Distribution Agreement In presenting this thesis as a partial fulfillment of the requirements for a degree from Emory University, I hereby grant to Emory University and its agents the non-exclusive license to archive, make accessible, and display my thesis in whole or in part in all forms of media, now or hereafter now, including display on the World Wide Web. I understand that I may select some access restrictions as part of the online submission of this thesis. I retain all ownership rights to the copyright of the thesis. I also retain the right to use in future works (such as articles or books) all or part of this thesis. Stephanie Pintas April 1, 2018 Antifungal Activity of Botanical Extracts against Malassezia furfur Implicated in Common Skin Conditions By Stephanie Pintas Dr. Cassandra Quave Adviser Department of Dermatology Dr. Cassandra Quave Adviser Dr. Meleah Hickman Committee Member Dr. Sarah Chisolm Committee Member 2018 Antifungal Activity of Botanical Extracts against Malassezia furfur Implicated in Common Skin Conditions by Stephanie Pintas Dr. Cassandra Quave Adviser An abstract of a thesis submitted to the Faculty of Emory College of Arts and Sciences of Emory University in partial fulfillment of the requirements of the degree of Bachelor of Arts with Honors Human Health Department 2018 Abstract Antifungal Activity of Botanical Extracts against Malassezia furfur Implicated in Common Skin Conditions By Stephanie Pintas Malassezia furfur is a commensal lipophilic fungus found on the surface of the skin that is implicated in the pathogenesis of several prevalent skin conditions, including Atopic Dermatitis (AD), Seborrheic Dermatitis (SD), and Tinea Versicolor (TV). With antifungal resistance becoming an increasing concern, it is important to identify new therapeutic options. While several studies have demonstrated anti-Malassezia activity with botanical extracts, the concentration tested is often high (>500 µg/mL) and not evaluated for mammalian cytotoxicity. The purpose of this study was to screen for potential anti-Malassezia activity of botanical extracts using a unique chemical library, the Quave Natural Products Library (QNPL), based on plants used in the traditional treatment of infectious and inflammatory skin disease. Initial screen of the QNPL (~1000 extracts) was conducted at 16 µg/mL, but failed to demonstrate anti- Malassezia activity. A second screen of the library was conducted at 128 µg/mL by broth microtiter plate dilution method in triplicate. The screen revealed potential activity for crude extracts 31, 55, 66, 637, and 1251 and pure compounds 448, 449, and 451. Further analysis via two-fold serial dilution in quadruplicate demonstrated 48-hour MIC90 at 128 µg/mL for extract 1251, Allium amethystinum, and 16 µg/mL for pure compound 451 as compared to 2 µg/mL for Ketoconazole control. Minimum fungicidal concentration (MFC) was determined by agar plate test. MFC was 256 µg/mL for both pure compound 451 and extract 1251. We also conducted two-fold serial dilution on extracts 158 and 237, ethanolic and methanolic crude extracts of Allium cepa (onion) in the same genus as A. amethystinum, but failed to observe any growth inhibition at either 24 or 48-hour optical density reads. To our knowledge, this is the first report of antifungal activity of Allium amethystinum. Mammalian cytotoxicity as determined by testing of human keratinocytes (HaCaTs) via LDH assay revealed that extract 1251 did not achieve an IC50 in HaCaTs at 512 µg/mL, twice the concentration that exhibited fungicidal activity. This provides promising evidence for potential topical treatments that include Allium amethystinum. Chemical inhibition of Malassezia furfur at lesional skin sites could support reduced allergenic responses and inflammation, improving symptoms of disease and decreasing risk of fungal resistance from current topical treatments. Future efforts to isolate the active compound(s) of extract 1251 via iterative bioassay guided fractionation are warranted. Antifungal Activity of Botanical Extracts against Malassezia furfur Implicated in Common Skin Conditions by Stephanie Pintas Dr. Cassandra Quave Adviser A thesis submitted to the Faculty of Emory College of Arts and Sciences of Emory University in partial fulfillment of the requirements of the degree of Bachelor of Arts with Honors Human Health Department 2018 Acknowledgements The idea for my thesis all began because of my own struggles with atopic dermatitis. I grew increasingly frustrated with the inefficacy of topical steroids at relieving my symptoms long-term. I started doing personal research online and found several “natural” remedies such as tea tree oil and apple cider vinegar. However, I wanted to put these ideas to the test and see if I could discover botanical extracts that truly exhibited antimicrobial effects in the lab. My hope is that this research will provide insights and information for future clinical testing and topical natural products that may help individuals who also suffer from chronic skin conditions. I would like to express my sincere appreciation and gratitude to Dr. Quave, for supporting my research, guiding me through this immense process, and pushing me to learn more about botanical medicine than I could ever imagine. Without her guidance and expertise, this project would not have been possible, and I am so appreciative that she was willing to accept me into her lab to pursue this project. I would also like to thank my thesis committee members, Dr. Chisolm, for providing knowledge on the skin conditions I researched and Dr. Hickman for her interest and expertise in fungal microbes. I would like to especially thank Kate Nelson for teaching me the microbiology techniques necessary for my experiments and Dr. Lyles for his expertise in phytochemistry. A huge thank you to Dr. Freeman for providing her support and endless tips for creating a thorough thesis and presentation. I am thankful to my parents, especially my mother who sparked my interest in this topic because of its clinical relevance in dermatology. I would also like to thank my father, grandparents, and brothers for supporting and encouraging me throughout the process. TABLE OF CONTENTS Chapter 1 Introduction........................................................................................................... 1 1.1 Skin Microbiome .................................................................................................................. 1 1.2 Characteristics of Malassezia spp. ........................................................................................ 7 1.3 Atopic Dermatitis .................................................................................................................. 9 1.4 The Role of Malassezia species in Atopic Dermatitis ........................................................ 11 1.5 Seborrheic Dermatitis ......................................................................................................... 16 1.6 Tinea Versicolor.................................................................................................................. 20 Chapter 2 Background ......................................................................................................... 22 2.1 Antifungal Resistance and Toxicity: The Need for New Solutions .................................... 22 2.2 Implications of Other Treatments ....................................................................................... 28 2.3 Current Research on Botanical Extracts ............................................................................. 31 2.4 In Vitro Research against Malassezia species .................................................................... 32 2.5 Clinical Research Utilizing Botanical Extracts................................................................... 42 2.6 Relevance of Studying Botanicals ...................................................................................... 52 Chapter 3 Materials and Methods....................................................................................... 53 3.1 Collection and Extraction ................................................................................................... 53 3.2 Growing Malassezia species: Agar and Broth Microdilution Method ............................... 56 3.3 Microbiological Assay: Initial Screen ................................................................................ 57 3.4 Serial Dilution and Fungicidal Plate Test ........................................................................... 59 3.5 XTT Assay .......................................................................................................................... 62 3.6 Cytotoxicity Assay .............................................................................................................. 66 Chapter 4 Results .................................................................................................................. 67 4.1 Screening Outcomes ........................................................................................................... 67 4.2 Antifungal Activities—MIC Results .................................................................................. 92 4.3 Fungicidal Plate Results ...................................................................................................... 98 4.4 LDH Cytotoxicity Results................................................................................................. 102 Chapter 5 Discussion