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Exploring the Antibacterial, Antioxidant, and Anticancerproperties of Lichen Metabolites Brigham Young University BYU ScholarsArchive Theses and Dissertations 2015-03-01 Exploring the Antibacterial, Antioxidant, and AnticancerProperties of Lichen Metabolites Gajendra Shrestha Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Biology Commons BYU ScholarsArchive Citation Shrestha, Gajendra, "Exploring the Antibacterial, Antioxidant, and AnticancerProperties of Lichen Metabolites" (2015). Theses and Dissertations. 4393. https://scholarsarchive.byu.edu/etd/4393 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Exploring the Antibacterial, Antioxidant, and Anticancer Properties of Lichen Metabolites Gajendra Shrestha A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Larry L. St. Clair, Chair Kim L. O’Neill Richard A. Robison Steven G. Wood Clinton J. Whipple Department of Biology Brigham Young University February 2015 Copyright © 2015 Gajendra Shrestha All Rights Reserved ABSTRACT Exploring the Antibacterial, Antioxidant, and Anticancer Properties of Lichen Metabolites Gajendra Shrestha Department of Biology, BYU Doctor of Philosophy Natural products have been a significant source of new drugs, especially in treating cancer, infectious diseases, hypertension, and neurological disorders. Although many natural metabolites have been screened and yielded pharmaceutically important drugs, many potential sources of natural product drug therapies still need to be investigated, including lichens. Lichens are symbiotic systems consisting of a filamentous fungus and a photosynthetic partner (an eukaryotic alga and/or cyanobacterium). Lichens produce an impressive variety of unique secondary compounds and have been used as ingredients in folk medicines for centuries. Demonstrated biological roles based on lichen chemistry include: antibiotics, anti-proliferative, antioxidants, anti-HIV, anti-cancer, immunomodulation, and anti-protozoans. Although North America is home to an impressive variety of lichen species, there is limited research to examine the biological potentials of these lichens. The core goal of this dissertation research has been to investigate some of the biological roles including, antibiotic, antioxidant, and anticancer potentials using lichen crude extracts and their metabolites collected from various locations in the United States. Antibiotic screening of crude extracts of 36 lichen species demonstrated inhibitory effects against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Methicillin- resistant S. aureus (MRSA). Generally, acetone extractions were found to be more effective than methanol extractions. It has also been shown that L. vulpina extract was bacteriocidal against MRSA with a relatively slow kill rate that disrupts cell membrane integrity and cell division as possible modes of action. Antioxidant screening of extracts from 11 lichen species, using the Oxygen Radical Absorbance Capacity (ORAC) assay, showed that lichen extracts inhibited the oxidative degradation of the fluorescent molecule (fluorescein-sodium salt) by the oxygen free radical initiator AAPH (2,2'-azobis(2-aminidopropane) dihydrochloride Acetone extracts as well as pure compounds from lichen species showed cytotoxic effects against Burkitt's lymphoma (Raji) cells and a colon cancer cell line (HT29 and SW620). They decreased proliferation, arrested cell cycle at various stages and force the cell to undergo apoptosis. The tested extracts or pure compounds were not toxic to normal cells. In colon cancer apoptosis took place independent of casapase-3. The results of this dissertation showed that lichen compounds merits for further investigation. Keywords: lichen, antibacterial, anticancer, antioxidant, immunomodulation, secondary metabolites, mode of action, cell cycle, apoptosis, MTT, bacteriolysis, transmission electron microscopy ACKNOWLEDGMENTS I owe many people my sincere thanks for their support, encouragement, and help over the last five years. I am indebted to my research advisor Dr. Larry L. St. Clair for providing me with the opportunity to pursue a research project of my own design. His constant guidance and support as my mentor have helped me to successfully complete my graduate research. I am also very thankful to Dr. Kim L. O’Neill for his guidance and allowing me to use his lab space to conduct my research. I also appreciate the time, assistance and guidance provided by the members of my graduate committee – Dr. Richard Robison, Dr. Clint Whipple, and Dr. Steven Wood. I am also thankful to Dr. Juan Arroyo for his invaluable support with one of my research projects. I would also like to thank those organizations that have supported parts of my research, including Brigham Young University Graduate Studies, the California Lichen Society, the Bean Life Science Museum, the Department of Biology, and the BYU Cancer Research Center. Last but certainly not least, I thank my wife, Saraswati Manandhar and our sons, Shreyash and Snehal Shrestha for their love, constant support, patience, and encouragement, without which none of this would have been possible. TABLE OF CONTENTS TITLE PAGE…………………………………………………………………………………..i ABSTRACT ............................................................................................................................... ii ACKNOWLEDGMENTS ......................................................................................................... iii LIST OF TABLES ................................................................................................................... vii LIST OF FIGURES ................................................................................................................. viii Chapter 1: Background ............................................................................................................... 1 Chapter 2: Biological roles of lichen-derived compounds: A review ........................................ 5 Introduction ......................................................................................................................................... 5 Conclusion ......................................................................................................................................... 29 Chapter 3: Screening antibiotic properties of acetone and methanol extracts of thirty-six lichen species ............................................................................................................................ 31 Introduction ....................................................................................................................................... 31 Materials and methods ....................................................................................................................... 32 Collection of Lichen species ......................................................................................................... 32 Extraction of lichen metabolites .................................................................................................... 32 Identifying lichen metabolites ....................................................................................................... 32 Microbial Cultures ......................................................................................................................... 33 Micro-well Dilution assay ............................................................................................................. 33 Results ............................................................................................................................................... 36 Identification of lichen metabolites ............................................................................................... 36 Antimicrobial activities ................................................................................................................. 37 Discussion ......................................................................................................................................... 39 Conclusions ....................................................................................................................................... 41 Chapter 4: Lichen compound targets cell membrane and cell division processes in methicillin- resistant Staphylococcus aureus ............................................................................................... 42 Introduction ....................................................................................................................................... 42 Materials and methods ....................................................................................................................... 43 Bacterial strain. .............................................................................................................................. 43 Lichen materials. ........................................................................................................................... 43 Minimum inhibitory concentration (MIC) assays. ........................................................................ 43 Bacteriolysis. ................................................................................................................................
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