IDENTIFICATION OF MARINE ANTIOXIDANTS by BRIGITTE E. TOWNSEND (Under the Direction of Lyndon M. West) ABSTRACT Pharmaceutical science has benefited from the variety of bioactive lead compounds discovered in marine organisms over the past several decades. The remarkable structural diversity of marine secondary metabolites brings marine natural product chemistry to the forefront of drug discovery. In search of novel therapeutic interventions, marine natural products research has converged with antioxidant studies to form an exciting new research focus. Phenolic antioxidants and indole alkaloids are among the most bioactive classes of marine metabolite, but relatively little has been done to thoroughly investigate the biomedical potential of organisms containing these types of metabolites. In this study, several compounds representing both of these structural classes were identified in marine sponges collected from the Western Atlantic. Several of the compounds were found to have high antioxidant potential when screened with the ferric-reducing antioxidant power (FRAP) assay. Antioxidant activity of marine samples was detected primarily in shallow and intermediate-water sponges, but no correlation was found between activity and fraction polarity. INDEX WORDS: Marine antioxidant, FRAP assay, chromazonorol, brominated aplysinopsin, Smenospongia sp. IDENTIFICATION OF MARINE ANTIOXIDANTS by BRIGITTE E. TOWNSEND B.S., Liberty University, 2007 A thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2008 © 2008 Brigitte E. Townsend All Rights Reserved IDENTIFICATION OF MARINE ANTIOXIDANTS by BRIGITTE E. TOWNSEND Major Professor: Lyndon West Committee: Phillip Greenspan Timothy Long Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2008 ACKNOWLEDGEMENTS First of all I would like to thank my major advisor, Dr. West, for assistance throughout this project. I am grateful for the patient instruction to a student who previously knew nothing about natural products chemistry! Your ability to stay calm and to bring laughter to many conversations helped remind me that research need not be stressful. All the best as you start the new position in Florida! Thanks to Maia, for encouraging me to stay in Athens and see this degree through. I would not have stuck it out without your support and encouragement. Thank you for helping my project take shape, and for your valuable input towards experiments along the way. It was great to have someone working through calculations and new procedures along with me. Congratulations on your Ph.D! I would also like to thank my committee members Dr. Greenspan and Dr. Long for your helpful suggestions, for being flexible with early graduation deadlines, and for the interesting conversations on numerous occasions. To Prasoon, for your incredible skill in NMR analysis! Thank you for all the assistance you provided in the research. Also to Phillip—for the fun, and often off-topic study times we had last year, and for all the conversations in which we battled through obstacles and doubts as new grad students. I know you will finish strong! I am grateful to Joy Wilson for providing suggestions, encouragement, and solutions to problems since the very beginning when I was stuck in the airport trying to make it to the iv interview weekend! Stopping by your office always brightened my day. Thank you as well to Libby Rice for going out of your way in offering to help me take care of details! It is only fitting to thank my mentor and former advisor, Dr. David DeWitt, for providing the first opportunity for me to be involved in research. The lessons and principles you taught me and modeled by your own work will stay with me the rest of my life. To Dr. Dallas—your influence has been an incredible and timely blessing in my life. I am excited about the new direction my career is taking, and look forward to joining a team that is reaching out to prepare our hospitals and communities for whatever lies ahead. I admire your many achievement and abilities, but most of all I am thankful for the unwavering Christian principles that guide your life at school, at work, at home. Additionally, the love and support your family has shown me has been one of my biggest sources of encouragement. A special thanks to Lt. Daniel, for helping me keep in mind that there is more to life than sponges. Thank you for your willing service to defend our country and freedoms. To my friends: Melissa, I am truly blessed to have you for my best friend. Our conversations have been a constant source of inspiration and laughter, and I look forward to years to come of fun and meaningful memories! Celia—I wish the last half of my degree could have included as many memorable excursions for coffee with you as the beginning! I missed having you nearby. Amy—thank you so much for your patience, understanding, and helpful suggestions. Our study breaks for story-telling will be highlights of my graduate memories. Autumn—your sense of intrigue in my work was a great source of inspiration. Thank you for always being ready to listen and reminding me by your encouragement to keep a sunny disposition. v Finally, without the faithful support of my family I would not be who I am today. To my parents, brothers, and grandparents: your prayers and understanding have been a source of motivation I should never take for granted. Thank you for the reminders that work and responsibilities faced with perseverance and diligence brings a much deeper reward than may be realized on the surface. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF SEPARATION SCHEMES ............................................................................................ ix LIST OF FIGURES .........................................................................................................................x CHAPTER 1 History of Natural Products Chemistry ..........................................................................1 Marine natural products in current drug research .....................................................5 Marine natural products in the Western Atlantic .....................................................9 Sponges (Phylum Porifera) .....................................................................................10 Identification and isolation of bioactive compounds ..............................................12 Emerging methods in natural products chemistry ...................................................16 Specific aims ...........................................................................................................16 2 Antioxidant Activity of Marine Organisms from the Western Atlantic ......................18 Antioxidants in the marine environment .................................................................18 Bioassays for antioxidant activity ...........................................................................19 Antioxidant activity in sponges ...............................................................................22 Specific aims ...........................................................................................................23 Results and discussion .............................................................................................24 3 Bioactive Compounds from the Bahamian Sponge Smenospongia sp. .......................28 Isolation and identification of chromazonorol ........................................................29 vii FRAP activity of chromazonorol ............................................................................32 Isolation and identification of aplysinopsin derivatives ..........................................35 Bioactivity of brominated aplysinopsin derivatives ................................................39 Indole alkaloid antioxidants ....................................................................................40 4 Experimental Procedures .............................................................................................42 General experimental procedures ............................................................................42 Collection and preservation of marine biomaterial .................................................43 Solid-phase extraction .............................................................................................43 Extraction and isolation ...........................................................................................44 FRAP assays ............................................................................................................45 DCFH-DA assays ....................................................................................................45 REFERENCES ..............................................................................................................................47 viii LIST OF SEPARATION SCHEMES Page Scheme 1: .......................................................................................................................................30 Scheme 2: .......................................................................................................................................36 ix