CHEMISTRY AND PHARMACOLOGY OF KINKÉLIBA (COMBRETUM MICRANTHUM), A WEST AFRICAN MEDICINAL PLANT By CARA RENAE WELCH 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 Medicinal Chemistry written under the direction of Dr. James E. Simon and approved by ______________________________ ______________________________ ______________________________ ______________________________ New Brunswick, New Jersey January, 2010 ABSTRACT OF THE DISSERTATION Chemistry and Pharmacology of Kinkéliba (Combretum micranthum), a West African Medicinal Plant by CARA RENAE WELCH Dissertation Director: James E. Simon Kinkéliba (Combretum micranthum, Fam. Combretaceae) is an undomesticated shrub species of western Africa and is one of the most popular traditional bush teas of Senegal. The herbal beverage is traditionally used for weight loss, digestion, as a diuretic and mild antibiotic, and to relieve pain. The fresh leaves are used to treat malarial fever. Leaf extracts, the most biologically active plant tissue relative to stem, bark and roots, were screened for antioxidant capacity, measuring the removal of a radical by UV/VIS spectrophotometry, anti-inflammatory activity, measuring inducible nitric oxide synthase (iNOS) in RAW 264.7 macrophage cells, and glucose-lowering activity, measuring phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in an H4IIE rat hepatoma cell line. Radical oxygen scavenging activity, or antioxidant capacity, was utilized for initially directing the fractionation; highlighted subfractions and isolated compounds were subsequently tested for anti-inflammatory and glucose-lowering activities. The ethyl acetate and n-butanol fractions of the crude leaf extract were fractionated leading to the isolation and identification of a number of polyphenolic ii compounds. Some of these compounds, the catechins and glycosylflavones, were previously reported in kinkéliba. Other compounds, the flavans and galloylated C- glycosylflavone derivatives, are being reported for the first time in this species and family. Finally, a group of major constituents in the kinkéliba leaves were discovered to be a series of compounds with a new skeleton, a flavan-piperidine alkaloid. The four kinkéloids, as they are named here first, were isolated and structurally elucidated by 1- and 2-D NMR spectroscopy and HRMS spectrometry. The catechins and flavans were the active compounds by antioxidant capacity and epicatechin was identified as a glucose-lowering compound by PEPCK inhibition. The positive glucose-lowering activities led to an animal study that tested the activity of the crude extract and ethyl acetate and n-butanol fractions in mice fed a high-fat diet, resulting in the development of a diabetic model. After six weeks of daily treatments, the treated groups showed lowered baseline blood glucose levels as well as decreased PEPCK levels in the liver, strongly suggesting that kinkéliba constituents may be beneficial in the treatment of Type 2 diabetes. iii Dedication to my husband, Joel iv Acknowledgements I would like to begin by thanking Dr. James Simon and Dr. Qing-Li Wu for their direction, assistance and encouragement throughout this research project. They had a vision 5 years ago of what this project could become and without their support, it would not have materialized. Additionally, I owe many other scientists my gratitude for helping throughout my graduate school career. Dr. Emmanuel Bassene, of the Université Cheikh Anta Diop, Dakar, Senegal, for sharing his extensive knowledge of kinkéliba throughout the project. Dr. Babou Diouf and Dr. Malainy Diatta, of ASNAPP-Senegal, for their assistance with kinkéliba collections and taxonomical authentication. Here at Rutgers, I thank all the NUANPP lab group members, specifically Drs. H. Rodolfo Juliani and Adolfina Koroch, for their support, both in and out of the lab. Dr. Diandian Shen, thank you for paving the road ahead of me and being an excellent office mate; Julia Coppin and Yanping Xu, thank you both for coming behind me to give the extra push when I was stranded and entertaining me during the long days in the lab. I owe huge thanks to Dr. Slavko Komarnytsky (and Dr. Ilya Raskin) for joining the project at the perfect time to provide the technical expertise in the application of cell culture assays and animal studies. He helped steer my project to completion, gave it a promising future, and did all with a good sense of humor. This work was conducted in part by the Partnership for Food and Industry in Natural Products (PFID/NP) project and ASNAPP with funds from the Office of Economic Growth, Agriculture and Trade of the USAID (Leader Contract Award No. AEG-A-00- 04-00012-00) and an Associate Award (Associate Cooperative Agreement No. 690-A-00- 06-00126-00) from the USAID-Regional Center for Southern Africa. I thank Jerry v Brown, Carol Brown, Larry Paulson, and Bob Hedlund, USAID Cognizant Technical Officers for their active involvement, support and encouragement to begin examining the potential development of kinkéliba as a bush tea with funding from the USAID (Contract Award No. HFM-O-00-01-00116). This research was also conducted in support of the National Botanical Center for Age-Related Diseases (NIH grant OD-00-004) in searching for alternative sources of anti-inflammatory products. Funds and support were also provided by the NUANPP, the New Jersey Agriculture Experiment Station and the School of Environmental and Biological Sciences. And, of course, my gratitude is extended to those Senegalese communities and African small farmers who were gracious to provide the kinkéliba plant materials to ASNAPP-Senegal, who have always opened their doors to us and for whom it is our hope that this research may benefit and expand their economic opportunities by increasing the market interest in this natural product. There are so many people outside of my research who encouraged me, it’s too many to mention. But I thank Dr. James Gloer for advising me at the beginning of my graduate career and helping to foster a love of natural products. The Gloer group members, specifically Dr. Stephen Deyrup and Dr. Ani Jordan, who graciously invited me to Gloer group functions long after I left Iowa, I thank you. Chad and Rachel Bareither, Dean and Kathleen Macke and Charlie and Olivia Florek, I thank you all for giving me nonstop encouragement, allowing me to escape from the lab and, most of all, making my life in New Jersey wonderful. I love you all so much. Finally and most importantly, I must thank my family. To my Welch family, you have welcomed me into your arms and I thank you for offering your endless encouragement. You’ve done this before and still, you managed to stand by another child through the trials of graduate school. I thank my vi parents, who deserve the credit for whatever I’ve accomplished. Dad, you’ve taught me a great deal inside of school and even more outside. Mom, you’re an inspiration for me to be a strong woman. Rachel, you are the most amazing (real!) doctor I know and the best sister I could have hoped for. Steve and Angie, thank you for your support and the gift of the best nephews and niece a girl could ask for; Skyler, Isaac, and Josie, I love you all so much. And finally Joel, I dedicated this work to you because I couldn’t have done it without you. You’ve supported me in too many ways to count. You’ve forged the way before me but hang back to pull me along...you complete me. I love you and I thank you! vii Table of Contents Abstract of the Dissertation ii Dedication iv Acknowledgements v Table of Contents viii List of Tables xi List of Figures xii 1. Introduction 1 1.1. Traditional Medicines of West Africa – specifically Combretaceae 3 1.1.1. Botany 3 1.1.2. Traditional Medicinal Uses and Modern Research 3 1.2. Combretum micranthum 7 1.2.1. Botany 8 1.2.2. Traditional Medicinal Uses 9 1.2.3. Modern Research on Natural Products Chemistry and Biological Activities 10 1.3. Polyphenolic Compounds and the Impact on Medicinal Chemistry 14 1.3.1. Background of Polyphenols 14 1.3.2. Antioxidant Capacity 18 1.3.3. Anti-Inflammatory Activity 19 1.3.4. Glucose-Lowering Activity 20 1.4. Dissertation Hypothesis and Specific Objectives 21 1.5. References 23 2. Identification of Known Polyphenolic Compounds 33 2.1. Methodology 35 2.1.1. General Experimental Procedures 38 2.1.2. Plant Material 39 2.1.3. Extraction and Isolation 39 2.2. Structural Elucidation 48 2.2.1. MS, NMR, Optical Rotation 48 2.3. Discussion 51 2.4. References 55 3. Identification of the New Ring Structures, Piperidine-Flavan Alkaloids 58 3.1. Methodology 60 3.1.1. General Experimental Procedures 60 3.1.2. Extraction and Isolation 62 3.2. Structural Elucidation 65 3.2.1. HRMS, 1-D and 2-D NMR 65 3.3. Discussion 74 viii 3.4. References 77 4. Bioactivity – Validation of Traditional Medicinal Uses 79 4.1. Methodology 80 4.1.1. Antioxidant Capacity 80 4.1.2. Anti-Malarial Activity 82 4.1.2.1. Parasite Culture and Assay 83 4.1.3. Anti-Inflammatory Activity 83 4.1.3.1. Cell Culture 83 4.1.3.2. LPS-induced COX-2 and iNOS Enzyme Activities 84 4.1.3.3. Nitrite Assay 84 4.1.3.4. Western Blotting 85 4.2. Results 86 4.2.1. Antioxidant Capacity 86 4.2.2. Anti-Malarial Activity 90 4.2.3. Anti-Inflammatory Activity 91 4.2.3.1. Nitrite Assay 91 4.2.3.2. Western Blotting 93 4.3. Discussion 95 4.4. References 98 5. Bioactivity – Glucose-Lowering Activity 101 5.1.