Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2008 Understanding the biological activity of Hypericum species through metabolomics studies Matthew Lee iH llwig Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, and the Plant Biology Commons Recommended Citation Hillwig, Matthew Lee, "Understanding the biological activity of Hypericum species through metabolomics studies" (2008). Retrospective Theses and Dissertations. 15775. https://lib.dr.iastate.edu/rtd/15775 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Understanding the biological activity of Hypericum species through metabolomics studies by Matthew Lee Hillwig A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Biology Program of Study Committee: Eve Syrkin Wurtele, Major Professor George A. Kraus Basil Nikolau Reuben J. Peters Mark Widrlechner Iowa State University Ames, Iowa 2008 Copyright © Matthew Lee Hillwig, 2008. All rights reserved. 3337386 3337386 2009 ii TABLE OF CONTENTS CHAPTER 1: Introduction 1 Dissertation Organization 1 General Introduction 2 Literature Cited 8 CHAPTER 2: Characterizing the Metabolic Fingerprint and Anti-inflammatory Activity of Hypericum gentianoides 10 Abstract 10 Introduction 10 Materials and Methods 13 Results 17 Discussion 21 Literature Cited 24 Figures 27 CHAPTER 3: Identification of Compounds from Hypericum gentianoides that Reduce Prostaglandin E2 Levels in cellulo 35 Abstract 35 Introduction 35 Materials and Methods 36 Results 38 Discussion 42 Literature Cited 45 Tables and Figures 47 CHAPTER 4: Hypericum gentianoides Metabolite Extract Reduces Human Immunodeficiency Virus Infectivity 62 Abstract 62 Introduction 62 Materials and Methods 64 Results 67 Discussion 69 Literature Cited 71 Figures 73 CHAPTER 5: A brief Communication about the Analgesic Properties of Hypericum Extracts via the TRPV1 Receptor 79 Summary 79 Literature Cited 80 Figures 82 iii CHAPTER 6: Distribution of Bioactive Compounds in Hypericum gentianoides 85 Introduction 85 Materials and Methods 85 Results and Discussion 86 Literature Cited 88 Figures 89 CHAPTER 7: General Conclusions 93 APPENDIX A: Pseudohypericin is Necessary for the Light- Activated Inhibition of Prostaglandin E2 Pathways by a 4 Component System Mimicking an Hypericum perforatum Fraction 100 APPENDIX B: Inhibition of Prostaglandin E2 Production by Anti- inflammatory Hypericum perforatum Extracts and Constituents in RAW 264.7 Mouse Macrophage Cells 132 ACKNOWLEDGEMENTS 167 1 CHAPTER 1. INTRODUCTION Dissertation Organization This dissertation includes a general introduction to plant specialized metabolites found in the Hypericum plant genus and their medicinal properties. The specific focus of this dissertation is on acyl-phloroglucinols and drug discovery with studies related to biologically active compounds from the medicinal plant Hypericum gentianoides (H. gentianoides). Included are chapters adapted from published works, manuscripts in review, and unpublished findings. Chapter 2, modified from a publication in the Journal of Agricultural and Food Chemistry, focuses on introducing the chemical diversity found in Hypericum species and describes several bioactivities that may be associated with one class of polyketides found in several Hypericum species, the acyl-phloroglucinols. Chapter 3, a manuscript in preparation for publication, focuses on the bioactivity guided fractionation of a H. gentianoides extract, the subsequent identification of anti-inflammatory compounds from the active fractions using various analytical methods, the discussion of their potential as a drug discovery lead, and the hypothesis that these compounds may behave synergistically to enhance biological activity. Chapter 4, a manuscript in preparation for publication, focuses on the investigation of the effect of treating human immunodeficiency virus (HIV) infected HeLa37 cells with H. gentianoides extracts and fractions containing acyl-phloroglucinols, and the unusual cytotoxicity of these fractions on HeLa cells. Furthermore, it discusses the affects of these acyl-phloroglucinols in another viral infectivity model, equine infectious anemia virus (EIAV) in equine dermis cells. Chapter 5 briefly discusses preliminary investigations of Hypericum extracts effects on the physiology of transient receptor vanilloid potential 1 2 (TRPV1) human pain receptors expressed in frog oocytes and human embryonic kidney cells. Chapter 6 briefly discusses the distribution of bioactive acyl-phloroglucinols in flowering H. gentianoides plants. Chapter 7, General Conclusions, ties together the hypotheses from these different studies and draws conclusions based on the findings and other data from primary literature. The Appendices include two publications related to this dissertation research. A unified hypothesis for the small-molecule signaling ecological and mechanistic role of acyl-phloroglucinols is provided. General Introduction Natural Product Diversity in Hypericum The genus Hypericum L. contains approximately 450 species (Robson, 1977) and is currently classified in the family Clusiaceae, also known as Guttiferae Juss. 1789, nom. et cons. However, this genus was previously classified taxonomically under its own family named Hypericaceae Juss., nom. et cons. Hypericum is well known for containing several species with pharmacological activity and typically have yellow flowers of varying size and petal number; some examples are shown in Figure 1. The most heavily used and researched species is Hypericum perforatum, commonly referred to as St. John’s Wort (SJW). Used since the time of the ancient Greeks, this popular herbal supplement has been studied extensively over the past few decades with much progress on understanding the effects of its large host of bioactive compounds has on humans and mammalian cell model systems (APPENDIX A: Hammer, 2008; APPENDIX B: Hammer, 2007.) 3 H. perforatum H. crux-andreae H. androsaemum H. gentianoides H. hypericoides H. denticulatum Figure 1. Comparison of flower size in six different species of Hypericum. Many classes of plant metabolites accumulate in Hypericum species including monoterpenes, diterpenes, triterpene sterols, phenylpropanoids, aglycone flavonoids, glycosylated flavonoids, biflavones, benzophenones, xanthones, acyl-phloroglucinols, anthrones, dianthrones, and porphorins (Ernst, 2003). In particular, two compounds from H. perforatum that have been extensively studied. The first is the dianthrone, hypericin (see Figure 1, p. 25), which was identified as the compound responsible for the light-induced photosensitivity caused by the ingestion of SJW (Knox, 1985). Also, hypericin has been linked with the inhibition of human- immunodeficiency virus by extracts from SJW (Meruelo, 1988). The second heavily studied compound from H. perforatum is hyperforin. This acyl-phloroglucinol is the suspect compound responsible for the anti-depressive activity of SJW (Mennini, 2004). With that said, there is still much that is unknown about how other metabolites in plant extracts are modulating or acting synergistically along side hypericin and hyperforin, altering their function in the human body. When the context is expanded from just SJW to the entire 4 genus, it becomes clear how little we know about what small-molecule signaling and other chemical ecological interactions specialized metabolites from Hypericum may have. The number of specialized secondary small molecules/compounds identified from Hypericum species grows each year. This list is further complicated by the fact that these compounds are often enzymatically modified to form a large variety of “hyphenated” class compounds. One example is the addition of terpenoid biosynthesis using polyketides as substrates, creating a large class of polyketide-terpenoid compounds. This is especially prevalent in the modification of acyl-phloroglucinols, causing phloroglucinol-terpenoid natural product discovery to continue to grow every year. In Hypericum species alone, there are now approximately 60 identified compounds (Inder Pal Singh, 2003). In all plant species surveyed, over 700 acyl-phloroglucinols have been identified (Inder Pal Singh, 2003). Although this is only a small subset of terpene class compounds, considering how little biochemical and molecular biology research has been completed on the 450 known species of Hypericum and related taxa, this number will most likely exponentially increase over the next several years. The Known Biological Activities of Specialized Compounds from Hypericum The Hypericum species is known to contain numerous flavonoids. Flavonoids are present throughout the plant kingdom. Due to their commonality, we focus on classes of compounds more specific to Hypericum and its parent family, Clusiaceae. Xanthones Xanthones are derived from benzophenones via a cyclization reaction forming a third heteronuclear six-member ring. The Clusiaceae family, including Hypericum contains 5 numerous xanthones. These compounds are believed to be defense compounds against fungi, which is now