Analysis of Marine Samples in Search of Bioactive Compounds (eBook)

Armando C. Duarte, Teresa Rocha-Santos (Herausgeber)

eBook Download: PDF | EPUB

2014 | 1. Auflage
312 Seiten
Elsevier Science (Verlag)
978-0-444-63381-1 (ISBN)

Comprehensive coverage of analytical techniques and applications
Clear diagrams to adequately support important topics
Real examples of applications of analytical techniques in the search for new bioactive compounds

Seas and oceans offer a wide range of temperature, pressure, light and chemical conditions thus allowing a wide diversity of marine organisms from shallow coastal waters to the deep ocean. These resources can be used to obtain new products and develop services, and in turn help to provide solutions to the challenges that affect our planet, including offering a sustainable supply of food and energy, new industrial materials and processes, new bioactive compounds, and new health treatments. Marine compounds have been identified as having antibacterial, anticoagulant, antifungal, antimalarial, antiprotozoal, antituberculosis, and antiviral activities. The major sources of these bioactive compounds are marine sponges, coelenterates, and microorganisms, followed by algae, echinoderms, tunicates, molluscs, and bryozoans. The discovery of bioactive compounds from marine samples is a hot topic considering the current need for sustainable use of marine resources. This book is a comprehensive overview of the analytical techniques employed in the discovery and characterization of bioactive compounds isolated from (all possible) marine samples and gives future perspectives of analytical methodologies. This overview includes an assessment of the sampling and preparation of extracts, the separation and isolation of bioactive compounds, their structural characterization and the application of bioassays in the discovery of bioactive compounds. Comprehensive coverage of analytical techniques and applications Clear diagrams to adequately support important topics Real examples of applications of analytical techniques in the search for new bioactive compounds

Cover 1
Title page
4
Copyright 5
Contents 6
Contributors to Volume 65 12
Series Editor’s Preface 14
Preface 16
Chapter 1 - Introduction to the Analysis of Bioactive Compounds in Marine Samples 18
1.1 - Importance of Bioactive Compounds 18
1.2 - Sources of Bioactive Compounds 19
1.3 - Classes of Bioactive Compounds 22
1.3.1 - Polyketides 23
1.3.2 - Terpenes 25
1.3.3 - Peptides 25
1.3.4 - Alkaloids 26
1.3.5 - Shikimates 26
1.3.6 - Sugars 26
1.4 - General approaches for screening bioactive compounds 27
References 28
Chapter 2 - Prospection, Collection, and Preservation of Marine Samples 32
2.1 - Introduction 32
2.2 - Bioprospecting in Extreme Environments 34
2.3 - Collection of Marine Samples 36
2.4 - Sustainable Production of Bioactive Compounds 44
2.5 - Preservation of Marine Samples 47
2.6 - Final Considerations 47
Acknowledgements 48
References 48
Chapter 3 - Classical Methodologies for Preparation of Extracts and Fractions 52
3.1 - Sample Preparation of Bioactive Compounds from the Marine Environment 52
3.1.1 - Extraction of Bioactive Compounds 63
3.1.1.1 - Extraction by Solvents 64
3.1.1.2 - Extraction by Soxhlet Methodology 66
3.1.1.3 - Extraction by Other Methodologies 67
3.1.2 - Fractionation of Extracts to Obtain Fractions of Increasing Purity in Bioactive Compounds 68
3.1.2.1 - Fractionation by Solvent Partition 70
3.1.2.2 - Separation and Purification by Chromatography 70
3.2 - Final Considerations 71
References 71
Chapter 4 - Green Analytical Methodologies for Preparation of Extracts and Analysis of Bioactive Compounds 76
4.1 - Green Extraction Techniques to Obtain Bioactive Compounds 76
4.1.1 - Supercritical Fluid Extraction (SFE) 79
4.1.2 - Pressurized Liquid Extraction (PLE) 84
4.1.3 - Ultrasound Assisted Extraction (UAE) 85
4.1.4 - Microwave Assisted Extraction (MAE) 86
4.1.5 - Enzyme Assisted Extraction (EAE) 87
4.1.6 - Solid Phase Microextraction (SPME) 89
4.1.7 - Solid Phase Extraction (SPE) 90
4.2 - Direct analysis of untreated samples to obtain bioactive compounds 92
References 94
Chapter 5 - Bioassays for Bioactivity Screening 96
5.1 - Introduction 96
5.2 - Screening Models 100
5.2.1 - Antimicrobial Assays 100
5.2.1.1 - Antibacterial Assays 100
5.2.1.2 - Antifungal Assay 101
Diffusion 101
Dilution 103
Diffusion and Dilution 103
5.2.2 - Anticancer Assays 103
5.2.2.1 - In Vitro Anticancer Activity 104
Cellular assays 104
Sulforhodamine B (SRB) assay 104
Propidium iodide (PI) assay 105
MTT assay 105
5.2.2.2 - In Vivo Anticancer Activity 106
5.2.3 - Antiviral Assays 106
5.2.4 - Anti-Inflammatory Assays 107
5.2.4.1 - Acute Assay 108
5.2.4.2 - Chronic assay 108
5.2.5 - Analgesic Assays 108
5.2.5.1 - Writhing Syndrome Method (or Acetic acid-induced Writhing Syndrome Method) 109
5.2.5.2 - Hot Plate Method 109
5.2.6 - Antiallergic Activity 109
5.2.6.1 - Antiasthmatic Assay 110
Milk-induced leucocytosis and eosinophilia 110
Egg albumin induced activity 110
5.2.6.2 - Allergic Pleurisy 111
5.2.7 - Diuretic Activity 111
5.2.8 - Adaptogenic Assays 112
5.2.8.1 - Antistress Activity 112
5.2.9 - Immunomodulation Activity 113
5.2.9.1 - Determination of Cell Mediated Immune Response 114
5.2.9.2 - Determination of Phagocytic Ability 115
5.2.9.3 - Measurement of Nitric Oxide Production 115
5.2.10 - Choleretic and Anticholestatic Activities 115
5.2.11 - Acute Toxicity and CNS Activities 116
5.2.11.1 - Effect on Hexabarbital Sleeping Time 116
5.2.11.2 - Acute Toxicity Study 116
5.2.12 - Isolated Tissues 117
5.3 - Toxicity Evaluation 117
5.3.1 - Reproductive Toxic Evaluation Studies 117
5.3.1.1 - Teratological Toxic Evaluation Studies 118
5.3.1.2 - Pre- and Postnatal Toxic Evaluation Studies 118
5.3.2 - Carcinogenic Evaluation Studies 118
5.3.3 - Mutagenic Toxicity Evaluation Studies 118
5.4 - Use of Animals 118
5.5 - Clinical Trials 119
5.5.1 - Definition and Phases of Clinical Trials 119
5.5.2 - Status of Marine Bioactive Molecules in Clinical Trials or Market 120
5.5.2.1 - Molecules Targeting ion Channels 123
5.5.2.2 - Compounds Targeting Enzymes 123
5.5.2.3 - Microtubule-interfering Agents 123
5.5.2.4 - DNA Interactive Agents 124
5.5.2.5 - Oxidative Stress Inducers 124
5.5.2.6 - Lysosomotropic Compounds 124
5.5.2.7 - Immunostimulatory Agents 124
5.5.2.8 - Calcium-binding Protein Antagonists 124
5.6 - Conclusions and Future Perspectives 124
Acknowledgements 125
References 126
Chapter 6 - Vibrational Spectroscopy for Structural Characterization of Bioactive Compounds 132
6.1 - Vibrational Spectroscopy 132
6.2 - Introduction to Infrared (IR) and Raman Spectroscopy 134
6.2.1 - Fourier Transform IR Spectrometers 135
6.2.2 - Analytical Information 135
6.2.3 - Functional Groups 136
6.2.3.1 - The Functional Group Region, 4000 to 1300.cm-1 138
6.2.3.2 - The Fingerprint Region, 1300 to 910.cm-1 138
6.2.3.3 - The Aromatic Region, 910 to 650 cm 1 138
6.3 - Selection Rules in Vibrational Spectroscopy 138
6.4 - Structure Characterization by Vibrational Spectroscopy 139
6.4.1 - Polysaccharides 140
6.4.1.1 - Chitin and Chitosan 140
6.4.1.2 - Sulfated Polysaccharides 142
6.4.2 - Amino Acids and Peptides 143
6.4.3 - Proteins 144
6.4.4 - Alkaloids 147
6.4.5 - Terpenes 149
6.4.6 - Flavonoids 150
6.4.7 - Sterols 151
6.4.7.1 - Functional Group Analysis 152
Carbonyl stretching of ketones 152
Esters: Carboxylic acid esters and lactones 153
Amides 153
Methylene Groups: C=C and C=CH Stretching 154
Hydroxyl Absorptions 154
6.4.7.2 - Raman Spectra for Steroids 155
6.4.8 - Siderophores 155
6.4.9 - Melanin 157
6.5 - Conclusion 158
Acknowledgements 159
References 159
Chapter 7 - Nuclear Magnetic Resonance Spectroscopy for Structural Characterization of Bioactive Compounds 166
7.1 - Introduction 166
7.2 - Proton Resonances Assignment 167
7.3 - Establishment of Carbon Skeleton 172
7.4 - Stereochemistry 175
7.5 - Hyphenated NMR Techniques 188
7.6 - Quantitative NMR 191
7.7 - Combined NMR Techniques and Different Families of Compounds 194
7.8 - Revised Structures 204
Acknowledgements 205
References 205
Chapter 8 - Mass Spectrometry for Determination of Bioactive Compounds 210
8.1 - Introduction 210
8.2 - Mass Spectrometry (MS) 211
8.3 - Tandem Mass Spectrometry (MSn) 212
8.3.1 - Product or Daughter Ion Scanning 213
8.3.2 - Precursor or Parent Ion Scanning 213
8.3.3 - Constant Neutral Loss Scanning 213
8.3.4 - Selected/Multiple Reaction Monitoring 214
8.4 - Liquid Chromatography–Mass Spectrometry (LC-MS) 214
8.5 - Application of MS 215
8.6 - Peptides 215
8.6.1 - Case Study: Cyclodepsipeptides, Kahalalides 216
8.7 - Alkaloids 221
8.7.1 - Case Study: Bromotyrosine Alkaloids 221
8.8 - Lipids 225
8.8.1 - Case Study: Molecular Species of Ceramides 226
8.9 - Conclusion 230
Acknowledgements 231
References 231
Chapter 9 - Chromatography Coupled to Various Detectors as a Tool for Separation and Determination of Bioactive Compounds 236
9.1 - Introduction 236
9.2 - One-Dimensional Chromatography for Bioactive Compound Analysis 238
9.2.1 - Setting the Scene and General Trends on the Use of One-Dimensional Liquid Chromatography 238
9.2.1.1 - Stationary Phases 239
9.2.1.2 - Mobile Phases 251
9.2.1.3 - Detection Systems 252
9.2.2 - Application of One-Dimensional Gas Chromatography-Based Methods for Bioactive Compound Analysis 253
9.3 - Multidimensional Chromatography for Bioactive Compounds Discovery 255
9.3.1 - Concepts and Considerations 255
9.3.1.1 - Fundamentals and Instrumental Aspects of Comprehensive Two-dimensional Liquid Chromatography 256
9.3.1.2 - Graphical Representation and Data Processing in Comprehensive Two-dimensional Liquid Chromatography 258
9.3.2 - Exploring the Use of Comprehensive Two-Dimensional Liquid Chromatography 261
9.4 - Concluding Remarks 265
Acknowledgments 266
References 266
Chapter 10 - Online Combination of Bioassays with Chemical and Structural Characterization for Detection of Bioactive Compounds 270
10.1 - Introduction 270
10.2 - High-Throughput Screening Methods 271
10.2.1 - High-Throughput Screening Based on Precolumn Methodologies 274
10.2.2 - High-Throughput Screening Based on Postcolumn Methodologies 277
10.3 - High-Resolution-Based Screening Methods 284
10.3.1 - Online Postcolumn Antioxidant Assays 284
10.3.2 - Online Postcolumn Affinity/Activity BCD Methods 288
10.3.3 - Online MS-Based Assays for Identification and Bioassay Readout 290
10.4 - Conclusions and Perspectives 291
Acknowledgments 292
References 293
Index 296

Chapter 1

Introduction to the Analysis of Bioactive Compounds in Marine Samples

Teresa Rocha-Santos1

Armando C. Duarte2
1 ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, Lordosa, Viseu, Portugal; Department of Chemistry & CESAM, University of Aveiro, Campus de Santiago, Aveiro, Portugal
2 Department of Chemistry & CESAM, University of Aveiro, Campus de Santiago, Aveiro, Portugal

Abstract

Marine natural resources can be explored providing an alternative and sustainable source of new compounds with biological properties (bioactive compounds) that may help to develop new potential health treatments. Therefore, this chapter highlights the importance of bioactive compounds and describes the explored marine sources/samples including microorganisms such as microalgae, fungi, and bacteria; macroorganisms such as sponge, macroalgae, coelenterates, tunicates (ascidians), and mollusks; and marine by-products and fisheries waste streams. Furthermore, the state-of-the-art in terms of classes of bioactive compounds and its bioactivity is presented. The general approaches used for the screening of bioactive compounds are also described and discussed.

Keywords

Bioactive compounds

marine natural compounds

marine sources

microorganisms

bioactivity

1.1. Importance of bioactive compounds

The marine environment occupies approximately three quarters of the surface of the Earth and offers a wide range of conditions of temperature, pressure, light, and chemistry, as wells as a wide diversity of marine organisms, from shallow coastal waters to the deep ocean [1]. These resources can be used to obtain new products and to develop new services, presenting solutions regarding the challenges that affect our planet. Potential solutions include a sustainable supply of food and energy, new industrial materials and processes, new bioactive compounds, and new health treatments. The use of these resources not only creates jobs and improves health, but it can also contribute to the development of greener and smarter economies.

Marine bioactive compounds are natural compounds, derived and isolated from biological sources, that have biological activity and are isolated from marine sources. The natural products also refer to secondary metabolites; that is, small molecules with molecular weight (MW) less than 2 kDa, produced by an organism that is not strictly essential for its survival [2]. The first discovery of a bioactive marine natural product was reported in the late 1950s by Bergmann [3]. The arabino and ribo-pentosyl nucleosides extracted from marine sponges were the first demonstration that naturally occurring nucleosides could contain sugars besides ribose and desoxyribose. Chemical synthesis allowed the development of two derivatives (vidarabine and cytarabine) that are two nucleosides with antiviral activity. These two nucleosides have been applied in clinical treatments for decades [3].

Concerning their applications, the marine bioactive compounds have been used mainly due to their antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, antioxidant, antidiabetic, anticancer, and antiviral activities [4]. Additionally, marine compounds have been found to affect the immune and nervous systems [4,5]. Schumacher et al. [5] reviewed the marine compounds acting on the six hallmarks of cancer; that is, the compounds that abolish self-sufficiency in growth signals, compounds that reestablish sensitivity to growth-inhibitory signals, compounds that lead to apoptosis, antiangiogenic compounds, compounds that reduce the replicative potential, and compounds that prevent tissue invasion and metastasis, demonstrating that the marine environment is rich with organisms useful for the isolation of compounds with anticancer activities.

Mayer et al. [4] reviewed the marine compounds used in pharmacology accordingly with their origins (organisms) and chemical classes. They found that the studies about the molecular mechanisms of action of discovered bioactive compounds are lacking, remaining undetermined in the major works at the time of publication. Since the mechanism of action is unknown, and due to the loss of the biological activity when tested in vivo, the majority of bioactive compounds isolated from marine organisms do not attain the clinical trials. According to Sawadogo et al. [6], concerning the anticancer compounds from marine origin, among the 83% of the compounds tested in vitro, the biological mechanisms of action of about 45% are unknown, with only 2% in clinical trial and 14% already tested in vivo. Thus, such specific biochemical interaction through which a drug substance produces its pharmacological effect is often unclear for the newly discovered compounds, which represents a challenging task.

1.2. Sources of bioactive compounds

Marine organisms that constitute approximately one half of the total global biodiversity are rich reservoirs of structurally diverse biofunctional components. So far, more than 22,000 compounds have been isolated from marine organisms [7] such as sponges, coelenterates, microorganisms, algae, echinoderms, mollusks, bryozoans, and from other sources such as marine processing wastes. Figure 1.1 gives an overview of marine derived sources used from 2010 to 2013 for the discovery of new bioactive compounds. Roughly more than one-third of all new bioactive compounds reported from marine sources are derived from microorganisms. About two-fifths of new marine bioactive compounds are derived from microorganisms, including bacteria, microalgae, and fungi, and about one-fifth of the new bioactive compounds are from other marine sources such as fish processing waste and ascidian (tunicates).

Figure 1.1 Marine-derived sources used on the search of new bioactive compounds.

Table 1.1 shows a selection of examples [8–16] of bioactive natural products from sources such as bacteria, fungi, sponge, microalgae, starfish, algae, ascidian and fish waste. There are several reviews covering the marine natural products from differences sources [7,17–21].

Table 1.1

Selected Examples of Bioactive Marine Natural Products from Different Sources

Compound

Source

Bioactivity

Reference

Veromycin E

Bacteria/Streptomyces sp

Antibacterial Antifungal
Anticancer

[8]

Penicitide A

Fungus/Penicillium chrysogenum

Cytotoxicity

[9]

Simplextones A

Sponge/Plakortis simplex

Cytotoxicity

[10]

Bouillonamide

Microalgae/Moorea bouillonii

Cytotoxicity

[11]

Astrosteriosides A

Starfish/Astropecten
monacanthus

Anti-inflammatory

[12]

Fucoxanthin

Algae/Sargassum siliquastrum

Antioxidant

[13]

S. plicata dermatan sulfate

Ascidian/Styela plicata

Anti-inflammatory

[14]

Tyr-Asn

Fish waste/clam/Meretrix lusoria

ACE inhibitory

[15]

Eusynstyelamide B

Bryozoan/Tegella cf. spitzbergensis

Antimicrobial

[16]

The marine sponges are multicellular invertebrates attached to solid substrates in benthic habitats, and they have been isolated as sources of sterols, such as steroids, terpenoids, alkaloids, cyclic peptides, and unsaturated fatty acids [18].

The marine coelenterates include the sea anemones, the true jellyfish, and the hydroids [22]. Coelenterates have been a source of several bioactive compounds such as steroids, terpenes, and glycosides. These compounds showed antitumor activity against cancer cell lines.

The microorganisms group, and in particular the marine bacteria, have been used to isolate bioactive secondary metabolites with pharmacodynamic properties such as antitumor, antivirus, and enzyme inhibitor properties, which are important in the area of the drug discovery [23]. More than 600 marine bioactive compounds have been isolated from marine bacteria, mostly from cyanobacteria, and actinobacteria due to their particular metabolic and physiological capabilities [24]. The marine cyanobacteria, which are photosynthetic prokaryotes, are considered as excellent sources of antineoplastic, antibacterial, antiviral, and antifungal compounds such as toxins. Recently, Pagliara and Caroppo [25] have investigated the bioactive properties of cyanobacterial strains that belong to the Leptolyngbya and Synechococcus genera, isolated from the Mediterranean sponge Petrosia ficiformis. The bioassays performed with the...

Erscheint lt. Verlag	26.8.2014
Sprache	englisch
Themenwelt	Naturwissenschaften ► Biologie ► Limnologie / Meeresbiologie
	Naturwissenschaften ► Chemie ► Analytische Chemie
	Naturwissenschaften ► Chemie ► Organische Chemie
	Naturwissenschaften ► Geowissenschaften ► Hydrologie / Ozeanografie
	Technik
ISBN-10	0-444-63381-2 / 0444633812
ISBN-13	978-0-444-63381-1 / 9780444633811

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