Zebrafish: A Model for Marine Peptide Based Drug Screening [1st ed.] 978-981-13-7843-0;978-981-13-7844-7

Table of contents :
Front Matter ....Pages i-xi
Biomedical Importance of Marine Peptides/Toxins (Saravanan Ramachandran, Senthilkumar Rajagopal)....Pages 1-14
Teratogenic Activity of Peptides in Zebrafish Model (Saravanan Ramachandran, Senthilkumar Rajagopal)....Pages 15-25
Teratogenic Activity of Toxins in Zebrafish Model (Saravanan Ramachandran, Senthilkumar Rajagopal)....Pages 27-42
Anticancer Properties of Marine Peptides/Toxins Using Zebrafish Model (Saravanan Ramachandran, Senthilkumar Rajagopal)....Pages 43-53
Protective Effect of Marine Peptides/Toxins in CVD Using Zebrafish Model (Saravanan Ramachandran, Senthilkumar Rajagopal)....Pages 55-73

Citation preview

Saravanan Ramachandran  Senthilkumar Rajagopal

Zebrafish: A Model for Marine Peptide Based Drug Screening

Zebrafish: A Model for Marine Peptide Based Drug Screening

Saravanan Ramachandran Senthilkumar Rajagopal

Zebrafish: A Model for Marine Peptide Based Drug Screening

Saravanan Ramachandran Native Medicine and Marine Pharmacology Laboratory, Faculty of Allied Health Sciences Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education (Deemed to be a University) Kelambakkam, Tamil Nadu, India

Senthilkumar Rajagopal Department of Biochemistry Rayalaseema University Kurnool, Andhra Pradesh, India

ISBN 978-981-13-7843-0    ISBN 978-981-13-7844-7 (eBook) https://doi.org/10.1007/978-981-13-7844-7 © Springer Nature Singapore Pte Ltd. 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Foreword by Balasubramanian

The field of marine peptide research is vast and evolving rapidly in both basic research and clinical therapeutics. When I was asked to write a foreword to this book, my immediate thought was that there are many monographs and comprehensive textbooks focusing on zebrafish model for utilizing drug discovery from marine peptides that cover a wide variety of topics at various levels of detail, so why the need for another book in this increasingly crowded field? Although quite a lot of information is available on the zebrafish model for drug discovery, the present monograph is different from the earlier as the authors provide a comprehensive information on the basic mechanisms of peptides/toxins and how these mechanisms are linked with the emergence of common, devastating pathological disorders. The authors build on current knowledge to detail the link between zebrafish models for discovery of more drugs. They explain these complex concepts with straightforward language that allows greater accessibility to a wide audience. The succinct text will assist the novice in understanding marine peptide complexity research, while the up-to-date information on the current state of this work and pathophysiology will be of interest to the experts in the field. The authors provided all the relevant information in an update fashion in five different chapters. Chapter 1 highlights the introduction, classification, and biomedical importance of peptide, toxins, alkaloids, polysaccharides, and phenolic compounds of macro- and microorganisms and other marine organisms for various human diseases and disorders. Chapter 2 emphasizes the isolation, structural

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Foreword by Balasubramanian

characterization techniques, and de novo sequencing of peptide from ascidians and its teratogenic activity in the zebrafish embryo. Chapter 3 accentuates the separation, purification, and sequencing techniques of toxin from the posterior salivary gland of cuttlefish—furthermore, the maximum tolerated dose and teratogenic activity of toxin by using the embryo of the zebrafish. Chapter 4 draws the attention to the anticancer properties of cuttlefish toxin and the development of xenotransplantation model of zebrafish for anticancer property marine drug screening. Chapter 5 represents the protective effect of marine peptides for cardiovascular diseases in zebrafish model; each chapter of this book contains an insight that will be useful to the scientists at all levels. I congratulate the authors on producing a straightforward text that can be useful to the researchers with different levels of expertise. I hope that this work will essentially help to expand interest in the field of the marine peptide using drug discovery.

Vice Chancellor Chettinad Academy of Research and Education Kelambakkam, Tamil Nadu, India

T. Balasubramanian

Foreword by Madeswaran

It is a distinct honor to have been invited by my colleagues in marine peptide-based drug screening arena to write the foreword to this very articulate and scientifically state-of-the-art book entitled Zebrafish: A Model for Marine Peptide-Based Drug Screening. The book covers all the areas required to create a robust category and perform read-across. I am certain that the readers, including the faculties, researchers, and students, will find this book extremely informative, interesting, and inspiring. Hence, I hope that you will find this book possess sufficient disclosure and adequate utility. This book aims to simplify the revolution and to fortify the researcher with the information needed to use marine protein/peptides/toxins with complete confidence and the best compound that can be applied for therapy of the individual. The book explores in many ways and makes a good sense to investigate further on the isolation of these peptides from marine species. Moreover, this valuable text opens the doors for the progression that occurs when one discovers a fact, becomes interested, and then begins investigating and discovering the natural process. I am a scientist G in the Ministry of Earth Sciences (MoES), who is presently working at the National Centre for Coastal Research (NCCR), Chennai. Since 1994, I have handled the following four major research and development programs, viz., (i) Development of Potential Drugs from Ocean (Drugs from Sea); (ii) Integrated Coastal and Marine Area Management (ICMAM); (iii) Marine Living Resources (MLR); and (iv) Coastal Ocean Monitoring and Prediction System (COMAPS). In vii

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addition, I have handled two international programs, i.e., (i) Commission for Conservation of Antarctic Marine Living Resources (CCAMLR), Hobart, Australia, and (ii) South Asia Co-operative Environment Programme (SACEP), Colombo, Sri Lanka. As a consequence, a large number of books, thick and thin, are being published continuously on various aspects of beneficial usage of marine peptides. The central theme of this monograph is giving fundamental mechanisms of marine peptides/ toxins on physiological activities in our body. The authors have made an effort to unify all the content of scattered research literature in this area of research and tried to provide thorough information. By and large, this monograph is not just a collection of papers, but it is an essence of the diverse marine peptides-/toxins-based drugs screened using the zebrafish model. In all chapters, they have provided the basic information relevant to the topics, and at the same time, they have described the perspective knowledge about using the zebrafish model. I believe that this monograph could be an informative resource in the form of condensed handbook for both fundamental and advanced researchers. Consequently, this monograph covers information on marine pharmacognosy. The authors, Saravanan Ramachandran and Senthilkumar Rajagopal, have brought through understanding the milieu of clinical research, experimental research, marine omics, and pharmacology which, collected revenue, focused on the most active areas of cancer research in the zebrafish model. Best wishes to the authors.

Scientist G, National Centre for Coastal Research Ministry of Earth Sciences, NIOT Campus Chennai, Tamil Nadu, India

P. Madeswaran

Contents

1 Biomedical Importance of Marine Peptides/Toxins�������������������������������������� 1 1.1 Introduction�������������������������������������������������������������������������������������������� 1 1.2 Marine Environment ������������������������������������������������������������������������������ 4 1.3 Toxins������������������������������������������������������������������������������������������������������ 9 1.4 Toxins and Therapeutics ������������������������������������������������������������������������ 9 1.5 Classification of Toxins������������������������������������������������������������������������ 10 1.6 Marine Toxins �������������������������������������������������������������������������������������� 12 1.7 Conclusion�������������������������������������������������������������������������������������������� 12 References ������������������������������������������������������������������������������������������������������ 12 2 Teratogenic Activity of Peptides in Zebrafish Model���������������������������������� 15 2.1 Introduction������������������������������������������������������������������������������������������ 15 2.2 Ascidians���������������������������������������������������������������������������������������������� 16 2.3 Morphology of Ascidians �������������������������������������������������������������������� 17 2.4 Importance of Ascidians ���������������������������������������������������������������������� 18 2.5 Results and Discussion ������������������������������������������������������������������������ 20 2.5.1 FT-IR Spectroscopy���������������������������������������������������������������� 20 2.5.2 MALDI-TOF and Mascot Analysis���������������������������������������� 21 2.5.3 Embryo Toxicity of Peptide���������������������������������������������������� 22 2.6 Conclusion�������������������������������������������������������������������������������������������� 24 References ������������������������������������������������������������������������������������������������������ 24 3 Teratogenic Activity of Toxins in Zebrafish Model������������������������������������ 27 3.1 Introduction������������������������������������������������������������������������������������������ 27 3.2 Cephalopods ���������������������������������������������������������������������������������������� 28 3.3 Cephalopod Toxins ������������������������������������������������������������������������������ 29 3.4 Results and Discussion ������������������������������������������������������������������������ 30 3.4.1 Structural Characterisation of PSG Toxin������������������������������ 31 3.4.2 CD Spectroscopy of PSG Toxin���������������������������������������������� 32 3.4.3 K2D Analysis �������������������������������������������������������������������������� 34 3.4.4 MALDI-TOF/MS�������������������������������������������������������������������� 34

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3.4.5 MASCOT Analysis ���������������������������������������������������������������� 36 3.4.6 Teratogenicity of PSG Toxin�������������������������������������������������� 39 3.4.7 Zebrafish Embryotoxicity Assay �������������������������������������������� 39 3.5 Conclusion�������������������������������������������������������������������������������������������� 40 References ������������������������������������������������������������������������������������������������������ 41 4 Anticancer Properties of Marine Peptides/Toxins Using Zebrafish Model���������������������������������������������������������������������������������� 43 4.1 Introduction������������������������������������������������������������������������������������������ 43 4.2 Cancer �������������������������������������������������������������������������������������������������� 44 4.3 Causes of Cancer���������������������������������������������������������������������������������� 44 4.4 Marine Peptides������������������������������������������������������������������������������������ 46 4.5 Bioactive Cyclic Peptides from Marine Sources���������������������������������� 47 4.6 Bioactive Toxins from Marine Species������������������������������������������������ 47 4.7 Cytotoxicity of PSG Toxin Against MCF-7 Cancer Cell Line by MTT Assay������������������������������������������������������������������������������ 48 4.8 Zebrafish as a Model for Cancer Research ������������������������������������������ 49 4.9 Conclusion�������������������������������������������������������������������������������������������� 51 References ������������������������������������������������������������������������������������������������������ 51 5 Protective Effect of Marine Peptides/Toxins in CVD Using Zebrafish Model���������������������������������������������������������������������������������� 55 5.1 Introduction������������������������������������������������������������������������������������������ 55 5.2 Prevention �������������������������������������������������������������������������������������������� 56 5.2.1 Diet������������������������������������������������������������������������������������������ 57 5.2.2 Dietary Supplements �������������������������������������������������������������� 58 5.3 Medication�������������������������������������������������������������������������������������������� 59 5.4 Physical Activity ���������������������������������������������������������������������������������� 62 5.5 Exercise������������������������������������������������������������������������������������������������ 62 5.6 Adverse Effects of Smoking and Tobacco Usage�������������������������������� 63 5.7 Maintain a Healthy Weight ������������������������������������������������������������������ 63 5.8 Quality Sleep���������������������������������������������������������������������������������������� 63 5.9 Manage Stress�������������������������������������������������������������������������������������� 64 5.10 Health Monitoring�������������������������������������������������������������������������������� 64 5.10.1 Screening of Blood Pressure �������������������������������������������������� 64 5.10.2 Examine the Cholesterol �������������������������������������������������������� 64 5.10.3 Observation of Diabetes���������������������������������������������������������� 64 5.11 Cardiovascular Disease Treatment�������������������������������������������������������� 64 5.12 Zebrafish: An Excellent Model for Study of CVD ������������������������������ 66 5.13 Summary���������������������������������������������������������������������������������������������� 67 References ������������������������������������������������������������������������������������������������������ 70

About the Authors

Saravanan Ramachandran is an Assistant Professor of Marine Pharmacology at the Faculty of Allied Health Sciences, Chettinad University, Chennai, India. He completed his Ph.D. in Marine Biotechnology at Annamalai University, India. His research interests focus on drugs from marine organisms. He has received two research awards from the Indian Council of Medical Research and the Centre for Marine Living Resources & Ecology, Kochi, India, and has published 40 research articles in international and national journals. He is a Member of the Society for Biotechnologists (India) and the International Neurotoxin Association, holds two patents and one copyright, and has submitted 15 gene sequences to the NCBI. Currently, he is serving as the Principal Investigator of a research project funded by the Government of India Department of Biotechnology.  

Senthilkumar  Rajagopal is an Assistant Professor at the Department of Biochemistry, School of Life Sciences, at Rayalaseema University, Kurnool, India. He completed his Ph.D. in Biochemistry at Annamalai University, India, and his postdoctoral fellowship at various universities in the USA, including the University of Virginia, Harvard University, and Virginia Commonwealth University. He is a Recipient of the Rameshwardasji Birla Smarak Kosh Endowment Award from Mumbai Medical Trust and the Ramalingaswami Re-entry Fellowship from the Department of Biotechnology, Ministry of Science and Technology, Government of India. His research has addressed protein kinase C modulation of calcium channel currents and was the Principal Investigator of a major project entitled “G-protein coupled receptor-mediated intracellular mechanisms by alcohol-induced digestive disorders in the gastrointestinal tract.” Other interests include G-protein-coupled receptor signaling pathways in smooth muscle physiology and neurotransmitter transporters to the central nervous system. He is a Life Member of a number of respected scientific associations, such as the Indian Society for Atherosclerosis Research (ISAR), the Indian Science Congress Association (ISCA), the Society for Free Radical Research-India (SFRR), the Indian Society of Cell Biology (ISCB), the American Association of Pharmaceutical Scientists (AAPS), and the Science Advisory Board (SAB), USA.  

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Biomedical Importance of Marine Peptides/Toxins

Abstract

Peptides transfer the information from cell to cell and regulate the life which is involved in biological signalling mechanism. Peptides consist of a combination of two or more amino acids which are linked together to form a polypeptide. Peptides are unable to enter into the cell since they are water soluble molecule. The DNA receives the peptide’s signal through the membrane receptor and these peptides are important bioactive natural products. Based on peptide’s broad spectrum bioactivites, these peptides have high medicinal and are present in many marine species and they have high potential nutritional and medicinal values. The divergence activities of these marine peptides fascinated towards the consideration of the pharmaceutical industry, which endeavours to intention them for use in the remedy or anticipation of various diseases. Various marine peptides and their derivatives have high cost-effective values and had reached the pharmaceutical and nutraceutical perseverance. Keywords

Antimicrobial peptides · Bioactive compounds · Neuroprotective · Nutraceutical · Peptides · Toxins · Trabectedin

1.1

Introduction

Bioactive peptides are very momentous ingrained products that are present in many marine species. Proteins can be degraded either by digestion or by processing to produce 2–20 amino acid residues which are known as bioactive peptides (Di-Bernardini et al. 2011). Two elements can impact the type of bioactive peptides produced: (1) the principal sequence of the protein substrate; (2) the specific substrate of the enzyme(s) used to produce such peptides. Additionally, bioactive peptides can be obtained by hydrolysis of protein (by acid or alkaline treatment), © Springer Nature Singapore Pte Ltd. 2019 S. Ramachandran, S. Rajagopal, Zebrafish: A Model for Marine Peptide Based Drug Screening, https://doi.org/10.1007/978-981-13-7844-7_1

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1  Biomedical Importance of Marine Peptides/Toxins

fermentation or cooking. These peptides have an array of biomedical activities comprising antiviral, antimicrobial, immunomodulatory, antithrombotic and antihypertensive activities (Murray and FitzGerald 2007). They are deliberated greatly significant drugs like molecules. Investigation is being implemented to apprehend the structure of peptide and the configuration and sequence of amino acids. Bioactive marine peptides have numerous governing cellular functions on specific target formulations (Agyei and Danquah 2011). Marine bioactive peptides produced from different sources of protein through enzymatic hydrolysis or fermentation have been demonstrated to possess diverse physiological functions such as immune stimulation, blood pressure and lipid lowering; antidiabetic, antiobesity, skin protection and wound healing activities as well as memory and learning-enhancing property in an animal model or in human. Marine bioactive peptides are seem to have potential as a functional ingredient in the food product or nutraceuticals to increase consumer health and wellbeing. Voluminous scientists have emphasised on the development of pharmaceutical compounds from marine-originated peptides chiefly for ACE inhibition and antihypertensive function (Byun and Kim 2001). Rajapakse et  al. (2005) have reported the richest protein sources from crustaceans, molluscs and fish. Collage is a signficant protein of porcine and bovine essence and is used in various industries like food, cosmetics, pharmaceutics and biomedicine. Collagen, a connective tissue protein, obtained from marine animals is an outstanding source for bioactive peptides which is served as antihypertensive and antithrombotic agent besides inhibitors of brush border enzymes such as dipeptidyl peptidase-IV (Minkiewicz et al. 2011). There is a great quantity of technical and unreliable substantiation that suggests proteins are functional for a wide spectrum of useful concert enrichments, but a new baptism of designer supplementation of bioactive peptides, obtained from fractions of protein including higher amounts of growth factors—could become the fresh natural molecules of alternative for sportspersons in search of boosted recovery, advanced strength and lean body mass expansion. In 1800 century, the shine associate Stanislaus Bondzynski noticed natural “bioactive peptides” in the organ but did not recognise at that time the exact energy they acquired on function, prevention of disease, and their probable roles in tackling cancer. Those peptides, for example insulin and growth hormone, as, have transformed the countenance of medicine above the previous decades; though, their consumable in athletics is forbidden. For those in the nutritional complement world, conversely, bioactive peptides symbolise a new breed of complements that may impressive performance, decrease the time to healing owing to muscle inflammation, immune-role, post-effect recovery and most colourfully, increase the competence of muscle-mass growth (Chakrabarti et  al. 2018; Kamdem and Tsopmo 2017; Kitts and Weiler 2003). There is little doubt bioactive peptides are a major find and a possible infiltrate as a natural safe remedy in a group of human complaints. Many biotech industries have begun scrutinising supplementary gains away from the treatment and disease prevention, and some have generated products that may support in not only improvement but also diminution of pain management and inflammation.

1.1 Introduction

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More than 7000 naturally occurring peptides have very important functions in individual physiology, embracing roles as neurotransmitters, hormones, ion channel ligands, growth factors and anti-infective agent (Padhia et al. 2014; Robinson et al. 2014). These peptides are unique and potential signalling particles which attach to the particular cell surface receptors, for example G protein-combined receptors (GPCRs) or ion channels, where they activate the intracellular functions. Based on their chemical and pharmacological reactions, these peptides will provide important message for propose of new remedial candidate and their reductivism has been observed to interpret into exceptional safety, permissibility and efficacy silhouettes in individuals. This characteristic might also be the chief distinguishing factor of peptides evaluated with habitual small molecules. Additionally, peptide-based medical treatment is normally correlated with lower production difficulty contrasted with protein-based biopharmaceuticals and, consequently, the production costs are also less, usually approaching those of small particles. Thus, in many types, peptides are in the sugary dot stuck between small molecules and biopharmaceuticals (Craik et al. 2003; Lee and Hur 2017; Rizzello et al. 2016). Bioactive peptides are regularly not openly appropriate for use as expedient treatments as they have essential limitation, counting weak chemical and physical immovability, and a limit circulating plasma half-life. These characteristics must be proposed for their applications in medicines. Few of these limitations have been effectively determined throughout the “traditional design” of curative peptides as illustrated below (Fig.  1.1). Moreover conventional peptide proposes, a series of

Fig. 1.1  Analysis of the strengths, weaknesses, opportunities and threats (SWOT) of naturally occurring peptides in their use as therapeutics

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1  Biomedical Importance of Marine Peptides/Toxins

peptide technologies has been materialising which correspond to the opening the chances and future directions in the proteomics. These contain multifunctional and cell-targeting peptides, with peptide-based drug and technologies spotlighting on alternative administration routes. Natural bioactive molecules are derived from marine organisms which comprised roughly one-half of the total global biodiversity, and these organisms have gained substantially in importance as a supply of new compounds (De Vries and Beart 1995). To identify the sample marine organisms in deep water is so difficult. Therefore, the previous marine studies were conducted in shallow water, but development of submarines or remotely operated vehicles simplify an assessment of sample marine organisms in deep water (Dias et  al. 2012; Montaser and Luesch 2011). The availability of marine peptides are very less and sizeable quantity of raw materials is needed for purification, especially when the peptides of interest are not facile to purify or present in only trace amounts in the marine organisms. Therefore, the supply of marine raw material is prerequisite (Molinski et al. 2009). These problems can be fixed by mariculture (cultivation of marine organisms in the open ocean) or aquaculture (cultivation of marine organisms in artificial conditions) (Naylor 2001). The peptides are derived from two main pathways such as heterologous peptide expression in cultured microorganisms in combination with fermentation techniques and chemical peptide synthesis or total synthesis (Martins et  al. 2014; Rosenberg 2008). Broad-spectrum bioactivities of these peptides such as antimicrobial, antiviral, antitumour, antioxidative and neuroprotective activities have potential applications in the pharmaceutical industry, and therefore, more companies are willing to invest in this area. The above-mentioned methods can produce the marine sources or the peptides effectively, and with minimal environment impact.

1.2

Marine Environment

Marine bioresources are valuable source of bioactive compounds with industrial and nutraceutical potential. Marine milieu has always developed as an alternative suitable source of pharmaceutics to the existing pharmacopoeia comprising various structures with irreplaceable mechanism of action. About 16,000 natural products from marine organisms have been reported in 6800 pamphlets worldwide so far (Mayer and Gustafson 2008). Reen and coworkers (Rennekamp and Peterson 2015) discussed the promising potential of bioactive compounds from marine sources as next group drugs for a wide range of diseases. Moreover, bioactive peptides from marine sources have many beneficial effects on human health. Besides, numerous studies reported that marine peptides exhibit various anti-infective activities, such as antimicrobial, antifungal, antimalarial, antiprotozoal, anti-tuberculosis and antiviral activities (Fig.  1.2). Presently, 13 different classes of bioactive compounds from various marine sources have arrived the clinical pipeline (Table 1.1). Cytarbine and vidarbine are nucleosides obtained from the marine sponge Tethya sp. are commended drugs for cancer and viral infections correspondingly.

1.2 Marine Environment

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Fig. 1.2  Micro and macro algae products and their usages

Trabectedin is an alkaloid family obtained from the tunicate Ecteinascidia turbinata, which has been approved to enter clinical trials phase IV against cancer. Eribulin mesylate is a macrolide from sponge Halichondria okadai, developed for the treatment of deadly disease like cancer, which is currently in clinical trials phase III. Ziconotide, a peptide from the cone snail, Conus sp. has been approved as a drug for the treatment of inflamation. Another durg soblidotin, a peptide from the marine bacterium Sympioca sp. has been approved for phase III clinical trials against cancer (Meyer et al. 2003). In recent years, it has been recognised that bioactive peptides derived from plant and animal food protein can exert a wide range of physiological or hormone-like biological activities beyond their nutritional value (Hartmann and Meisel 2007). The 60% of the global biodiversity comprise with marine species and marine bioactive peptides are obtained from sea resources and these resources to be explored a lot (Kim and Mendis 2006; Rustad 2003). Marine fish collagen is hydrolysed by enzyme to produce the marine collagen peptide (MCP), which is low-­ molecular-­weight peptide. This specific fragment has been found to have a broad diversity of functional and biological properties containing antioxidant (Nishimoto et  al. 2008), antihypertensive (Felician et  al. 2018) and anti-skin-aging activities (Barzideh et al. 2014; Nagai et al. 2002). Therefore, these fragments are used as

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Table 1.1  Marine pharmaceuticals in clinical pipeline Clinical status Approved

Phase III Phase II

Phase I

Compound name Cytarbine, Ara-C Vidarbine, Ara-A Ziconotide Trabectedin (ET-743) Eribulin mesylate (E7389) DMXBA (GTS-21) Pinabulin (NPI-2358) Plitidepsin Elisidepsin PM1004 Tasidotin, Synthadotin (ILX-651) Pseudopterosins Bryostatin1 Hemiasterlin (E7974) Marizomin (Salinosporamide A; NPI-0052)

Trademark Cytosar-U® Vira-A® Prialt® Yondelis®

Source Sponge Sponge Cone snail Tunicate

Chemical class Nucleoside Nucleoside Peptide Alkaloid

Disease area Cancer Antiviral Pain Cancer

NA

Sponge

Macrolide

Cancer

NA

Worm

Alkaloid

NA

Fungus

Diketopiperazine

Cognition schizophrenia Cancer

Aplidin® Irvalec® Zalypsis® NA

Tunicate Mollusc Nudibranch Bacterium

Depspeptide Depspeptide Alkaloid Peptide

Cancer Cancer Cancer Cancer

NA

Soft coral

NA NA

Bryozoa Sponge

Diterpine glycoside Polyketide Tripeptide

Wound healing Cancer Cancer

NA

Bacterium

β-Lactonegamma lactam

Cancer

Adapted from Odyssey of marine pharmaceuticals. (Source: Mayer and Gustafson 2008)

ingredients of functional foods or pharmaceuticals because of their bioactive properties or health enhancement (Felician et al. 2018; Nagai et al. 2002). One half of the collagen tissues, including skin, bone and scale, are fish processing waste products (Nagai et al. 2010). In order to identify the tolerability and potential chronic adverse effects of marine collagen peptides, the dose response studies can be carried out in animals and clinical trials, which might be exerted by the collagen peptides themselves or their by-­ products during the processing. In addition, there are unique differences in physiochemical properties and peptide composition between collagen peptides derived from marine fish and livestock animals (Nagai et al. 2010; Silva et al. 2014). Current investigations have revealed that assessments of safety profiles of marine collagen peptides are insufficient (Silva et al. 2014). The molecules which are important constituents of the innate immunity as immune modulators in certain organisms are known as antimicrobial peptides (AMPs) (Zanetti 2004). AMPs play an important function in the living organisms, and they conflict the attacking pathogens to the host through a wide array of

1.2 Marine Environment

7

Table 1.2  Examples for antimicrobial peptides Type Anionic peptides Linear cationic α-helical peptides

Cationic peptide enriched for specific amino acid Anionic and cationic peptides that contain cysteine and form disulphide bonds

Characteristic Rich in glutamic and aspartic acid Lack in cysteine

Rich in proline, arginine, phenylalanine, glycine, tryptophan Contain 1~3 disulphide bonds

AMPs Maximum H5 from amphibians; dermcidin from humans Cecropins, andropin, moricin, ceratotoxin and melittin from insects; magainin, dermaseptin, bombinin, brevinin-1, esculentins and buforin II from amphibians; CAP18 from rabbits; LL37 from humans Abaecin, apidaecins from honeybees; prophenin from pigs; indolicidin from cattle

1 bond: Brevinins; 2 bonds: protegrin from pig; tachyplesins from horseshoe crabs; 3 bonds: defensins from humans; more than 3:drosomycin in fruit flies

antimicrobial activity that can differ concurrency to the type of pathogens (Lehrer et al. 1983; Zasloff 1987). AMPs are distinguished in their structures that vary from alpha helix to beta strands of peptides, and they play a defensive role for producing organisms (Hancock 2001). To study the specific toxicity of AMPs, these peptides have distinguished between the lipid component of the membrane of prokaryotic and eukaryotic creature which act as their target delivery sparing the host from injurious. However, AMPs are based on their identical net charge and hydrophobicity which are characteristic grades for them. The greater part of these molecules have been illustrated to be transmitting a consistent positive charge between +2 and + 9, with only few accounted to be anionic in nature (Table 1.2) (Zasloff 1987). AMPs are able to binding to the anionic nature of lipopolysaccharides by electrostatic attractive forces in the membrane of invading pathogen because of their cationic in nature. AMPs obtain a bipolar substantiation that minimise the attaching to the pathogen’s membrane during their interaction. This type of AMPs are categorised as membrane-penetrating peptides due to their ability of developing apertures in the membrane subsequent to their electrostatic communication, thus consequence in discharge of cellular substance and following cell death (Sengupta et al. 2008). Furthermore, to the earlier revealed mechanism, AMPs can act by impeding with some critical cellular progressions inhibiting them subsequent to their movement through the cellular membrane (Brogden et al. 2005; Kondejewski et al. 1999). In past one decade, AMPs are a fascinating field of study for prospective drug molecules due to their wide array of function and more essentially to the information that AMPs can prevail over the antimicrobial confrontation. Because AMPs mark the lipid constituents in the membrane of the entry of pathogens, this intervention with such an essential configuration in microbes generates a barrier against resistance improvement (Fig. 1.3).

Fig. 1.3  Different marine sources with major bioactivity area discovered. (Adapted from Rushikesh et al. 2017)

8 1  Biomedical Importance of Marine Peptides/Toxins

1.4 Toxins and Therapeutics

1.3

9

Toxins

Toxins are biomolecules, comprised of proteins, peptides or glycoproteins, that are predominantly produced by animals for the persistence of escape or predation from prediators which can cause infection or poisonous effects. Venom or toxins are highly complex mixtures combination of highly active biomolecules capable of perpetrating a range of biochemical reactions. Toxins are composed of proteins, peptides, enzymes, neutral sugars, serotonin, enzyme inhibitors, histamine etc. (Casewell et al. 2013). These toxins may be affecting gated ion channels and causes neurotoxicity, hemotoxicity or cytotoxicity, killing the cells. Myotoxins are principally peptide based which root the necrosis of muscles (Nagai 2012).

1.4

Toxins and Therapeutics

For the past three decades, the FDA has given approval for 1355 new drugs from natural sources (Newman and Cragg 2012) and 1453 in all years up to 2013 (Kinch et al. 2014). Several lead drug candiadates like eptifibatide, atracurium, captopril, tirofiban, ziconotide and botulinum toxin products have been derived from toxins or venom elements. Because of their increased specificity and ability, venom and toxin “biologics” are supported over unadventurous small biomolecule pharmaceutical and drug-governing agencies. For the past 10 years, the global tally of commended new chemical entities (NCEs) have lingered stable, and the authorisation of number of therapeutic agents has transmited to others such as vaccines and protein-based diagnostic candidates is not on a regularly ascending trend from 2004 to 2012 (Fig. 1.4). Moreover, the figure of pharmaceutical and biotechnology unit apprehensions subordinated with FDA-approved medicines has facaded a gradual fall in recent years (Kinch et al. 2014). It is also identified that biomolecular patterns, for example presence of intramolecular disulphide bridges, have reduced firmness to toxin-related peptides, permitting large-scale production and profitable productivity

Fig. 1.4  FDA approved new drugs

10

1  Biomedical Importance of Marine Peptides/Toxins

(King 2011). Though, much of these improvements of toxin-related peptides are yet to be employed headed fors drug development and clinical training.

1.5

Classification of Toxins

The marine toxins are classified in to two types, namely peptides and non-peptides. Peptides are synthesised by marine animals, whereas non-peptides are synthesised by marine microbes, plants and fungi (Fig.1.5). These have been associated with a number of poisoning cases which leads to paralysis, vomiting, pruritus, respiration difficulties, foaming in mouth, bloody diarrhoea and finally death. Characterisations of these toxins such as jellyfish toxin, sea anemone toxin, coral toxin, hydra toxin, starfish toxin, sea urchin toxin and molluscan toxins have not been carried out due to the proteinaceous nature and structural complexity. The toxins from these marine species are unstable and hence difficult to characterise (Nagai 2012) (Fig.  1.6). However, over the past decade, research on peptide-based marine toxins has gained momentum due to their utility in biomedical and pharmacological applications. A wide range of fishes such as stone fish, striped eel fish, rabbit fish, scorpion fish, weaver fish and sting ray are known to secrete proteinaceous toxins as protective measures against predators. Stone fish are considered the most dangerous of all the fishes worldwide. These toxins from the stone fish when gets injected through

Toxins

Peptides

Marine animals

Active - injections

Act as a venom by spray, sting & specialized apparatus

Fig. 1.5  Classification of toxins from marine organism

Non-peptides

Marine microorganism & plants

Active – orally

Act as a poisons incase of swallowing

1.5 Classification of Toxins

11

Fig. 1.6  Isolation, purification, structural characterisation and bioapplication of toxins from marine species

spine into humans generate terrible pain, localised oedema and necrosis of the tissues. Ghadessey and co-workers (Ghadessy et  al. 1996) isolated and completely characterised the dimeric stonustoxin of 71 and 79 kDa from the stone fish Synanceia horrida. Neoverrucotoxin, a dimeric 166 kDa stonustoxin is isolated and characterised from the venom of Synanceia verrucosa. Ueda et al. (2006) also reported the unstable nature of stonustoxins and the need of use of buffers with higher ionic strength for optimum extraction of protein toxins. Stingrays are group of fish which secrete low-molecular-weight vasoconstrictor peptide, which on envenomation result in immediate intense pain and local oedema followed by skin necrosis. Other toxins from scorpion fish such as Scorpaena guttata, S. horrida, Kenyan S. verrucosa, Japanese S. verrucosa have also been isolated and characterised (Conceicao et al. 2006). The isolation, purification and characterisation of toxins from marine organisms are clearly depicted in Fig. 1.6.

12

1.6

1  Biomedical Importance of Marine Peptides/Toxins

Marine Toxins

The marine atmosphere has fast overhauled the extra boulevards of bio-prospecting in the direction of anticancer molecules, exactly breast cancer owing to its immense nature and inimitable collection of biomolecules. Marine toxins and peptides have been observed to display antiproliferative action by several mechanisms. Jasplakinolide, which is a cyclic depsipeptide obtained from marine sponge Jaspis johnstoni, is investigated to induce cell death by Bcl-2 regulation in breast cancer. Dolastatin-10 developed from the marine mollusc Dolabella auricularia with peptide units induces apoptosis by downregulation of Bcl-2 expression. Pardaxinis is polypeptide (composed of 33 amino acids) from the Red sea moses solitary convinces apoptosis by up regulation of gene caspase 3/7 and interference of mitochondrial membrane potential. Marine peptides also form paramount components of a number of toxins which are structurally well characterised by sequence of amino acids, conformation and threedimensional spatial configurations (Conceicao et al. 2006).

1.7

Conclusion

Marine milieu has fast become the technical terminus for the exploration of bioactive compounds for their passionate, raging nature with biota boastful of bio-­ macromolecules unique in structure and biological applications. The maritime reservoir has become vast foundation of peptides/toxins rich in biomedical promises against a wide range of diseases and clamps promise as antioxidants, antiviral, anti-inflammatory, anticancer and antibacterial agents. Marine toxins, peptides in nature, at sub-lethal applications have shown immense potentials in reducing tumour growth with increased specificity and minimised toxicity in suitable model with less adverse effects. This opens up a budding alternate therapeutic strategy as a pharmacological agent of the future. Acknowledgement  Saravanan Ramachandran gratefully acknowledges the Department of Biotechnology, Ministry of Science and Technology, Government of India (BT/PR15676/ AAQ/03/794/2016) for providing the facilities and financial support. Senthilkumar Rajagopal gratefully acknowledges the support provided by the Department of Biotechnology, Ministry of Science and Technology, Government of India (No: BT/RLF/Re-entry/42/2012).

References Agyei D, Danquah K (2011) Industrial-scale manufacturing of pharmaceutical-grade bioactive peptides. Biotechnol Adv 29:272–277 Barzideh Z, A L, CY G, Benjakul S, Karim A (2014) Isolation and characterisation of collagen from the ribbon jellyfish (Chrysaora sp.). Int J Food Sci Technol 49:1490–1499 Brogden K, Guthmiller J, Salzet M, Zasloff M (2005) The nervous system and innate immunity: the neuropeptide connection. Nat Immunol 6:558–564

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Byun H, Kim S (2001) Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragrachalcogramma) skin. Process Biochem 36:1155–1162 Casewell N, Wuster W, Vonk F, Harrison R, Fry B (2013) Complex cocktails: the evolutionary novelty of venoms. Trends Ecol Eval 28:219–229 Chakrabarti S, Guha S, Majumder K (2018) Food-derived bioactive peptides in human health: challenges and opportunities. Nutrients 10:1738 Conceicao K, Konno K, Melo R, Marques E, Hiruma-Lima C, Lima C, Richardson M, Pimenta D, Lopes-Ferreira M (2006) Orpotrin: a novel vasoconstrictor peptide from the venom of the Brazilian sting ray Potamotrygong orbignyi. Peptides 27:3039–3046 Craik D, Daly N, Saska I, Trabi M, Johan Rosengren K (2003) Structures of naturally occurring circular proteins from bacteria. J Bacteriol 185:4011–4021 De Vries D, Beart P (1995) Fishing for drugs from the sea: status and strategies. Tredens Pharmacol Sci 16:275–279 Dias D, Urban S, Roessner U (2012) Historical overview of natural products in drug discovery. Meta 2:303–336 Di-Bernardini R, Harnedy P, Bolton D, Kerry J, O’Neill E, Mullen A, Hayes M (2011) Antioxidant and antimicrobial peptidichydrolysates from muscle protein sources and by-products. Food Chem 124:1296–1307 Felician F, Xia C, Qi W, Xu H (2018) Collagen from marine biological sources and medical applications. Chem Biodivers 15:e1700557 Ghadessy F, Chen D, Kini R, Chung M, Jeyaseelan K, Khoo H, Yuen R (1996) Stonustoxin is a novel lethal factor from stonefish (Synanceja horrida) venom cDNA cloning and characterization. J Biol Chem 271:25575–25581 Hancock R (2001) Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 1:156–164 Hartmann R, Meisel H (2007) Food-derived peptides with biological activity: from research to food applications. Curr Opin Biotechnol 18:163–169 Kamdem J, Tsopmo A (2017) Reactivity of peptides within the food matrix. J Food Biochem:e12489 Kim S, Mendis E (2006) Bioactive compounds from marine processing byproducts—a review. Food Res Int 39:383–393 Kinch M, Haynesworth A, Kinch S, Hoyer D (2014) An overview of FDA-approved new molecular entities: 1827–2013. Drug Discov Today 19:1033–1039 King G (2011) Venoms as a platform for human drugs: translating toxins into therapeutics. Expert Opin Biol Ther 11:1469–1484 Kitts D, Weiler K (2003) Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Curr Pharm Des 9:1309–1323 Kondejewski L, Jelokhani-Niaraki M, Farmer S, Lix B, Kay C, Sykes B, Hancock R, Hodges R (1999) Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity. J Biol Chem 274:13181–13192 Lee S, Hur S (2017) Antihypertensive peptides from animal products, marine organisms, and plants. Food Chem 228:506–517 Lehrer R, Selsted M, Szklarek D, Fleischmann J  (1983) Antibacterial activity of microbicidal cationic proteins 1 and 2, natural peptide antibiotics of rabbit lung macrophages. Infect Immun 42:10–14 Martins A, Vieira H, Gaspar H, Santos S (2014) Marketed marine natural products in the pharmaceutical and cosmeceutical industries: tips for success. Mar Drugs 12:1066–1101 Mayer A, Gustafson K (2008) Marine pharmacology in 2005–2006: antitumour and cytotoxic compounds. Eur J Cancer 44:2357–2387 Meyer B, Mann N, Lewis J, Milligan G, Sinclair A, Howe P (2003) Dietary intakes and food sources of omega-6 and omega-3 polyunsaturated fatty acids. Lipids 38:391–398

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Minkiewicz P, Dziuba J, Michalska J  (2011) Bovine meat proteins as potential precursors of biologically active peptides-a computational study based on the BIOPEP database. Food Sci Technol Int 17:39–45 Molinski T, Dalisay D, Lievens S, Saludes J (2009) Drug development from marine natural products. Nat Rev Drug Discov 8:69–85 Montaser R, Luesch H (2011) Marine natural products: A new wave of drugs? Future Med Chem 3:1475–1489 Murray B, FitzGerald R (2007) Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production. Curr Pharm Des 13:773–791 Nagai H (2012) Marine protein toxins. Springer, Dordretch Nagai T, Araki Y, Suzuki N (2002) Collagen of the skin of ocellate puffer fish (Takifugu rubripes). Food Chem 78:173–177 Nagai T, Suzuki N, Tanoue Y, Kai N, Nagashima T (2010) Characterization of acid-soluble collagen from skins of surf smelt (Hypomesus pretiosus japonicus Brevoort). Food Nutr Sci 1:59–66 Naylor R (2001) ECOLOGY: aquaculture–a gateway for exotic species. Science 294:1655–1656 Newman D, Cragg G (2012) Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 75:311–335 Nishimoto S, Goto Y, Morishige H, Shiraishi R, Doi M, Akiyama K, Yamauchi S, Sugahara T (2008) Mode of action of the immunostimulatory effect of collagen from jellyfish. Biosci Biotechnol Biochem 72:2806–2811 Padhia A, Senguptaa M, Senguptaa S, Roehmb K, Sonawane A (2014) Antimicrobial peptides and proteins in mycobacterial therapy: current status and future prospects. Tuberculosis 94:363–373 Rajapakse N, Jung W, Mendis E, Moon S, Kim S (2005) A novel anticoagulant purified from fish protein hydrolysate inhibits factor XIIa and platelet aggregation. Life Sci 76:2607–2619 Rennekamp A, Peterson R (2015) 15 years of zebrafish chemical screening. Curr Opin Chem Biol 24:58–70 Rizzello C, Tagliazucchi D, Babini E, Rutella G, Saa D, Gianotti A (2016) Bioactive peptides from vegetable food matrices: research trends and novel biotechnologies for synthesis and recovery. J Funct Foods 27:549–569 Robinson S, Safavi-Hemami H, McIntosh L, Purcell A, Norton R, Papenfuss A (2014) Diversity of Conotoxin gene Superfamilies in the venomous snail, Conus victoriae. PLoS One 9:e87648 Rosenberg A (2008) Aquaculture: the price of lice. Nature 451:23–24 Rushikesh S, Pravin P, Seetharama J  (2017) Peptides, Peptidomimetics, and polypeptides from marine sources: A wealth of natural sources for pharmaceutical applications. Mar Drugs 15:124 Rustad T (2003) Utilisation of marine by-products. Electron J  Environ Agric Food Technol 2:458–463 Sengupta D, Leontiadou H, Mark A, Marrink S (2008) Toroidal pores formed by antimicrobial peptides show significant disorder. Biochem Biophys Acta 1778:2308–2317 Silva T, Moreira-Silva J, Marques A, Domingues A, Bayon Y, Reis R (2014) Marine origin collagens and its potential applications. Mar Drugs 12:5881–5901 Ueda A, Suzuki M, Honma T, Nagai H, Nagashima Y, Shiomi K (2006) Purification, properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolyticlethal factor from the stonefish Synanceia verrucosa venom. Biochim Biophys Acta 1760:1713–1722 Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75:39–48 Zasloff M (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci U S A 84:5449–5453

2

Teratogenic Activity of Peptides in Zebrafish Model

Abstract

Proteins and peptides are vital elements in food production both for their dietetic properties and for their functional properties that impact quality and customer acceptance of food. Peptides are significant bioenergetic natural products which exist in many marine organisms. These marine peptides have high budding nutraceutical and therapeutic values as of their wide spectra of bioactivities. The crude peptides are extracted from ascidians using organic solvents, fractionated to gel chromatography and then purified by using reversed-phase high-performance liquid chromatography (RP-HPLC). The presence of peptides and their functional groups is confirmed by SDS-PAGE and Fourier transform infrared spectroscopy (FT-IR), respectively. The molecular weight and de novo sequencing of purified peptide are confirmed by matrix-assisted laser desorption/ionisation-­ time of flight (MALDI-TOF) and Mascot analysis, respectively. Finally, the lethal dose (LD50) and maximum tolerated dose (MTD) and teratogenicity of purified peptide from ascidians are reported by using zebrafish embryo model. This chapter emphasises on nutraceutical and pharmaceutical improvement into arcaded products. Keywords

Ascidian peptides · RP-HPLC · FT-IR · MALDI-TOF · Zebrafish · Teratogenicity

2.1

Introduction

Marine organisms comprise half of the world’s phenomenal biodiversity, which offer a wide range of biologically active principle components currently used in the production of functional foods. They include bioactive peptides, omega fatty acids, oligosaccharides, enzymes, pigments, minerals and biopolymers. Amongst the bioactive compounds, proteins are of most important nutritional compounds and © Springer Nature Singapore Pte Ltd. 2019 S. Ramachandran, S. Rajagopal, Zebrafish: A Model for Marine Peptide Based Drug Screening, https://doi.org/10.1007/978-981-13-7844-7_2

15

16

2  Teratogenic Activity of Peptides in Zebrafish Model

tremendous source of all essential amino acids. They are in authority for maintaining, building and repairing of body tissue and are one of the core energy sources. Moreover, some of the proteins in nutritional regimens have various biological properties which improve the health. With respect to this, it has been reported that proteins and peptides from marine milieu can lower blood pressure and lipid contents and therefore decrease the risk of atherosclerosis and cardiovascular diseases (CVDs) (Saravanan 2011). During food digestion, proteins are hydrolysed by protease enzymes producing an extensive series of peptides, consisting of 2–20 amino acids joined by peptide bonds. Some of these peptides have distinct structures which permit them to deliberate particular biomedical functions. All these released form of peptides exhibits numerous biochemical reactions such as antioxidant, anticancer, antimicrobial, immune stimulatory, blood pressure, glucose and lowering the lipid activities due to their bioactive features (Karthik et al. 2016). More than 30,000 marine bioactive compounds have been screened so far from nautical species; nearly 30 are in clinical trials, in which only 10 have been moved for production in the pharma industries (Hartmann and Meisel 2007). This type’s marine bioactive compound includes polyketides, peptides, terpenes, sterols, fatty acids and alkaloids. The infusion of the bioactive compound, which has been categorised to have a unique biological property, can be practiced either unaided or aided as major constituent in the synthesis of organic compounds. It can also be used within the structure of its active sites to develop new drug candidates with programmed properties. Owing to the differing milieu both chemically and biologically, it shadows that marine species will yield different types of natural products from terrestrial strains. This has formerly been confirmed with many marine natural creation structures that have no example from the terrestrial atmosphere and indication of new toxicological properties. These unique chemical entities are proven to exist in marine invertebrates (Karthik et al. 2016). Peptides have gained attention as therapeutics during recent years. More than 60 peptide-based drugs have been influenced to the pharmacy for the revenue of patients’ health, and a number of new therapeutic peptides are in stage of preclinical trail and clinical trial for drug development. The key supplier to this triumph is the powerful and specific, yet harmless, approach of reaction of peptides. Amongst the prevalent range of biologically active peptides, naturally transpiring marine-isolated cyclopolypeptides reveal a broad range of unusual and potent biomedical activities (Karthik et al. 2017).

2.2

Ascidians

Ascidians are one of the sessile organisms amongst the marine invertebrates that are commonly called “sea squirts”, because of their habit of squirting a jet of water when disturbed. They are found distributed from the littoral zone to the deep sea. Almost 3000 ascidians species are reported so far, which range from miniature ones 1 mm long above 10 cm. Similar to sponges, ascidians exist in a variety of forms, consistency (soft, rough and tough) and colours (Fig. 2.1). Ascidians settle on hard

2.3 Morphology of Ascidians

17

Fig. 2.1  Phallusia arabica (selected ascidians)

surfaces such as hard rocks, stones, hull of ships, branches and roots of sea weeds in tropical marine habitation, which affords favourable features of milieu such as apt bedrock for attachment and temperature which stimulates nonstop breeding of ascidians. They are the major components of fouling community. They epitomise the most highly developed group of animals normally investigated by marine natural product biochemists. Ascidians (also known as tunicates) are governing and always surviving group of animals amongst benthic creatures in tropical and temperate regions (Sahade et al. 1998). This class of animals has been recognised universally for the manifestation of effective subordinate metabolites and rapid invasion. The simplicity of the ascidians is, they replicate together at different sites of the entire embryo, as well as at the genetic/molecular level (lesser genome with scarcer genes). Whether ascidians are more simple because of a basal point or to an ancillary popularisation (or, almost certainly, as amalgamation of the two, conditional on the specific trait) is of not more importance than the preservation of simple cellular developments.

2.3

Morphology of Ascidians

Ascidians are often referred to as “tunicates” as their body is fully covered by a sac-­ like structure. The oral siphon marks the anterior end of the animal. The posterior end is arbitrarily designated and lies somewhere beyond the gut loop. The tunic encases the mantle which houses the brachial sac, within which is present the digestive tract, the “kidney”, the nervous system, the circulatory system and the reproductive organs. Ascidians are hermaphrodites; they have independent male and female gonads within the same body. Sperms are released into the sea; mature eggs

18

2  Teratogenic Activity of Peptides in Zebrafish Model

may also be released into the sea or they may be harboured within the animal until fertilisation is completed and they have other characteristic features like tailed free-­ swimming larva during development and the notochord in the tail region of the larva (Keller 2002, 2006). They are broadly classified into two groups – the solitary ascidians, which are living singly, and the colonial ascidians, in which many individuals called zooids live together to form a colony.

2.4

Importance of Ascidians

Worldwide, ascidians are well known for their speedy incursions and also for the existence of promising biomolecules for biomedical applications. They are a leading and ever existing group between benthic organisms in humid and temperate areas (Sahade et al. 1998). Many ascidian species contain abundant, potently toxic secondary metabolites that are implicated in chemical defence (Paul et  al. 1990; Vera and Joullie 2002). Therefore, marine chemists characterise the utmost greatly developed class of animals and regularly examine them for natural product isolation for biomedical functions (Faulkner 1984). Six biomolecules have been derived from marine species which have been brought to clinical trials for antitumour agents; amongst the six, three bioactive compounds are isolated from ascidians, as indication of the great prospective of these species as a new foundation of antitumour drugs. The bioactive compounds of ascidians have become one of the maximum energetic meadows of marine pharmacology (Fig. 2.2); it has been sufficiently regulated and marine creatures are productive protocols of strange structures with important bioactivities. Most of these marine drugs are used for cancer remedy (Menna et al. 2011). In the current scenario, 10% of the world’s biodiversity has been identified for prospective biological actions and applications, few more therapeutic natural principal biomoles expect innovation with the task being how to contact this natural biochemical diversity. Polyclinidae is one of the most diverse families in the class Ascidiacea and genus Aplidium, which have been the subject of extensive chemical and biological investigations (Zubia and Salva 2005). Ascidians are leading animals in many marine communities, having a large geographic dissemination. They encompass approximately 3000 labelled species, established in all marine environments: from shallow water to the deep sea (Shenkar and Swalla 2011). Various new bioactive compounds are isolated from ascidians, majority of which are amino acids, peptides and protein derivatives. Recently, the most prominent samples of ascidian bioactive compounds include dehydrodidemnin B, didemnin B, sulcatin, ecteinascidin-743, stolonic acids A and B and bistramides A, B, C, D and K (Gopalakrishnan et al. 2011). Secondary metabolites produced from the ascidians have important defensive roles besides predation. The United States National Cancer Institute has shown that marine invertebrates display a higher frequency of cytotoxic compounds and tunicates are amongst the most hopeful animals as sources of novel active lead compounds for drug development (Faulkner 2001).

2.4 Importance of Ascidians

Fig. 2.2  Unique chemical structures derived from ascidians. (Source: Arumugam et al. 2017)

19

20

2.5

2  Teratogenic Activity of Peptides in Zebrafish Model

Results and Discussion

Live ascidian Phyllostachys nigra is collected by scuba diving during the low tide of the intertidal area at Rameswaram and Thoothukudi coast, Tamil Nadu, Southeast coast of India. It is identified with the help of a marine taxonomist, Zoological Survey of India, Chennai, and used for further experiment. It exhibited highest concentration of protein (55.23 mg/g) when compared to the result of Edler et al. (2002), who had reported a protein concentration of 10.2 mg and 12.5 mg of a cyclic peptide vitilevuamide derived from the ascidians Didemnum cuculliferum and Polysyncraton lithostrotum, respectively, obtained through purification using organic solvents. The difference in yield of the protein concentration may be attributed to the variation in solventbased extraction procedures. The crude ascidian peptides upon purification by RP-HPLC yielded a chromatogram characteristic of peptides from marine sources. The ascidian peptide of P. nigra on HPLC yielded a distinct active fraction at a retention time of 6.14 min (Fig. 2.3). The purified active peptide from P. nigra is labelled P1 and is lyophilised and stored at − 20 °C for further study. The SDS-PAGE of the fractionated and purified ascidian peptide revealed prominent bands of confirmation of protein, with two bands [Lane 1 corresponds to protein marker (20–204 kDa range); Lanes 2 and 3 correspond to fractionated and purified peptides] (Fig. 2.4).

2.5.1 FT-IR Spectroscopy The FT-IR spectrum of the purified peptides P1 from P. nigra revealed the presence of vital functional groups corresponding to characteristic nature of the peptide. Strong absorption stretch at 3500–3000 cm−1 resulted in a broad peak representative of the presence of O-H stretch (Fig. 2.5). Moderate intensity absorption peak in the range of 2000–1000  cm−1 is observed due to the N-H amines. Presence of a moderate intensity peak at 1076.67 cm−1 is a characteristic of the aliphatic amine stretch (-CN) within 1020–1250 cm−1.

Fig. 2.3  RP-HPLC chromatogram of purified peptides from ascidians

2.5 Results and Discussion

21

Fig. 2.4  SDS-PAGE of fractionated and purified peptides

Fig. 2.5  FT-IR spectrum of purified peptides from ascidians

2.5.2 MALDI-TOF and Mascot Analysis The molecular weight (recorded as 666.24 Da) and oligomerisation pattern of the purified peptides are shown in Fig.  2.6. Mascot analysis revealed the de novo sequence of amino acids of the purified peptide from ascidian to be MAKFLIRTNPVNTTMMMRN LFLVNKSFVRKMWSKHYFGK, which resembles to that of enzyme peptidoglycan transglycosylase. Further, it has to be confirmed by in vivo assay. The coupled techniques of liquid chromatography with mass spectroscopy (LC-­ MS) metabolomics have been practised currently to categorise 71 metabolites in the hostile ascidian Styela plicata. Active fractions are analysed for anticancer and apoptosis-inducing properties, illuminating various biomolecules with possible waiting further investigation (Palanisamy et  al. 2017a). From 1994 to 2014, Palanisamy and co-workers have reported a widespread treatise on roughly 580 bioactive compounds isolated and purified from ascidians (Palanisamy et al. 2017b),

22

2  Teratogenic Activity of Peptides in Zebrafish Model

Fig. 2.6  MALDI-TOF/MS of purified peptides from ascidian

which mainly explained their chemical structure and examined their various biological activities, such as antibacterial, antiparasitic, antiviral, anti-inflammatory, antiproliferative and antidiabetic effects.

2.5.3 Embryo Toxicity of Peptide Zebrafish embryos are exposed to increased concentration of peptide for embryo toxicity in control group, 0.5, 1, 1.5, 2 and 2.5 mg/ml in 24-well plates (Fig. 2.7). The LC50 of peptide is determined as 1.5  mg/ml and MTD obtained is 1  mg/ml (Fig. 2.8). Embryos started to hatch out after 48 h; the hatching rate is about 5.6% in control group and 5% in 1 mg/ml. At 96 h, 100% of embryos had hatched in the control group and 100% in the 0.5 mg/ml treatments. Significant delay is observed in embryos exposed to 2 mg/ml of peptide recorded for 30% hatching. After 24 h of exposure in control group, there are no malformations or significant delay. In other concentrations, which are exposed to peptide, major malformations like heart beat abnormalities, cardiac oedema, short body length, abnormal pigmentation, tail bent, pericardial oedema and trunk curvature are also observed. It can be noticed that abnormal heart beating is due to increase in peptide concentration and decrease in heartbeat rate. However, less heartbeat rate is noted in embryos treated with higher peptide concentration (2 and 2.5 mg/ml) and subsequently arrested the growth of embryo. Similarly, with the percent of hatchability, heartbeat rate is also displayed to be dependent on the concentration of peptide.

2.5 Results and Discussion

23

Fig. 2.7  Teratogenicity of zebrafish embryos treated in peptide and control groups

Fig. 2.8 LD50, hatchability, heart beat rate and body length of zebrafish embryos treated with peptides and control groups. (a) Lethal concentration of the Zebrafish larvae exposed to peptide. (b) Hatchability rate of Zebrafish larvae exposed to peptide. (c) Heart beat rate of Zebrafish larvae exposed to peptide. (d) Body length of Zebrafish larvae with peptide treated

24

2.6

2  Teratogenic Activity of Peptides in Zebrafish Model

Conclusion

This chapter discussed the outcomes of the isolation and purification of peptide from selected Indian ascidian P. nigra, collected from the Gulf of Mannar. Spectrophotometric estimation showed that the ascidian is rich in peptide content. The molecular weight of purified peptide is observed as 666.24 Da. Mascot analysis revealed the de novo sequence of amino acids of the purified peptide from ascidian to be MAKFLIRTNPVNTTMMMRN LFLVNKSFVRKMWSKHYFGK, which resembles to that of enzyme peptidoglycan transglycosylase. The FT-IR spectrum of the ascidian peptide revealed the presence of vital functional groups such as phenols, amines and amides as for their teratogenic activity. Exposures of peptides are embryotoxic, provoking developmental changes in D. rerio embryos, namely delayed hatching, coagulation, bent spine and abnormal heart beating. Acknowledgement  The author gratefully acknowledges the Department of Biotechnology, Ministry of Science and Technology, Government of India (BT/PR15676/AAQ/03/794/2016).

References Arumugam V, Manigandan V, Saravanan R, Umamaheswari S (2017) Bioactive peptides from marine ascidians and future drug development–a review. Int J Pept Res Ther 24:13–18 Edler MC, Fernandez AM, Lassota P, Ireland CM, Barrows LR (2002) Inhibition of tubulin polymerization by vitilevuamide, a bicyclic marine peptide, at a site distinct from colchicine, the vinca alkaloids, and dolastatin 10. Biochem Pharmacol 63(4):707–715 Faulkner DJ (1984) Marine natural products: metabolites of marine invertebrates. Nat Prod Rep 14(3):5–51 Faulkner DJ (2001) Marine natural products: metabolites of marine invertebrates. Nat Prod Rep 17(1):7–55 Gopalakrishnan S, Meenakshi VK, Shanmugapriya D (2011) Antipyretic and analgesic activity of Phallusia nigra Savigny, 1816. Ann Biol Res 2(4):192–196 Hartmann R, Meisel H (2007) Food-derived peptides with biological activity: from research to food applications. Curr Opin Biotechnol 18:163–169 Karthik R, Manigandan V, Saravanan R, Rajesh RP, Chandrika B (2016) Structural characterization and in  vitro biomedical activities of sulfated chitosan from Sepia pharaonis. Int J  Biol Macromol 84:319–328 Karthik R, Manigandan V, Saravanan R (2017) Toxicity, teratogenicity and antibacterial activity of posterior salivary gland (PSG) toxin from the cuttlefish Sepia pharaonis, Ehrenberg (1831). J Chromatogr B 1064:28–35 Keller R (2002) Shaping the vertebrate body plan by polarized embryonic cell movements. Science 298:1950–1954 Keller R (2006) Mechanisms of elongation in embryogenesis. Development 133:2291–2302 Menna M, Aiello A, Aniello FD, Imperatore C, Luciano P, Vitalone R, Irace C, Santamaria R (2011) Conithiaquinones a and B, tetracyclic cytotoxic meroterpenes from the Mediterranean ascidian Aplidium conicum. Eur J Org Chem 15:3241–3246 Palanisamy SK, Trisciuoglio D, Zwergel C, Del Bufalo D, Mai A (2017a) Metabolite profiling of ascidian Styela plicata using LC–MS with multivariate statistical analysis and their antitumor activity. J Enzyme Inhib Med Chem 32:614–623

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Palanisamy SK, Rajendran NM, Marino A (2017b) Natural products diversity of marine ascidians (tunicates; Ascidiacea) and successful drugs in clinical development. Nat Prod Bioprospect 7:1–111 Paul VJ, Lindquist N, William F (1990) Chemical defences of the tropical ascidian Atapozoa sp. and its nudibranch predators Nembrothasp. Mar Ecol Prog Ser 59:109–118 Sahade R, Tatian M, Kowalke J, Kuhne S, Esnal GB (1998) Benthic faunal associations on soft substrates at potter cove, King George Island, Antarctica. Polar Biol 19(2):85–91 Saravanan R (2011) Isolation of low molecular weight heparin/heparan sulfate from marine sources. Food Nutr Res 72:45–60 Shenkar N, Swalla BJ (2011) Global diversity of Ascidiacea. PLoS One 6(6):1–12 Vera MD, Joullie MM (2002) Natural products as probes of cell biology: 20 years of didemnin research. Med Res Rev 22:102–145 Zubia E, Salva J (2005) Natural products chemistry in marine ascidians of the genus Aplidium. Mini Rev Org Chem 2:389–399

3

Teratogenic Activity of Toxins in Zebrafish Model

Abstract

Toxins from the posterior salivary gland (PSG) of cuttlefish are known toxins with pronounced toxicity. In this chapter, crude toxins from Sepia pharashadi are fractionated by ion-exchange chromatography and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The yield protein and carbohydrate contents of the PSG toxin are estimated to be 1.61  mg/g and 0.06 mg/g, respectively. Fourier transform infrared spectroscopy (FT-IR) of PSG toxin affirmed the incidence of CO-NH, CH and conjugated alkyl, alcoholic OH and primary NH functional groups. Circular dichroism (CD) spectroscopy and K2D analysis of the PSG toxin authenticated the attendance of secondary structure with 37% α-helix, 26% β sheet and 38% random coil. Teratogenicity of PSG toxin against Zebrafish embryo exhibited evolving malformations and premature hatching at a maximum tolerated dose of 1.0 μM. These findings strongly exhibit the toxicity of the ionic peptide-rich PSG toxin from S. pharashadi and its utilisation for its promise as a prospective cytotoxic agent of the future. Keywords

PSG toxin · S. pharashadi · RP-HPLC · FT-IR · CD · Zebrafish · Teratogenicity

3.1

Introduction

Toxins are bioactive compounds mainly comprised of biomolecules like proteins, peptides or glycopeptides which are mainly secreted by cell or gland of species for the purpose of escapism or predation from their predicators that can origin of disease or deleterious effects or can even be fatal. Toxins or venom are complex mixtures of highly active biochemical compounds accomplished of inflicting an array of biological responses. Toxins are mainly composed of proteins, peptides, enzymes, enzyme inhibitors and, in addition to this, trace amount of carbohydrates, serotonin, © Springer Nature Singapore Pte Ltd. 2019 S. Ramachandran, S. Rajagopal, Zebrafish: A Model for Marine Peptide Based Drug Screening, https://doi.org/10.1007/978-981-13-7844-7_3

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3  Teratogenic Activity of Toxins in Zebrafish Model

histamine etc. (Casewell et al. 2013). Toxins may be neurotoxin, affecting gated ion channels; haemotoxins, causing lysis of RBCs or cytotoxins, killing the cells. Myotoxins are predominantly peptide based which cause the necrosis of muscles (Nagai 2012). Phycotoxins can account in various marine species such as crabs, fish or filter-feeding bivalves, for example mussels, oysters, scallops and clams (Mackessy 2009). Marine milieu has constantly developed as an alternative suitable natural source of pharmaceutics to the existing pharmacopoeia consisting of diverse structures with unique mechanism of action. So far, 16,000 marine natural products have been reported in 6800 publications worldwide (Saravanan and Karthik 2016). Marine toxin extraction has always remained a challenging task over the years primarily due to difficulties in optimum extraction and variations in stability ever since Ghiretti, in 1951, isolated a proteinaceous toxin, cephalotoxin, from Sepia officinalis, using 0.5% cold potassium chloride solution followed by precipitation with sodium hydroxide, which is toxic to crabs and crustaceans (Ghiretti 1959). Erspamer and Anastasi in 1962 have ventured to study endecapeptide and eledoisin from the posterior salivary gland of Eledone. They employed an aqueous-based extraction followed by ammonium sulphate precipitation, dialysis and fractionation by amberlite CG-50 ion-exchange chromatography (Erspamer and Anastasi 1962). Salivary gland toxins are directly extracted from the cephalopods using the envenomation technique proposed by Ballering et al. (1972). The technique involves the use of a plastic bag which is used to collect the saliva when the mollusc is provoked to bite. The methodology, induced envenomation or milking, ensures repetitive collection of cephalotoxin, but demands excessive care and expertise (Ballering et al. 1972). A series of purification steps was carried out using cation-exchange FPLC on a mono S HR5/5 column and reversed-phase HPLC on TSKgel phenyl 5PW RP column (Shiomi et al. 2002). It has been established that when compared to ultrafiltration, ion-exchange chromatography was more suitable for concentration of the toxins. It was also known that storage of the purified samples in a high-salt concentration solution (0.8 M sodium chloride) was a better alternative to freeze-drying (Nagai 2012). Ueda et al. (2006) formalised an extraction protocol with phosphate buffer containing 0.5  M NaCl and purification by gel filtration and hydroxyapatite HPLC. Cornet et al. (2014) studied the proteome of the cephalotoxin from S. officinalis using 0.1% heptafluorobutyric acid.

3.2

Cephalopods

Cephalopods are the most advanced class of the molluscs which comprise nautilus, cuttlefish, squid and octopods. Cuttlefish are free-swimming organisms inhabiting the shallow waters of the continental shelf varying in depth from 200 to 1000 m. Their streamlined body is divided into a distinct head containing the mantle, succulent body, tapering into a tail. The mantle is characterised by the presence of 5 or

3.3  Cephalopod Toxins

29

6 suckers in transverse rows aiding in locomotion and a couple of tentacles or radula for capturing the prey. Shell, which is a characteristic feature of molluscs, is replaced with cuttlebone in cuttlefish, which is highly calcareous, primarily made up of calcium carbonate. The cuttlebones are responsible for providing neutral buoyancy by regulating the gas and water content and assist the fins for stabilisation in mid-­ waters. The cuttlefish are known to express camouflage, through pigmentation on the skin containing chromatophores (Karthik and Saravanan 2014). Sepia, of the family Sepiidae, is one of the important geniuses of cephalopods widely consumed around the world for their high nutritive content, rich in protein. Species of Sepia such as Sepia esculenta, S. officinalis, S. inermis, Sepia pharaonis and S. aculeata are exported worldwide as fresh, frozen, canned and processed foods rich in taste and nutrition. The posterior salivary glands of the cuttlefish secrete toxic polypeptides primarily to paralyse the prey and engulf them. Species of Sepia from around the world have been widely explored for their nutritive role and small molecules with potential biomedical applications.

3.3

Cephalopod Toxins

Cuttlefish, squids and octopus constitute the cephalopods, which are predatory carnivorous molluscs that feed on bivalves, crustaceans and smaller fishes. The posterior salivary glands of the cephalopods secrete toxins primarily for hunting and capturing their prey. The toxins either paralyse the prey or kill them to be engulfed and swallowed. Toxins from the blue-ringed octopus, Hepalochlaena maculosa, have been reported as the most toxic of cephalopods which has resulted in deadly envenomation in humans. Tetrodotoxin, a toxic compound present in numerous marine species, has been found to be responsible for the toxic effects caused due to envenomation by H. maculosa toxin. Proteinaceous toxins form the major toxic component of cephalopod venom. The anterior salivary glands of the molluscs are too small to collect, and hence, the previously reported salivary gland toxins are extracted from the posterior salivary glands of cephalopods (Ueda et al. 2006). Cephalotoxin is the toxic component from the posterior salivary gland of cephalopods such as S. officinalis and octopus species, Octopus macropus and Octopus vulgaris, that causes paralysis and death when injected into crabs. Cephalotoxins have also been isolated and purified from the posterior salivary gland of octopus Eledone cirrhosa and Octopus dofleini. Paralysis-inducing cephalotoxin with two subunits α and β-cephalotoxin have been isolated and purified from the salivary gland of O. vulgaris. SE-cephalotoxin, a 100 kDa monomeric glycoprotein, which is toxic to crabs, was isolated from the salivary gland of the cuttlefish S. esculenta. SO-cephalotoxin, the posterior salivary gland toxin of common cuttlefish S. officinalis has been isolated, purified and its proteome has been extensively studied. Figure 3.1. shows the global diversity and collection site of selected cephalopod for the present study.

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3  Teratogenic Activity of Toxins in Zebrafish Model

Fig. 3.1  Global distribution of the cephalopods and collection centre of S. pharaonis. (Source: Karthik 2016)

3.4

Results and Discussion

The selected cephalopod (S. pharaonis) is collected from Kasimedu landing centre, Chennai, washed with water, preserved in ice box and brought to laboratory for further study. Biochemical composition and the yield of PSG toxin is shown in Table 3.1. Gel filtration chromatography yielded a total of 32 fractions, of which 26 are found to be active (Fig. 3.2). RP-HPLC of the purified toxin yielded 13 distinct fractions, 5 of which showed considerable yield and toxicity.

3.4  Results and Discussion

31

Table 3.1  Yield and biochemical composition of PSG toxin S. no. 1. 2.

Nature of purification Crude extract Gel filtration chromatography RP-HPLC

Absorbance @ 280nm

3.

Protein (mg/g) 3.51 3.36

Carbohydrate (mg/g) 0.56 0.37

Molecular weight (kDa) 30–100 30–100

1.61

0.06