B., Iten, M., Grether-Buhler, Y., Kaminsky, R. and Brun, R. (1997) The Alamar Blue assay to determine drug sensitivity of African trypanosomes ( T.B. rhodesiense and T.B. gambiense ) in vitro. Acta Tropica , 68 , pp. 139-147.

Rezanka, T., and Dembitsky, V. M. (2001) Polyhalogenated Homosesquiterpenic Fatty Acids from Plocamium cartilagineum . Phytochemistry , 57 , pp. 607-611.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evens, C. (1999) Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization Assay. Free Radical Biology & Medicine , 26 , pp. 1231-1237.

Rinehart, K. L. (2000) Antitumor compounds from tunicates. Medicinal Research Reviews , 20 , pp. 1-27.

Rocha, F. D., Soares, A. R., Houghton, P. J., Pereira, R. C., Kaplan, M. A. C., and Teixeira, V. L. (2007) Potential Cyctotoxic Activity of Some Brazilian Seaweeds on Human Melanoma Cells. Phytotheraphy Research , 21 , pp. 170-175.

Rotterdam Convention. (2009) Annex III: Operation of the prior informed consent procedure for banned or severely restricted chemicals, Tributyltin compounds. [Online] Available from: http://www.pic.int/en/Table7Files/En/TBT DGD E.doc [Accessed 5 th October 2010]

Rizvi, M. A., and Shameel, M. (2005) Pharmaceutical Biology of Seaweeds from the Karachi Coast of Pakistan. Pharmaceutical Biology , 43 , pp. 97-107..

Ruchonnet, D. (2007). The Microbial Chemical Ecology of the Brown Alga Halidrys siliquosa . Ph.D. thesis. University of Aberdeen

236 References

Saifah, E., Kelley, C. J., and Leary, J. D. (1983) Constituents of the Leaves of Cissus rheifolia. Journal of Natural Products , 46 , pp. 353-358.

Saitou, N., and Nei, M. (1987) The neighbour joining method: a new method for reconstructing phylogenitic trees. Molecular Biology and Evolution , 4, pp. 406-425.

Salvador, N., Garreta, A. G., Lavelli, L., and Ribera, M. A. (2007) Antimicrobial activity of Iberian macroalgae. Scientia Marina , 71 , pp. 101-113.

San-Martin, A., Negrete, R., and Rovirosa, J. (1991) Insecticide and Acaricide Activities of Polyhalogenated Monoterpenes from Chillean Plocamium cartilagineum . Phytochemistry , 30 , pp. 2165-2169.

Sand-Jensen, K. (1977) Effects of epiphytes on eelgrass photosynthesis. Aquatic Botany , 3, pp. 55-63.

Satuito, C. G., Shimizu, K., and Fusetani, N. (1997) Studies on the factors influencing larval settlement in Balanus amphitrite and Mytilus galloprovincialis . Hydrobiologia , 358 , pp. 275-280.

Segawa, M., Enoki, N., Ikura, M., Hikichi, K., Ryoichu Ishida, Shirahama, H., and Matsumoto, Y. (1987) Dictymal, a new seco -fusicoccin type diterpene from the brown alga Dictyota dichotoma . Tetrahedron Letters , 28 , pp. 3703-3704.

Shakespeare, W. (1977) Much Ado About Nothing . [Play]. London: Book Club Associates

Shakespeare, W. (1977) Orthello . [Play]. London: Book Club Associates

Shoeib, N. A., Bibby, M. C., Blunden, G., Linley, P. A., Swaine, D. J., Wheelhouse, R. T., and Wright, C. W . (2004) In-vitro Cytotoxic Activities of the Major Bromophenols of the Red Alga Polysiphonia lanosa and Some Novel Synthetic Isomers. Journal of Natural Products , 67 , pp. 1445-1449.

Siamopoulou, P., Bimplakis, A., Iliopoulou, D., Vagias, C., Cos, P., Berghe, D. V., and Roussis, V. (2004) Diterpenes from the Brown Alga Dictyota dichotama and Dictyota lineraris . Phytochemistry , 65 , pp. 2025-2030.

Sieburth, J. M., and Conover, J. T. (1965) Sargassum tannin, an antibiotic which retards fouling. Nature , 208 , pp. 52-53.

Silberstein, K., Chiffings, A. W., and McComb, A. J. (1986) The loss of seagrass in Cockburn Sound, Western Australia. III. The effect of epiphytes on productivity of Poisidonia australis Hook. f. Aquatic Botany , 24 , pp. 355-371.

Spavieri, J., Kaiser, M., Casey, R., Hingley-Wilson, S., Lalvani, A., Blunden, G., and Tasdemir, D. (2010) Antiprotozoal, Antimycobacterial and Cytotoxic Potential of Some British Green Algae. Phytotherapy Research , 24 , pp. 1095-1098.

237 References

Stratmann, K., Boland, W., and Muller, D. G. (1992) Pheromones of Marine Brown- Algae – A New branch of the Eicosanoid Metabolism. Angewandte Chemie-International Edition in English, 31 , pp. 1246-1248.

Stierle, D. B. and Sims, J. J. (1979) Marine Natural Products-XV Polyhalogenated cyclic monoterpenes from the Red Alga Plocamium cartilagineum of Antarctica. Tetrahedron , 35 , pp. 1261-1265.

Sullivan, B. W., Albizati, K. F., and Faulkner, D. J. (1986) The diterpenes of Dictyota dichotoma from the Indian Ocean. Journal of Organic Chemistry , 51 , pp. 2736-2742.

Sun, Y., Zhan, Y-C., Sha, Y., and Pei, Y-H. (2007) Norisoprenoids from Ulva lactuca . Journal of Asian Natural Products Research , 9, pp. 321-325.

Svensson C. J., Pavia, H., and Toth, G. B. (2007) Do plant density, nutrient availability, and herbivore grazing interact to affect phlorotannin plasticity in the brown seaweed Ascophyllum nodosum . Marine Biology , 151 , pp. 2177-2181.

Swerdlow, J. L. (2000a) Chemicals from Plants. In: Nature’s Medicine, Plants that Heal. Washington DC. National Geographic Society. pp 9

Swerdlow, J. L. (2000b) Herbal Medicine of India and China. In: Nature’s Medicine, Plants that Heal. Washington DC. National Geographic Society. pp 36-37

Tyler-Walters, H. and Pizzolla, P. (2008) Halidrys siliquosa . Sea oak. Marine Life Information Network: Biology and Sensitivity Key Information Sub-programme [online]. Plymouth: Marine Biological Association of the United Kingdom. Available from: [Accessed on 21/12/2010]

Unni, K. (2004) Hyphenated Techniques Breathing New Life into the Analytical Instrumentation Industry [online] Available at http://www.frost.com/prod/servlet/market- insight-top.pag?docid=9298168 [Accessed on 10 th August 2011]

Voet, D., and Voet, J. G. (1995) Biosythesis of Physiologically active Amino Acids. In: Biochemistry. 2 nd ed. New York. John Wiley & Sons. pp 758-760

Wahl, M. (1989) Marine epibiosis. I. Fouling and antifouling: some basic aspects. Marine Ecology Progress Series , 58 , pp. 175-189.

Walker, R. B., and Everette, J. D. (2009) Comparative Reaction Rates of Various Antioxidants with ABTS Radical Cation. Journal of Agriculture and Food Chemistry , 57 , pp. 1156–1161

Wang, D.-Z., (2008) Neurotoxins from Marine Dinoflagellates: A Brief Review. Marine Drugs, 6, pp. 349-371

Wiedenfeld, H., Knoch, F., and Koch, M. (1985) Die Kristallstruktur von Spiro-bis- pinnaketal, einer neuen Spiroverbindung aus Laurencia pinnatifida Lamouroux. Archiv der Pharmazie , 318 , pp. 289-293.

238 References

Welch, J. T., and. Samartino, J. S. (1985) Dictyoxetane, a Novel Diterpene from the Brown Alga Dictyota dichotoma from the Indian Ocean. Journal of Organic Chemistry , 50 , pp. 3665-3666.

Wessels, H., Hagem, W., Molis, M., Wiencke, C., and Karsten, U. (2006) Intra- and interspecific differences in palatability of Arctic macroalgae from Kongsfjorden (Spitsbergen) for two benthic sympatric invertebrates. Journal of Experimental Marine Biology and Ecology , 329 , pp. 20-33.

Wieses, J., Theil, V., Nagel, K., Staufenberger, T., and Imhoff, J. F. (2009) Diversity of Antibiotic-Active Bacteria Associated with the Brown Alga Laminaria saccharina from the Baltic Sea. Marine Biotechnology , 11 , pp. 287-300.

Wink, M. (1987) Quinolizidine Alkaloids: Biochemistry, Metabolism, and Function in Plants and Cell Suspension Cultures. Planta Medica , pp. 509-514.

World Health Organization. (2001) Quinine, Quinidine and Related Alkaloids. In: The Use of Anti-Malarial Drugs. Part II. 1.6

Wright, A. 1998. Isolation of Marine Natural Products. In: R. J. P. Cannell, ed. 1998. Natural Product Isolation. Totowa. Humana Press Inc.

Xu, N. J., Fan, X., Yang, Y. C., and Shi, J. G. (2003a) A New Poly Brominated Dibenzylphenol from Rhodomela confervoides . Chinese Chemical Letters , 14 , pp. 807- 809.

Xu, N. J., Fan, X., Yan, X., Li, X., Niu, R., and Tseng, C. K. (2003b) Antibacterial Bromophenols from the Marine Red Alga Rhodomela confervoides . Phytochemistry , 62 , pp. 1221-1224.

Xiaojun Y., Nagata, T., and Fan, X. (1998) Antioxidative Activities in some common seaweeds. Plant Foods for Human Nutrition , 52 , pp. 253-262.

Yuan, Y. V., Bone, D. E., and Carrington, M. F. (2005) Antioxidant activity of dulse (Palmaria palmata ) extract evaluated in vitro. Food Chemistry , 91 , pp. 485-494.

Yvin, J.-C., Chevolot-Magueur, A.-M., Chevolot, L., Lallemand, J.-Y., Potier, P., and Guilhem, J. (1982) Delesserine, a New Metabolite of Mixed Biogenesis from the Red Marine Alga Delesseria sanguinea (Lamouroux). Journal of the American Chemical Society , 104 , pp. 4497-4498.

Zhang, W.-W., Duan, X.-J., Huang, H.-L., Zhang, Y., and Wang, B.-G. (2007) Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae). Journal of Applied Phycology , 19 , pp. 97-108.

Zhao, J., Ma, M., Wang, S., Li, S., Cao, P., Yang, Y., Lü, Y., Shi, J., Xu, N., Fan, X., and He, L. (2005) Bromophenols Coupled with Derivatives of Amino Acids and

239 References

Nucleosides from the Red Alga Rhodomela confervoides . Journal of Natural Products , 68 , pp. 691-694.

Zhuo, J., Fan, X., Wang, S., Li, S., Shang, S., Yang, Y., Xu, N., Lu, Y., and Shi, J. (2004) Bromophenol Derivatives from the Red Alga Rhodomela confervoides. Journal of Natural Products , 67 , pp. 1032-1035.

Zubia, M., Robledo, D., and Freile-Pelegrin, Y. (2007) Antioxidant activities in tropical marine macroalgae from the Yucatan Peninsula, Mexico. Journal of Applied phycology , 19 , pp. 449-458.

Zubia, M., Fabre, M.-S., Kerjean, V., and Deslandes, E. (2009a) Antioxidant and cytotoxic Activities of some Red Algae (Rhodophyta) from Brittany Coasts (France), Botanica Marina , 52 , pp. 268-277.

Zubia, M., Fabre, M.-S., Kerjean, V., Le Lann, K., Stiger-Pouvreau, V., Fauchon, M., and Deslandes, E. (2009b) Antioxidant and antitumoural activites of some Phaeophyta from Brittany coast. Food Chemistry , 116 , pp. 693-701.

240 Appedix I

Appendix I

Figure A.1. 1H NMR of Sub-fraction Ca

241 Appedix I

Figure A.2. 1H NMR of Sub-fraction Ca

242 Appedix I

Figure A.3. 13 C NMR of Sub-fraction Ca

243 Appedix I

Figure A.4. 13 C NMR of Sub-fraction Ca

244 Appedix I

Figure A.5. 13 C NMR of Sub-fraction Ca

245 Appedix I

Figure A.6. HSQC NMR of Sub-fraction Ca

246 Appedix I

Figure A.7. HSQC NMR of Sub-fraction Ca

247 Appedix I

Figure A.8. HSQC NMR of Sub-fraction Ca

248 Appedix I

Figure A.9. HMBC NMR of Sub-fraction Ca

249 Appedix I

Figure A.10. HMBC NMR of Sub-fraction Ca

250 Appedix I

Figure A.11. HMBC NMR of Sub-fraction Ca

251 Appedix I

Figure A.11. 1H NMR of Sub-fraction Cb

252 Appedix I

Figure A.12. 1H NMR of Sub-fraction Cb

253 Appedix I

Figure A.13. 1H NMR of Sub-fraction Cb

254