©2011 John Peter Munafo, Jr ALL RIGHTS RESERVED NATURAL PRODUCTS CHEMISTRY OF LILIUM LONGIFLORUM: STRUCTURAL ELUCIDATION, QUANTIFICATION, BIOLOGICAL ACTIVITY AND FUNGAL METABOLISM OF STERODAL GLYCOSIDES by JOHN PETER MUNAFO JR A Dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Plant Biology written under the direction of Professor Thomas J. Gianfagna and approved by ________________________ ________________________ ________________________ ________________________ New Brunswick, New Jersey May, 2011 ABSTRACT OF THE DISSERTATION Natural Products Chemistry of Lilium longiflorum: Structural Elucidation, Quantification, Biological Activity and Fungal Metabolism of Steroidal Glycosides By JOHN PETER MUNAFO JR Dissertation Director: Professor Thomas J. Gianfagna The Easter lily (Lilium longiflorum Thunb., Liliaceae) has beautiful white flowers and a delicate aroma and is appreciated worldwide as an attractive ornamental plant. In addition to its economic importance and popularity in horticulture, lily bulbs are regularly consumed in Asia, as both food and medicine. The Easter lily is a rich source of steroidal glycosides, a group of compounds that may be responsible for some of the traditional medicinal uses of lilies and may play a role in the pant-pathogen interaction. This research project was designed to: 1) Isolate and characterize new steroidal glycosides from the bulbs of L. longiflorum, 2) quantify their contents in all of the organs of L. longiflorum, and 3) perform studies on the antifungal activity and fungal metabolism of the compounds. A phytochemical investigation conducted on the bulbs resulted in the discovery of several novel steroidal glycosides. A novel acetylated steroidal glycoalkaloid and two novel steroidal furostanol saponins, along with three other steroidal glycosides were isolated from the bulbs of L. longiflorum for the first time. A LC-MS/MS method performed in multiple reaction monitoring (MRM) mode was developed for the ii simultaneous quantitative analysis of the five steroidal glycosides in the different organs of L. longiflorum. The highest concentrations of total steroidal glycosides were detected in flower buds, lower stems, and leaves. The steroidal glycoalkaloids were detected in higher concentrations as compared to the furostanol saponins in all of the plant organs except for the fibrous and fleshy roots. The proportions of steroidal glycoalkaloids to furostanol saponins were higher in the plant organs exposed to light and decreased in proportion from the aboveground organs to the underground organs. The highest concentrations of the steroidal glycoalkaloids were detected in flower buds, leaves, and bulbs. Purified steroidal glycosides were evaluated for fungal growth inhibition activity against the plant pathogenic fungus, Botrytis cinerea. All of the compounds showed weak fungal growth inhibition activity; however, the natural acetylation of C-6′′′ of the terminal glucose in the acetylated steroidal glycoalkaloid, increased the antifungal activity by inhibiting the rate of metabolism of the compound by the fungus. A model system was developed to generate fungal metabolites of the steroidal glycoalkaloids and this system led to the discovery of several new fungal metabolites. The fungal metabolites characterized from the model system were subsequently identified by LC-MS and found to naturally occur in Easter lily tissues infected with the fungus. iii ACKNOWLEDGEMENTS I would acknowledge my major advisor, Professor Thomas Gianfagna, for his guidance and helpful insight throughout this research. I would also like to thank my faculty committee members, Professor Richard Merritt and Professor Chee-Kok Chin and my outside member Dr. John Didzbalis. I would like to acknowledge my collaborators, colleagues and friends; Professor Leslie Jimenez, Professor Edward Durner, Dr. Ahalya Ramanthan, Dr. Marshall Bergen, Dr. Christopher Johnson, Dr. Mark Kelm, Dr. Catherine Kwik-Uribe, Thomas Collins, Jeanne Peters, Nimmi Rajmohan, Dr. Mahdu Aneja, Bob Carhart, Jadwiga Leonczak, Professor Ilya Raskin, Dr. Slavko Komarnytsky, and Debroa Esposito. I would like to give a special thanks to my family and especially my wife, Kristin, for their constant encouragement and support. Most of all, I would like to thank God, for creating such a wonderful Universe for us to explore and ponder. iv TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION ...................................................................... II ACKNOWLEDGEMENTS ........................................................................................... IV TABLE OF CONTENTS ................................................................................................ V LIST OF TABLES ........................................................................................................... X LIST OF FIGURES ........................................................................................................ XI CHAPTER 1: GENERAL INTRODUCTION ............................................................... 1 1.1. INTRODUCTION .......................................................................................................... 1 1.2. BOTANICAL CLASSIFICATION .................................................................................... 2 1.3. BOTANICAL DESCRIPTION ......................................................................................... 2 1.4. NATURAL PRODUCTS FROM LILIACEAE ..................................................................... 5 1.5. SAPONINS IN GENERAL .............................................................................................. 6 1.5.1. Steroidal Saponins ............................................................................................. 9 1.5.1.1. Commercially Important Steroidal Saponins ............................................ 12 1.5.1.2. Dietary Sources of Steroidal Saponins ..................................................... 16 1.5.1.3. Steroidal saponins isolated from Lilium ................................................... 18 1.6 STEROIDAL ALKALOIDS ........................................................................................... 27 1.6.1 Steroidal Alkaloids in Liliaceae ....................................................................... 27 1.6.1.1. Classification of Isosteroidal Alkaloids of Liliaceae ................................ 28 1.6.1.2. Classification of Steroidal Alkaloids of Liliaceae .................................... 30 1.6.1.3. Steroidal Alkaloids in Lilium .................................................................... 31 1.6.2. Steroidal Glycoalkaloids ................................................................................. 32 1.6.2.1. Dietary Sources of Steroidal Glycoalkaloids ............................................ 35 v 1.6.2.2. Steroidal Glycoalkaloids in Lilium ........................................................... 37 1.7. PLANT ORGAN DISTRIBUTION OF STEROIDAL GLYCOSIDES ....................................... 40 1.8. STEROIDAL GLYCOSIDES IN PLANT DEFENSE ............................................................ 42 1.9 DETOXIFICATION OF STEROIDAL GLYCOSIDES .......................................................... 45 CHAPTER 2: ISOLATION AND STRUCTURAL DETERMINATION OF STEROIDAL GLYCOSIDES FROM THE BULBS OF EASTER LILY (LILIUM LONGIFLORUM THUNB.) ........................................................................................... 48 2.1. ABSTRACT ............................................................................................................... 48 2.2. INTRODUCTION ........................................................................................................ 49 2.3. MATERIALS AND METHODS ..................................................................................... 51 2.3.1. Plant Material.................................................................................................. 51 2.3.2. Chemicals. ....................................................................................................... 52 2.3.3. Isolation and Purification of Steroidal Glycosides 1 – 5 from L. longiflorum.53 2.3.3.1. Sequential Solvent Extraction of Lyophilized L. longiflorum Bulbs ........ 53 2.3.3.2. Gel Permeation Chromatography (GPC) .................................................. 54 2.3.3.3. Semipreparative Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) ................................................................................ 56 2.3.4. Structural Elucidation ..................................................................................... 59 2.3.4.1. Acid Hydrolysis of Compounds 1 – 5. ..................................................... 61 2.3.4.2. Aglycone Analysis .................................................................................... 61 2.3.4.3. Sugar Composition Analysis .................................................................... 62 2.3.4.4. Determination of Sugar Absolute Configurations .................................... 62 2.3.4.5. Thin Layer Chromatography (TLC) ......................................................... 63 vi 2.4. RESULTS AND DISCUSSION ...................................................................................... 64 2.4.1 Structure Elucidation of Compounds 1 – 5. ..................................................... 64 2.3.4.1.