Pharmacognostic Investigation of Black Cohosh (Cimicifuga racemosa (L.) Nutt.) BY DANIEL S. FABRICANT B.S., University of North Carolina-Chapel Hill, 1997 DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacognosy in the Graduate College of the University of Illinois at Chicago, 2005 Chicago, Illinois DEDICATION This dissertation is dedicated to my family, Wendy, Michael, Rachel, and Loretta. Thank you for your support, without which this work would not have been possible. ii ACKNOWLEDGEMENTS Much gratitude is due to Dr. Guido Pauli who shared lab space, equipment, philosophy and his enthusiasm. Special thanks to all of those brave souls who volunteered to serve on my committee: Drs. Norman R. Farnsworth, major and dissertation advisor, Guido F. Pauli, Judy L. Bolton, Richard B. van Breemen, Jim Wang and Dean Rosalie Sagraves. I am indebted to a number of people who have helped make this work possible. Thanks to Joanna Burdette for her skilled and exhaustive bioassay work, Cassia Overk, Drs. Vivian Zheng, Jianghua Liu, Rachel Ruhlen and the project leader, Dr. J.L. Bolton for their help during the bioassay phases of this research. To Dr. van Breemen and the Botanical Center’s Core C, especially Dr. Dejan Nikolic, thank you for the wonderful mass spectral work provided herein. I am very grateful to Drs. Shao-Nong Chen and Linda Lu for all their exceptional assistance and expansive knowledge with many phytochemical aspects of this research. Much gratitude is due to Dr. Guido Pauli, the Project Leader, who shared lab space, equipment, philosophy and his enthusiasm. I am much obliged to Drs. Dejan Nikolic, David Lankin, Steve Totura, James Graham, Colleen Piersen, Aleksej Krunic, Eduardo Callegari, Paul W. Buehler, Chad Haney, Hongjie Zhang, Jose Fausto-Rivero, Amanda Koch and Wenkui Li for cheerful collaboration along the way. Thanks to the Department of Medicinal Chemistry and Pharmacognosy, the Program for Collaborative Research in the Pharmaceutical Sciences, the ODS/NCCAM/NIH center for Botanical Dietary Supplement Research in iii Womens’ Health (grant # P50 AT00155) and the office of the Dean, College of Pharmacy, UIC, for teaching and research assistantships during the course of my studies. Thanks to Dr. Qun Yi Zheng of Pure World Botanicals and to our collaborators at Pharmavite. Much gratitude is due to NAPRALERT for funding and data that made this work possible, especially Mary Lou Quinn, and Norman R. Farnsworth. Thanks to Aubrey Neas, Dr. Gwynn Ramsey, Bambi Teague and Keith Langdon at the National Park Service with their assistance in acquiring plant material. Thanks to Drs. Andreas Constantinou, Andrew Mesecar, Steven Swanson and Bernard Santarsiero for kindly sharing lab space and equipment during the PCR-phase of this research. Thanks to Donna Webster for her hard work with the RAPD-PCR work. I am truly grateful to my mother, Loretta Mae Fabricant, for her support. I would like to individually thank my brother and sister, Michael and Rachel Fabricant, for their continuous support, friendship and humor. To Wendy, thanks for keeping me on task, you make me want to be a better person. Special thanks to the Boss, Professor Norman R. Farnsworth and his better half Priscilla, for their input, patience, kindness and guidance. iv PREFACE This dissertation is concerned with the standardization of a botanical dietary supplement for use in a clinical trial. It is the intent of the author, for this document to be used as a resource for future work with Cimicifuga racemosa extracts and its constituents. As well as to provide a sound review of the chemical, biological and botanical aspects of the literature of C. racemosa. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ii PREFACE iv LIST OF SYMBOLS AND ABBREVIATIONS x LIST OF TABLES xiv LIST OF FIGURES xv SUMMARY xviii 1.0 INTRODUCTION AND SCOPE OF STUDY 2 2.0 STATEMENT OF PROBLEM 6 3.0 LITERATURE REVIEW 7 3.1 TAXONOMY OF BLACK COHOSH 7 3.1.1 Family Ranunculaceae 7 3.1.2 Genus Cimicifuga L. 8 3.1.3 Species Cimicifuga (L.) Nutt. 9 3.2 BLACK COHOSH IN TRADITIONAL MEDICINE 10 3.3 EFFECTS ON CLIMACTERIC SYMPTOMS 11 RELATED TO MENOPAUSE 3.3.1 Conclusion of Clinical Study of Black Cohosh 18 on Climacteric Symptoms Related to Menopause 3.4 PHARMACOLOGY AND BIOCHEMISTRY OF 20 CIMICIFUGA RACEMOSA EXTRACTS 3.4.1 Competitive Estrogen Receptor Binding 21 3.4.2 Receptor Expression 22 3.4.3 Plasma Hormone Levels 23 3.4.4 Hormonal Secretion 24 3.4.5 Osteopenia Inhibition 24 3.4.6 Uterine Weight/Estrous Induction 25 3.4.7 MCF-7 Cell Proliferation Inhibition 25 vi 3.4.8 CNS Effects and Neurotransmitter Binding 26 3.4.9 Miscellaneous 27 3.4.10 Conclusion on Extract Activity 28 3.5 PHYTOCHEMISTRY OF BLACK COHOSH 35 3.5.1 Triterpene Glycosides 36 3.5.2 Phenolic Acid Derivatives 36 3.5.3 Flavonoids 37 3.5.4 Miscellaneous 37 4.0 EXPERIMENTAL 39 4.1 PLANT MATERIAL 39 4.1.1 Procurement of Plant Material 39 4.1.2 RAPD-PCR Plant Identification 39 4.1.3 Microscopic Analysis 41 4.1.3.1 Light Microscopy 41 4.1.3.2 Scanning Electron 41 Microscopic (SEM) Analysis 4.2 CHROMATOGRAPHY 42 4.2.1 Solvent Partition and Fractionation of Black Cohosh 42 4.2.2 Column Chromatography 42 4.2.3 Thin-Layer Chromatography (TLC) 43 4.2.4 High-Performance Liquid Chromatography (HPLC) 44 4.2.4.1 Analytical HPLC 46 4.2.4.2 Semi-preparative HPLC 50 vii 4.3 SPECTROSCOPIC METHODS 52 4.3.1 Nuclear Magnetic Resonance (NMR) Spectroscopy 52 4.3.2 Mass Spectrometry (MS) 52 4.3.3 Other Techniques 53 4.4 BIOLOGICAL ACTIVITY 53 4.4.1 Estrogenic Activity 53 4.4.2 Serotonin (5-HT) Binding Activity 54 4.4.3 Other Biological Activities 54 5.0 RESULTS 55 5.1 PLANT PROCUREMENT 55 5.2 RAPD-PCR ANALYSIS 58 5.3 MICROSCOPY 63 5.3.1 Light Microscopy 63 5.3.2 Scanning Electron Microscopy (SEM) 64 5.4 CHROMATOGRAPHY 66 5.4.1 Solvent Extraction and Partitioning 66 of non-Polar Fractions 5.4.2 Solvent Extraction and Partitioning 67 of Polar Fractions 5.4.3 Amberlite XAD-2 69 5.4.4 MCI gel CHP20P 72 5.4.5 Sephadex LH-20 81 5.4.6 Silica Gel Separation 83 5.5 ISOLATION OF CONSTITUENTS 85 viii 5.5.1 Isolation of 1 (cimipronidine) 85 5.5.2 Isolation of 2 (fukinolic acid) 88 5.5.3 Isolation of 3 (actein(R/S)) 88 5.5.4 Isolation of 4 (isoferulic acid) 90 5.6 CHARACTERIZATION OF CONSTITUENTS 91 5.6.1 Characterization of 1 91 5.6.2 Characterization of 2 104 5.6.3 Characterization of 3 108 5.6.4 Characterization of 4 113 5.6.5 Additional Characterization Studies 117 5.7 Preparation of the Clinical Extracts 120 5.7.1 Collaborator Formulation of Phase I 130 Clinical Extract 5.7.2 Collaborator Formulation of Phase II 130 Clinical Extract 5.7.3 Phase I clinical capsule analysis 130 5.7.4 Phase II clinical capsule analysis 132 5.7.5 Overview of Clinical Material 132 6.0 DISCUSSION 6.1 Clinical Extract Formulation and Biological Activity 136 6.2 Biological Activity of Black Cohosh 139 6.3 Chemical and Botanical Nomenclature 140 6.4 Preparative and Analytical Techniques 142 6.5 NMR spectroscopy 149 ix 7.0 CONCLUSIONS 152 7.0.1 Review the Pharmacognosy of Black Cohosh 152 7.0.2 Preparation of a biologically, chemically and 152 botanically standardized extract for clinical trial. 7.0.3 Chemical characterization of the active butanolic 152 Black Cohosh fraction 7.0.4 Development of preparative and analytical methods 153 7.0.5 Dereplication of Botanical Center isolates 154 7.1 Summary of Conclusions 154 8.0 FUTURE DIRECTIONS 155 9.0 REFERENCES 158 10.0 APPENDICES 174 10.1 Additional Structural Data for Novel Center Isolates 174 10.2 Mass spectral data supporting the presence 184 of additional nitrogenous constituents in Black Cohosh 10.3 Additional Black Cohosh documentation 188 11.0 VITA 205 x LIST OF ABBREVIATIONS ACN acetonitrile (HPLC grade) Ac acetyl AP alkaline phosphatase [α]D specific optical rotation APT attached proton test C5D5N deuterated pyridine CGI Clinician’s Global Impression Scale CHCl3 chloroform CHP20P mci-gel CHP20P resin 13C NMR carbon-13 nuclear magnetic resonance COSY COrrelation SpectroscopY cm-1 wave number δ chemical shift DEPT distortionless enhanced proton transfer DSHEA Dietary Supplement Health and Education Act d.d.i. distilled, deionized (d) doublet eV electron volt ε molar absorptivity EIMS electron impact mass spectrometry ELSD evaporative light scattering detector equiv. equivalent xi LIST OF ABBREVIATIONS(continued) ER estrogen receptor EtOH ethanol EtOAc ethyl acetate GPS global positioning system HAM-A Hamilton Anxiety Scale HMBC heteronuclear multiple-bond connectivity spectroscopy HMQC heteronuclear multiple-bond quantum coherence spectroscopy 1H NMR proton nuclear magnetic resonance HPLC high-performance liquid chromatography 5-HT serotonin (5-hydroxytryptamine) Hz hertz IPR isopropanol (2-propanol) IR infrared absorption IC50 inhibitory concentration (50%) J coupling constant KMI Kupperman Menopausal Index λmax maximum wavelength M molar concentration MHz megahertz MeOH methanol min minutes xii LIST OF ABBREVIATIONS (continued) mp melting point m/z mass-to-charge ratio MSS unspecified menopausal index using the Likert scale νmax maximum frequency n-BuOH 1-butanol NMR nuclear magnetic resonance Open Open-Labeled study ovx Ovariectomized ppm parts per million PDA photodiode array pet. ether petroleum ether POMS Profile of Mood States Scale (q) quartet SDS Self-Assessment Depression scale Si silica (s) singlet (t) triplet TLC thin-layer chromatography tR retention time UV ultraviolet absorption VMI Vaginal Maturity Index xiii VAS Visual Analog Scale v volume w weight XAD-2 amberlite XAD-2 ion exchange resin xiv LIST OF TABLES Table 1.