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. Black Cohosh clinical studies 12
Table 2. Commercially available products that have been evaluated 15 in clinically studies
Table 3. Compounds isolated from C.racemosa roots 30
Table 4. Standard TLC solvent systems 43
Table 5. Analytical HPLC-ELSD conditions and method parameters 47
Table 6. Botanical Center database of Black Cohosh collections 56
Table 7. 1H and 13C NMR data of 1 103
Table 8. 1H and 13C NMR data of 2 107
Table 9. 1H and 13C NMR data of 3 112
Table 10. 1H and 13C NMR data of 4 116
Table 11. Biological activity of a MeOH Black Cohosh Extract. 121
Table 12. Compound activity data of triterpenes. 122
Table 13. Average constituent levels of 14 C. racemosa Collections 123
Table 14. Geographic averages of constituent levels of 124 14 C. racemosa Collections
Table 15. Percentage of Active standards in extracts 126
Table 16.Serotonin binding activity of crude extracts 128
xv
LIST OF FIGURES
Figure 1. Flowering raceme of C. racemosa. 1
Figure 2. Triterpenes from Black Cohosh Roots/Rhizomes. 33
Figure 3. Phenolic constituents from Black Cohosh Roots/Rhizomes. 35
Figure 4. Mixed HPLC-ELSD standard trace. 48
Figure 5. Calculations for preparative HPLC scale-up. 51
Figure 6. RAPD-PCR of N. American Cimicifuga species. 61
Figure 7. PCR profiles utilizing different primers to distinguish 62 C. racemosa from potential adulterants.
Figure 8. Freehand microscopical drawings. 63
Figure 9. Scanning electron microscope pictures. 65
Figure 10. Solvent extraction and partitioning of plant material 67 to yield non-polar fractions for isolation from the EtOAc-soluble fraction
Figure 11. Solvent extraction and partitioning of plant material 68 to yield polar fractions for isolation from the BuOH-soluble fraction
Figure 12. Summary of XAD-2 fractionation and bioassay results 70
Figure 13. HPLC-ELSD of XAD-2 Fractions 71
Figure 14. Pilot CHP20P separation summary HPLC-ELSD 74 results F 21-23 to 21-29.
Figure 15. CHP20P separation of XAD-2 fractions 5 through 7 75
Figure 16. HPLC-ELSD results of CHP20P fractions 76 xvi
LIST OF FIGURES (continued)
Figure 17. LC-MS screening of CHP20P fractions for compounds 80 with an odd number of nitrogen atoms
Figure 18. HPLC-ELSD of sephadex LH-20 separation of F-12. 82
Figure 19. Semi-preparative RP-HPLC isolation of 1. 86
Figure 20. Qualitative analytical HPLC-ELSD results of 87 semi-preparative HPLC-ELSD of separation of fraction G-15.
Figure 21. Isocratic analytical HPLC-ELSD of 2. 89
Figure 22. QTOF-2 HREIMS of 1 with fragmentation pattern. 95 Figure 23. 13C-NMR APT of 1 96
13 Figure 24. Gated C-NMR spectrum of (1) in D2O 97
Figure 25. HMBC of cimipronidine (1) in D2O 98
Figure 26. HSQC of 1 in D2O 99
1 1 Figure 27. H- H gCOSY spectra of (1) in D2O 100
Figure 28. The NOESY correlations of 1 101
1 Figure 29. H-NMR spectra of 1 in d6-DMSO and D2O 102
Figure 30. Fukinolic acid (2) 1H-NMR spectrum in MeOH-d4 105
Figure 31. 13C-NMR spectrum of fukinolic acid (2) in MeOH-d4 106
Figure 32. LC-MS/MS identification of 3 109
Figure 33. Proton NMR spectrum of actein (3) in pyridine-d5 110
13 Figure 34. C-NMR spectra of actein (3) in pyridine-d5 111 xvii
LIST OF FIGURES (continued)
Figure 35. Carbon NMR of (4) isoferulic acid in pyridine-d5 114
Figure 36. Proton NMR of isoferulic acid (4) in pyridine-d5 115
Figure 37. Positive ESI LC-MS of a fraction containing 118 cimipronidine (1) and its analogues
Figure 38. Positive-ion electrospray tandem mass spectral 119 data to identify N-methyl cimipronidine
Figure 39. Phase II clinical capsule analysis standard curves 125
Figure 40. HPLC-ELSD overlay of Black Cohosh extracts 127 using different solvents
Figure 41. Positive-ion electrospray LC-MS chromatographic 128 overlay of Black Cohosh extracts using different solvents
Figure 42. Electrospray LC-MS (negative-ion mode) Total Ion 134 chromatogram (TIC) of the Phase I clinical extract
Figure 43. Clinical capsule comparison 135
Figure 44. Comparison of clinical starting material triterpene 138 content to Asian species
Figure 45. HPLC-EL:SD trace showing the polarity of the 147 BuOH fraction
Figure 46. Potential complexation of cimipronidine 148 and fukinolic acid
Figure 47. Other guanidine isolates from higher plants 151
xviii
SUMMARY
This work was conducted within Project 1 of the UIC/NIH Center for
Botanical Dietary Supplements Research. The focus of project one is the
standardization of botanicals for womens’ health for FDA-style Phase I and II
clinical studies. This work details primarily the botanical and chemical aspects of
botanical dietary supplement standardization. The specific aims of this work are;
1) To effectively review the existing literature on the chemical, biological and botanical aspects that would be essential for the standardization of black cohosh
(Cimicifuga racemosa (L.) Nutt.). 2) To botanically standardize a black cohosh extract using the appropriate microscopical, taxonomic and genetic fingerprinting
techniques available to the UIC/NIH Center, 3) To isolate and characterize
known and novel constituents for the chemical standardization and biological
standardization of a black cohosh extract, and to develop preparative
methodologies for isolation and characterization where needed. 4) To chemically standardize a Black Cohosh extract using the appropriate techniques such as
HPLC-ELSD, and develop methodologies where appropriate. 5) Ultimately, to apply the aforementioned aims to best formulate a standardized black cohosh extract for clinical study.
Figure 1. Flowering raceme of Cimicifuga racemosa (L.) Nutt. 1.0 INTRODUCTION
A component of the Dietary Supplement Health and Education Act
(DSHEA) of 1994 led to the establishment of the Office of Dietary Supplements
(ODS) and the National Center for Complementary and Alternative Medicine
(NCCAM) within the National Institutes of Health.1 The purpose of ODS and
NCCAM, as mandated by Congress, is to provide accurate information on alternatives to conventional means of healthcare to the public. One of the methods for achieving this goal is the funding of extramural research. In 1999,
ODS and NCCAM jointly funded two centers, one at the University of Illinois at
Chicago (UIC) and one at the University of California-Los Angeles (UCLA), for
research of botanical dietary supplements. The UIC/NIH Center was organized to
focus its research on botanicals traditionally used for womens’ health concerns
(i.e., menopause, PMS). Black cohosh, Cimicifuga racemosa (L.) Nutt. (syn.
Actaea racemosa L.),a was chosen as one of the plants to be developed for the
clinical component of the Center based on it’s widespread use for relief from
symptoms related to menopause.2-12 The unique purpose of the study being that it would be one of the first extract dosage forms standardized botanically, biologically and chemically to undergo FDA-style phase I and II clinical trials.
Menopause, a normal biological event, is generally regarded as the time in a woman’s life when she stops menstruating, usually between the ages of 48 and
52. In the year 2000 approximately 31.2 million American women were over the age of 55, that number is estimated to be 45.9 million by the year 2020.13
Because of the average life expectancy over the past few decades, it is
a Actaea racemosa is not an officially accepted synonym by the International Botanical Congress
2 estimated that most women will live one-third to one-half of their lives after menopause.
While menopause is a natural condition, the decrease in hormonal concentrations that occur during menopause and hereafter, can increase the risk of osteoporosis (bone loss) and heart disease. Additionally, more common throughout menopause are atrophic and psychic symptoms, which occur in pre- and peri-menopause. The development of menopausal symptoms involves many complex interactions between exogenous and endogenous factors. Menopausal symptoms result as the ovaries produce less estrogen and progesterone, these decreases initially result in irregular periods, and other symptoms such as hot flushes, vaginal dryness and sleep disturbances. Via this process, the pituitary gland is prompted to increase follicle stimulating hormone (FSH) production to try to stimulate the ovaries to produce more estrogen.14
Basic endocrinology research has elucidated several pathways for managing the climacteric symptoms related to menopause. Premarin™
(Conjugated Pregnant Mare Estrogens) was introduced in the 1960’s with the idea of managing climacteric symptoms (hot flushes, night sweats, bone loss and vaginal atrophy) through steroidal hormone therapy, commonly referred to as estrogen replacement therapy (ERT). Hormone replacement therapy (HRT), is estrogen replacement, like Premarin™, administered in conjunction with progesterone or synthetic progestagens. Currently 38% of postmenopausal women in the U.S. use HRT during menopause.14, 15 ERT is prescribed for women who have had a hysterectomy, others are generally prescribed combined HRT to
3 prevent endometrial hyperplasia. Oral estrogen preparations include conjugated equine estrogens (CEE), synthetically derived piperazine estrone sulfate, estriol, micronized estradiol, and estradiol valerate. Alternatively, transdermal estradiol is available in the form of a patch or gel, a slow-release percutaneous implant, or an intranasal spray. Intravaginal estrogens include topical tablets, rings, creams and pessaries. Oral estrogen preparations result in up to ten-fold higher concentrations of circulating estrone sulfate than do transdermally administered estradiol of comparable or even higher doses. The primary indication for systemic HRT is, therefore, the relief of moderate-to-severe vasomotor symptoms. Vaginal estrogens are effective for urogenital symptoms.
The Women’s Health Initiative, a multi-armed study, one of the study arms, using more recent HRT formulations, such as Prempro™ (conjugated estrogen and progesterone), demonstrated an increased risk toward development of breast cancer, endometrial cancer, gallbladder disease, coronary heart disease, stroke, and embolism.16-25 A review of all observational studies found that, in women taking estrogen alone, the relative risk of developing breast cancer was 1.32 26, for women taking estrogen plus progestogen, the relative risk was 1.41 for developing breast cancer, in comparison with women who did not take hormones.27 Thus, there is an imperative to develop safer alternatives to
HRT to relieve climacteric symptoms.28-31
Development of medical systems incorporating plants can be traced back as far as recorded history.32 According to the World Health Organization (WHO), approximately 65% of the world’s population use plants as their primary modality
4 of healthcare.33-37 The four primary ways plants are used in health care are: 1) to
isolate bioactive compounds for direct use as drugs, 2) to isolate compounds for
use as lead compounds in semi-synthesis, 3) to use isolated agents as
pharmacological tools and 4) to use the a) whole plant and/or b) plant part(s) as
a herbal remedy, and/or c) an extract of a) and b). According to estimates, the
use of herbal remedies in the US increased as much as 380% between 1990 and
1997.38 Despite recent data to suggest a decrease in usage of complementary and alternative medicines, the relatively of herbal remedies in the USA is currently a $4 billion dollar-per-year industry.39, 40 National surveys have shown
that the average user of herbal products is more inclined to be a college-
educated female between the ages of 35 and 49.38
5
2.0 STATEMENT OF PROBLEM
With over 50 years of clinical research, supporting the use of black cohosh
(Cimicifuga racemosa (L.) Nutt. Syn. Actaea racemosa L.) for climacteric
symptoms related to menopause,9, 41-46 (i.e. hot flushes, depression) it’s
mechanism of action is unclear. Black cohosh has long been believed to be
estrogenic, with estrogenicity attributed to the presence of estrogenic
isoflavones, commonly referred to as phytoestrogens. A study done at the
UIC/NIH Center for Botanical Dietary Supplements Research confirmed no direct
estrogenic effect of black cohosh.47 Additional studies have shown no presence
of estrogenic isoflavones.48, 49 A goal of the UIC/NIH Center for Botanical Dietary
Supplements Research is to prepare a standardized dosage form of black
cohosh for clinical studies. This extract will be standardized to specific biological,
botanical and chemical characteristics. The specific challenges addressed in this study are:
1. Chemical characterization of bioactive constituents of black cohosh
2. Development of methods to identify and isolate bioactive constituents
3. Revision and review of reference data for future investigations of bioactive
constituents
6
3.0 LITERATURE REVIEW
3.1 TAXONOMY OF BLACK COHOSH
Cimicifuga L., from the Latin “cimex” (bedbug) and “fugare” (to banish) has
been used as a medicine across several cultures and continents. Common
names include:
Americas: Black cohosh, Black Snake-root, Macrotys, Rattleweed,
Rattleroot, Snakeroot, Bugbane, Squaw root, Squaw weed
China: Sheng Ma
3.1.1 Ranunculaceae
Often referred to as the buttercup family, Ranunculaceae, comprises roughly 2000 species of plants across 50 genera, mainly spread in temperate countries. Herbs are perennial or annual, sometimes subshrubs, herbaceous or woody vines. Leaves are basal and cauline, alternate, rarely opposite or whorled, simple or variously compound, palmately nerved, rarely penninerved, with or without stipules. Inflorescence appears as a simple or compound monochasium, dichasium, as a simple or compound raceme, or with solitary flowers. Flowers are bisexual, sometimes unisexual, actinomorphic, rarely zygomorphic, and hypogynous. Sepals show 3–6 or more, they appear free, petaloid or sepaloid, imbricate or sometimes valvate in bud. Petals are present or absent, usually 2–8 or more, free, usually with nectaries. Stamens are numerous, rarely few, and free. Filaments appear linear or filiform. Anthers are latrorse, introrse, or extrorse, with some sterile stamens becoming staminodes. Carpels are numerous or few,
7 rarely singular. The carpels are free and rarely connate to varying degrees.
Ovaries can possess one or many ovules. Fruits are follicles or achenes, rarely capsules or berries. Seeds are small, with abundant endosperm and a minute embryo. A majority of the species are herbs and lianas having large flowers.50-52
3.1.2 Genus Cimicifuga
The genus Cimicifuga L. includes 18 species, one of which is native to
Europe, six from North America, and the remainder from northeast Asia. If the genus is expanded into Actaea L., as is proposed on the basis of recent morphological and DNA sequencing studies, it would include a total of 24 species.53 In the phylogenetic study in question, Actaea was reclassified to include Cimicifuga and Souliea. However, for the present account, the genus
Cimicifuga remains distinct on the basis of no such formal change by the World
Conservation Union (IUCN) International Botanical Congress.54, 55 In North
America, the genus is comprised of six species; three Western species (C. arizonica Wats., C. elata Nutt., C. laciniata Wats.) and three Eastern species (C. americana Michx., C. racemosa (L.) Nutt., C. rubifolia Kearney).
The genus is morphologically characterized by the following: The herbs, are perennial. The rhizomes are robust, and creeping, with fibrous roots. The stem is terete. The stem appears apically and frequently with several branches.
Leaves are 1–3 ternately sect or subpinnately compound with a long petiolate.
The Inflorescence appears densely racemose, sometimes spicate, simple or branched. The rachis is densely glandular, pubescent and hairy. The bracts are
8 small, subulate to narrowly triangular. Flowers are dense, small, actinomorphic,
hermaphroditic or rarely unisexual. The plants are dioecious. There are 4 or 5
sepals which appear petaloid, white, obovate-orbicular, or caducous. Petals are
elliptic to sub-orbicular, entire, slightly concave or forked-lobed with 2 empty
anthers, rarely with a nectary. Stamens are numerous and filaments narrowly
linear to filamentous, with yellow anthers. The anthers are broadly ellipsoid to
suborbicular. One to eight follicles appear either stalked or sessile, oblong-
ellipsoid to obovate-ellipsoid, adaxially convex veined, with a beaked apex.
Seeds are few, yellowish brown, ellipsoid to narrowly ellipsoid.50-52
3.1.3 Species
Cimicifuga racemosa is an erect, smooth-stemmed perennial 1-2.5 meters
in height. Large compound leaves are alternately arranged and triternate on short
clasping petioles. Basal leaf petioles are grooved in young specimens, this
shallow, narrow sulcus in C. racemosa disappears as the petiole enlarges and matures, where it remains present throughout the life of the related Eastern North
American species C. rubifolia and C. americana.56 Terminal leaflets are acute, glabrous with sharp serrated margins often trilobate, occasionally bilobed. Fruits are ovoid follicles occurring sessile on the pedicel. The flowering structure, the raceme, is a long wand-like structure with showy white flowers. Flowers possess numerous characteristic stamens with slender filaments with distinctive white anthers.57 The roots are branched and knotted structures. The roots have a dark
brown exterior, but are usually internally white and mealy and are rarely brown
9 and waxy. The upper surface of the plant has several buds and numerous large
stem bases that are terminated frequently by deep, cup-shaped, radiating scars,
each which show a radiate structure. These bases are less frequently terminated
by fibrous strands. The lower and lateral surfaces show numerous root scars and
a few short roots with horny fractures. The plant gives a slight odor and has a
distinctive, bitter and acrid taste.58
3.2 BLACK COHOSH IN TRADITIONAL MEDICINE
The use of black cohosh as a medicine is generally traced back to native
American groups. References point to its use by the Oklahoma,b Delaware,
Cherokee, Penobscots (Algonquins) and Micmac tribes.59, 60 The ethnomedical
uses of the roots by these indigenous peoples include, but are not limited to, use
as an emmenagogue to treat rheumatism, as a tonic, diuretic, or anodyne to treat
kidney trouble, and to treat fever.
Use was not limited to indigenous peoples. Black cohosh was a well
known home remedy and widely used in mainstream medical circles, it was
included in a secondary list of the first United States Pharmacopoeia (USP) in
1820 as Cimicfuga serpentaria (snakeroot). The uses that are generally cited are
as a relief from rheumatism, astringent, sudorific, anodyne, repellant,
emmenagogue and subtonic. With a shift in the materia medica of the USP to
‘mainstream’ medicine, the use of black cohosh and interest in its properties
shifted toward “eclectic” or herbal medicine. John King, author of The Eclectic
b This is taken from the text, the use of the herb this far to the west is out of the range of distribution of C. racemosa
10 Dispensatory of the USA, made significant note of black cohosh, or as he
referred to it, as Macrotys. King claimed that Macrotys was most beneficial as a remedy for “abnormal conditions of the principle organs of reproduction in the female.” The text further states that, the root is “very efficacious in the treatment of chronic ovaritis, endometriosis, and menstrual derangements, such as amenorrhea, dysmenorrhea, menorrhagia, frigidity, sterility, threatened abortion,
uterine sub-involution, and to relieve severe after pains.” King worked closely in
Cincinnati with the Lloyd brothers who later put forth a drug treatise on Macrotys.
In this treatise, the Lloyd brothers pointed to the use of Macrotys as a remedy for
rheumatism and for correcting the “wrongs” of menstruation.61
3.3 EFFECTS ON CLIMACTERIC SYMPTOMS RELATED TO MENOPAUSE
With a history spanning almost 50 years of clinical study, mainly in
Europe, black cohosh is one of the more popular alternatives to hormone
replacement therapy (HRT) or Estrogen Replacement Therapy (ERT).62 Most of
the clinical research has been carried out on the commercially available
Remifemin®.63 The formulation and dosage of Remifemin® used in human
studies has changed over time as shown in table 1. However, other commercial
formulations are available, and have been clinically studied as evident in table 2.
However, most commercial products have not been clinically trialed.46 The
results of clinical studies have been measured using a variety of parameters.
Self-assessments, physician assessments and physiological parameters are
usually used together when designing studies to measure
11
Table 1. Black cohosh clinical studies
Author Year Extract/ Study n Outcome Measure/Result Study Design Formulation length /Dosage
Hernandez- 2003 BNO 1055 12 months 136 Combination therapy with Open, Munoz et al.,64 tamoxifen (20 mg) reduced Randomized, severity and incidence of hot Patient self- flushes. assessment Baier- 1995 Cimisan® T 4-8 weeks 157 Open, Jagodinski65 Tropfen, Uncontrolled variable dose Wuttke et al.,66 2003 Klimadynon® 3 months 62 Equipotent to 0.6 CE for relief Randomized, /BNO1055 of climacteric complaints and double-blinded, for bone resorption. No effect placebo on endometrial thickness. controlled ,multi- center. MRS Schotten67 1958 Remifemin® 3-4 weeks 22 Alleviation of neurovegetative Case series 20 drops and psychic complaints associated with menopause and pre-menopause. Kesselkaul68 1957 Remifemin® 2 weeks 63 Alleviation of climacteric Case series 60 drops complaints in 95% of patients. Nesselhut69 1999 Remifemin® 3 Months 28 Good to very good alleviation of Open, Tablets, 10 menopausal symptoms in Postmarket equiv. to 80% of study participants Surveillance 136 mg dried herb/day Foldes70 1959 Remifemin®, Unknown 41 31 patients of the verum group Placebo 3 tablets/day responded to the treatment with Controlled, a decrease in menopausal Open, complaints. Crossover, Patient Self- Assessment Starfinger71 1960 Remifemin®, 1 year 105 Decreased climacteric Case series 3-20 complaints without incidence of drops/day side effects or resulting in unphysiological bleeding. Liske et al.,72 2000 Remifemin®, 6 months 57 Alleviation of symptoms in both Double blinded, equiv. to 39 groups. Results similar after 3 randomized, or 127 mg months Good-Clinical dried Practice herb/day compliant, KMI, SDS, CGI Schlidge73 1964 Remifemin®, Variable 135 Case series Fluid extract 60 drops/day
12 Table 1 (continued). Black cohosh clinical studies
Author Year Extract/ Study n Outcome Measure/Result Study Design Formulation length /Dosage
Daiber74 1983 Remifemin®, 12 weeks 36 Alleviation of climacteric Open, KMI, CGI Fluid extract complaints (hot flushes, 80 drops/day insomnia, sweating and restlessness). Stolze75 1982 Remifemin®, 6-8 weeks 629 Alleviation of neurovegetative Open, Physician Fluid extract and psychological menopausal and Patient Self- 80 drops/day symptoms in 80% of patients Assessment Vorberg76 1984 Remifemin®, 12 weeks 50 Significant or highly significant Randomized, Fluid extract alleviation of menopausal Open, KMI, CGI, 80 drops/day (neurovegetative and psychic) POMS complaints, study included subjects contraindicated to Hormone Therapy. Warnecke77 1985 Remifemin®, 12 weeks 20 Significant alleviation of Randomized, Fluid extract symptoms (psychic and Open, KMI, 80 drops/day neurovegetative) in the black HAM-A, SDS, cohosh, conjugated estrogen CGI, and diazepam groups. Vaginal Karyopyknosis cytology of treatment group index, Eosinophil was comparable to estrogenic index. stimulation.
Heizer78 1960 Remifemin®, 2-18 66 Alleviation of menopausal Case series tablets 3- months (neurovegetative and psychic) 6/day complaints in 47% of patients with intact uteri and 35% with hysterectomies
Jacobson, et 2001 Remifemin®, 60 days 42* No change in median number Double blinded, al.79 tablets equiv. or intensity of hot flushes randomized, to 40 mg placebo- dried controlled, herb/day patient self- assessment, VAS, MSS Duker et al.,43 1991 Remifemin®, 2 months 110 LH suppression In vitro study tablets equiv. using blood from to 40 mg menopausal dried women taking herb/day black cohosh Stoll80 1987 Remifemin®, 12 weeks 26 Significant alleviation of Double-Blinded, Tablets climacteric symptoms (vaginal Randomized, equiv. to 8 atrophy, neurovegetative and Placebo- mg psychic complaints) in Controlled, KMI, extract/day comparison with estrogen and HAM-A, VMI placebo groups. (vaginal epithelium)
13 Table 1 (continued). Black cohosh clinical studies
Author Year Extract/ Study n Outcome Measure/Result Study Design Formulation length /Dosage
Lehman- 1988 Remifemin®, 6 months 15 Significant alleviation of Randomized, Willenbrock et Tablets climacteric symptoms in black Open, KMI al.,42 equiv. to 8 cohosh and drug treatment mg groups. No significant change extract/day in gonadotropin (FSH, LH) levels. Petho81 1987 Remifemin®, 6 months 50 KMI decreased significantly Open, KMI, Tablets, from 17.6 to 9.2, correlates with Patient Self- Unspecified a significant reduction in Assessment dose neurovegetative symptoms. Severity of subjective self- assessments of subject’s physical and psychological symptoms decreased.
Gorlich82 1962 Remifemin®, Variable 41 Alleviation of climacteric and Case series tablets, (258) vascular symptoms in 85% of variable dose patients
Brucker83 1960 Remifemin®, Variable 87 Alleviation of menopausal Case series tablets, (517) complaints variable dose Mielnik84 1997 Uncharacteri 6 months 34 Alleviation of climacteric Open, KMI zed extract, (neurovegetative) symptoms in 4 mg daily 76% of patients after 1 month
Georgiev85 1997 Uncharacteri 3 Months 50 Alleviation of climacteric Open, KMI, zed extract, symptoms in 90% of patients. HAM-A, VMI Unspecified Increase in vaginal cell dose proliferation (VMI) in 40% of treated women. Liske et al.,72 2002 Unique C. 6 months 152 No direct systemic estrogenic Drug racemosa effect on serum levels of FSH, Equivalence preparation, LH, SHBG, prolactin, & 17-ß Trial, KMI, SDS, equiv. to 39 estradiol. No change in vaginal CGI or 127.3 cytology. Higher dose had a mg/day more significant reduction in KM index after 6 months. Significant reduction with both doses in neurovegetative and psychic complaints.
*-all with breast cancer history
14
Table 2. Commercially available products that have been evaluated in clinically studies.*
Brand Name/ Delivery Effective Indications Manufacturer form ingredients (Country)
Cefakliman® capsules Concentrated menopausal and premenstrual symptoms, mono/Cefak extract from dysmenorrhea (Germany) Cimicifuga Root Cefakliman® solution Ethanolic extract menopausal and premenstrual symptoms, mono/Cefak from Cimicifuga dysmenorrhea Root Cimipure-PE® capsules Dried climacteric symptoms related to menopause 2.5/ Pure World hydroalcoholic (USA) extract Cimisan®-T/ blister Concentrated premenstrual and dysmenorrheic as well as APS pack extract from neurovegetative symptoms from menopause (Germany) Cimicifuga Root Cimisan-T/APS drops Liquid extract premenstrual and dysmenorrheic as well as neurovegetative symptoms from menopause Femilla®/ tincture Hydroalcoholic neurovegetative symptoms with painful menstruation Steigerwald extract from (dysmenorrhea) as well as during menopause (Germany) Cimicifuga root Klimadynon®/ blister Concentrated menopause-caused neurovegetative symptoms Menofem/BNO pack extract from 1055/Bionorica Cimicifuga Root (Germany)
Klimadynon®/ solution Liquid extract menopause-caused neurovegetative symptoms Menofem/BNO 1055/ Bionorica Remifemin dragees Extract of menopausal symptoms; such as hot flushes, sweats, Plus®/ Hypericum depressive moods and psychovegetative problems Schaper & (aerial parts) and such as despondency, inner tension, irritability, lack Brümmer† Cimicfuga root of concentration, insomnia, fear and/or nervousness; (Germany) premenstrual vegetative symptoms Remifemin®/ tablets Concentrated menopausal symptoms; mild dysfunction after Schaper & extract from ovarectomy or hysterectomy; to aid treatment with Brümmer† Cimicifuga Root sexual steroids; premenstrual neurovegetative and emotional problems; juvenile menstrual irregularities Remifemin®/ solution‡ Percolate extract menopausal symptoms; mild dysfunction after Schaper & of Cimicifuga root ovarectomy or hysterectomy; to aid treatment with Brümmer sexual steroids; premenstrual neurovegetative and emotional problems; juvenile menstrual irregularities
*-independent confirmation as to the identity and/or quality of formulations is not publicly available. †-previously marketed in the US through GlaxoSmithKline, after 6/2005 will be distributed by Enzymatic Therapy. ‡-no longer marketed in this dosage form.
15 psychological, neurovegetative, somatic and physiological markers of
menopause or in the case of the treatment groups, relief from menopausal
symptoms. More significant when evaluating studies, is the study design. More
weight should be placed on studies that follow good clinical practice, are double-
blinded, placebo-controlled and use characterized plant material and/or
extracts.41, 86
The standard for clinical studies are randomized, double blinded, multi-
center, placebo-controlled designed studies. With that in mind, three studies on
black cohosh have been randomized, double blinded and placebo controlled.44, 66,
79 The Jacobson study, spanning only 60 days of treatment, suggests that study
length may have limited study findings.79 Additionally, the study participants all had a history of breast cancer. The outcome of the study was that the median number of hot flushes decreased 27% in both the placebo and black cohosh groups. No significant differences were observed between groups. Thus, black cohosh, on the basis of this study, was no more effective than placebo in the treatment of hot flushes. The source and formulation of the extract used in the
Jacobson study was not specified.
A more recent open-labeled study using breast cancer survivors, treated with either Tamoxifen or a combination of an unspecified BNO 1055 extract with
Tamoxifen, suggested a significant reduction in the number and severity of hot
flushes, in the combination treatment group.64 In another randomized, double-
blinded and placebo controlled clinical study, black cohosh was compared with
standard conjugated estrogen (CE) therapy (0.625 mg/daily) with a duration of 12
16 weeks.44 Patients’ physical and psychological symptoms were measured every four weeks. The end result of the study was that the black cohosh treated subjects demonstrated a significantly lower index score with both the Kupperman
Menopausal (KM) and the Hamilton Menopausal (HAM-A) scales compared with placebo. This indicated a decrease in severity and frequency of hot flushes following black cohosh treatment. In addition, an increase in the number of estrogenized cells in the vaginal epithelium were noted in this study.44
In 2003, a study compared two different preparations of BNO 1055 extract
(defined in table 2, page 15) to CE therapy (0.625 mg/daily).66 The study
outcomes were: a patient self-assessment (diary and menopause rating scale
[MRS]), crosslaps (to measure bone resorption), bone specific alkaline
phosphatase (marker of bone formation), and endometrial thickness (measured
by ultrasound). Both BNO 1055 extracts were equipotent to CE therapy and
significantly greater than placebo in reducing climacteric complaints. Additionally,
the study demonstrated that both BNO 1055 preparations had beneficial effects
on bone metabolism in serum, specifically an increase in bone-specific alkaline
phosphatase, and no reduction in bone resorption, thus an increase in bone
turnover formation. No change in endometrial thickness was observed in both
BNO 1055 treatment groups, but it was significantly increased with CE therapy.
However, an increase in superficial vaginal cells was observed in both the CE
and BNO 1055 treatment groups. The authors of this study hypothesized that the
activities of both BNO 1055 preparations were similar to the effects of Selective
17 Estrogen Receptor Modulator (SERM) i.e., raloxifene, therapy on bone and
neurovegetative climacteric symptoms, without any uterotrophic effects.66
A recent double-blinded, randomized good clinical practice compliant
study used two dosages (low 39 mg, high, 127 mg) of Remifemin® (unspecified
formulation).87 The effectiveness was measured using a KM index, a self-
assessment depression scale (SDS), the clinical global impression scale (CGI),
serum concentrations of luteinizing hormone (LH), follicle stimulating hormone
(FSH), sex hormone binding globulin (SHBG), prolactin, and 17-β-estradiol and vaginal cytology. Reductions in the KM and SDS indices were significant. Global efficacy (CGI) was scored at good to very good in 80% and 90% of the patients in the treatment groups.87 No effect on serum hormone concentrations or vaginal
cytology was shown, prompting the authors of the study to suggest that black
cohosh does not have a direct estrogenic effect on serum hormone concentration
or vaginal epithelium.72 Two recent open studies using unspecified types of
extracts showed reduced KM index scores.64, 66 One study reported a significant
reduction in one month.84 While another study, also using the HAM-A scale,
recorded a 90% improvement in climacteric symptoms in menopausal women
after three months administration of an unspecified black cohosh extract.85
3.3.1 Conclusions of Clinical Studies of Black cohosh on Menopausal
Symptoms
Recently, an analysis interpreting the safety data from published clinical
trials, case studies, post-marketing surveillance studies, spontaneous report
18 programs and phase I studies were reviewed.86 The data obtained from over 20
studies, including over 2,000 patients, suggest that adverse event occurrence
with black cohosh is rare. The events are mild and reversible, the most common
events reported being gastrointestinal upsets and rashes.86 Recently, a summary
of case reports regarding potential liver toxicity of black cohosh, has received
much attention.88 In response to this report, NCCAM held a Workshop on the
Safety of black cohosh in Clinical Studies on November 24, 2004
(http://nccam.nih.gov/news/pastmeetings/blackcohosh_mtngsumm.pdf). Present at this workshop were a team of experts on black cohosh, spanning industry and academia, as well as basic and clinical science. The findings of this workshop were that “the group cautioned that there were problems establishing a cause and effect relationship on the basis of information presented (from the case studies). The hepatotoxicity might be coincidental or correlated with this adverse event but not necessarily be the cause”, and “at this time, there is no known mechanism with biological plausibility that explains any hepatotoxic activity of black cohosh”. It should be noted that no identification and/or characterization of black cohosh in the herbal preparations in question was made in any of the case reports1. Additionally, all black cohosh case report subjects were concurrently
using varied regimens of prescription pharmaceuticals.88
Placebo effects for any treatment of hot flushes have been well
documented, and self-reported measures of this endpoint are problematic. It
appears that the placebo effect peaks at 12 weeks.46 Thus, any study must
weight placebo effects appropriately. When clinical study design limitations are
19 considered along with Hawthorne effects,89 small sample sizes,42, 44, 67, 69, 70, 74, 77, 79, 84 and heterogeneous samples, it is not surprising that the literature in aggregate yields equivocal results concerning efficacy.46 However, in light of these studies,
the fact remains that the majority of clinical data, spanning over 2,000
randomized subjects has been positive in terms of efficacy and has shown no
incidence of liver toxicity, permanent or otherwise1. Based on the data provided, at the minimum, black cohosh appears to be a relatively safe alternative therapy to ERT or HRT, with a significant scientific basis to support its efficacy as an alternative therapy.
3.4 PHARMACOLOGY AND BIOCHEMISTRY OF Cimicifuga racemosa
EXTRACTS
Despite the extensive aforementioned clinical research, the mechanism of action of Black cohosh remains unclear. A majority of the older literature on black cohosh suggests a direct estrogenic effect. More recent studies have targeted the limbic system (hypothalamus) or more specifically the neurotransmitters involved in regulation of this system, as being responsible for the activity of black cohosh. Serotonin selective reuptake inhibitors (SSRI) have been used successfully to treat hot flushes in women with breast cancer, and there appears to be a link between estrogen and the regulation of serotonin receptors in the brain and regulation of tryptophan hydroxylase.90-93 Data fall into the following
categories: competitive estrogen receptor binding, receptor expression, plasma
hormone levels, hormonal secretion, osteopenia inhibition, uterine weight/estrous
20 induction, MCF-7 cell proliferation inhibition, and CNS effects/neurotransmitter
binding.
3.4.1 Competitive Estrogen Receptor Binding
The first report of estrogen receptor binding pointed toward clarifying the
mechanism of action of black cohosh.94 Additional studies were carried out to
further the substantiation of endocrine activity.43, 95 One significant factor frequently overlooked regarding black cohosh in these studies is the relative lipophilic nature of the extract used in making this determination. Extracts and fractions displaying estrogen receptor binding activity are of a significantly different chemical nature than the typical hydroalcoholic (ethanolic, methanolic or isopropanolic) extracts available for human use. For the purposes of this review lipophilic will be defined as having an affinity for, or, tending to combine with, or capable of dissolving completely or partially in lipids, solvents such as CHCl3,
EtOAc, will be classified as lipophilic. These lipophilic extracts have not been
administered clinically to date. A lipophilic (CHCl3) extract of the plant showed
relatively weak (35 µg/ml) estrogen receptor binding in rat uteri.94 One study also
confirmed the estrogen receptor binding activity of an unspecified lipophilic sub-
fraction (originating from CHCl3) on ovariectomized (ovx) rat uterine cells, with no
binding activity seen with a hydroalcoholic extract.43
Recent reports have contradicted the estrogen binding affinity of black
cohosh extracts.47, 96, 97 Using an unspecified root extract in an in vitro competitive
cytosolic estrogen receptor (from ovx rat livers) binding assay with
21 diethylstilbesterol (DES), an inhibitor of estrogen binding, showed a significant
inhibition of estradiol binding in the presence of DES.96 No binding was
demonstrated with the extract. A hydroalcoholic extract (50% aqueous ethanol)
was assayed for ER-binding to intact human breast cancer cell lines, MCF-7 and
T-47-D. No binding affinity was shown for the black cohosh extract. However,
binding activity was evident for other hydroalcoholic plant extracts.97 Using a methanol extract of black cohosh at a high concentration (200 µg/mL) on
recombinant diluted ER-α and ER-β, no binding activity was evident.47 A recent study using BNO 1055 (an unspecified hydroalcoholic liquid extract) showed contrasting results.98 The extract displayed dose-dependent competition with radio-labeled estradiol in both a porcine and human endometrial cytosolic estrogen receptor ligand binding assay (ER-LBA) system. By comparison, the extract did not displace human recombinant ER-α and ER-β. These findings prompted the authors to suggest that their product contains estrogenic compounds that have binding affinity for a putative ER-γ receptor. Another potential explanation for the activity is the presence of estrogenic fatty acids in black cohosh.99-101 Fatty acids, like linoleic acid have been shown to bind to
estrogen receptors, these fatty acids would most likely be found in more lipophilic black cohosh extracts.102-106
3.4.2 Receptor Expression
A lipophilic C. racemosa extract, was studied using luciferase expression
in a MCF-7 α- and β-estrogen receptor expressing subclone.107 The extract at a
22 concentration of 35 µg/mL, was shown to activate the transcription of estrogen-
regulated genes; the hydrophilic (hydroalcoholic) extract showed no activity. 107 A recent study measuring a commercial hydroalcoholic extract (standardized to
2.5% triterpene glycosides) at a low concentration (4.75 µg/L), increased estrogen receptor levels, an effect also produced by estradiol in human MCF-7 cells.108 An unspecified black cohosh extract tested in a transient gene
expression assay using HeLa cells co-transfected with an estrogen dependent
reporter plasmid in the presence of human ER-α or ER-β cDNA failed to show transactivation of the gene.109
3.4.3 Plasma Hormone Concentrations
The effect of black cohosh on serum concentrations of FSH and LH has
been studied extensively. Crude methanol and ethanol extracts, suppressed
plasma LH, with no effect on FSH in ovx rats.94, 95 Further fractionation of the
crude fraction resulted in the activity residing in an unspecified lipophilic fraction,
with the aqueous soluble fractions devoid of this activity.94 A later study in rats using lipophilic and hydrophilic extracts at high doses (140 and 216 mg/rat, i.p.) resulted only in LH suppression with a single injection of the lipophilic extract.43
The authors of another study reported LH suppression in ovx rats with an unspecified dose and extract type of C. racemosa.110 A recent study compared the effect of C. racemosa (BNO 1055) with that of estradiol on LH concentrations.107 Reduced concentrations were reported for the black cohosh
treated animals at 60 mg/day administered subcutaneously (s.c.) for seven days.
23 However, another study reported no estrogen agonistic effects on FSH, LH or
prolactin concentrations in ovx rats (DMBA model) with daily administration of a
40% isopropanolic extract (Remifemin®) for seven weeks.111
3.4.4 Hormonal Secretion
The effect of black cohosh on prolactin secretion in pituitary cell cultures was assayed using an unspecified ethanolic extract of C. racemosa.112 Basal and
TRH-stimulated prolactin levels were reduced significantly at doses of 10 and
100 µg/mL of the ethanolic extract. This effect was reversed by the addition of
haloperidol (a D-2 antagonist) to the cell cultures, suggesting dopaminergic
regulation of hormone secretion by C. racemosa.
3.4.5 Osteopenia Inhibition
The BNO 1055 black cohosh extract (60 mg/rat, s.c.) has been shown to
increase the expression of collagen I and osteocalcin in rats equivalent to ovx
rats treated with estradiol (8 µg).107 An additional study using the BNO 1055 liquid
extract demonstrated an osteoprotective effect, a reduced loss of bone mineral
density in rat tibia after three months of administration.113 A study using an
unspecified isopropanol extract of C. racemosa showed reduced urinary
parameters of bone loss. The authors of this study suggested this action was
similar to that of the raloxifene.114 A follow-up study using BNO 1055 (liquid
extract) in comparison with conjugated estrogen therapy, showed beneficial
effects of the extract on bone metabolism in humans, specifically an increase in
24 bone-specific alkaline phosphatase in serum.66 While no direct correlation
between species has been established it is of note that studies of the Asian
Cimcifuga species have demonstrated similar activity and may be of importance
for further investigation of this biological activity.115, 116
3.4.6 Uterine Weight/Estrous Induction
Uterine and ovarian weight increase, cell cornification and an increased
duration of estrous are generally considered evidence of endometrial estrogenic
activity. However, recently it has been debated that uterine weight is a poor
marker for endometrial effects.117 Three studies demonstrating that black cohosh
extracts (unspecified hydroalcoholic) increased the uterine weight of ovx rats.62,
110, 118 Two of these studies used an undetermined root extract.110, 118 One study using immature mice reported similar findings.62 By contrast, two studies on ovx
rats,107, 119 as well as four studies of immature mice, reported the converse.107, 109,
111, 120 One of these studies found that despite no increase in uterine or ovarian
weight, the duration of estrous was significantly increased by black cohosh.120 A study demonstrated no attenuation in uterine weight at variable doses (4, 40 and
400 mg/kg day) of a 40% isopropanol extract in ovx rats.121
3.4.7 MCF-7 Cell Proliferation Inhibition
The MCF-7 assay is a straightforward test for detecting weakly estrogenic
compounds. In this assay, oestrogen-dependent cells are grown in the presence
of test compounds. As with the estrogen receptor ligand binding assays, the
25 nature of the extract or fraction is a decisive factor in the expression of estrogen receptors (Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens).122( Lipopophilic extracts and fractions may contain constituents that have been reported to interefere with the estrogenic ligand binding assays, which may be responsible for receptor expression activity as well. 104, 108, 123, 124 An unspecified black cohosh extract failed to induce growth of
MCF-7 cells significantly when compared with untreated control cells.109 A study using isopropanolic and ethanolic extracts also failed to induce the growth of
MCF-7 cells.125
3.4.8 CNS Effects and Neurotransmitter Binding
A study using an unspecified extract of black cohosh (25-100 mg/kg, orally), measured its effects on mice body temperature and ketamine-induced sleep time. The study used bromocriptine (D-2 agonist) as a positive control, pretreated with sulpiride (D-2 blocker) and this suggested a receptor mediated dopaminergic effect.112 Another study was carried out to characterize neurotransmitter levels in the striatum and hippocampus after pretreatment in mice with the unspecified extract for 21 days.126 Serotonin and dopamine concentrations in the striatum were substantially lower in comparison with the control group. These studies have helped lead to the hypothesis that it is a dopaminergic action, rather than estrogenic-like activity that is responsible for the success of black cohosh in reducing climacteric symptoms.127 A study in the
UIC/NIH center, pointed to the effects of black cohosh being mediated by
26 serotonin (5-HT) receptors.121 Three different extracts (100% methanol, 40%
isopropanol, 75% ethanol) bound to the 5-HT7 receptor subtype, the methanol
extract demonstrated the greatest affinity with an IC50 ≤ 3.12 µg/mL. The 40%
isopropanol extract inhibited [3H]-lysergic acid diethylamide (LSD) binding to the
3 5-HT7 receptor with greater potency than [ H]-8-hydroxy-2(di-N-propylamino) tetralin (positive control) to the rat 5-HT1A receptor. Analysis of ligand binding
data suggests that the methanol extract functioned as a mixed competitive ligand
of the 5HT7 receptor. Further testing of the methanol extract in 293T-5-HT7 transfected HEK cells revealed elevated cAMP concentrations. These concentrations were reversed in the presence of the 5-HT antagonist methiothepin, indicating a receptor mediated process and possible agonist activity local to the receptor.121
3.4.9 Miscellaneous
A Black cohosh methanol extract protected s30 breast cancer cells
against menadione-induced DNA damage at variable concentrations, and
scavenged DPPH free radicals at a concentration of 99 µg/mL.128 With the recent
reports of black cohosh hepatotoxicity, black cohosh extract (100% MeOH) found
in the presence of glutathione (GSH) were screened using ultrafiltration liquid
chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) for the
presence of glutathione (GSH) conjugates.129 The potential for toxicity stemmed
from the finding that catechols from black cohosh were activated to quinoid
metabolites. However, it should be noted that there appears to be no absorption
27 of these catechols across the intestinal epithelium by Caco-2 cells, only
triterpenoids demonstrated absorption by Caco-2 cells.130 A completed Phase I
study to measure the pharmacokinetics of black cohosh (75% EtOH extract) did
not detect any catechol metabolites in the blood or urine of ten women following
the oral administration of black cohosh extract (75% EtOH) at three dosages: 40,
80, and 120 milligrams.130
3.4.10 Conclusion on Extract Biochemistry and Pharmacology
While much research has been reported on the activity of black cohosh
extracts, there seems to be little consensus on exactly how the extracts, and as
important, the type of extract that best modulates the biochemical parameters
and targets of the climacteric symptoms related to menopause. It is of note that a majority of the research that supports direct estrogenic activity of black cohosh
(as indicated by ER binding assays, ER receptor expression and plasma hormone concentration) have been performed using lipophilic extracts which may contain other constituents, with estrogenic activity, not unique to black cohosh.
Additionally, these lipophilic extracts are not generally used in commercial products or clinical studies, thus limiting the relevance of these studies. With evidence to support minimal ER binding or receptor expression at high doses of non-lipophilic/hydroalcoholic extracts, it is difficult to maintain the position that these extracts mediate climacteric symptoms through a direct estrogenic effect.
In addition, the MCF-7 cell proliferation studies using hydroalcoholic extracts demonstrated no xenoestrogenic effect. Compounding the lack of estrogenic
28 data with the CNS activity that black cohosh extracts have shown, it seems more likely that black cohosh mediates menopausal symptoms by way of the CNS and a secondary messenger than by action at estrogen receptors.121, 131
3.5 PHYTOCHEMISTRY OF BLACK COHOSH
The natural product literature on Black Cohosh is vast and widely distributed. The literature holds an abundance of chemical information as well as the aforementioned pharmacological information pertaining to compounds and extracts. In order to quickly and thoroughly compile information on the analysis of natural products, the use of databases is necessary. The databases used for this work are Natural Products Alert (NAPRALERT),132 Chemical Abstracts (CA) and
Chemical Abstracts Registry, SCIFINDER, BEILSTEIN and MEDLINE among others.133 Isolated compounds from C. racemosa roots that have been reported in the literature are listed in table 3. The structures of some of these compounds are shown in figures 2 and 3.
29
Table 3. Compounds reported in the literature from C. racemosa roots based on NAPRALERT (percent yields in parentheses where available)
Isolate (yield) Reference Actaeaeposide-3-O-β-D-Xylopyranoside 134 Actein (0.0005-0.0156) 135, 136 Actein, 23-epi: 26-deoxy 137 Actein, 26(S): 137 Actein, 26-deoxy (0.0017) 137 Actein,2'-O-acetyl (0.0022) 138 Actein, deacetyl: 139 Acteol, 27-deoxy: acetyl: 140 Caffeic acid (0.01) 141 Caffeic acid methyl ester (0.00015) 142 Cimicifugic acid A 143 Cimicifugic acid A, dehydro: 144 Cimicifugic acid B 143 Cimcifugic acid B, dehydro 144 Cimicifugic acid C 145 Cimicifugic acid D 145 Cimicifugic acid E 143 Cimicifugic acid F 143 Cimicifugoside (0.0008) 139 Cimicifugoside H-1 (0.0034) 48 Cimicifugoside H-2 (0.01) 48 Cimicifugoside M (0.0005) 139 Cimicifugoside, 26-deoxy: (0.02) 138 Cimigenol, 12-β-21-dihydroxy: 3-O-α-L-arabinopyranoside 146 (0.00128) Cimigenol, 12-β-hydroxy: 3-O-α-L-arabinopyranoside 136 Cimigenol, 12-β-hydroxy: 3-O-β-D-xylopyranoside 136
Cimigenol,24(S): 3-O-α-L-arabinoside (0.0001) 48 Cimigenol,24(S): 3-O- β-D-xyloside (0.0009) 48 Cimigenol,25-anhydro: 3-O-α-L-arabinoside (0.0013) 138 Cimigenol,25-anhydro: 3-O- β-D-xyloside (0.0039) 138 Cimigenol,25-O-acetyl: 3-O-α-L-arabinoside (0.00024) 138 Cimigenol,25-O-acetyl: 3-O-β-D-xyloside (0.0079) 138
30 Table 3 (continued). Compounds reported in the literature from C. racemosa roots based on NAPRALERT (percent yields in parentheses where available) Isolate (yield) Reference Cimigenol, 25-O-acetyl-12- β-hydroxy: 3-O-α-L- 146 Arabinopyranoside (0.01) Cimigenol, 25-O-methyl: 3-O-α-L-arabinopyranoside (0.00196) 146
Cimigenol, 25-O-methyl: 3-O-β-D-xylopyranoside (0.01346) 147 Cimigenol-3-O-α -L-arabinopyranoside (0.00075) 146 Cimigenol-3-O-α-L-arabinoside (0.0019) 138 Cimigenol-3-O-β-D-xylopyranoside 134 Cimigenol-3-O-β-D-xyloside (0.0079) 138 Cimiracemate A (0.001) 142 Cimiracemate B (0.00006) 142 Cimiracemate C (0.00002) 142 Cimiracemate D (0.00002) 142 Cimiracemoside (0.0032) 148 Cimiracemoside A (0.05) 136 Cimiracemoside B 136 Cimiracemoside C 136 Cimiracemoside D 136 Cimiracemoside E 136 Cimiracemoside F (0.00076) 136 Cimiracemoside G (0.00019) 136 Cimiracemoside H (0.0009) 136 Cimiracemoside I (0.0008) 138 Cimiracemoside J (0.0019) 138 Cimiracemoside K (0.0025) 138 Cimiracemoside L (0.0018) 138 Cimiracemoside M (0.0016) 138 Cimiracemoside N (0.0079) 138 Cimiracemoside O (0.0013) 138 Cimiracemoside P (0.0013) 138 Cyclolanost-7-Ene, 9-19: 12-β-acetoxy-16-β-23;22- β -25- 147 diepoxy-3- β -23-24-trihydroxy: 3-O-β-D-xylopyranoside (0.0058) Cyclolanostan-3- β -ol, 9-19: 12- β -acetyl-oxy-16-β-23: 22-25- 146 diepoxy-23(R)-24(R)-dihydroxy: α-L-arabinopyranoside 22(R): (0.0003)
Cytisine,N-Methyl: 149
31 Table 3 (continued). Compounds reported in the literature from C. racemosa roots based on NAPRALERT (percent yields in parentheses where available) Isolate (yield) Reference Daucosterol-6'-linoleate (0.05) 138 Ferulic acid (0.01) 150 Formononetin* 94 Fukinolic acid 143 Glycerol-1-palmitate (0.013) 138 Isoferulic acid (0.07) Kaempferol* 151 Shengmanol, 23-acetoxy: 3-O-β-D-xyloside (0.22) 48 Shengmanol, 23-O-acetyl: 3-O-α-L-arabinopyranoside (0.0021) 152 Shengmanol, 23-O-acetyl: 3-O-β-D-xylopyranoside (0.01181) 147
Shengmanol, 23-O-acetyl: 3-O-β-D-xyloside (0.0019) 138 Shengmanol, 24-O-acetyl: (0.0012) 138 Shengmanol-3-O-α-L-arabinopyranoside (0.0012) 152
*-verification of these compounds from C. racemosa has not been confirmed
32 21 21 O OAc 24 22 O 18 OAc 18 24 20 27 20 27 23 25 23 25 17 17 19 O O 19 O O H OH H OH 26 26 H H 15 15 28 28 Xyl O Xyl O 30 29 30 29
actein (26S) actein (26R)
21 21 OAc O O 18 24 OAc 18 24 20 27 20 27 23 25 23 25 17 19 17 O O 19 O O H 26 H 26 15 15 28 28 Xyl O Xyl O 30 29 30 29
26-deoxyactein 23-epi-26-deoxyactein
21 21 O H 18 OAc 18 20 OAc 20 26 OH 17 17 19 O 19 O OH H H 15 O 27 15 28 OH 28 Xyl O Xyl O 30 29 30 29
cimiracemoside A 23-OAc-shengmanol-O-ß-D-xyloside
21 21 H 18 H O OAc 18 24 27 20 O 27 17 23 25 23 25 19 O R2 17 H 24 19 O O 15 O H 26 26
15 28 OH R O 28 1 Xyl O 30 29 30 29
26-deoxycimicifugoside R1= Xyl, R2= H, cimigenol-3-O-ß-D-xyloside (24S)
R1= Ara, R2=H, cimigenol-3-O-α-L-arabinoside (24S)
R1= Xyl, R2= Ac, 25-OAc-cimigenol-3-O-ß-D-xyloside (24S)
R1= Ara, R2= Ac, 25-OAc-cimigenol-3-O-ß-D-xyloside (24S)
Figure 2. Triterpenes isolated from black cohosh roots
33 O AcO O
O O O O
B-D-Xyl-O A-L-Ara-O
cimiracemoside I cimiracemoside N
O O AcO AcO 23R 23R O O O O O OH
Xyl-O 4'-O-acetyl-B-D-Xyl
cimiracemoside O cimiracemoside P
OAc O AcO 23R 24S 23R 24S O O O
OH OH RO RO
R= α-L-Ara, cimiracemoside J R=4’-O-acetyl-α-L-Ara, cimiracemoside L
R=ß-D-Xyl, cimiracemoside K R= 4’-O-acetyl-ß-D-Xyl, cimiracemoside M
21 21
18 18 HO 20 HO 20 OH O 17 O 17 O 19 O 19 O H H 15 OH 15 28 28 Xyl O Xyl O 30 29 30 29
cimicifugoside H-1 cimicifugoside H-2
Figure 2 (continued). Triterpenes isolated from black cohosh roots
34 O R1O CO2H R1 O CO2H R2O R2O HO CO2H R3 R1= H, R2= H, caffeic acid R = H, R = CH , isoferulic acid 1 2 3 OH R1= CH3, R2= H, ferulic acid
R1= OH, R2= H,R3=OH, fukinolic acid
R1= OH, R2= CH3, R3=OH, cimiciugic acid A
R1= OCH3, R2= H, R3=OH, cimicifugic acid B
R1= H, R2= H, R3=OH, cimicifugic acid C
R1= OH, R2= H, R3=H, cimicifugic acid D
R1= OCH3, R2= H, R3=H, cimicifugic acid E
R1= OH, R2= CH3, R3=H, cimicifugic acid F
O R3
O
O R1O OH
OR2 OH
R1= CH3, R2= H, R3=H, cimiracemate A
R1= H, R2= CH3, R3=H, cimiracemate B
R1= CH3, R2= H, R3=OCH3, cimiracemate C
R1= H, R2= CH3, R3=OCH3, cimiracemate D
Figure 3. Phenolic constituents that have been isolated from black cohosh roots
35 3.5.1 Triterpene Glycosides
Numerous chemical investigations of the roots of Cimicifuga have yielded
over 50 unique 9,19- cyclolanostane type triterpenes and glycosides.134, 135, 138, 140,
142, 146, 153-173 Actein was the first, discovered by Gennazi in 1952, as a result of his
experiments to explain the activity of Black Cohosh. Since that time over 40
triterpenes have been elucidated from C. racemosa.134, 136-139, 147, 148, 174, 175 The triterpene glycosides comprise approximately 0.5-2.0% of the dry weight of Black
Cohosh roots. The basic chemical composition of most of the triterpene glycosides is a 9,19-cycloartane triterpene skeleton, with different substitution patterns occurring. The position and variety of these chemical substitutions are further diversified by the stereochemical possibilities. Unique compounds like actein, which undergoes spontaneous mutarotation in solution, represent the cyclical or spiro- ketal variations of these constituents, which are of great interest to natural products hemists. Because of the distinctiveness of the triterpenes, and their relative abundance in commercially available extracts, they are generally regarded as the best choice as markers for chemical standardization.48,
144, 174
3.5.2 Phenolic Acid Derivatives
The cinnamic acid esters of fukiic and piscidic acid, fukinolic acid, cimicifugic acid, cimiracemates A-D, and cimicifugic acids A-F have been isolated from more-polar extracts and fractions of Black Cohosh roots. 142, 143, 150
Additional phenolic isolates include ferulic acid, isoferulic acid, caffeic acid and caffeic acid methyl ester. Esculetin, 4-O-acetyl-caffeic acid and sinapic acid have
36 been isolated from Asian Cimicifuga species, and are believed to be present in
C. racemosa on the basis of preliminary studies.176 Dehydrocimicifugic acid A,
and dehydrocimicifugic acid B were tentatively identified in C. racemosa by using
tandem LC-MS/MS.144
3.5.3 Flavonoids
Some debate surrounds the presence of the flavonoids biochanin A,
formononetin and kaempferol in the roots of Black Cohosh. An initial study in
1985, claimed the presence of formononetin in a lipophilic extract of the roots.94
An additional study in 1996 claimed to have found biochanin A in Black
Cohosh.177 The author of a study on isolation of kaempferol, later clarified that it
was only found in the aerial parts.151 Since those studies were published,
numerous other studies using more robust detection methods, have not detected
the purported flavonoids in the roots.48, 49 The presence of biochanin A and
formononetin would be considered significant because of their weak estrogenic
activity.
3.5.4 Miscellaneous
Fatty acids, tannins, resins, starches and sugars have also been reported
to occur Black Cohosh.132, 178 The sole report of the quinolizidine alkaloids cytisine
and N-methylcytisine from Black Cohosh is questionable, with no other reports of
these constituents found in Black Cohosh since that time.149 Concentrations of
the these alkaloids of Caulophyllum thalictroides (blue cohosh), a perennial herb
37 contained in dietary supplements marketed in the United States, have been well
established by analytical methods.179, 180 The teratogenic compounds anagyrine,
N-methylcytisine, baptifoline and taspine have been reported from Blue
Cohosh.179, 181 These constituents are known to interfere with the ability of a newly
fertilized ovum to implant in the uterus, damage the uterus and thyroid, and
cause severe birth defects in cattle and laboratory rats.181-189 Additionally, the compounds caulophyllosaponin and caulosaponin, appear to constrict coronary vessels, limiting blood flow to the heart and reducing its ability to pump.184, 186, 189
Population overlap with Black and Blue Cohosh is most likely responsible for this misidentification.182, 190
38
4.0 EXPERIMENTAL
4.1 PLANT MATERIAL
4.1.1 Procurement of Plant Material
The required National Park Service permits for collections in the Blue
Ridge Parkway and Great Smoky Mountains National Park are available in the appendix (section 10.3). Roots were separated from above ground parts on collection. Plant material was taken from the collection sites to the UIC
Pharmacognosy Field Station (Downers Grove, IL) to be washed, dried and milled to a coarse powder. Plant material was stored at the UIC Pharmacognosy
Field Station.
4.1.2 RAPD-PCR plant identification
Dried plant material (roots and aerial parts) was ground using a mortar and pestle under liquid nitrogen. Plant DNA extraction was carried out using the
QIAgen (Valencia, CA) DNeasy plant mini kit. The extraction procedure was based on the protocol provided by the supplier with modification to enhance overall yield of root material. Suspension and lysis buffers were added to the ground frozen plant material (10 mg), and the lysate was then applied to the
QIAshredder spin column to remove precipitates and cell debris by centrifugation. The flow-through fraction was then applied to the DNeasy mini spin column, centrifuged, washed, and eluted with the elution buffer (2 aliquots of
50 µL). Samples were all stored at -20ºC. The polymerase chain reaction (PCR)
39 was initiated by combining the following in each tube to make a total volume of
50 µL: 5 µL DNA template to PCR tubes (concentration and volume adjusted for
25 ng DNA in final volume of 50 µL), 37 µL DEPC water or nuclease-free water,
5 µL 10X Clontech (Mountain View, CA) Advantage 2 PCR Buffer, 1 µL 10 mM dNTP from invitrogen (Carlsbad, CA), 1 µL of working primer (1:10 stock primer solution, OPA-19: CAAACGTCGG, OPC-15: GACGGATCAG, OPA-05:
AGGGGTCTTG, Operon Ltd., [Huntsville, AL]), 1.0 µL Clonetech Advantage 2
Platinum Taq, final Volume is 50 µL. Then 50 µL Mineral Oil was added to each tube, vortexed and spundown. The positive control contained all of the above with 1.0 µL of Saccaromyces cerevisiae or mammalian DNA used as the template. The negative control contained all of the above without template DNA.
When preparing more than two samples, a master mix of the above ingredients was prepared without template DNA. Template was then added individually to each tube. Tubes were placed in a thermocycler (Perkin-Elmer (Wellesley, MA)
GenAmp PCR 2400) and run according to the following procedure: Repeat for 34 cycles: Denature: 94˚C for 1 min., Anneal: 36 ˚C for 1 min., Extension: 72˚C for 2 min. Final Extension: 72 ˚C for 10 min. All DNA was stored at -20˚C. Samples were run on a 2% agarose gel in 1X TBA containing Vistra Green NA Stain
(Amersham-Pharmacia Biotech, Inc. (Piscataway, NJ)). Loading dye was added to each sample in a ratio of 1:5 and 5.0 µL of each sample or control was added to a well. These samples were run with two wells that each contained 2.0 µL of
DNA ladder at 100 mV for 45 min to 1 hour. The resultant gel was digitally photographed under UV light.
40 4.1.3 Microscopic analysis
4.1.3.1 Light microscopy
The powdered plant material (BC 001) was placed on a microscope slide and treated with either water and washed with iodine to indicate the presence of starch, 10% phloroglucinol/HCl to indicate lignin, and/or chloral hydrate to verify calcium oxalate crystals.191 A cross sectional slice of roots was mounted on a slide with water. Cellular structures were documented by freehand drawings.
4.1.3.2 Scanning electron microscopic analysis
Powdered plant material (BC 001 and 010) and cross-sectional slices of dried plant material were placed on an SEM-aluminum stub with an adherent pad. Scanning electron microscope images were obtained on a JEOL-JSM 35C in secondary electron imaging (SEI) scanning mode with both platinum-palladium
(Pt-Pd) sputter coated using the secondary electron detector and uncoated stubs for observation in low vacuum mode. The waveform monitor was adjusted for the best exposure. The exposure settings were: RAPID 1, Photo horizontal @ 6, vertical @ 7, collector @ 5, load current 60-100 µA, magnification 1,000x-
10,000x.
41
4.2 CHROMATOGRAPHY
4.2.1 Solvent Partition and fractionation of Black Cohosh
HPLC grade methanol and acetonitrile were obtained from Fisher
Scientific (Fair Lawn, NJ) and used for all TLC and HPLC experiments. USP
grade (200 proof) ethanol was used for extracts. The bulk solvents chloroform, 1-
butanol, EtOAc, acetone and petroleum ether were distilled before use in
fractionation, column chromatography or TLC. Distilled, deionized, ultrafiltered milliQ™ water (Waters, MA) was obtained from the Bioassay Research Facility,
UIC College of Pharmacy (Chicago, IL).
4.2.2 Column Chromatography
For the isolation of compounds from the polar n-BuOH soluble fractions and the non-polar EtOAc fractions, either Amberlite XAD-2 (Sigma, St. Louis,
MO), MCI gel CHP20P (Mitsubishi, Tokyo, Japan), silica gel (Merck 60Å, 70-230 mesh), Sephadex LH-20 (Amersham Biosciences, Piscataway, NJ) and reverse phase HPLC were used to separate fractions. Once assay results were obtained for ‘active’ fractions, they were pooled or individually separated using one of the aforementioned techniques. The subfractions were subsequently submitted for bioassay. From this point due to the large generation of subfractions produced from these chromatographic techniques, the fractions were purified by semi- preparative RP-HPLC. All subfractions were analyzed by analytical RP-HPLC with inline PDA and ELSD detection.
42
4.2.3 Thin-layer chromatography (TLC)
Fractions and pure isolates were spotted on 0.25 mm thick pre-coated F254 normal phase glass-backed silica gel plates (20 x 20 cm; Merck, Darmstadt,
Germany). Plates were cut to 10 cm in length, and various widths. The solvent systems in table 4 were used. TLC chromatograms were scanned at 600 dpi (.tif file). Most TLC chromatograms were sprayed with either sulfuric acid (10% in
EtOH), ninhydrin, or anisaldehyde and subsequently heated by a heat gun or a
Fisher oven. Heating continued until spots ranging throughout the spectrum of available colors appeared on the white background of the plate. Additionally UV
(λ-254 and 365 nm) light was used for general chemical detection.
Table 4. Standard TLC Solvent Systems used for fraction control, isolation and structure elucidation.
SSE= EtOAc based
SSE6 EtOAc-MeOH 90:10 SSE7 EtOAc-MeOH-H2O 87:13 SSE8 EtOAc-MeOH-H2O 77:15:8 SSE9 EtOAc-MeOH-H2O 67:25:8
SSC= CHCl3 based
SSC6 CHCl3-MeOH-H2O 85:15:0.5 SSC7 CHCl3-MeOH-H2O 80:19:1 SSC8 CHCl3-MeOH-H2O 75:24:1 SSC9 CHCl3-MeOH-H2O 60:39:1
43 4.2.4 High-Performance Liquid Chromatography (HPLC)
HPLC was carried out using a Waters Delta 600 convertible unit with an inline 996 photodiode array detector (PDA), Waters 717 autosampler,
Millennium32™ chromatography manager, YMC-ODS AQ columns were used for both analytical (4.6 x 250mm, 5 micron) and semi-preparative (20 x 250 mm, 10 micron) purposes, as indicated. A Waters Delta-Pak RP-18 guard column was used for the analytical HPLC. An inline Sedex 75 Evaporative Light Scattering
Detector (ELSD) (Sedere, France) was used at variable nitrogen pressure and variable nebulizing temperature, with the output signal (mV) connected to the
Millennium32™ software through a SAT/IN analogue box. MeOH, ACN, both
HPLC grade were purchased from Fisher Scientific. TFA was purchased from
Sigma. Water was drawn from a Millipore reverse osmosis NANOpure™ unit.
HPLC conditions for analysis are summarized in table 5.
44 Table 5. Analytical HPLC-ELSD conditions and method parameters
Conditions for triterpene quantitative analysis (method A):
ELSD Nebulization Temperature : 43 °C Gain: 11 N2 Pressure: 3.4 Bar
Column: YMC ODS 250 x 4.6 mm, 5 micron Flow 1.6 ml/min
A- ACN, B-H2O, C-0.05% TFA/H2O
Gradient Elution (all gradient curves linear)
Time %A %B %C 0 20 80 8 20 80 15 32 68 55 64 36 65 85 15
Conditions for phenolic acid/polar constituent analysis (method B):
ELSD Nebulization Temperature : 61 °C Gain: 11 N2 Pressure: 2.9 Bar
Column: YMC ODS 250 x 4.6 mm, 5 micron Flow 1.0 ml/min
A- 0.05% TFA/5% MeOH/ H2O, B- 0.05% TFA/MeOH
Gradient Elution (all gradient curves linear) Time %A %B 0 97 3 50 25 75 61 25 75 62 10 90 67 10 90
45 4.2.4.1 Analytical HPLC analysis
Milled authenticated plant material (BC009) Black Cohosh roots collection
material was extracted with MeOH, 40, 60, 75% EtOH and 40% Isopropanol to
determine the optimal conditions for the clinical extract. Extracts were prepared
by exhaustively extracting ca. 1 g of plant material in 10 ml of the previously
mentioned solvents. Extracts were concentrated in vacuo until dry. A mass of
extract, usually 100 mg, was weighed in a 10 mL class A volumetric flask, 9 mL
of HPLC-grade MeOH was added. The sample was sonicated for 5 min. Each
sample was appropriately diluted then syringe filtered with 0.22 µm sterile filters.
Additionally, clinical extracts were provided in bulk by our collaborators from
BC009 for the Phase I clinical extract. Quantitative analysis of clinical samples
was performed in duplicate. Standards for quantitative analysis of the clinical
samples were made from serial dilutions of 1.0 mg/mL stock solution of 23-epi-
26-deoxyactein. A working stock was prepared by making a 1:5 dilution of the
stock solution in MeOH. The dilutions for the standard curve were prepared by
diluting the 400, 300, 200, 100, 50, and 25 µL of working stock solution in HPLC
MeOH to make the final volume 0.5 mL. The standards were syringe filtered
through 0.22 µm sterile filters. Standards were injected in duplicate. The dried
extracts were submitted for bioassay in the 5HT7 binding assay performed by
Project 2 of the Center. ELSD output is measured in mV, a power function based on droplets analyzed through the detector. Thus, the formula to calculate the percentage of 23-epi-26 deoxyactein in the samples is:
Log(c) = −m × Log(AUC) + b
46 Where c is the mass of sample per 10 µL injection, m is the slope of the standard curve, AUC is the area under the curve, and b is the y-intercept of the standard curve. From the standard curve the r2 value must be greater than 0.995,
or linearity between peaks cannot be assumed. Once this criterion was
established the other marker triterpene glycosides present in the extract, as
specified by the UIC Center, specifically 26-deoxyactein, actein-(26R) and actein-
(26S) were calculated as 23-epi-26-deoxyactein using a 1:1 ELSD response.192,
193 Retention times of the UIC Center marker compounds were determined by
running a mixed standard including 26-deoxyactein, actein-(26R) and actein-
(26S) were calculated as 23-epi-26-deoxyactein.
For assessing diurnal and geographical variation of Black Cohosh roots,
nineteen reference standards were used. The reference standard
cimiracemoside A was purchased from Chromadex Inc., (Laguna Hills, CA). The
reference caffeic acid was obtained from Sigma (St. Louis, MO). The reference
standards kaempferol and formononetin were obtained from Indofine Chemical
(Somerville NJ). The reference standards actein-(26S), actein-(26R), ferulic acid,
isoferulic acid, cimicifugoside H-1, cimicifugoside H-2, 26-deoxycimicifugoside,
23-epi-26-deoxyactein, 23-OAc-shengmanol-3-O-β-D-xyloside, 26-deoxyactein,
25-OAc-cimigenol-3-O-α-L-arabinoside, 25-OAc-cimigenol-3-O-β-D-xyloside,
cimigenol-3-O-α-L-arabinoside, cimigenol-3-O-β-D-xyloside were isolated and
purified from Cimicifuga racemosa roots. An HPLC-ELSD chromatographic trace
of the standards is detailed in figure 4. The identities of the isolates were
characterized by means of spectral (NMR and MS) analyses and/or X-ray
47 200. 200. 400. 600. 400. 600. 00 00 mV 0. 0. 0. 0. 00 00 00 00 00 00 00 00
0. 0. 0. 0. 0. 0. (24 x xyloside(24 cimigenol-3-O- ß-D-xylos 10) 26-deoxycimic cimiracemoside A,7)formononetin,8) ferulic acid,3)isoferulic4) compounds, elutedusingmethodA.The Figure 4 00 00 00 00 00 00 y loside R ,25 5. 5. 5. 5. 5. 5. 00 00 00 00 00 00
R 1 ), 15)25-OAc-cimigenol i . HPLC-ELSDchromatogramus de, 13)23- 10. 10. 10. 10. 10. 10. S 00 00 00 00 00 00
), 18)ci 2
α 3 15. 15. 15. 15. 15. 15. -L-arabinos 00 00 00 00 00 00 i fugoside, 20. 20. 20. 20. 20. 20. migenol-3- epi 00 00 00 00 00 00 -26-deoxyactein (24 25. 25. 25. 25. 25. 25. ide(24
44 11) actein(26 00 00 00 00 00 00 O 30. 30. 30. 30. 30. 30. 5 R - 00 00 00 00 00 00 6 α ), 17)25-OAc-cimigenol-3-O-ß-D- 7 -L-arabinoside, 19)cimigenol-3-O-ß-D- -3-O-ß-D-xylos
cimicifugoside H-2, 8 M M M M M M 48 35. 35. 35. 35. 35. 35. i i i i i i nut nut nut nut nut nut 9 cimicifugoside H-1,9)actein(26 00 00 00 00 00 00 es es es es es es 10
compounds are:1)caffeicacid,2) 11
S 1212 40. 40. 40. 40. 40. 40. ing themixedstandardof19 ), 12)23-OAc-shengmanol-3-O- 1313 00 00 00 00 00 00 S ,25 45. 45. 45. 45. 45. 45. 14 ide(24 00 00 00 00 00 00 S ), 14)26-deoxy 50. 50. 50. 50. 50. 50. 00 00 00 00 00 00 R 5) kaempferol,6) ), 16)25-OAc- 55. 55. 55. 55. 55. 55. 1515 00 00 00 00 00 00 1616 60. 60. 17 60. 60.
60. 17 60. 17 00 00 00 00 00 00 a 65. 65. 65. 65. 65. 65. ctein R 00 00 00 00 00 00 1818
), 19 70. 70. 70. 70. 70. 70. 00 00 00 00 00 00
crystallography. The purity was characterized by HPLC. The structures of these
reference standards are shown in figures 2 and 3 (pages 33-35). All reference
standards were dissolved in 10 mL of methanol at ranges of concentrations from
0.10–0.50 mg/mL. Serial dilutions were prepared as needed for the standard
curve. Standard stock solutions were stored at -20 ˚C and brought to room
temperature before use. Fourteen wild-crafted root collections (BC001, 005, 007,
010, 012, 014, 031, 034, 036, 038, 072, 089, 090, 092) were extracted exhaustively with MeOH, concentrated in vacuo to yield dried, powdered extracts. Each sample was prepared by dissolving approximately 100.0 mg ± 5.0 mg in 10 mL of MeOH, and syringe filtered with a 0.22 µm filter. Again, the
formula to calculate the percentage of the nineteen standards in the samples is:
Log(c) = −m × Log(AUC) + b
Where c is the mass per 10 uL injection, m is the slope of the standard curve, AUC is the area under the curve, and b is the y-intercept of the standard curve. From the standard curve the r2 value must be greater than 0.995, or linearity between peaks cannot be assumed.
49
4.2.4.2 Semi-Preparative HPLC
Semi preparative HPLC to purify compounds was performed using a
YMC-ODS AQ (20 x 250 mm, 10 µm) column. Once a retention time and the
corresponding elution conditions were established for a peak of interest in the
aforementioned analytical gradient elution method, an isocratic analytical method
was developed to optimize the separation and resolution of the peak/constituent
of interest. Once the isocratic gradient conditions were established, the equations
in figure 5 were used for scaling up the preparative column and determining the
appropriate flow rate and concentration of analyte.194 The amount of sample injected on to the column was dependent on the solubility of the analyte in the injection solvent and the relative polarity of the injection solvent comparable to the mobile phase. If the injection solvent is substantially more or less polar in comparison, the column will be volume overloaded and the sample resolution will broaden at injection. In an isocratic system this broadening problem, is not improved as it can be in gradient systems. Thus, for optimal resolution the injection solvent should be slightly less polar than the chromatographic mobile phase in a reverse phase HPLC system.195
50 L1
r1 ƒ1 Column 1-Analytical
L2
r2 ƒ2
Column 2-Preparative
2 ƒ1 r1 x1 x2 1 = 2 2 = 2 • ƒ2 r2 π •r1 π •r2 CL
flow= ƒ, column radius=r, column length =L, concentration or amount= x, ratio of column lengths= L2/L1= CL
Figure 5. The calculations necessary for preparative HPLC scale-up method development from analytical HPLC. The optimal flow rate is determined from an analytical HPLC method, this rate is used to determine the sample concentration and flow rate for the optimization of preparative HPLC.
51
4.3 SPECTROSCOPIC METHODS
4.3.1 Nuclear magnetic resonance spectroscopy
NMR spectra were recorded on Bruker Avance™(Billerica, MA) 300, 360,
400 or 500 MHz instruments where indicated. Dr. David C. Lankin in the
Department of Medicinal Chemistry and Pharmacognosy maintained the 300 and
400 MHz instruments. The 360 and 500 MHz instruments were maintained by Dr.
Robert Kleps of the Research Resources Center (RRC, UIC). Spectra were
referenced internally to TMS (Pyridine-d5, MeOH-d4, DMSO-d6). Offline
processing was performed with either NUTS™ (NMR utility transform software;
Acorn NMR Inc.) or MestR-C™ (www.mestrc.com). Line resolution of
experimental data was enhanced by Lorentz-Gauss (LG) transformation where
indicated. Online data processed with Bruker Software. Simulation experiments
to determine relative stereochemistry were carried out with Perch (Kuiopo,
Finland) and ACD™ software (www.acdlabs.com). NOESY relaxation delay time
was D1=1.5 seconds, mixing time D8=0.60 seconds.
4.3.2 Mass spectrometry
Drs. R.B. van Breemen and D. Nikolic (Core C of the UIC/NIH Center) in the UIC Botanical Center, Project 3, herein provided all mass spectra, including exact mass measurements and tandem mass spectra. Nominal mass, exact mass and LC-MS data were obtained using a high-resoultion Waters Micromass
(Manchester, UK) Q-Tof2 resolution hybrid orthogonal angle time-of-flight tandem
52 LC mass spectrometer equipped with electrospray ionization (ESI) and featuring
a quadrupole mass filter and collision cell. Argon was used as the collision gas.
The LC system was a Waters Alliance™ HPLC. Mass spectra were analyzed
using Masslynx™ software version 3.4.
4.3.3 Other Techniques
Optical rotations were measured with a Perkin-Elmer™ 241 polarimeter
(Perkin-Elmer, Inc. MA.). UV spectra were obtained with a Beckman™ DU-7
Spectrophotometer. Samples were loaded into a quartz cell (path length 1.0 cm).
FTIR spectra were obtained with a Jasco™ 4390 (Tokyo, Japan) equipped with a
Golden Gate™-ATR.
4.4 BIOLOGICAL ACTIVITY
Drs. Judy L. Bolton, J.E. Burdette, J. Liu, R. Ruhlen and C. Celimine in the
UIC Botanical Center, Project 2, herein provided all biological activity data.
4.4.1 Estrogenic Activity
Percent estrogenic values at given concentrations, for both α- and β-
estrogen receptors (ER) were determined by Project 2 within the center, for
extracts, chromatographic fractions and purified compounds.47 Two distinct estrogen receptors in our bodies: ER-α and ER-β. While they both bind estrogen as well as other agonists and antagonists, the two receptors have distinctly different localizations and concentrations within our body. Structural differences
53 also exist between the two allowing for a wide range of diverse and complex processes to take place. The cell binding assay to assess the binding affinities of botanical extracts followed a standard pattern. The receptors are infused with
17β-estradiol, and a binding curve is obtained based on varying concentrations of
17β-estradiol present. Then, the extract in question is added in varying concentrations, acting as a competitive inhibitor, and its ability to bind is plotted against 17β-estradiol.
4.4.2 Serotonin (5-HT) Binding Activity
Either IC50 values or percent inhibition of receptor binding in transfected human CHO- cells for fractions and compounds were determined by personnel in
Project 2 of the Center.121 Serotonin activity was first assessed by the inhibition of radioligand binding to cell membrane preparations containing recombinant human serotonin receptor (5-HT) subtypes. The botanical extracts, fractions or purified compounds in question are assayed for competitive binding against
[3H]lysergic acid diethylamide (LSD) in human 5-HT7 receptor or [3H]8-hydroxy-
2-(di-N-propylamino)tetralin to the rat 5-HT1A receptor. Ligand binding data was then analyzed to determine if the extract, fraction or purified compound behaved as a competitive ligand of the serotonin receptor (5-HT) subtypes.
4.4.3 Other Biological Activities
AP induction, PS2 induction on Ishikawa cells and antioxidant activity
(DPPH) were provided by personnel in Project 2.47, 196
54 5.0 RESULTS
5.1 PLANT PROCUREMENT
Twenty-seven wild collections of C. racemosa roots and aerial parts were
made from N. Carolina to Pennsylvania. In addition, 20 collections of C.
americana and C. rubifolia were gathered in order to validate the chemical and
genetic authenticity of the C. racemosa collections. Collection records included
date of collection, genera, species, part, mass of collection (grams), collection
location and GPS coordinates, where indicated, are presented in table 6. Due to
overlap in distribution of C. racemosa and C. americana, morphological field
identification was essential for distinguishing collections. The major
morphological characteristic in the flower spike to distinguish between C.
racemosa and C. americana is that C. americana possesses three glabrous pistils vs. only one in C. racemosa; and the seeds are covered with broad, lacerate scales vs. no scales in C. racemosa.
Initial collections made in June of 1999 with assistance of the North
American botanical expert for the genus, G.W. Ramsey, Ph.D. Voucher specimens are housed at the Searle Herbarium, Field Museum of Natural History
(Chicago, IL) and the Ramsey-Freer Herbarium, Lynchburg College (Lynchburg,
VA).
55
Table 6. Botanical Center Database Summary of Black Cohosh and related species collections (PX=aerial parts, RR=roots).
Accession Date Genera Species Part Mass in Location number grams (GPS coordinates) BC001 6/28/1999 Cimicifuga racemosa RR 4827 Rockbridge County, Va. (37 48.27 N & 79 18.67 W)
BC002 6/28/1999 Cimicifuga americana RR 175 Rockbridge County, Va. BC003 6/29/1999 Cimicifuga rubifolia RR 177 Scott County, Va. BC004 6/29/1999 Cimicifuga rubifolia RR 280 Hancock County, Tn. BC005 6/29/1999 Cimicifuga racemosa RR 2106 Sevier County, Tn. (35 38.48 N & 83 28.47 W) BC006 6/30/1999 Cimicifuga americana RR 95 Sevier County, Tn. BC007 6/30/1999 Cimicifuga racemosa RR 800 Sevier County, Tn. BC008 6/30/1999 Cimicifuga americana RR 165 Swain County, NC. BC009 5/30/1999 Cimicifuga racemosa RR 10173 Somerset Co., PA BC010 8/26/1999 Cimicifuga racemosa RR 4359 Sevier County, Tn. BC011 8/26/1999 Cimicifuga americana RR 87 Sevier County, Tn. BC012 8/26/1999 Cimicifuga racemosa RR 829 Sevier County, Tn. BC013 8/26/1999 Cimicifuga americana RR 214 Swain County, NC. (35 35.30 N & 83 21.52 W)
BC014 8/26/1999 Cimicifuga racemosa RR 721 Swain County, NC. BC015 8/27/1999 Cimicifuga rubifolia RR 75 Scott County, VA, BC016 8/27/1999 Cimicifuga rubifolia RR 95 Scott County, VA, BC017 8/26/1999 Cimicifuga racemosa PX 498 Sevier County, Tn. BC018 8/26/1999 Cimicifuga racemosa PX 336 Sevier County, Tn. BC019 8/26/1999 Cimicifuga americana PX 167 Sevier County, Tn. BC020 8/26/1999 Cimicifuga americana PX 52 Swain County, NC. BC021 8/27/1999 Cimicifuga rubifolia PX 71 Scott County, VA, BC022 8/27/1999 Cimicifuga rubifolia PX 86 Scott County, VA, BC023 6/29/1999 Cimicifuga rubifolia PX 97 Scott County, Va. BC024 6/29/1999 Cimicifuga rubifolia PX 171 Hancock County, Tn.
56 Table 6 (continued). Botanical Center Database Summary of Black Cohosh and related species collections
Accession Date Genera Species Part Mass in Location number grams
BC025 6/29/1999 Cimicifuga racemosa PX 259 Rockbridge County, Va.
BC026 6/29/1999 Cimicifuga racemosa PX 351 Sevier County, Tn.
BC027 6/29/1999 Cimicifuga racemosa PX 111 Sevier County, Tn.
BC028 6/28/1999 Cimicifuga americana PX 88 Rockbridge County, Va.
BC029 6/30/1999 Cimicifuga americana PX 160 Sevier County, Tn.
BC030 6/30/1999 Cimicifuga americana PX 150 Swain County, NC.
BC031 6/22/1999 Cimicifuga racemosa RR 1471 Butler Co., PA
BC032 6/22/1999 Cimicifuga racemosa PX 650 Butler Co., PA
BC034 10/16/1999 Cimicifuga racemosa RR 295 Westmorland Co., Cook Townshi p, PA.
BC035 10/16/1999 Cimicifuga americana RR 844 Westmorland Co., Cook Townshi p, PA.
BC036 10/16/1999 Cimicifuga racemosa RR 710 Elk township, PA.
BC037 10/16/1999 Cimicifuga racemosa RR 582 Somerset, Co.,Summit Townshi p PA.
BC038 10/18/1999 Cimicifuga racemosa RR 2098 Butler Co., Little Buffalo Townshi p, PA.
BC043 5/30/1999 Cimicifuga racemosa PX 791 Somerset, Co.,PA
BC072 5/7/2000 Cimicifuga racemosa RR 5711 Somerset Co., PA
BC089 11/7/2000 Cimicifuga racemosa RR 452 Bedford Co. ,PA
BC090 11/7/2000 Cimicifuga racemosa RR 235 Bedford Co. ,PA
BC091 11/7/2000 Cimicifuga americana RR 32 Forbes State Forest, PA. BC092 11/7/2000 Cimicifuga racemosa RR 320 Rockbridge County, Va.
BC093 11/7/2000 Cimicifuga americana RR 37 Rockbridge County, Va.
BC094 11/7/2000 Cimicifuga racemosa RR 23510 Forbes State Forest, PA. BC095 11/7/2000 Cimicifuga racemosa RR 123 Sevier Co. TN, Smokey
BC102 4/16/2001 Cimicifuga racemosa RR 170000 Somerset Co, PA
57 In addition, C. americana flowers from August until October in the southern
states, roughly one month after C. racemosa (June-September).56, 57 With climate changes flowering characteristics are not always readily available, thus non- flowering characteristics must be used, the most distinguishing of which is the scar present on the stem at the point of branching on C. americana.56, 57 The roots
of C. americana also appear to be yellow and relatively smaller when compared
with the larger, whiter roots of C. racemosa. Roots were separated from aerial
parts on collection. Asian Cimicifuga species, C. acerina (BC164) (Sieb. Et
Zucc.) Tanaka, C. simplex (BC165) (DC.) Wormsk. ex Tursk., C. heraclefolia
(BC167) Kom., and C. dahurica (BC166) (Turcz) Maxim, were provided through
Dr. Norman R. Farnsworth, who secured the material through Toyama Medical
and Pharmaceutical University (Toyama, Japan) where voucher specimens are
maintained.
5.2 RAPD-PCR ANALYSIS
Initial studies to verify the species C. racemosa in comparison with the
other Eastern North American species (C. rubifolia and C. americana) have been
previously reported by the author and collaborators.197 This work is shown in
figures 6 A & B. Additional RAPD-PCR work was used to differentiate the Asian
Cimicifuga species (usually C. simplex), believed to be substituted for
C.racemosa in commercial products for economic reasons, as well as
C.americana, commonly named, yellow or white cohosh, which grows alongside
C. racemosa in wild populations. 192,56, 57 This work was undertaken, based on
58 information from members of the American Herbal Pharmacopoeia, believed
commercial Asian Cimicifuga material was being mislabeled and sold to
manufacturers as C. racemosa.198, 199 With the use of different primers, we were
able to distinguish the C. racemosa clinical extract starting-material provided by
our commercial collaborator, Pure World Botanicals, from the Asian Cimicifuga
species and C. americana. The PCR profiles are pictured in figures 7 A, B & C.
We conclude from figure 7 that the commercial root sample (BC163) was not
adulterated with any of the Asian species C.simplex, C. dahurica, and C. acerina.
The commercial sample, BC163, is consistent with C. racemosa. Possibly, the
commercial sample (BC163) may represent a subspecies or another subtype of
C. racemosa. This may explain the slight PCR band differences between the
authenticated material collected and used in this study by the UIC/NIH Center, in
contrast to the commercial material provided by our collaborators. Although there
is no report in the literature, these data match the observations made.
It was difficult to reproduce results using RAPD-PCR on root/rhizome
material. Extraction was performed using Qiagen’s plant DNA extraction kit,
which only provided DNA from Pure World’s C. racemosa (BC163) C. racemosa
(BC001), and C. americana leaf (BC030). A small quantity of DNA was available
from C. acerina (BC164), C. simplex (BC 165) and C.dahurica (BC166). The
DNA quality from these samples proved insufficient to produce reliable PCR
profiles. As a net result, it was extremely difficult to visualize high-resolution
profiles of poor quality DNA on an electrophoresis gel. The RAPD profiles of the commercial sample (BC 163) are consistent with those of C.racemosa (BC001).
59 DNA extraction is the limiting step. Unfortunately, only root material of the commercial sample (BC163) was available. The problems we observed in obtaining quality DNA from stored, hard pieces of root material match the experiences and reports of other groups.200
These data indicated that none of the Asian species of Cimicifuga that were tested, could be found in the C. racemosa material, which was the purpose of the investigation from the outset. Only primers OPA-05, OPC-15 and OPA-19 were used and therefore a more thorough analysis is needed using several more primers to ensure that these primers are not amplifying regions of the plant that are identical to all species.
60 Figure 6. RAPD-PCR of N. A American Cimicifuga species
6A. RAPD-PCR profiles of Cimicifuga species amplified by OPA-10 primer. Lanes 1- 6, C. racemosa; Lanes 7-14, C. americana; lanes 15-20, C. rubifolia. M-100 bpdna ladder
6B. RAPD-PCR profiles of Cimicifuga species amplified by OPA-15 primer. Lanes 1- 3, C. racemosa; Lanes 4-6, C. americana; lane 7 commercial C. racemosa & lane 8 C. rubifolia, M-100 bp DNA ladder, x- unknown
M 1 2 3 x M 4 5 6 M 7 8 B
61 100bp ladder - + + 12345 6 A Figure 7. PCR profiles utilizing different primers to distinguish C. racemosa from potential adulterants. The following samples were assayed: commercially supplied roots (BC163), authenticated C. racemosa roots (BC001), authenticated C. simplex roots (BC 165), authenticated C. dahurica roots (BC 166), authenticated C. acerina roots (BC 164), authenticated C. americana leaf (BC030).
7A. RAPD-PCR profiles of Cimicifuga species amplified by OPA-05 primer. M + - + 121 2233 M Lane 1- C. acerina, 2-C.heraclefolia, 3- C. dahurica, 4- C. simplex, 5- C. B racemosa, 6- Pure world C. racemosa material, +-positive control dna (S. cerevisae and ), - -negative control
7B. RAPD-PCR profiles of Cimicifuga species amplified by OPC-19 primer. Lane 1- C. americana, 2- C.racemosa, 3- Commercial C.racemosa material+- positive control DNA (S. cerevisae and ), - -negative control
7C. RAPD-PCR profiles of Cimicifuga species amplified by OPC-15 primer. M 12 333 4 5 - + M Lane 1-C. dahurica, 2-C. simplex, 3- C. americana, 4-C. racemosa, 5- C Commercial C. racemosa material +- positive control DNA (S. cerevisae), - - negative control
62
5.3 MICROSCOPY
5.3.1 Light microscopy
The following characteristics were observed in light microscopy:
parenchyma, trachea in secondary xylem, broad-wedge shaped medullary rays,
wood fibers, tabular cells, simple and compound, starch grains.58 These are
shown in figure 8 below. No distinct microscopic features of either coarsely-
powdered, finely-milled, or cross sectional slices of eastern North American
Cimicifuga species could be determined to distinguish one from another.
phloem
cortex a
epidermis a
medullary rays xylem
endodermis
b
b
Figure 8. Freehand microscopic drawings a) Cross sectional drawing of root/rhizome (BC001); b) root/rhizome (BC001) vessels and fibers.
63 5.3.2 Scanning-electron microscopy (SEM)
These aforementioned cell structural characteristics were also identified by scanning electron microscope (SEM). The advantage of SEM was in the magnification strength, up to 10000 times, to observe the cortex and starch granules in the coarse-ground samples (BC010). From the finely-milled powder phloem fibers were observed. SEM pictures representing these structures are presented in figure 9. Scanning electron microscopy is a central tool for identification of ground plant material, where no voucher specimen is available. It was important in this study to identify some of the morphological characteristics of Cimicifuga spp. at the cellular level.
64 A B
SG
C D
E F PF
G H RE
Figure 9. Scanning electron microscope pictures in decreasing magnification (5A-5C and 5F-5H are coarsely ground plant material (BC010), 5D & 5E are finely ground material (BC001)). Observed characteristics include phloem fibers (PF), starch granules (SG), root/rhizome exodermis (RE)
65 5.4 CHROMATOGRAPHY
5.4.1 Solvent extraction and partitioning to yield fractions for triterpene/standard isolation
The initial fractionation of C. racemosa was necessary for the development of less polar fractions and the subsequent isolation of triterpene glycosides to be used as marker compounds for the clinical extract. The dried, milled roots of C. racemosa (8 kg, BC001) were exhaustively extracted with
MeOH. The MeOH extract was concentrated in vacuo to yield 1250 g of a syrup residue. A sample (625 g) of the residue was suspended in water-MeOH (9:1,
1500 mL) and fractionated by successive partitions with EtOAc (2000 mL x 3) followed by n-BuOH (2000 mL x 5, sodium sulfate was added to the separatory funnel to enhance separation) to produce EtOAc-soluble (262 g) and n-BuOH- soluble fractions (100 g). This fractionation is detailed in figure 10 (page 67).
While the directive of this fractionation was to yield a large, relatively non-polar,
EtOAc-soluble fraction, to yield as many triterpene constituents as possible to properly identify and characterize a clinical black cohosh extract all resultant fractions (EtOAc, BuOH and H2O-soluble) were bioassayed. The 5-HT7 bioassay results indicated that the BuOH-soluble fraction produced from this separation demonstrated the greatest activity of all of the extracts and fractions generated by the Center at that time.201
66 C. racemosa roots (BC001) ca. 8.0 kg, MeOH ext. (1250 g) IC50 14.0 µg/mL, conc. in vacuo, re-suspended H2O/MeOH (9:1)
EtOAc
EtOAc (262 g) Mother Liquor IC50 21.0 µg/mL
BuOH Aqueous IC50 4.78 µg/mL 14% inhibition
Figure 10. A) Solvent extraction and partitioning of plant material to yield polar fractions for isolation from the butanol soluble fraction. 5HT7 bioassay data reported with the respective fractions. B) Fractionation of black cohosh roots for isolation of non-polar constituents from the EtOAc-soluble fraction. 5-HT7
5.4.2 Solvent extraction and partitioning of plant material to yield polar fractions
The previously published activity of the BuOH-soluble extract, led to the selection of the BuOH-soluble extract as the best candidate for further separation and isolation efforts.121 In order to increase the yield of the BuOH-soluble fraction
CHCl3:IPR (3:2) was used to initially partition the MeOH soluble extract.
The dried, milled roots of C. racemosa (3.75 kg, BC010) were further milled to a fine powder by a tissue homogenizer (ultra-turrax) and extracted with
MeOH (10 x 8,000 mL). The crude organic extract was concentrated in vacuo (<
40˚ C), to yield ca. 1.1 kg of a syrupy residue, the residue was reconstituted in d.d.i. H2O (1:9, 4 x 2000 mL) and subsequently partitioned with CHCl3:IPR (3:2, 4 x 2000 mL) and BuOH (4 x 2000 mL), to give >500 g and 150 g of CHCl3-soluble
67 and BuOH-soluble extracts, respectively. The fractionation scheme with bioassay
results is detailed in figure 11. The bioassay data that was generated for these
C.racemosa fractions showed decreased activity of the resultant BuOH-soluble fraction.
C. racemosa roots (BC010) ca. 3.75 kg, MeOH ext. (900 g) 39% inhibition, conc. in vacuo, re-suspended H2O/MeOH (9:1)
CHCl3/IPR
CHCl3/IPR Mother Liquor 44% inhibition
BuOH (150 g) from 40+ L Aqueous 35% inhibition 0% inhibition
Figure 11. A) Solvent extraction and partitioning of plant material to yield polar fractions for isolation from the butanol soluble fraction. 5HT7 bioassay data reported with the respective fractions bioassay data reported with the respective fractions.
68 5.4.3 Amberlite XAD-2, Second Level of Separation
For the first chromatographic separation of the n-BuOH soluble fraction,
150 g of the butanol-soluble extract from BC010 was loaded to 1.735 kg of
Amberlite XAD-2 (Sigma) Column. Column specifications are as follows: i.d. 50 mm, length: 1200mm, total volume: 2400 mL, density of XAD-2: 0.736 g/cm3 .
The column was washed with acetone until no residue was present upon evaporation, then it was subsequently washed with MeOH and water at a flow rate of 5 ml/min regulated by the MPLC Isco (Lincoln, NE) unit. The syrupy butanol-soluble extract (138 g) was suspended and sonicated in 600 mL of d.d.i. water and added to the top of the column. The columns was resealed at both ends, inverted and eluted with water, water with increasing percentages of
MeOH, 100% MeOH, MeOH with increasing acetone, and 100% acetone. This yielded 10 unique fractions, F01-F10 as determined by TLC and HPLC-ELSD.
The results are summarized in figures 12 and 13 (pages 70, 71). Bioassay data for these fractions indicated that the best candidates for further fractionation were
F05-F07.
69 step a 01-75-15 (150 gms .)
01-90-15 % 5HT 7 binding @ 100 µg/ml F-01 0 01-90-16 F-02 0
01-90-17 F-03 15.7
01-90-18 F-04 32.4
01-90-20 F-05 83.2
01-90-21 F-06 79.3
01-90-22 F-07 90.4
01-90-23 F-08 72.3
01-90-24 F-09 49.8
01-90-25 F-10 64.4
Figure 12. Summary of XAD-2 fractionation and bioassay results. Step a is the elution of the column with water, water with increasing percentages of MeOH, 100% MeOH, MeOH with increasing acetone, and 100% acetone. 01-75-15 is the butanol-soluble fraction from Figure 9. B on the TLC refers to the same entity. IFA and FKA represent isoferulic acid and fukinolic acid standards, respectively.
70 BuOH Fraction
F-01
F-02
F-03
F-04
F-05
F-06
mV F-07
F-08
F-09
F-10
Cimicifugic acid A
Fukinolic acid
Isoferulic acid
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 13. HPLC-ELSD chromatograms of the resultant XAD-2 fractions (F-01 through F-10), the fractions are overlayed with cimicifugic acid, fukinolic acid and isoferulic acid standards to show the relative polarity of the fractions, the capacity of XAD-2 to separate the polar butanol-soluble fraction, and served as a level of ad-hoc dereplication. Fraction concentrations ca. 10 mg/mL ran by method B. Injection volume was 10 µL.
71 5.4.4 MCI gel CHP20P
For the third level of separation of the n-BuOH soluble fraction (from
BC010), MCI gel® CHP20P was selected. The initial study to test the feasibility of this resin was on 4.3 grams of F-08 dissolved in 40 mL of 20% aq. MeOH into
100 g of a CHP20P MPLC column at a flow rate of 1 ml/min. The summary of the pilot column is depicted in figure 14. Column specifications for the pilot column are as follows: i.d. 20 mm, length: 500 mm, total volume: r2h= 157079.5 mm3 or
157.1 cm3, density of CHP 20P: 0.636 g/cm3. The sample was eluted with 30%
MeOH, at a 10% gradient until it reached 100% MeOH, then eluted with 100%
EtOH and 100% acetone, subsequently.
For the scaled-up column, the resin was initially washed with acetone followed by EtOH, MeOH, and d.d.i. water. The wash flow-through was evaporated in vacuo until no CHP20P residue eluted from the column. After the column wash was completed. Fractions F-05 to F-07 (8.6 g) were resuspended in
5% MeOH and loaded onto 400 gm of CHP20P. The extract was eluted with water, water with increasing percentages of MeOH, 100% MeOH, MeOH with increasing acetone, and 100% acetone. This yielded 35 unique fractions (G01-
G35) as determined by TLC fraction control and HPLC-ELSD. These results are summarized in figures 15 and 16. The HPLC-ELSD results of the resultant G- fractions, show that the use of CHP20P, the separation followed a ‘predictable’ pattern. The earlier fractions possessed primarily more polar, unknown constituents, gradually the pattern shifted to predominately the known phenolic acid esters (i.e. fukinolic acid). Because of the large number of fractions, it would
72 have been a poor use of resources to obtain bioassay data on all fractions.
Chemical screening of resultant fractions was used to better characterize the fractions. The use of LC/MS provided a targeted screening strategy that indicated the possible presence of compounds possessing an odd number of nitrogens in the resultant fractions, these fractions were then selected to pursue for further isolation work. Additional LC-MS chromatograms of fractions possessing compounds with an odd number of nitrogens are depicted in figure 17 and the appendix (section 10.2).
73 step a 01 - 75 - 15 (150 gms .)
01 - 90 - 23 F - 08 (4.3 g)
21-23 02- 21 - 23 F - 11 (1.25 g)
02 - 21 - 24 21-24 F - 12 (1.08 g)
02 - 21 - 25 21-25 F - 13 (0.63 g)
21-26 02 - 21 - 26 F - 14 (0.04 g) 21-27 02 - 21 - 27 F - 15 (0.26 g) 21-28 02 - 21 - 28 F - 16 (0.24g) 21-29 02 - 21 - 29 F - 17 ( 0.05 g)
02 - 21 - 30/31 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Minutes F - 18 (0.02 g)
90-2321-23 21-24 21-26 21-25 21-27 21-28 21-29 21-30 21-31 90-23
Figure 14. Pilot column CHP20P separation summary of F-08 (72.3 % inhibition of 5HT-7 at 100ug/ml). The HPLC and TLC results of the resultant pilot column CHP20P fractions show the proficiency of the resin to provide a means for separating a complex polar fraction. These results led to the selection of CHP20P as the stationary phase for further large scale separation.
Step a-2.0 kg XAD-2 eluted with water, aq. MeOH, MeOH, Acetone Step b-100 gm CHP20P eluted w/ water, aq. MeOH, MeOH, EtOH, acetone HPLC-Method B TLC-Anisaldehyde, SSC 7 74
3 distinct fractions (F-05 through F-07), over 10 distinct spots (Anisaldehyde SSC8) 1 2 3 4 5 6 7 8 9 10
ca. 35 distinct fractions (g-1 through g-36), over 100 distinct spots (UV-254, SSC8)
Figure 15. Large Scale Separation CHP20P separation of 01-90-20 ,-21 and -22 (F-05 through –07, 8.6 g) by 400 gm CHP20P eluted w/ water, aq. MeOH, MeOH, EtOH, Acetone. The ‘active’ XAD-2 fractions were further separated by CHP20P. The complexity of the resultant fractions, on the 3rd level of separation can be seen by the large number of distinct spots on the UV(254) developed TLC plate.
75 2
3
4
5 mV 7
9
10
11
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 16. HPLC-ELSD results of CHP20P separation. Fractions G-2, 3, 4, 5, 7, 9, 10, 11 by method B. The chromatograms show the complexity of the third level fractions, and the potential for isolation of new constituents from C. racemosa. Fraction concentration ca. 5 mg/mL. The separation followed a ‘predictable’ pattern. The earlier fractions possessed primarily more polar, unknown constituents, gradually the pattern shifted to predominately the known phenolic acid esters (i.e. fukinolic acid).
76 12
13
14
15
mV 16
17
18
19
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 16 (continued). HPLC-ELSD results of CHP20P separation. Fractions G-12 through - 19 by method B. The chromatograms show the complexity of the third level fractions, and the potential for isolation of new constituents from C.racemosa. Fraction concentration ca. 5 mg/mL. The separation followed a ‘predictable’ pattern. The earlier fractions possessed primarily more polar, unknown constituents, gradually the pattern shifted to predominately the known phenolic acid esters (i.e. fukinolic acid).
77 21
22
23
24
mV 25
26
27
28
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 16 (continued). HPLC-ELSD results of CHP20P separation. Fractions G-21 through -28 by method B. The chromatograms show the complexity of the third level fractions, and the potential for isolation of new constituents from C. racemosa. Fraction concentration ca. 5 mg/mL. The separation followed a ‘predictable’ pattern. The earlier fractions possessed primarily more polar, unknown constituents, gradually the pattern shifted to predominately the known phenolic acid esters (i.e. fukinolic acid).
78
29
30
31
32
mV 33
34
35
36
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 16(continued). HPLC-ELSD results of CHP20P separation. Fractions G 29-36 by method B. The chromatograms show the complexity of the third level fractions, and the potential for isolation of new constituents from C. racemosa. Fraction concentration ca. 5 mg/mL. The separation followed a ‘predictable’ pattern. The earlier fractions possessed primarily more polar, unknown constituents, gradually the pattern shifted to predominately the known phenolic acid esters (i.e. fukinolic acid).
79 Sample g7 on amide column CRJULY150405A TOF MS ES+ 9.87 166 100 166.1 1.45e3
% 16.12 130.1 2.90 229.1 0 CRJULY150405A TOF MS ES+ 10.35 186 100 186.1 2.40e3
%
12.01 186.1 0 CRJULY150405A TOF MS ES+ 13.52 172 100 172.1 8.70e3 Relative abundance
%
0 CRJULY150405A TOF MS ES+ 13.52 TIC 100 172.1 1.04e4
3.23 15.07 2.90 337.1 154.1 229.1 % 9.21 10.35 12.85 3.87 4.25 314.1 14.30 186.1 11.29 136.1 163.0167.15.19 6.22 7.82 330.2 16.99 344.2 19.30 254.1 210.1 246.2 20.67 132.1 316.2 316.2 0 Time 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Minutes
Figure 17. LC-MS screening for N-bearing compounds possessing an odd number (n=1,3,5,…) of nitrogens. Random chemical screening by LC/MS indicated the possible presence of compounds bearing an odd-number of nitrogen atoms in fraction G-7, indicated by the even m/z in positive mode. With a large number (over 30) of 3rd level fractions bioassay-guided fractionation would be a poor use of resources, thus this technique proved useful to identify fractions for isolation of potentially new constituents, as a method of dereplication.
80 5.4.5 Sephadex LH-20
Gel permeation chromatography offered by Sephadex LH-20 was selected for the third level of separation in parallel with MCI gel CHP20P. Gel permeation chromatography is a particular type of liquid/liquid chromatography for the separation of substances according to their differing molecular sizes. The column was washed with MeOH and H2O. Sample was dissolved in aq. MeOH and added to the gravimetric column through a 0.45 micron paper filter. Column specifications for the sephadex LH-20 column are as follows: i.d. 20 mm, length:
1000 mm. F-12 (0.5 g) was loaded onto 50 gm of Sephadex LH-20 and eluted with 100% MeOH. Five combined fractions were observed; SF-1 (0.05 g), SF-2
(0.15 g), SF-3 (0.11 g), SF-4 (0.07 g), and SF-5(0.02 g). Results and HPLC-
ELSD of resultant fractions are summarized in figure 18.
81 F-12 (0.5 g)
SF-1
SF-2
SF-3 SF1 SF2 SF3 SF4 SF5 SF-4 SF-5
Cimicifugic acid A
Fukinolic acid Isoferulic acid
mV SF-1
SF-2
SF-3
SF-4
SF-5
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 Minute Figure 18. Fractionation and TLC of Separation of F-12 (02-21-24) by 50 g of Sephadex LH-20 eluted with MeOH HPLC-ELSD of Sephadex LH- 20 separation of F-12 (02-21-04) by method B compared with standards of cimicifugic acid, fukinolic acid and isoferulic acid. Fraction concentration ca. 5 mg/mL.
82
5.4.6 Silica Gel Separation
For the initial separation of the EtOAc soluble fractions to generate
triterpene standards (figure 10, page 67) silica gel column chromatography (SiO2,
2.0 kg) was used. Column specifications are as follows: .i.d. 60 mm, length: 1000
mm, total volume: 2800 mL, density of silica gel: 0.708 g/cm3. A portion of the
EtOAc-soluble fraction (262 g) was eluted with CHCl3, CHCl3-MeOH (10%),
CHCl3-MeOH (20%), CHCl3-MeOH (30%), CHCl3-MeOH (40%), CHCl3-MeOH
(50%), CHCl3-MeOH (75%), and MeOH (100%) to yield eight combined fractions:
S-1 (oil), S-2 (3.0 g), S-3 (1.2 g), S-4 (2.3 g), S-5 (23 g), S-6, S-7 (60 g), S-8 (120 g) as depicted in figure 22 as previously cited.137 Column cuts were taken every
500 mL until the resultant eluent was colorless. The approximate volume eluted was 35 L. These eight fractions were used to produce thirty (30) compounds by project 1 of the Center.137, 138, 142 Further separation of fraction S-4 (2.3 g) afforded; glyceryl-1-palmitate (5 mg, 0.0013% of dried plant material); daucosterol-6'- linoleate (400 mg, 0.05%). In addition to the novel isolates 4',23-O-
diacetylshengmanol-3-O-α-L-arabinopyranoside, cimiracemoside M (6.1 mg,
0.0016%); 4'-O-acetyl-26-deoxyactein, cimiracemoside O (5.0 mg, 0.0013%);
4',23-O-diacetylshengmanol-3-O-β-D-xylopyranoside, cimiracemoside L (7.0
mg, 0.0018%): 2'-O-acetylactein (7.0 mg, 0.0022%), cimiracemate A (8 mg),
cimiracemate B (5 mg), cimiracemate C (2.1 mg) and cimiracemate D (1.8
mg). Fraction S-5 (23 g) was used to produce 23-O-acetylshengmanol-3-O-α-L- arabinoside (8.1 mg, 0.0021%), 23-O-acetylshengmanol-3-O-β-D-xyloside (7.3
83 mg, 0.0019%), cimigenol-3-O-β-D-xyloside (24 mg, 0.0063%), cimigenol-3-O- -L- arabinoside (7.1 mg, 0.0019%), cimigenol-3-O-β-D-xyloside (6 mg, 0.0016%), 26-
deoxycimicifugoside (3.0 mg 0.00079%), 25-anhydrocimigenol-3-O-β-D-xyloside
(30 mg, 0.0039%), 25-anhydrocimigenol-3-O-α-L-arabinoside (5.1 mg, 0.0013%),
25-O-acetylcimigenol-3-O-β-D-xyloside (40 mg, 0.01%), 25-O-acetylcimigenol-3-
O-α-L-arabinoside (30 mg, 0.0079%), cimicifugoside H-2 (8 mg, 0.0021%), cimicifugoside H-1 (13 mg, 0.0034%), 26-deoxyactein (210 mg) 40 mg of actein
(R,S) and the novel isolates 12-O-acetyl-25-anhydrocimigenol-3-O-α-L- arabinopyranoside, Cimiracemoside J (7.3 mg, 0.0019%), 12-O-acetyl-25-
anhydrocimigenol-3-O-β-D-xylopyranoside, Cimiracemoside K (9.4 mg,
0.0025%); 16-β:23;24:25-diepoxy-12-O-β-acetyl-3-hydroxy-9,19-cyclolanost-
23,26-olide-O-β-D-xylopyranoside, Cimiracemoside P (5.1 mg, 0.0013%); 23-
epi-26-deoxyactein (5.1 mg), 7-dehydro-23-epi-12,26-dideoxyacteol-3-O-β-D-
xylopyranoside, Cimiracemoside I (3.2 mg, 0.0008%), and 23-epi-acetylacteol-
3-O-α-L-arabinopyranoside, cimiracemoside N (30 mg, 0.0079%). The structural
data for the fourteen new constituents (in bold type above) from these fractions,
are available in the appendix (section 10.1).
84 5.5 ISOLATION OF CONSTITUENTS
5.5.1 Isolation of 1 (cimipronidine)
Compound 1 was isolated by semi-preparative RP-HPLC from fraction G-
15 resulting from the CHP20P fractionation (figure 13, page 71) of the combined
XAD-2 fractions F-5 through F-7 (figure 15, page 75) Thus, compound 1 was isolated from the fourth level of separation of the butanol-soluble fraction.
Isolation of 1 occurred following elution with 10% aqueous methanol. The analytical HPLC-ELSD separation of G15 (figure 16, page 77) indicated 5 major peaks. Eight peaks were collected as indicated in figures 19 and 20 (page x).
The PDA was used as a detector, the scan setting was from 190-410 nm. Six injections of 50 mg of fraction G-15 in 300 µL of MeOH were made. Column flow rate was 10 ml/min, HPLC stationary phase was 10% MeOH. Compound 1 was eluted isocratically. The six injections yielded a total of 25 mg of compound 1.
85
Wash out 1234 567 8 (mostly fukinolic acids) 350.00
300.00 nm
250.00
200.00
10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes Figure 19. Semi-preparative RP-HPLC isolation of 1 (cimipronidine-P2) from fraction G-15 , PDA detector scan from (300 mg-6 runs), flow rate- 10.0 ml/min, isocratic 10% MeOH. Dead time of 2.0 minutes was used to reduce dead volume.
86 1
2
3 4 mV 5
6
7
8
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 20. Qualitative analytical HPLC-ELSD results of semi-preparative HPLC- ELSD of separation of fraction G-15, peaks 1 through 8. Chromatograms reveal additional peaks, than that of the initial HPLC-ELSD separation. Additional peaks were realized following preparative separation of fraction G-15 than were apparent from the initial HPLC-ELSD analysis (figure 16, page 77).
87 5.5.2 Isolation of 2 (fukinolic acid)
Compound 2 was isolated by semi-preparative RP-HPLC of fraction G-15 from the CHP20P fractionation (figure16, page 79) of the combined XAD-2 fractions F-5 through F-7 (figure 13, page 77). Thus, compound 2 was also isolated from the fourth level of separation of the butanol-soluble fraction from figure 11 (page 68). Isolation of 2 occurred when G-15 was eluted with 10% aqueous methanol. Fukinolic acid eluted approximately 25 min after cimipronidine from the same g-15 subfraction. Many polar compounds and plant extracts are routinely separated by RP-HPLC with small volumes of acid or base
(0.1-1.0%) added to improve retention time and peak shape of pH-sensitive analytes such as polyphenolics and alkaloids, respectively. in this instance, added TFA reduced the retention time of the highly polar material. Potentially complicating the effort further, complexation between nitrogenous constituents and highly conjugated phenolic acids have been previously reported.202, 203 The analytical HPLC-ELSD separation of G-15 (figure 14) indicated 5 major peaks.
Eight peaks were collected as indicated in figures 19 and 20 (pages 86, 87). A total of 65 mg of 2 were collected from the semi-preparative HPLC. Additional fukinolic acid was isolated in the production of fraction G-19 (Figure 16) from
CHP20P (31 mg).
5.5.3 Isolation of 3 (actein-(R/S))
Compound 3 was isolated by normal phase silica gel chromatography.
Compound 3 was eluted with CHCl3:MeOH (6:4), fraction S-6 from figure 22
88 (page 87). A total of 1.47 g of compound 3 was isolated from silica gel column chromatography, the 2nd level of fractionation of the EtOAc soluble fraction (figure
10, page 67). Actein was isolated as a mix of the 26-R and 26-S anomers
(stereoisomers). The yield of R and S was determined to be 20 and 80% respectively by HPLC-ELSD as shown in figure 21. This mixture was subjected to clean-up by semi-preparative RP-HPLC using a isocratic ACN:0.05% TFA in H2O
(65:35) mobile-phase, with a flow rate of 16.0 mL/min. A chiral column was not used. A flow splitter to utilize the ELSD for detection was graded at 10%. The
ELSD parameters were run according to method A. The additional preparative
HPLC step offered no further purification of the anomers due to mutarotation of the actein E ring in solution.
26-S
26-R
Figure 21. Isocratic analytical HPLC-ELSD of actein (26-R) and actein (26-S). Eluted with ACN:0.05% TFA in H2O (60:40) mobile-phase at 1.0 mL/min. flow rate. ELSD parameters according to method A.
89
5.5.4 Isolation of 4 (isoferulic acid)
Compound 4 was isolated by normal phase silica gel chromatography.
Compound 4 was eluted with CHCl3:MeOH (5:5), fraction S-7. A total of 1.34 g of compound 4 was isolated by silica gel column chromatography, the 2nd level of fractionation of the EtOAc soluble fraction shown in figure 10 (page 67).
90 5.6 CHARACTERIZATION OF ISOLATED COMPOUNDS
5.6.1 Characterization of 1 (cimipronidine)
The high-resolution exact mass measurement of compound 1, gave a protonated molecule of m/z 172.1014 indicating a molecular formula of
13 C7H14N3O2. Seven C-NMR resonances established the presence of a single carboxylic acid functionality (δC 179.826) and a single imine functionality (δC
154.573), the latter of which was confirmed by the MS/MS fragment ion of m/z
+ 13 130.0887 [MH-CN2H2] (figure 22). Both of the low field C NMR signals were shown to be quaternary carbon resonances (figure 23). One methine resonance and four methylene resonances were present in the APT 13C-NMR spectrum.
These assignments were confirmed by the gated 13C NMR experiment. (figure
24). Since there are no additional sites of unsaturation, the third double-bond equivalency requires a ring. HMBC data obtained for 1 failed to reveal all of the correlations necessary for full assignment of the structure. Contrary to expectations, no HMBC correlations could be observed between C-5 and the protons at H-2, H-3, and H-4. In addition, the expected correlations between C-2 and the protons H-3 and H-4b were undetectable as well (figure 25). Missing
HMBC correlations have previously been noted in the course of elucidation of guanidine structures.204 The absence of these correlations combined with broad, undefined peaks in the proton spectrum, the exceptions being the signals for H-
8a and H-8b, presented a difficult problem in terms of structure elucidation.
A probable explanation for the absence of cross peak correlations from the HMBC spectrum of 1 is the combination of the zwitterionic nature of the
91 2 compound 1 in conjunction with the fact that (i) typical JH,C couplings are generally small in magnitude and (ii) for the expected 3-bond correlations (C-5,
H-2) and (C-5, H-3) the dihedral angle between these protons and the C-5 carbon approach 90o and, therefore, the expected correlation are too weak or completely absent from the spectrum. Further complicating the situation is the chemical exchange phenomenon associated with the guanidine unit as well as the rapid fluctuations associated with the 5-membered ring, which are known to occur. Appropriate HMBC correlation cross peaks between the methylene protons at C-8 and carbons C-5 (two-bond) and C-4 (three-bond) were observed, while the expected HMBC correlations cross peaks used for defining the 5- membered ring were absent. These observations are consistent with the proposed structure. At this point the ACD database was used to generate potential structures that best fit the available data.205 Gradient HSQC data further confirmed the carbon and proton shift assignments of C/H-3 as well as C/H-4.
The C-4 protons appear as two broadened multiplets in the 1D-1H experiments
(D2O) at ~1.97 and ~2.20 ppm and are obscured by the two-proton multiplet around 2.15 ppm of the C-3 protons (figure 26). The 1H-1H-COSY data showed the following correlations, indicating abundant long-range coupling in the molecule and, thus, confirming a cyclic structure: H-2 (a,b) with both H-3 (a,b) and H-4 (a,b); H-3 (a,b) with H-2 (a,b), H-4 (a,b), and H-5; H-4 (a,b) with H-2
(a,b), H-3 (a,b), and H-5; H-5 with H-3 (a,b), H-4 (a,b), H-8a, and H-8b; and H-8a and H-8b with H-5 (figure 27).
92 The gradient NOESY results (D2O) provided further evidence consistent with structure 1. In addition to the presence of strong cross-peaks between the
C-2, C-3, C-4, and C-8 geminal protons, correlations consistent with vicinal relationships were observed between H-5 and both C-8 protons, as well as between H-5 and the C-4 protons. This is consistent with structural features inferred from the observed gHMBC data. In addition, there were strong cross- peaks between both of the C-2 protons and the 2H multiplet of the H-3 protons
(H ~2.15). Weaker cross-peaks were observed between one of the C-8 protons
(the higher field doublet-of-doublets at H 2.410) and both C-4 protons. H-5 exhibited a weak cross-peak in the gNOESY spectrum to the high-field C-2 proton multiplet (H 3.594). Its geminal partner, the low-field multiplet at H 3.467, showed a weak cross-peak to the low-field C-8 proton (doublet-of-doublets at H
2.692). A summary of the observed NOESY data obtained in D2O, except for the geminal NOE correlations, is shown in figure 28 and is consistent with proposed
1 structure 1. The H-NMR data for (1) in D2O permitted observation of all the protons except for overlapping C-3 protons and the low-field multiplet of one of the C-4 protons, which could be differentiated by the gHSQC, gNOESY, and gCOSY results. This served to confirm the relative stereochemistry of 1. The
1 absence of the guanidine protons in the H-NMR spectrum of 1 in D2O is clearly attributed to exchange of the guanidine protons with deuterium of the solvent.
Following the tentative assignment of structure 1, additional 1H-NMR experiments were performed on a dilute solution of 1 in DMSO- d6, in order to further confirm the presence of the guanidine moiety shown in figure 29. The spectrum showed
93 two (2H) broadened singlets centered at δH 9.291 and 7.828, respectively attributed to the guanidine zwitterions. A 1H- NMR spectrum of dilute 1 in DMSO- d6 with added trifluoroacetic acid (TFA), showed a broad singlet with all guanidine protons appearing at δH 13.578. Relevant NMR data for 1 are summarized in table 7. Off-white powder (H2O); [α]D: 36.21 (c 0.1), H2O; UV
-1 (H2O)λmax (log ε):285 (1.04), 333 (1.62) nm; IR (ITR-neat) νmax (cm ) 3365,
1631.
Compound 1 was given the trivial name cimipronidine({1-
[amino(imino)methyl]pyrrolidin-2-yl}acetic acid), unique to plants. The novel β- amino acid type structure, exhibiting zwitterionic behavior is not typical of the previously reported guanidine structures obtained from plants.206-208 Most of these guanidines have been shown to possess longer aliphatic side chains.206-208
94 100 172.1014 [M+H]+
CO H 2 CO2H N N N H H NH H2N 2 e
% 130.0933 [M-CN2H2] bundanc A e v i 70.0584 [Pyrolidine] lat e R
154.1106 [M-H2O] 94.0658 112.0831 113.0794 137.0804
0 m/z 70 90 110 m/z 130 150 170
Figure 22. High resolution positive-ion electrospray tandem mass spectrum of the protonated molecule of m/z 172.1014 (cimipronidine (1)) obtained utilizing a quadrupole time-of-flight mass spectrometer.
95 179. 154. 57. 41. 31. 22. 47 .
123 213 289 682 361 78 68 0 5 C - 5
ppm C O C N 2 H 3 H 3 C - 8 C - 2 C C - - 4 3
13 Figure 23. C-NMR APT of cimipronidine (1) in D2O (500 MHz). The low field 13C NMR signals were shown to be quaternary carbon resonances. One methine resonance and four methylene resonances were confirmed with a gated-carbon NMR experiment.
96 y it C-3 360 MHz). The ( O ity for each carbon. C-4 2 D ) in 1 he multiplic 97 C-8 rmation that was used to confirm the multiplic
C-2 C-NMR spectrum of ( 13
. Gated C-NMR experiment revealed t 13
C-5 gated of the carbon chemical shifts from the APT experiment. Figure 24 Important structural info
H H - - 3 4 a a
H H - - H H H H H 8 8 a b - - - - - 2 2 4 3 5
a b b b
0
C-3 C-4 C-8 C-2 50 C-5
100
150
ppm (t1
5.00 4.50 4. 00 3.50 3.00 2.50 2.00 ppm ppm (t2)
Figure 25. HMBC of cimipronidine (1) in D2O (360 MHz) no HMBC correlations could be observed between C-5 and the protons at H-2, H-3, and H-4. In addition, the expected correlations between C-2 and the protons H-3 and H-4b were also undetectable.
98
20.0 3
25.0
4 30.0
35.0
8 40.0
2 45.0
50.0
5 55.0
4.00 3.50 3.00 2.50 2.00
Figure 26. HSQC in D2O (400 Mhz). Gradient HSQC data confirmed the carbon shifts. Additionally, the HSQC experiment was used to confirm the chemical shifts of both H-4 protons and both H-3 protons, which appeared as a two broad singlets in the 1D-1H experiments with the peak at 1.75 ppm to be one proton by integration and the peak at 1.97 ppm to be 3 protons by integration .
99
1.50
2.00
2.50
3.00
3.50
4.00
4.00 3.50 3.00 2.50 2.00 1.50
1 1 Figure 27. H- H gCOSY spectra of (1) in D2O (400 MHz). The gCOSY shows the contiguity and connectivity of the ring. The H- H-COSY data showed the following correlations, indicating abundant long-range coupling in the molecule and, thus, confirming a cyclic structure: H-2 (a,b) with both H-3 (a,b) and H-4 (a,b); H-3 (a,b) with H-2 (a,b), H-4 (a,b), and H-5; H-4 (a,b) with H-2 (a,b), H-3 (a,b), and H-5; H-5 with H-3 (a,b), H-4 (a,b), H-8a, and H-8b; and H-8a and H- 8b with H-5.
100 H N H H Hb O O H 8 5 N N H 1 H 4 H H 2 Ha b 3 H
H a H NOE
Figure 28. The NOESY correlations of 1 (tmix = 1.5 seconds, D2O, 400 MHz) except for the observed geminal NOE contacts, which are left out to improve the clarity of presentation.
101 34 9 2 5 COOH 1 N 8
6 7 1 HN NH2
A
A
B
B
10 9 8 7 6 5 4 3 2 1 ppm
4a+4b 3a 8a 8b 5 3b 2a 2b
4.0 3.5 3.0 2.5 2.0 ppm
1 Figure 29. H-NMR spectra of 1 in d6-DMSO (A, B) and D2O (C) demonstrating the broad chemical shifts, the pH variability of NMR solvents and the presence of guanidine protons and the proton multiplicities of all resonances (D2O), respectively. The data also illustrate why D2O is the preferred NMR solvent for dereplication of 1 and its congeners.
102 1 13 Table 7. H and C NMR data of cimipronidine (1) (400/500 and 125/100 MHz, respectively, D2O and DMSO-d6)
a a 1 1 δC mult δH δH mult. [H] J [Hz] H- H COSY Position (D2O) [C] (D2O) (DMSO-d6) (D2O) (D2O) (D2O) 3.594 (2a) 3.36 (2a) ddd/mc 2.5 (3), 7.6 (3), 10.4 (2b) 2 47.31 CH 3a, 3b, 4a, 4b 2 3.467 (2b) 3.25 (2b) ddd/mc ~1 (3), 9.3 (3), 10.4 (2a) 2.17 (3a) 1.91 (3a) 3b 31.13 CH m 2a, 2b, 4a, 4b, 5 2 2.13 (3b) 1.87 (3b)
103 4b 2.20 (4a) 1.92 (4a) 22.61 CH m 2a, 2b, 3a, 3b, 5 2 1.97 (4b) 1.68 (4b) ~1 (4),~5.4 (8a), ~7 (4), 8.4 5 56.99 CH 4.331 4.163 dddd/mc (8b) 6 154.57 C – – – – 2.692 (8a) 2.272 (8a) dd 5.4 (5a), 15.1 (8b) 8 41.02 CH 5 2 2.410 (8b) 2.127 (8b) dd 8.4 (5b),15.1 (8a) 9 179.83 C – – – –
a H,C correlations established by HSQC experiment b The determination of δH from the 1D spectrum was hampered by the broadening of the resonances and by the overlap in the AB-type signal of protons H-3 and 4. However, the HSQC data gave correlations that permitted the determination of the chemical shifts of these protons. c Apparent ddd and dddd multiplicities, respectively, under first-order assumptions. However, additional long-range coupling and higher order effects give rise to a more complex signal multiplicity.
5.6.2 Characterization of 2 (fukinolic acid)
The proton-NMR resonance signals at δH 7.72 and 6.42 (each 1H, d, J
=16.1 Hz) indicated the presence of a trans-ethylene group conjugated with the
aromatic ring. Two groups of typical ABX spin system signals for 1,2,4-tri-
substituted aromatic ring protons were observed the first at δH 7.10 (1H,d J=2.3
Hz), 7.01 (1H, dd, J=2.3, 8.3 Hz), and 6.80 (1H,d J=8.3 Hz). The second at δH
6.74 (1H,d J=8.0 Hz), 6.64 (1H,d J=2.0 Hz), and 6.59 (1H,dd J=2.2, 8.2 Hz). One methylene signal at δH 3.08 (d, J=13.5 Hz) and the other δH 2.95 (d, J=13.5 Hz) were consistent with the expected proton signals for fukinolic acid (figure 30).
The 13C-NMR spectra showed 20 carbon atoms, signals ascribable to the trans-
ethylene group at δC 115.32 (C-2’’’) and 146.85 (C-3’’’), as well as two signal groups for 1,2,4 tri-substituted aromatic rings at δC 127.79 (C-1’’’), 115.32 (C-2’’’),
146.85 ( C-3’’’), 149.83 (C-4’’’), 116.55 (C-5’’’),123.25 (C-6’’’); with the other ring
signals δC 128.04 (C-1’ ), 118.83 (C-2’), 145.71 (C-3’), 145.30 (C-4’), 115.94 (C-
5’) and 123.05 (C-6’) depicted in figure 31. Additionally, the spectra also showed
one methylene carbon δC 42.22 (C-4), two carboxylic groups δC 174.78 (C-4),
170.90 (C-1) and one esterified group δC 168.29 (C-1’’). The NMR spectral data
compiled in table 8 are were consistent with the literature values.143, 159, 209
104 2''' 3'' HO 3''' 1''' 2''
1'' 6''' HO 4''' OO 5''' 2 1 HO CO2H 3 CO2H 5 2' 4 3' OH 1' 4' 6' OH 5'
H-4a/4b
7 6 5 4 3
H-5’’’ H-5’ H-6’’ H-2’’ H-2’’’ H-2’
H-6’’’
ppm 7.00 6.50
Figure 30. Compound 2 (fukinolic acid) 500 MHz 1H-NMR spectrum in MeOH- d4. Chemical shifts indicate the presence of two (2) tri-substituted aromatic rings.
105
150 100 50 ppm
5’ 2’’’
6’’’ 3’’ 6’ 5’’’ 1’ 3’ 2’ 2’’ 3’’’ 1’’’ 4’’’ 4’
4’’’
150. 140. 130. 120. ppm
Figure 31. Compound 2 (fukinolic acid) 500 MHz 13C-NMR spectrum in MeOH-d4. The data confirmed the presence of two tri-substituted aromatic rings as indicated in the 1H-NMR experiment.
106
Table 8. Spectroscopic 13C and 1H-NMR data of fukinolic acid (500 MHz, MeOD-d4)
Position signal δ H δ C m J (Hz)
1 C (COOH) - 170.90 - -
2 CH 5.65 78.28 s -
3 C - 80.44 d 15.89
4 CH2 2.95a, 42.22 d 13.5 3.08b d 13.5
5 C (COOH) - 174.78 - -
1’ C - 128.04 - -
2’ CH 6.74 118.83 d 2.2
3’ C - 145.71 - -
4’ C - 145.30 - - 5’ CH 6.64 115.94 d 8.2
6’ CH 6.59 123.05 dd 8.2, 2.2
1’’ C - 168.29 - -
2’’ CH 6.42 114.26 d 16.0
3’’ CH 7.72 148.22 d 16.0
1’’’ C - 127.79 - -
2’’’ CH 7.10 115.32 d 2.3
3’’’ C - 146.85 - -
4’’’ C - 149.83 - -
5’’’ CH 6.80 116.55 d 8.3
6’’’ CH 7.01 123.25 dd 8.3, 2.3
107 5.6.3 Characterization of 3 (actein (26-R and S))
The high-resolution exact mass measurement of compound 3, gave a m/z
703.3704 indicating a molecular formula of C37H58O11Na (figure 32). From the
1H-NMR spectrum, the cyclopropane methylene signals (H-19) of 3 were
observed at δH 0.21 and 0.55 (each 1H, d, J = 4.1 Hz). The spectrum also
showed seven methyl groups at δH 0.77 (H-28), 0.97 (H-21, J =6.45 Hz), 0.98 (H-
30), 1.31 (H-29), 1.35 (H-18), 1.71 (H-27), and 2.11 (-OAc), the anomeric proton
at 4.85 (H-1’, J = 7.6 Hz) and the distinctive hemi-acetal signal δH 5.74 (H-26,
split due to stereoisomeric-anomeric mixture, one R and one S), indicative of
spontaneous interconversion in solution, also known as mutarotation as is seen
in figure 33. The 13C and DEPT NMR spectra of 3 showed signals ascribable to
four oxygen-bearing methine carbons at 88.1 (C-3), 77.1 (C-12), 73.01 (C-16),
and 63.47 (C-24), two oxygen-bearing quaternary carbons at 105.85 (C-23) and
67.16 (C-25). With the field strength of the 500 MHZ instrument two hemi-acetal peaks were distinguishable at δC 98.45 (C-26 a) and (C-26 b) for the anomers actein (26-S) and (26-R), respecitvely as seen in figure 34. The spectra also
posessed five oxygenated carbons assignable to the xylopyranose moiety 107.5
(C-1’), 75.6 (C-2’), 78.7 (C-3’), 71.3 (C-4’), 67.1 (C-5’) and an acetyl group
(170.6, 20.7 for -OAc). The NMR spectral data compiled in table 9 matches the
literature reference values.137
108 Relative Abundance
time-of-flight massspectrometer. protonated moleculeof Figure 32. Highresolutionpositive-ionelec m/ z 685.3549(actein(
109 m/z
tros pray tandemmassspectrumofthe 3 )) obtainedutilizingaquadrupole
H - 2 6
a
&
b B A O H H H - - - 2 2 H H A - 2 7 1 H c - - 1 3 8 - 2 8 0 A 9 H-1’-anomeric
B
Figure 33. Proton NMR spectrum of actein (3) in pyridine-d5 (500 MHz). The distinctive hemi-acetal signal H-26 is seen at the top. The spectrum also showed seven methyl groups (A), the anomeric proton at 4.85 (H-1’, J = 7.6 Hz) (B).
110
ppm 2 2 6 6 S R
ppm
13 Figure 34. C-NMR spectra of actein (3) in pyridine-d5 (500 MHz) instrument two hemi-acetal peaks were distinguishable at δC 98.45 (C-26 a) and (C-26 b) for the anomers actein (26-S) and (26-R).
111 1 13 Table 9. Anomeric actein (26-R and 26-S) H and C NMR data in Pyridine-d5 (500 MHz). The data represents the 26-R anomer as the more abundant constituent. However the signal at 26 indicates a doublet representing mutarotation position signal δ H δ C m J (Hz)
1 CH2 1.10, 1.54 31.92 m,m -
2 CH2 1.88, 2.19 30.21 m,m -
3 CH 3.44 88.10 dd 4.4, 11.2
4 C - 41.23 - - 5 CH 1.22 47.01 m -
6 CH2 0.77, 1.45 20.40 m,m -
7 CH2 0.92, 1.21 25.71 m,m -
8 CH 1.61 45.66 m -
9 C - 19.55 - -
10 C - 26.80 - -
11 CH2 1.20, 2.69 36.71 m, dd 9.1, 16.0
12 CH 5.09 77.10 dd 3.9, 8.6
13 C - 47.88 - -
14 C - 48.81 - -
15 CH2 1.50, 1.68 43.62 m,m -
16 CH 4.59 73.01 dd 7.4,14.7
17 CH 1.76 56.44 m -
18 CH3 1.35 13.52 s -
19 CH2 0.21, 0.55 29.50 d,d (4.3), (4.0) 20 CH 1.84 26.12 m -
21 CH3 0.97 21.65 d 6.45
22 CH2 1.65, 2.22 37.67 m -
23 C - 105.85 - -
24 CH 3.94 63.47 s -
25 C - 67.16 - -
26 CH 5.74 98.45 d 4.6
27 CH3 1.71 15.32 s -
28 CH3 0.77 19.51 s -
29 CH3 1.31 25.71 s -
30 CH3 0.97 15.32 s -
1’ CH 4.85 107.56 d 7.5
2’ CH 4.02 73.63 t
3’ CH 4.15 78.67 t 8.7
4’ CH 4.21 71.28 dd 5.25, 9.24
5’ CH2 3.73, 4.38 67.15 t, dd (10.2), (5.1, 11.2)
-OAc Ac 7.01 170.54, 21.02 dd 8.3, 2.3
112 5.6.4 Characterization of 4 (isoferulic acid)
13 The C NMR spectral data at δC 148.41(C-3) and 118.41 (C-2) was indicative of a trans-ethylene group. One signal group was ascribable as a 1,2,4 tri-substituted aromatic ring the conjugated quaternary signals at δC 150.98 (C-7),
148.84 (C-6), 129.11 (C-4), and the conjugated methine resonances at δC 121.49
(C-9), 115.67 (C-8), 112.42 (C-5). Additionally, the δC169.84 (C-1) was
ascribable to a carboxyl group, with the methoxy- group at δC 56.05 as depicted
in Figure 42. The proton resonances for the trans-ethylene group were observed
at δH 8.34 (H-3, J=15.9) and 7.12 (H-2, J=15.89). The conjugated ring proton
shifts were 7.89 (H-5, J=8.6), 7.42 (H-9, J=8.3), 7.18 (H-8, J=8.3) with the
methoxy- shift at 3.59. NMR data are compiled in table 10. 1H-and 13C-NMR data
are consistent with data in the literature.143
113
9 3 1 HO 4 CO2H 8 2
5 MeO 7 6 C- 8 C- C- 5 9 C- 3 C- O-M 2 e C- C- C- 4 7 6
ppm
Figure 35. Carbon NMR of (4) isoferulic acid in pyridine-d5 (500 MHz). The data revealed ten (10) carbons and one methoxy group.
114
O- Me H- 9 H- H 5 - 8 H- H- 3 2
ppm
Figure 36. Proton NMR of isoferulic acid (4) in pyridine-d5 (500 MHz). The ABX spin system signals for 1, 2, 4-trisubstituted aromatic ring protons were observed at δH 7.89, 7.42 and 7.18.
115 Table 10. Isoferulic acid 1H and 13C-NMR data in Pyridine-d5 (500 MHz)
Position signal δ H δ C m J (Hz)
1 C - 169.84 - -
2 CH 7.12 118.41 d 15.89
3 CH 8.34 148.84 d 15.89
4 C - 129.11 - -
5 CH 7.89 112.42 d 8.6
6 C - 148.84 - -
7 C - 150.98 - -
8 CH 7.18 115.67 d 8.3 9 CH 7.42 121.49 d 8.3
-OCH3 CH3 3.97 56.05 s -
116 5.6.5 Additional Characterization Studies
Review of the RP-HPLC on resultant fraction G-11 from the CHP20P
fractionation (figure 16, page 76) of the combined XAD-2 fractions F-5 through F-
7 (page indicated the possible presence of additional nitrogen bearing
compounds (figure 17, page 80). Additional high resolution LC-MS/MS analysis
by project 4 of the center indicated the presence of an additional guanidine
analog with the protonated molecule of m/z 186.1818 (figure 37), consistent with
the molecular composition of C8H16N3O2, the product ion tandem mass spectrum
of this analog contained a fragment ion of m/z 130 (figure 38) corresponding to
the loss of N-methyl carbondiimine from the guanidine group, strongly indicating that an additional methyl is present. Supporting LC-MS data of other potential nitrogenous constituents are shown in the appendicies (section 10.2). The compound was identified based on quasimolecular ions and MS-MS fragmentation pattern, at least four methylated and one dehydrated cimipronidine exist at the fourth level of separation of the butanol-soluble fraction from figure
10. The mass of fraction G-11 was 135 mg.
117 Sample g11 on amide column crjuly150403 TOF MS ES+ 9.71 100 268 268.2 6.29e3
%
6.11 137.1 0 crjuly150403 TOF MS ES+ 16.87 100 166 166.1 1.56e4
%
0 crjuly150403 TOF MS ES+ 14.16 100 172 172.1 1.73e4
% 16.65 172.2
0 crjuly150403 TOF MS ES+ 10.94 100 186 186.2 9.15e3 12.58 % 186.2 10.20 13.58 200.2 144.1 0 crjuly150403 TOF MS ES+ 14.16 16.85 TIC 100 166.1 3.06 172.1 2.79e4 5.90 163.1 3.89 12.93 15.67 137.1 9.71 10.94 15.19 163.1 5.35 12.16 144.1 154.1 % 268.2 186.2 180.1 334.2 6.83 8.56 297.2 298.2 19.09 21.68 27.30 28.17 169.2 196.1 290.2 342.3 316.2 0 Time 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Figure 37. Positive ESI LC-MS of a 5-HT7 active fraction containing cimipronidine (1) and its analogues. Following from their quasimolecular ions and MS-MS fragmentation pattern, at least four methylated and one dehydrated cimipronidine (1) analogue are contained in the active fraction. Compounds with no obvious structural correlation are marked with * and molecular weights provided based on MH+ quasi molecular ion calculations.
118
OH CO2H Me N N N
N NH HN NH2 CH 2 70.1059 2 10 0 Me
84.1201
154.1461 186.1818 % 126.1369 e 112.1220 130.1339 71.1139 86.1004 113.1053 abundanc e
v i 95.1023 t 83.07 145.1429 168.1620 a l
e R 0
m/z
CO2H CO H 2 N N N 70.1059 100 H H
HN NH2 130.1246 Me
% e
130.0966 abundanc
e 186.1818 v i t 95.0904 a l 113.1053 168.1620 e 71.0864 R 86.1080 137.1125 155.1259 169.1535 0 m/z 70 90 110 130 150 170 190
Figure 38. Positive ion electrospray tandem mass spectral (MS-MS) data to identify N-methyl cimipronidine. The variability with respect to the structural data of a mesomeric guanidine constituent. While further study is needed to confirm the N-methyl cimipronidine structure, the proposed fragmentation above, based on the structural fragmentation data of (1), is supportive of the N-methyl structure.
119 5.7 Preparation of the Clinical Extracts
Methanol extracts of Black Cohosh (BC001) were initially evaluated for
estrogenic activity using a battery of biological assays by Project 2 of the Center
as previously reported.47 These data are available in table 11. Without a direct estrogenic binding effect to explain the clinical activity of Black Cohosh, a methanol extract of Black Cohosh was sent out to a contract lab (PanLabs) to be assayed for serotonin (5-HT) binding activity, where the extract demonstrated
121 significant activity in receptor subtypes 5-HT1A and 5-HT7. The compounds
isolated from Black Cohosh by Project 1 of the center,137, 138, 142 were then tested
for their serotonin binding activity. The activities of some triterpenes isolated by
Project 1 are detailed in table 12. The triterpenes did not demonstrate strong
activity (ca. 50 µg/mL) especially in comparison to some of the phenolic
constituents (i.e. cimicifugic acids A (IC50 0.52 µg/mL) and B (0.91 µg/mL)).
However, it was determined by center personnel that the compounds 26-
deoxyactein, 23-epi-26-deoxyactein, actein (26-R) and actein (26-S) would be
the best candidates for marker compounds based on their abundance in the
black cohosh in combination with their biological activity. These markers were
used to confirm the identity of Cimicifuga racemosa in the extract as well as
correlate the activity of the extract with the relative abundance of these
constituents. in A previous study to evaluate diurnal compound variability was
used to determine the relative abundance of these active/marker compounds in a
methanol extract of Black Cohosh (tables 13 and 14).210 Standard curves for the
clinical capsule analysis and additional variation data are available in figure 39.
120 Additionally no formononetin or kaempferol were detected in the extracts as previously reported.48, 210 Ethanolic (40%, 60% and 75 %) and a 40% isopropanolic extract were analyzed by HPLC for the % of ‘active’ marker compounds. These data, (table 15, figures 40 and 41) and the bioassay data for these extracts, in table 16, led to the selection of a 75% ethanolic extract for the clinical study formulation.
Table 11. Biological activity of a MeOH Black Cohosh extract (from BC001), ER binding, AP induction, PR and pS2 mRNA expression and cytotoxicity. (Adapted from Liu, et al., 2001). Black Cohosh demonstrated no direct estrogenic effect.
ER-α-binding IC50 >> 50 µg/mL
ER-β-binding IC50 >> 50 µg/mL
AP induction, Ishikawa cells IC50 >> 50 µg/mL PR expression, Ishikawa cells NA (Intensity ratio)
Toxicity, Ishikawa cells ED50 >> 20 µg/mL pS2 expression, S-30 cells NA (Intensity ratio)
Toxicity, S-30 cells ED50 >> 20 µg/mL
121 Table 12. Serotonin (5HT) binding activity data of compounds isolated from within the UIC/NIH Center. The italicized compounds were selected by the
Center as standards to correlate the chemical fingerprint with biological activity of the extract. Actein (26R/S), 23-epi-26-deoxyactein, and 26-deoxyactein were selected based on their activity and relative abundance to the other constituents which displayed similar or greater activity. The biological activity refers to the percent at which the extract inhibits [3H]-lysergic acid diethylamide (LSD) binding to the human 5HT-7 receptor. The polyphenolics cimicifugic acids A and B demonstrated the greatest activity in this assay. Despite the activity of these constituents, they are less abundant in a 75% ethanolic extract than the triterpenes. These compounds are abundant in the BuOH-soluble fraction.
Compound 5HT-7 % inhibition IC50 @ 100 µg/ml Cimicifugic acid A 0.53 µg/ml or 1.2 µM
Cimicifugic acid B 0.91 µg/ml or 1.2 µM
Fukinolic Acid 53
Cimifugate A 45
Actein (R,S)* 50
23-epi-26-deoxyactein* 31
26-deoxyactein* 55
25-anhydrocimigenol-3-O-ß-D- 55 xyloside
25-O-acetylcimigenol- 3-O-ß-D- 46 xyloside
2-O-Acetylactein 47
Cimiracemoside I 69
Cimiracemoside M 48
Cimiracemoside O 50
122 Table 13. Average standard constituent concentrations in µg/g of 14 Cimicifuga racemosa root/rhizome methanol extracts determined by HPLC- ELSD (injected at 10 mg/ml). This study was used to determine the relative concentration of the major constituents in Black Cohosh. These collections were made in different locations and at different periods throughout the growth season, to analyze the degree of variation within the botanical. From the data the trends observed were the variability at which the concentration of the major constituents appeared influenced by geographical and diurnal factors, evident from the standard deviations. In addition, to the relative order of average abundance of the standards: actein (26S) > 23-OAc-shengmanol- xyloside > cimiracemoside A >23-epi-26-deoxyactein> isoferulic acid > 26- deoxyactein > 26-deoxycimicifugoside > actein (26-R) > caffeic acid > cimicifugoside H-1 > ferulic acid.
Standard Average Concentration
Caffeic acid 133 ± 63 Ferulic acid 66 ± 98
Isoferulic acid 452 ± 277
Cimiracemoside A 927 ± 939
Cimicifugoside H-1 102 ± 165
Actein (26-R) 264 ± 174
26-deoxycimicifugoside 370 ± 368
Actein (26-S) 2153 ± 1088
23-epi-26-deoxyactein 878 ± 338
26-deoxyactein 379 ± 168
23-OAc-shengmanol-xyloside 1573 ± 558
123 Table 14. Average constituent levels in µg/g of 14 Cimicifuga racemosa root methanol extracts determined by HPLC-ELSD (injected at 10 mg/ml). A) 7 southern states collections (NC, VA and TN) B) 7 Northern Appalachian Collections (PA). These collections were made within their respective, southern and northern regions at different periods of the growth season. The analysis of the standard deviations demonstrates similar variability to the analysis in table 13, this trend indicated that variability in the growing season may have more influence on constituent concentration than geographic origin.
Average Concentration A Standard Caffeic acid 105 ± 79 Ferulic acid 46 ± 122 Isoferulic acid 528 ± 383 Cimiracemoside A 1355 ± 1208 Cimicifugoside H-1 184 ± 202 Actein (26R) 305 ± 226 26-deoxycimicifugoside 384 ±353 Actein (26S) 2393 ± 1174 23-epi-26-deoxyactein 778 ± 339 26-deoxyactein 418 ± 186 23-OAc shengmanol-xyloside 1682 ± 608
B Standard Average Concentration
Caffeic acid 161 ± 22 Ferulic acid 87 ± 70 Isoferulic acid 388 ± 144 Cimiracemoside A 499 ± 150 Cimicifugoside H-1 21 ± 55 Actein (26R) 223 ± 103 26-deoxycimicifugoside 357 ± 412 Actein (26S) 1913 ± 1026 23-epi-26-deoxyactein 978 ± 330 26-deoxyactein 339 ± 151 23-OAc shengmanol-xyl 1465 ± 526
124 Standard curve of 23-epi-26 deoxy 03-12-03
7 6.9 6.8
a 6.7 e r 6.6 Linear (Standard curve 23- epi - A
g 6.5 26)
Lo 6.4 6.3 6.2 y = 1.6978x + 1.3416 6.1 R2 = 0.9982 2.8 2.9 3 3.1 3.2 3.3 Log Concentration
Figure 39. Phase II clinical capsule analysis standard curve. Chemical standardization of the extract was dependent on traditional analytical experimental design to the application of new methodology. The four (4)-point standard curve was generated to determine the concentration of 23-epi-26-deoxyactein in the clinical capsules by calibration of the ELSD response. The four points represent the logarithm of four different duplicate injections by concentration in the the HPLC- ELSD system. The analysis was only considered valid if the r2 value was greater than 0.995. The concentration of the other standards in the capsule (actein (26R), actein (26S) and 26-deoxyactein) were determined by calculating their respective ELSD response as 23-epi-26-deoxyactein. The calibration of the signal was inferred to be 1:1 for this analysis.
125
Table 15. Percentage of center standards in extracts determined by
HPLC-ELSD analysis. All were produced from BC 001. Samples ran in triplicate. Standard deviations for this analysis were within ± 5% of the average constituent concentrations. From the data, to 40% and 75% EtOH extracts appeared to be the most logical choice for selection to formulate the Center clinical extract.
Compound IPR 60% EtOH 40% EtOH 75% EtOH Commercial Extract Actein (26R) 0.21 0.23 0.31 0.26 0.05
Actein (26S) 2.40 2.55 3.34 3.26 0.75
23-epi-26- 0.80 0.90 1.08 1.01 0.19 deoxyactein 26-deoxyactein 0.62 0.37 0.92 0.48 0.20
Total 4.03 4.05 5.65 5.01 1.19 Percentage
*-supplied by PureWorld Botanicals™, PE 2.5% extract
126 40% EtOH
60% EtOH
75% EtOH mV
40% IPR
MeOH
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 Minutes
Figure 40. HPLC-ELSD chromatographic overlay of Black Cohosh (BC001) extracts produced with different solvents. The overlay revealed the variability in extraction efficiency of the “marker” constituents of Black Cohosh in addition to the data provided in table 15. The solvent used in the preparation of the clinical extract was 75% EtOH, which also demonstrated slightly greater biological activity than the 40% isopropanol and 60% ethanol extracts. Biological activity refers to the percent at which the extract inhibits [3H]-lysergic acid diethylamide (LSD) binding to the human 5HT-7 receptor.
127 60% EtOH
40% IPR
40% EtOH
MeOH Relative Abundance
75% EtOH
BuOH
Minutes
Figure 41. Positive-ion electrospray LC-MS chromatographic overlay of Black Cohosh (BC001) extracts produced with different solvents. Observation of m/z 685 a molecular-ion of deoxyacteins, acteins and related, unidentified cimigenols demonstrated the variability in extraction efficiency of the “marker” constituents of Black Cohosh. The solvent used in the preparation of the clinical extract was 75% EtOH, which also demonstrated slightly greater biological activity than the 40% isopropanol and 60% ethanol extracts. Biological activity refers to the percent at which the extract inhibits [3H]-lysergic acid diethylamide (LSD) binding to the human 5HT-7 receptor
128
Table 16. Serotonin binding activity of crude Black Cohosh extracts (BC001). The biological activity refers to the percent at which the extract inhibits [3H]-lysergic acid diethylamide (LSD) binding to the human 5HT-7 receptor. The methanol extract displaced radioligands from the 5-HT1A (IC50 = 2.5 ± 0.6 g/mL) and 5-HT7 (IC50 = 2.2 ± 0.2 g/mL) receptors equally well. While the crude methanol extract exhibited activity greater than or equivalent to the other extracts, it was not a candidate for the clinical extract due to the potential for toxicity with residual solvent. This biological data in conjunction with the chemical fingerprinting studies was used to select the 75% ethanolic extract as the specification for clinical study.
Extract 5HT7 % inhibition IC50 µg/mL @ 100 µg/mL
Crude MeOH 90
40% Isopropanol 89 14
60% EtOH 17
75% EtOH 12
129 5.7.1 Collaborator Formulation for the Phase I Clinical Extract
The specifications were given to our corporate collaborators at Pure World and Pharmavite, where they formulated the extracts and capsules for clinical trial and analysis. Due to the unexpected binding/interference of maltodextrin in the serotonin bioassay, rice flour was used as a flow-through, filler material. The plant material (BC001) was extracted with 75% EtOH. The resultant extract was spray dried. The Information provided from Pure World and Pharmavite is as follows; additional documentation is available in the appendix:
Phase I extract Production Lot #01I-2810 CR-01-49-10
Pure world notebook # KH-13-164, 02584-208
UIC received 8 kg from Pure World on 02/04/02 and sent 4.7 kg to Pharmavite via FedEx on 02/11/02 for encapsulation.
5.7.2 Collaborator Formulation for the Phase II Clinical Extract
The plant material (9-1744) was extracted with 75% EtOH. The resultant extract was spray dried. Plant material from Pure World Lot # 9-1744. Again, rice flour was used as a flow-through/filler.
5.7.3 Phase I Clinical Capsule Analysis
The mass of capsule was 0.511 gm ± 0.012. The mass of contents was
0.429 gm ± 0.010. The calculated mass of standards per capsule was found to be; 23-epi-26-deoxyactein: 1.41 mg ± 0.33; actein-(26S): 0.73 mg ± 0.14; actein-
(26R): 0.12 mg ± 0.02. The percentage of standards per capsule extract was
130 (2.26 mg ± 0.49 triterpene glycosides per 32 mg extract): 7.04%. The combined averages are given with standard deviations of 5 capsules run in triplicate. Rice flour was filtered out with a 0.22 µm syringe filter. LC-MS studies as depicted in figure 42 have shown stability of the most abundant mass ions of the extract used for the clinical capsule from time zero through one year.
Phase 1 Extract Activity (estimated error limit ±10%)
Estrogenic Activity
α-receptor binding >50% @ 200 µg/mlβ-receptor binding >50% @
200 µg/ml
PS-2 induction- no up-regulation @ 20 µg/ml
Ishikawa PR induction-no up-regulation @ 20 µg/ml
Antiestrogenicity:
Ishikawa cell assay >50% @ 20 µg/mL
DNA Damage:
Comet assay* 7.7% @ 20 µg/ml
Antioxidant Activity:
DPPH (IC50-76 µg/ml)HL-60* 88% @ 200 µg/ml
Serotonergic Activity:
5-HT7 receptor binding IC50 13 µg/ml
Cytotoxicity: @ 20 µg/mlIshikawa Cells- no reduction
s30 Cells- no reduction
LC-MS Assays:
131 GSH adduct formation* (m/z-460.5)
*Negative in PUF estrogen assay
5.7.4 Phase II Clinical Capsule Analysis
HPLC-ELSD analysis of the phase II clinical capsules found the average
percentage of dry weight of capsule content and standard deviation of ‘active’
standards per two capsules (128 mg) to be.: 23-epi-26-deoxyactein 2.84% ±
0.08, 26-deoxyactein 0.72% ± 0.02, actein-(26S) 2.77% ± 0.07, Actein-(26R)
0.94% ± 0.02. This gave a total of 5.67% active standards per two clinical
capsules. The combined averages are listed with standard deviations of five (5)
capsules run in duplicate. Rice flour was filtered out with a 0.22 µm syringe filter.
Phase II extract activity (estimated error limit ±10%)
Serotonergic Activity:
5-HT7-receptor binding IC50 18.1 ± 6.45 µg/mL
Phase II capsule activity:
5-HT7-receptor binding @ 10 µg/mL 61.153% ± 9.25
5.7.5 Overview of Clinical Material
The Phase II clinical extract was botanically authenticated (by PCR, vouchers, microscopy), chemically standardized to 5.6 % active triterpene glycosides and biologically standardized to competitively inhibit the 5-HT7 receptor subtype with an IC50 of 18 µg/mL. This 75% EtOH extract is being
132 evaluated in accelerated stability studies for a minimum of three years. The
chemical standardization and specification the clinical extracts display (figure 43)
appeared consistent. In comparison with a popular commercial product, the
commercial extract only demonstrated roughly 1/20th the concentration of ‘active’ constituents as the Black Cohosh extracts created for use within the Center for clinical study. Heavy metals, pesticide, bacterial counts and mutagenic assay data for the clinical extract were performed in independent contract labs
(appendix section 10.3). Heavy metals, pesticide and bacterial values of the extract were all within acceptable limits. 211, 212
133 Relative Abundance
extract intermsoftheabundantmolecularions. bottom afteroneyear.Theoverlayed was analyz chromatogram (TIC)ofthePhaseI Figure 42. ed fortriterpeneglycosidesattimezeroand Electrospray
Minutes LC-MS (negative-ionmode)totalIon 134 clinical extract. TIC’s validateth The TIConthetop e stabilityofthe t he TIConthe
Phase I extract
Phase I clinical cap
mV
Remifemin™ (200 mg/ml)
Phase II clinical cap
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 43. Clinical capsule HPLC-ELSD triterpene comparison. Phase I extract (01- 73-03), Phase I clinical capsule (Cap 3 102401), Remifemin(101141)*, Phase II clinical capsule (group 5 032303). Concentration 10 mg/mL.
135 6.0 DISCUSSION
6.1 Clinical Extract Formulation and Biological Activity
The major undertaking of the Center and the dissertation was to formulate
a botanically, biologically and chemically standardized extract for administration
in FDA-style clinical trials. Therefore, the three potential areas for difficulties are:
i) Botanical identification
ii) Chemical Standardization
iii) Biological significance of the formulation
Initially plant material collected by Center personnel to ensure the
research end of development would use the appropriate plant material. However,
with large scale production taking place outside of Project 1 and the University,
the authenticity of material was not guaranteed. Generally speaking, voucher
specimens, which could be used to positively identify a botanical used for
production of an extract are not available from raw material and finished product
manufacturers alike. Therefore, PCR methods as previously discussed were
used to evaluate starting material. These methods were shown to have some
difficulty with dried root material. For our purposes, however, they were sufficient to authenticate the starting plant material as shown in the results. On the other hand, with Black Cohosh increasing in popularity, the amount of biomass to fill the demand will be an area of concern. With most black cohosh commercial products using wild-crafted material for production, the issue of economically adulterated products is becoming an issue. Products using related Cimicifuga
136 species are considerably less expensive than C. racemosa and would be the logical candidates to be used as adulterants. PCR methods are generally not employed by commercial manufacturers to authenticate plant material.
Additionally, classical pharmacognosy techniques such as microscopy will not offer any selectivity between root material cellular characteristics among different species, and TLC which might not be sensitive or selective enough to establish differences among species. Preliminary HPLC-ELSD data on related Asian species (figure 44) shows distinctive differences between methanolic extracts of the commercial material and these Asian species shown. Only C.racemosa, C. foetida and C.heraclefolia appear to possess the triterpene 23-epi-26- deoxyactein. This methodology can be developed further for potential use to distinguish adulterated C. racemosa extracts by assaying additional samples and identifying other significant marker compounds that may be unique to Cimicifuga species. Development a library of chromatographic and spectral data, using a variety of standard compounds and extracts from the Cimicifuga species used in commerce, using the methodology herein will most likely provide unique fingerprinting data to properly identify and authenticate the species of the extract.
Chemical standardization of the extract was performed by HPLC-ELSD and is discussed in section 6.3. It is of significance to note that, based on our work in this area members of Project 1 in the center were selected as experts to contribute to the USP expert committee panel on the standardization of Black
Cohosh (PF 28(5)) and the American Herbal Pharmacopoeia monograph on
Black Cohosh.
137
1
2
3
mV
4
5
6
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 Minutes Figure 44. Comparison of Pure World (1) starting material to Asian species; Cimicifuga foetida (2), C. acerina (3) , C. heraclefolia (4), C. dahurica (5), C.simplex (6) Arrow indicates the presence of 23-epi-26-deoxyactein. The presence or absence of a peak in this instance is used as a biomarker to identify chemical differences within species. In this instance the three species without any detectable 23-epi-26-deoxyactein would be difficult to verify as C. racemosa, due to their lack of a substantial constituent of the botanical in question.
138 6.2 Biological Activity of Black Cohosh
Next to soy, black cohosh is the most widely studied botanical for menopausal symptoms. At the inception of the Center in 2000, the mechanism of action for Black Cohosh remained somewhat of an enigma. Long believed to have a direct estrogenic effect, studies produced by the Center as well as other research groups, has results to the contrary. The timing of this research fortunately coincided with the largest Women’s Health Study using estrogens to date, the Women’s Health Initiative (WHI). The risk factors discovered in the
WHI, increased the concern of healthy women towards using ERT and HRT for relief of climacteric symptoms. In addition, the use of selective-serotonin re- uptake inhibitors (SSRI), to relieve climacteric symptoms was awarded that indication by the FDA. A short period after the Center produced results indicating serotonin binding activity of Black Cohosh extracts. These extracts displayed competitive binding activity to the 5HT-7 and 5HT-1A cell lines in comparison to
[3H]-lysergic acid diethylamide (LSD) and [3H]-8-hydroxy-2-(di-N- propylamino)tetralin, respectively. Analysis of the ligand binding data indicated that components of a black cohosh methanol extract functioned as a mixed competitive ligand of the 5-HT7 receptor. Additionally, a black cohosh methanol extract elevated cAMP levels in 293T-5-HT7-transfected HEK cells, suggesting the extract acted as a partial agonist at the receptor. The elevation in cAMP mediated by the black cohosh extract could be reversed in the presence of the antagonist methiothepin, indicating a receptor-mediated process.121
139 The Center data implies that Black Cohosh is at least a partial serotonin agonist.
However, the compound(s) responsible for this activity in humans and if black
cohosh actually reaches serotonin receptors in the brain and has effects on hot
flashes in women is still unknown. While the BuOH-soluble fraction, in addition to
some of its constituents (i.e. cimicifugic acids A and B) have shown significant
activity within the Center, it is doubtful that a) these polyphenolic constituents would reach the CNS based on metabolic considerations, and b) the administration of a BuOH-soluble extract, would be unlikely, due to potential toxicity from residual solvent.
With the discovery of the guanidine constituents in Black Cohosh, the potential for complexes with the polyphenolics exists. This discovery may potentially provide an abundant number of possible novel structures for bioassay.
These guanidines, exhibit mesomeric chemical behavior, depending on the pH of their chemical environment, this may offer additional stability to the polyphenolics in the body, when complexed, possibly presenting the feasibility to reach the
CNS.
6.3 Chemical and Botanical Nomenclature
The proposed nomenclatural change of 27-deoxyactein (β-D- xylopyranoside, (3β,12β,16 β,23S,24R,25R)-12-(acetyloxy)-16,23:23,26: 24,25- triepoxy-9,19-cyclolanostan-3-yl) to 23-epi-26-deoxyactein was published by the
UIC/NIH Center. The basis for the change was that the designation for actein (β-
D-xylopyranoside,(3β,12β,16β,23R,24R,25S,26S)-12-(acetyloxy)16,23:23,26:
140 24,25-triepoxy-26-hydroxy-9,19-cyclolanostan-3-yl) has the hydroxyl group in
located at C-26. The corresponding reduced derivative, the former, 27-
deoxyactein is missing the hydroxyl group at that same position. Hence, 27-
deoxyactein is an incorrect name. In addition, the name 26-deoxyactein to the
former 27-deoxyactein would imply that the compound possesses the same
stereochemistry as actein, which would also be incorrect. The former 27-
deoxyactein is an epimer at C-23, thus the name 23-epi-26-deoxyactein was
applied to the former 27-deoxyactein in the effort to lend consistency to the
chemical literature.137 While this distinction was resolved as a result of a structure
elucidation effort, confusion still surrounds the stereochemistry, and consequently the nomenclature of other black cohosh triterpenes. The best example, actein (26-R/S), first isolated in the 1950’s, and the most abundant consitiuent of black cohosh, is near identical to 26-deoxyactein, yet no report in the literature exists of an epimer, stereochemically related to 23-epi-26- deoxyactein.
With the correction in the chemical nomenclature of 27-deoxyactein to 23- epi-26-deoxyactein by the Center, the issue of botanical nomenclature also needs to be addressed. Based on the evidence of one paper cross referencing the genetic similarities between Cimicifuga and Actaea, some within the scientific community now refer to Black Cohosh as Actaea racemosa,53 which is a
synonym that has not been formally adopted by the International Code of
Botanical Nomenclature (IUCN). Avoidable nomenclatural changes based on
genetic data should be avoided in order to maintain nomenclatural stability for
141 global communication about plant genetic resources. If the provisions of the
International Code of Botanical Nomenclature are followed, then very large
numbers of new combinations (names) will be needed at the species and
infraspecific level, which would lead to much confusion. Therefore the use of
Actaea racemosa for Cimicifuga racemosa is inconsistent with the intent of
Article 14 of the International Code of Botanical Nomenclature, which aims to
provide the means, by which the interests of nomenclatural stability may be best
served and in this instance preserved.54 Unless the conversion of all Cimicifuga species to Actaea is officially recognized and adopted by the authoritative bodies of botanical nomenclature it is best to use the generic name Cimicifuga to ensure the consistency of the literature and communication.
6.4 Preparative and Analytical Techniques
Based on the early study within the UIC Center, the n-BuOH fraction demonstrated the greatest activity To better characterize the n-BuOH soluble fraction, the isolated constituents, which include cimicifugic acids A (tR 42.63
min), B (43.26) and F (45.91), fukinolic acid (39.31), ferulic acid (34.58), isoferulic
acid (35.58), and cimipronidine (21.12), were analyzed individually by HPLC-
ELSD to determine their relative reverse-phase HPLC retention times. Individual
UV spectra were recorded during HPLC using a photodiode array (PDA) detector
to confirm the identity of the known constituents ferulic acid, isoferulic acid,
fukinolic acid, cimicifugic acids A, B, and F in the fraction, all with known
chromaphores. The HPLC-ELSD chromatogram in figure 45 also demonstrates
the very high polarity of the active fraction.
142 The butanol-soluble fraction contains phenolic acids and more polar constituents than the previously reported phytochemical studies of Cimicifuga.134,
135, 137, 138, 140, 142, 143, 146-148, 152-158, 160-171, 173, 213-228 The polarity of this fraction is demonstrated in Figure 50. The standard phytochemical isolation strategies usually incorporate silica as a ’normal phase’ stationary support. The use of silica with polar constituents is problematic, the more polar the compounds are, the more they are adsorbed, maybe even absorbed. Thus, the use of the standard phytochemical isolation strategies to obtain constituents from the more polar fractions and extracts is undesirable. Alternative chromatographic methods were considered to effectively isolate compounds from the more polar fractions/extracts. These included weak ion-exchange (XAD-2), size-exclusion
(LH-20), and other non-silica, reverse-phase methodologies (MCI® gel CHP20P).
These methodologies, while not entirely novel are underutilized by the natural products research community, with most of the research focused on relatively non-polar plant extracts and fractions. However, these methods as supported by this work may be the most efficient and advantageous way to isolate polar constituents from higher plants. The most significant advantage is the capability of these stationary phases to produce diverse fractions with polar starting material in a semi-predictable manner as shown in figures 15 and 16.
Amberlite® XAD-2 was selected as a bulk resin based on its reported ability to separate phenolics, and relative low cost compared to C18, MCI® Gel
CHP20P, Sephadex® LH-20 and zirconium (Zr) resins that were considered.
XAD-2 is a styrene based sized exclusion resin that must be washed thoroughly
143 prior to use with all eluting solvents. With the resultant fractions from XAD-2,
which was monitored by TLC (with UV and spray reagents (anisaldehyde and
sulfuric acid (10 % in EtOH) for detection) and HPLC-ELSD, we were able to
observe 35-45 distinctive spots and peaks, respectively, after the 2nd level of
fractionation, enhancing the initial solvent fractionation by roughly 10-fold. MCI
gel® CHP20P and Sephadex® LH-20 were used for the 3rd level of fractionation.
From the TLC and HPLC-ELSD fraction control results, 100 distinct spots and
peaks, representing numerous fractions and compounds, were detected. With
semi-preparative HPLC purification of the resultant fractions, as shown with
fraction G15, one 3rd level fraction, additional peaks were detected from the semi-preparative separation in comparison to what was visible in the analytical
HPLC due to analytical column overload. If 3rd level separation efforts were
initiated on ALL second level XAD-2 fractions (figure 13), HPLC purification of
ALL of theses resultant 3rd level fractions (figure 15), would yield 4th level separation that could potentially yield thousands of constituents. With the complexity of the 3rd and 4th level fractions the development of ‘universal’
detection methods was essential for the production of a relatively humble number
of fractions and compounds. In order to make comprehensive characterization of
a Cimicifuga racemosa extract a reality, the limiting factor will be the
development of ‘universal’ detection methods using higher throughput techniques
to avoid replication of isolation efforts.
Routine isolation of plant derived natural products, when polyphenolic
constituents are present generally requires the use of acids.229, 230 Zwitterionic
144 compounds such like cimipronidine are pH-sensitive and, thus, with the addition
of acid may introduce significant variability in the preparative HPLC retention
properties of the zwitterionic constituents as was observed in this semi-
preparative HPLC isolation of cimipronidine. In this instance, the addition of TFA reduced the retention time of the highly polar material. This may also result in a complexation of the zwitterionic components with the polyphenolic constituents found in the polar extracts.202, 231 This possibility presented it self in this study as
depicted in figure 51 which shows the elution of both fukinolic acid and
cimipronidine in fraction 6 (from G15). While this figure does not serve as a
confirmation of complexation, it does provide basis for additional experiments to
determine complexation.
Analytical HPLC evaluation of hydroalcoholic commercial extracts of
C.racemosa has been relatively limited to the detection and quantification of
triterpene glycosides.48, 210 Identification and quantitiation of triterpenoids presents
a challenge in detection of major compounds that are generally considered poor
chromophores, thus the application of HPLC-UV detection is not appropriate.
The recent use of in-line ELSD and MS detection has made the reliable and
robust quantitation of a commercial product available. However, the majority of
these studies only address one class of constituents, i.e., the triterpenes, in a complex matrix including aromatic acids, oils, sugars and resins, which have been reported from hydroalcoholic extracts of Cimicifuga racemosa. In addition,
the With greater serotonergic activity present in the more polar, butanol-soluble
fractions, standardization methods will need to be developed to rapidly separate,
145 quantify and identify known constituents of polar extracts and fractions, as well
as having the ability to separate new structures from these fractions. As with any
multi-component product, which includes all botanicals, the development of
methodology that best correlates to some biological activity is the most desired in
terms of commercially available products, offering the consumer some level of
assurance of quality. Botanical dietary supplement analysis does not follow the
same straight ahead approach as pharma QA/QC models (i.e. single point standard curves) to standardize a product. While achieving this model is not a possibility, using the best components of a pharma QA/QC model, such as standard-spike recoveries, would benefit the analyses of the complex matricies encountered in botanical analytical chemistry
Positive-ion electrospray was shown to be an excellent ionization technique for the mass spectrometric detection of guanidine constituents and polar phenolic acids. Compounds with an odd number (n=1,3,5,7,9…) of nitrogen atoms were detected as even numbered mass protonated molecules.
146 R1 R2 R3 Fukinolic Acid H H OH 34 9
2 5 COOH Cimicifugic Acid A CH3 H OH 1 N 8 1000 d B Cimicifugic Acid C H CH OH i 3
6 c 7 A 1 d Cimicifugic Acid F H CH3 H 900 2 HN NH c ci gi A fu c mV i 800 i O l d A c i i o c n
m R i
A 1
700 k O c Ci Fu
d F COOH ugi i HO f
600 ) R i c 2 COOH 1 c ( i A d i
e R d
c 3 m i 500 n i ci Ac C d ugi A i c f i i c i c on
400 l ul OH mi pr ru er i i of C m
300 Fe s I Ci
200
100
0
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 min
140.00 1000.00 Phenolics Triterpene glycosides 120.00 800.00 and ??? 100.00
600.00 80.00
V V m m 60.00 400.00 40.00
200.00 20.00
0.00 0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes Minutes Isoferulic acid in the butanol-soluble fraction Isoferulic acid in the clinical extract Method B Method A
Relative Polarity of C. racemosa Constituents
Resins unknown known phenolic phenolic triterpenes and ??? compounds compounds
Figure 45. HPLC-ELSD trace of the BuOH-soluble fraction (01-75-15), from which 1 was isolated (solvent A: 0.05% TFA/5% MeOH/H2O, solvent B: 0.05% TFA/MeOH; elution gradient: 0.0 min A=97%, 50.0 min. A=25%, 61.0 min A=25%, 62.0 A=10%), Retention times: 34.587-ferulic acid, 35.584-isoferulic acid, 39.309-fukinolic acid, 42.632-cimicifugic acid A, 43.263-cimicifugic acid B, 45.913-cimicifugic acid F. Additional HPLC-ELSD traces are present to make evident the difference in polarity between the clinical extract and BuOH-soluble
fraction.
147 Cimpronidine (1) signature Fukinolic acid (2) signature
10 9 8 7 6 5 4 3 2 1 ppm
Figure 46. P6 (from G15)1H-NMR spectrum (500 MHz) in MeOH-d4. Potential complexation of cimipronidine and fukinolic acid. While not conclusive evidence this is a semi-preparative fraction that was eluted between 1 and 2, approximately 20 minutes after 1 and 10 minutes before 2 in fraction G-15 .
148 6.5 NMR spectroscopy
The relatively simple structure of cimipronidine (1) offered quite a complex
structure elucidation problem. Guanidine compounds have only been
reported within a very limited number of plant genera.206-208 Among the
constituents pictured in figure 47, most have a long aliphatic component, thus
the 1H-NMR data will be of a greater merit in terms of providing information for
elucidating the structure. The principle difficulty with 1 is that with the
guanidine moiety centralized on the ring, the ring protons appear quite broad
in the 1H-NMR spectra, offer little spectral information (i.e. lack of coupling
constants) and do not offer long range HMBC correlations. The reasons for
this may be the following: (i) rapid fluctuations associated with 5-membered
ring dynamics, which are known to occur and which can lead to broadening of
the proton resonances with concomitant introduction of short T2’s that affect
the efficiency of polarization transfer from proton to carbon; (ii) the fact that
2 typical JH, C couplings are generally reduced in magnitude relative to 3-bond
couplings; (iii) for the expected 3-bond correlations C-5/H-2, C-5/H-3, C-4/H-
2, and C-3/H-5 the dihedral angle between these protons and the indicated
carbon atoms in the ring approach a dynamically averaged value of 90o, and,
therefore, expected correlations are too weak to be observed or are
completely absent (effective coupling is zero). This latter alternative is
probably the most reasonable explanation. The guanidine carbon NMR shift
ca. 150 ppm will be the one constant observable trend in these structures.
While a higher field strength instrument will usually offer higher resolution with
149 many structure elucidation issues, they offered no additional benefit in
elucidating 1 and will offer the same frustration with other guanidines. The
higher resolution, improved signal-to-noise ratio and greater sensitivity that
will solve many elucidation problems will offer no benefit with the rapid proton fluctuations relative to the NMR timescale. Solubility is an important challenge with guanidine protons. The use of a variety of NMR solvents with various concentrations of both, acid and base are necessary in confirming the presence of guanidine protons, as was needed for 1 (figure 30). Additionally, the possibility of complexation of nitrogen bearing compounds with substituted phenolics appeared during the isolation of 1. To further resolve the proton signals and for confirmation of complexation with phenolics by spectroscopic techniques, the stoichiometry of the complexes still remains to be determined, and may be easily rectified by qNMR. This complexation may be significant to the biological activity of Black Cohosh.
150 O H N NH N 2 H NH HO p-coumaroyl-agmatine HO
segetalin H
NH O O H H N N O H2N NH HN H O H N OH galegine H N N N 2 H O NH Ph OMe MeO H NH N N NHR O H
caracasanamide G1 (R = iso-C6H15) caracasanamide G5 (R = H) H NNHR H2N NH caracasanamide G3 (R = iso-C6H15) caracasanamide G6 (R = H)
Figure 47. Other guanidine isolates from higher plants
151 7.0 CONCLUSIONS
7.0.1. Review of the pharmacognosy of Black Cohosh
The introductory material (pages 1-38) serves as a comprehensive survey
of the clinical, biological and pharmacological literature to date of Black Cohosh.
7.0.2. Preparation of a biologically, chemically and botanically standardized
extract for clinical trial.
To this end, the use of RAPD-PCR, HPLC-ELSD and numerous bioassay
systems within the Center were used to select a formulation. The formulation is
unique in that the techniques above were used to verify the authenticity of the
plant material, select the best extract type of a number of extracts for a clinical
trial based on quantitation of the active constituents and the biological activity.
The result was:
-75% ethanolic extract
-Chemically standardized to 5% of the active triterpene glycosides as determined
by the UIC Center (actein (26R), actein (26S), 23-epi-26-deoxyactein and 26- deoxyactein)
-Biologically standardized to competitively inhibit 5HT-7 binding of 3H-LSD at 18
µg/ml.
7.0.3. Chemical characterization of the active butanolic Black Cohosh fraction
The initial butanol soluble fraction (figure 10) demonstrated significant
activity in the 5HT7 (IC50 4.78) bioassay used to characterize the anti-climacteric
152 activity of Black Cohosh. The constituents that make up this activity were isolated and elucidated within the center. HPLC-ELSD methodology has been developed to identify the presence of these constituents in the fraction. Isolation work on this fraction led to the first report of an alkaloid from Black Cohosh, a novel cyclic guanidine alkaloid. The compounds identified from this fraction are:
-Cimicifugic acids A, B, C, D, F
-Cimipronidine
-Ferulic acid
-Fukinolic Acid
-Isoferulic acid
7.0.4. Development of preparative and analytical methods
Chromatographic methods comprising non-silica (XAD-2 and CHP20P column chromatography), silica (column chromatography) and RP-HPLC have been developed for the serotonergic constituents from Black Cohosh. The results of the chromatographic experiments have been digitized and deposited on the
Center file server (humulus.pharm.uic.edu) to assist in future isolation efforts on
Black Cohosh. The following methods/experimental information are deposited on
Humulus:
- XAD-2 separation of the butanol-soluble fraction
- MCI gel CHP20P separations of
- TLC fraction control of XAD-2, MCI gel CHP 20P and Sephadex fractions
using SSC7-SSC9, and a variety of detection methods
153 - HPLC-ELSD data for clinical extracts, fractions and pure compounds
7.0.5. Dereplication of Botanical Center isolates
The guanidine compound that was identified (1) has not been previously catalogued in either the Chemical Abstracts or Beilstein databases. Thus, it is a new compound, the first report of an alkaloid from Black Cohosh. The processed spectral data have been digitized and deposited on the Center fileserver
(humulus.pharm.uic.edu) to assist in future structure elucidation efforts on Black
Cohosh. Data on the following chemical classes can be distinguished by comparison with the data herein:
- cyclic guanidines
- fukiic acid esters
- 9,19 cycloartane triterpenes
- cinnamic acid derivatives
7. 1 Summary of Conclusions
The work presented in this dissertation can serve as a reference for producing chemically, botanically and biologically standardized botanical extracts for clinical study. More specifically it can serve as a guide for future production and chemical analysis of a standardized Black Cohosh formulation. The chromatography and elucidation work provided herein provide a general compass for future efforts to isolate guanidine compounds, additional triterpene and phenolic compounds from black cohosh.
154 8.0 Future directions
The Center-developed standardized Black Cohosh extract is currently in a
Phase II clinical study at UIC. Thus, the issue of efficacy surrounding the extract will be resolved on completion of the study. However, other research issues have yet to be addressed:
- Production of novel methods to determine identity, strength and purity of
Black Cohosh commercial products. While current methods are
acceptable, they can be improved by adding spike recoveries, studying
the effects of different matricies and solvents on the major constituents. In
addition to determining what exactly the parameters of a quality
commercial product is on the basis of chemistry and biology. Only extracts
have been clinically trialed, still there are numerous products that are
powdered Black Cohosh, yet no equivalence of powdered material to
extracts exists in the compendial literature.
- Preliminary work provided herein shows the qualitative difference between
the Asian and North American Cimicifuga spp. by HPLC-ELSD. While
standards for C. racemosa are widely available to verify the quality and
quantitate commercial Black Cohosh products, little work has been done
to identify the major constituents in Asian Cimicifuga, which are
purportedly used as economic adulterants. In addition the use of ELSD is
not a reality in all academic or commercial labs that may work with Black
Cohosh, additional low cost techniques need to be developed at the very
155 least to ensure the identity of Black Cohosh genetic material and plant
extracts.
- Based on NMR data, the stereochemistry of actein (R/S), the most
abundant triterpene has not been resolved to the degree of 23-epi-26-
deoxyactein and 26-deoxyactein and should be further studied. Actein is
frequently used as a standard in Black Cohosh chemical analysis.
Mutarotation of this compound translates to the use of an uncalibrated,
variable standard for analysis. The mutarotation will vary in different
solvents, therefore effecting the ratio of R:S isomer.
- The interaction between guanidine compounds and phenolics in Black
Cohosh to form complexes in polar solutes, like water, should be studied.
LC-MS/MS indicated the presence of other guanidine compounds yet to
be isolated and characterized. Determination of the isoelectric points of
the guanidines will assist in determination of new structures.
- As additional bioassays are developed that correlated to the alleviation of
hot flushes and other climacteric symptoms, further bioassay guided
fractionation and structure elucidation efforts will be necessary. Future
studies will need to be conducted to determine if black cohosh actually
reaches serotonin receptors in the brain and has effects on hot flashes in
women.
- Reproducible and robust protocols for DNA isolation from dried root, not
only for Black Cohosh, but for other botanicals, can be developed to make
156 the use of genetic fingerprinting more widely available. This will alleviate much of the concern of adulterated material.
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173
10.0 Appendices
10.1 Additional Structural Data for Novel Center Isolates.
Structural data of 14 Cimicifuga racemosa novel constituents from silica
fractions (pages 83, 84). Proton NMR data given as; chemical shift (multiplicity, J
value(s) where applicable in Hz, proton position). Carbon NMR data given as;
chemical shift (multiplicity, carbon location).
Cimiracemate A: light brown powder, mp 94–96 °C. 1H NMR spectral data (500
MHz, CD3OD): δ 6.36 (d, 15.9, H-2), 7.61 (d, 15.9, H-3), 7.09 (d, 2.1, H-5a), 6.94 (d, 8.3, H-8a), 7.06 (dd, 2.1, 8.3, H-9a), 4.86 (s, H-1’), 3.63 (s, H-3’), 6.68 (d, 2.0, H-5b), 6.73 (d, 8.0, H-8b), 6.57 (dd, 2.0, 8.0, H-9b), 3.88 (s, MeO-7); 13C NMR
spectra data (125 MHz, CD3OD): δ 168.1 (s, C-1), 115.2 (d, C-2), 147.4 (d, C-3), 128.8 (s, C-4), 114.8 (d, C-5) 148.0 (s, C-6), 151.7 (s, C-7), 112.5 (d, C-8), 123.0 (d, C-9), 68.5 (t, C-1’), 204.5 (s, C-2’), 46.3 (t, C-3’), 126.0 (s, C-4’), 117.6 (d, C- 5’), 146.6 (s, C-6’), 145.7 (s, C-7’), 116.5 (d, C-8’), 122.0 (d, C-9’), 56.4 (q, MeO-
7). UV λmaxMeOH nm (log ): 210.5 (4.26), 291.0 (3.77), 325.0 (3.96). IR νmax NaCl cm−1: 3403, 2940, 1705, 1608, 1512, 1442, 1264, 1162, 1131, 1023.Negative ESI–MS m/z (relative intensity %): 357 (18)[M]−, 193 (100), 178 (20), 163 (90),
149 (25), 134 (48); HR-ESIMS m/z 357.0974 (calc. 357.0974 for C19 H17O7).
Cimiracemate B: light brown powder, mp 86–88.5 °C. 1H NMR spectral data
(500 MHz, CD3OD): δ 6.42 (d, 16.0, H-2), 7.65 (d, 16.0, H-3), 7.21 (d, 1.9, H-5), 6.83 (d, 8.3, H-8), 7.09 (dd, 1.9, 8.2, H-9), 4.86 (s, H-1′), 3.62 (s, H-3′), 6.68 (d, 2.0, H-5′), 6.73 (d, 8.0, H-8′), 6.57 (dd, 2.0, 8.0, H-9′), 3.89 (s, MeO-6); 13C NMR
spectra data (125 MHz, CD3OD): δ 168.3 (s, C-1), 114.5 (d, C-2), 147.4 (d, C-3), 127.6 (s, C-4), 116.5 (d, C-5), 150.9 (s, C-6), 149.4 (s, C-7), 111.7 (d, C-8), 124.3 (d, C-9), 68.5 (t, C-1′), 204.6 (s, C-2′), 46.3 (t, C-3′), 126.0 (s, C-4′), 117.6 (d, C- 5′), 146.6 (s, C-6′), 145.7 (s, C-7′), 116.5 (d, C-8′), 122.0 (d, C-9′), 56.4 (q, MeO- −1 6). UV λmax MeOH nm (log ): 208.5 (4.28), 327.5 (3.92). IR νmax NaCl (cm ):
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3396, 2930, 1708, 1600, 1519, 1448, 1217, 1172, 1125, 1030. Negative ESI–MS m/z (relative intensity %): 357 (23)[M]−, 313 (10%), 193 (100), 179 (45), 163 (70),
149 (70), 134 (68), HR–ESIMS m/z 357.0974 [M]− (calc. 357.0974 for C19
H17O7).
Cimiracemate C: light brown powder, mp 88–90 °C. 1H NMR spectral data (500
MHz, CD3OD): δ 6.35 (d, 15.9, H-2), 7.59 (d, 16.0, H-3), 7.07 (d, 2.0, H-5), 6.94 (d, 8.4, H-8), 7.06 (dd, 2.0, 8.4, H-9), 4.94 (d, 17.5, H-1′a), 5.00 (d, 17.5, H-1′b), 4.79 (s, H-3′), 6.78 (d, 2.0, H-5′), 6.79 (d, 8.1, H-8′), 6.72 (dd, 2.0, 8.1, H-9′), 3.89 13 (s, MeO-7), 3.33 (s, MeO-3′). C NMR spectra data (125 MHz, CD3OD): δ 168.1 (s, C-1), 115.2 (d, C-2), 147.4 (d, C-3), 128.8 (s, C-4), 114.8 (d, C-5) 148.1 (s, C- 6), 151.7 (s, C-7), 112.5 (d, C-8), 123.0 (d, C-9), 66.9 (t, C-1′), 203.6 (s, C-2′), 88.2 (d, C-3′), 127.9 (s, C-4′), 115.5 (d, C-5′), 147.4 (s, C-6′), 147.0 (s, C-7′),
116.5 (d, C-8′), 120.7 (d, C-9′), 56.4 (q, MeO-7), 57.1 (q, MeO-3′). UV λmax MeOH −1 nm (log): 212.0 (4.35), 292.9 (3.87), 326.0 (4.09). IR νmax NaCl (cm ): 3399, 2927, 1710, 1608, 1515, 1442, 1270, 1156. Negative ESI–MS m/z (relative intensity%): 387 (8)[M]−, 355 (33), 340 (10), 296 (30), 193 (100), 178 (45), 161
(38), 149 (27), 134 (37). HR–ESIMS m/z 387.1073 [M]− (calc. 387.1080 C20
H19O8).
Cimiracemate D: light brown powder, mp 100–102 °C. 1H NMR spectral data
(500 MHz, CD3OD): δ 6.40 (d, 15.9, H-2), 7.63 (d, 16.0, H-3), 7.19 (d, 1.9, H-5), 6.94 (d, 8.1, H-8), 7.06 (dd, 1.9, 8.1, H-9), 4.94 (d, 17.5, H-1′a), 5.00 (d, 17.5, H- 1′b), 4.79 (s, H-3′), 6.79 (d, 1.9, H-5), 6.80 (d, 7.9, H-8′), 6.72 (dd, 1.9, 7.9, H-9′), 13 3.88 (s, MeO-6), 3.35 (s, MeO-3′). C NMR spectra data (125 MHz, CD3OD): δ 168.5 (s, C-1), 114.5 (d, C-2), 147.8 (d, C-3), 127.6 (s, C-4), 116.5 (d, C-5), 150.8 (s, C-6), 149.4 (s, C-7), 111.8 (d, C-8), 124.4 (d, C-9), 66.9 (t, C-1′), 203.7 (s, C-2′), 88.2 (d, C-3′), 127.9 (s, C-4′), 115.5 (d, C-5′), 147.4 (s, C-6′), 145.0 (s,
C-7′), 116.6 (d, C-8′), 120.0 (d, C-9′), 56.5 (q, MeO-6), 57.2 (q, MeO-3′). UV λmax −1 MeOH nm (log): 212.0 (4.35), 326.0 (4.09). IR νmax NaCl (cm ): 3387, 2979, 1710, 1601, 1520, 1481, 1271, 1156, 1029. Negative ESI–MS m/z (relative
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intensity %): 387 (12)[M]−, 355 (22), 337 (22), 296 (22), 193 (95), 178 (100), 175
(52), 161(40), 149 (20), 134 (40), HR–ESIMS m/z 387.1084 (calc. 387.1080 C20
H19O8).
Cimiracemoside I: pale yellow powder, mp >300 °C (began decomposing at 250 20 1 °C), [α] D -13.13 (c 0.267, CHCl3); H-NMR spectral data (500 MHz, pyridine-
d5): δ 5.08 (d, 7.2, H-7), 4.87 (d, 7.4, H-1'), 4.38 (dd, 4.5, 10.5, H-5'), 4.31 (dd, 6.7, 12.0, H-16), 4.24 (dd, 6.0, 9.0,H-4'), 4.18 (t, 6.9, H-3'), 4.05 (overlapped, H- 2'), 4.04 (d, 10.2, H-26), 3.76 (t, 10.3, H-5'), 3.70 (s,H-24), 3.61 (d, 10.2, H-26), 3.48 (brd, 7.5, H-3), 2.33 (m, overlapped, H-2), 2.26 (overlapped, H-20), 2.11 (brt, 8.6, H-15), 2.09 (m, H-11a),1.96 (dd, 6.3, 11.4, H-15), 1.80 (m, H-6), 1.70 (m, H-1), 1.66 (2H, m, H-12), 1.58 (brd. 13.5, H-22a), 1.56 (t, 13.5, H-17), 1.46 (s, H-27), 1.46 (m, H-6), 1.40 (brt, 14.7, H-22b), 1.35 (s, H-29), 1.30 (m, overlapped, H-2), 1.30 (m, H-1), 1.26 (s, H-18), 1.24 (overlapped, H-5), 1.11 (m, H-11b), 1.10 (s, H-28), 1.04 (s, H-30), 1.00 (d, 6.4, H-21), 0.97 (d, 3.5, H-19), 13 0.46 (d, 3.5, H-20), C-NMR spectral data (500 MHz, pyridine-d5) 149.2 (s, C-8), 113.5 (d, C-7), 107.6 (d, C-1'), 106.2 (s, C-23), 88.1 (d, C-3), 78.7 (d, C-3'), 75.6 (d, C-2'), 74.9 (d, C-16), 71.3 (d, C-4'), 68.0 (t, C-26), 67.2 (t, C-5'), 62.6 (d, C- 24), 62.1 (s, C-25), 56.9 (d, C-17), 49.8 (s, C-14), 44.1 (s, C-13), 43.0 (t, C-15), 42.7 (d, C-5), 40.4 (s, C-4), 37.5 (t, C-22), 32.9 (t, C-12), 30.9 (t, C-1), 29.6 (t, C- 2), 28.3 (t, C-19), 26.9 (q, C-28), 25.8 (q, C-29), 25.3 (t, C-11), 23.7 (s, C-10), 23.7 (d, C-20), 22.9 (q, C-18), 21.8 (t, C-6), 21.0 (s, C-9), 20.8 (q, C-21), 14.3 (q, −1 C-27), 14.3 (q, C-30), IR νmaxNaCl (cm ): 3485, 3409, 2923, 1454, 1379, 1038,
966; HRESIMS m/z 623.3581 (calc. 623.3560 for C35H52O8Na).
20 Cimiracemoside J: pale yellow powder, mp 138-140 °C, [α] D -14.23 (c 0.260, 1 CHCl3); H-NMR spectral data (500 MHz, pyridine-d5): δ 5.36 (brs, H-26), 5.27 (brd, 7.8 H-12), 4.89 (brs, H-), 4.79 (d, 6.8, H-1'), 4.44 (t, 7.8, H-2'), 4.42 (s, H- 15), 4.32 (overlapped, H-4'), 4.31 (overlapped, H-5'), 4.31 (overlap, H-23), 4.17 (overlapped, H-3'), 4.12 (brs, H-24), 3.79 (brd, 11.3, H-), 3.49 (dd, 3.1, 7.9, H-3),
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2.94 (dd, 9.6, 15.4, H-22a), 2.32 (m, H-2), 2.29 (m, H-11), 2.22 (m, H-7), 2.13 (s, OAc), 1.85 (s, H-27), 1.77 (brd,12.4, H-5), 1.65 (overlapped, H-17), 1.64 (overlapped, H-20), 1.58 (brd. 12.2, H-1), 1.56 (m, H-6), 1.37 (s, H-18), 1.33 (m, H-2), 1.29 (overlapped, H-8), 1.28 (s, H-29), 1.20 (s, H-28), 1.17 (brd, 15.3, H- 22b), 1.12 (m, H-7), 1.11 (brd. 14.4, H-1), 1.03 (m, H-11), 1.01 (s, H-30), 0.94 (d, 6.5, H-21), 0.75 (m, H-6), 0.61 (d, 4, H-19), 0.32 (d, 4, H-19), 13C-NMR spectral data (500 MHz, pyridine-d5) δ 170.6 (s, OAc), 145.8 (s, C-25), 113.2 (t, C-26), 112.3 (s, C-16), 107.4 (d, C-1'), 88.3 (d, C-3), 86.5 (d, C-24), 79.3 (d, C-15), 77.3 (d, C-12), 74.7 (d, C-23), 74.7 (d, C-3'), 73.0 (d, C-2'), 69.5 (d, C-4'), 66.7 (t, C- 5'), 59.6 (d, C-17), 48.4 (s, C-13), 47.2 (d, C-5), 47.2 (d, C-8), 46.1 (s, C-14), 41.3 (s, C-4), 38.5 (t, C-11), 37.5 (t, C-22), 32.4 (t, C-1), 30.9 (t, C-19), 30.1 (t, C-2), 26.8 (s, C-10), 26.0 (t, C-7), 25.7 (q, C-29), 23.9 (d, C-20), 21.7 (q, O-Ac), 20.8 (t, C-6), 20.1 (s, C-9), 19.8 (q, C-21), 18.1 (q, C-27), 15.4 (q, C-30), 12.7 (q, C-18), −1 12.0 (q, C-28), IR νmaxNaCl (cm ): 3468, 2933, 2869, 1731, 1454, 1377, 1240,
1067, 755; HRESIMS m/z 683.3763 (calc 683.3771 for C37H56O10Na).
20 Cimiracemoside K: pale yellow powder, mp 142-143 ˚C, [α] D -59.32 (c 0.147, 1 CHCl3); H-NMR spectral data (500 MHz, pyridine-d5): δ 5.35 (brs, H-26), 5.28 (brd, 7.6, H-12), 4.89 (brs, H-26), 4.84 (d, 7.4, H-1'), 4.42 (s, H-15), 4.34 (dd, 4.8, 9.8, H-5'), 4.30 (d, 8.5, H-23), 4.21 (m, H-4'), 4.18 (brs, H-24), 4.15 (t, 8.4, H-3'), 4.03 (dd, 8.0, 15.4, H-2'), 3.72 (dd, 9.8, 9.1, H-5'), 3.49 (dd, 3.5, 10.8, H-3), 2.94 (dd, 9.5, 16.1, H-22), 2.30 (m, H-2), 2.26 (m, H-11), 2.19 (m, H-7), 2.12 (s, OAc), 1.90 (m, H-2), 1.85 (s, H-27), 1.76 (brd, 12.2, H-5), 1.64 (overlapped, H-17), 1.64 (overlapped, H-20), 1.56 (brt, 12.4, H-1), 1.53 (m, H-6), 1.32 (s, H-18), 1.31 (s, H- 29), 1.30 (overlapped, H-8), 1.20 (s, H-28), 1.16 (brd, 16.1, H-22), 1.10 (m, H-7), 1.08 (brd. 14.4, H-1), 1.04 (s, H-30), 1.02 (m, H-11), 0.95 (d, 6.5, H-21), 0.73 (m, H-6), 0.60 (d, 3.4, H-19), 0.32 (d, 3.4, H-19), 13C-NMR spectral data (500 MHz,
pyridine-d5) δ 170.6 (s, OAc), 145.8 (s, C-25), 113.2 (t, C-26), 112.3 (s, C-16), 107.5 (d, C-1'), 88.3 (d, C-3), 86.5 (d, C-24), 79.3 (d, C-15), 78.6 (d, C-3'), 77.3 (d, C-12), 75.6 (d, C-2'), 74.6 (d, C-23), 71.6 (d, C-4'), 67.1 (t, C-5'), 59.6 (d, C-
177
17), 48.4 (s, C-13), 47.2 (d, C-5), 47.2 (d, C-8), 46.1 (s, C-14), 41.3 (s, C-4), 38.5 (t, C-11), 37.5 (t, C-22), 32.4 (t, C-1), 30.9 (t, C-19), 30.0 (t, C-2), 26.8 (s, C-10), 26.0 (t, C-7), 25.7 (q, C-29), 23.9 (d, C-20), 21.7 (q, OAc), 20.8 (t, C-6), 20.1 (s, C-9), 19.8 (q, C-21), 18.1 (q, C-27), 15.4 (q, C-30), 12.7 (q, C-18), 12.0 (q, C-28), −1 IR νmaxNaCl (cm ): 3425, 2935, 2869, 1732, 1456, 1376, 1233, 1042, 760;
HRESIMS m/z 683.3745 (calc 683.3771 for C37H56O10Na).
20 Cimiracemoside L: white powder, mp 125-128 ˚C, [α] D -41.11 (c 0.450, 1 CHCl3); H-NMR spectral data (500 MHz, pyridine-d5): δ 5.61 (brs, H-4'), 5.42 (brt, 8.4, H-23), 4.82 (d, 7.1, H-1'), 4.48 (brt, 8.1, H-2'), 4.35 (s, H-15), 4.28 (d, 12.6, H-5'a), 4.21 (d, 7.3, H-3'), 3.85 (d, 12.6, H-5'b), 3.53 (dd, 4.3, 11.7, H-3), 3.05 (d, 8.4, H-24), 2.69 (brt 12.2, H-22a), 2.42 (m, H-2a), 2.35 (d, 6.5, H-17), 2.14 (overlapped, H-20), 2.12 (s, OAc), 2.10 (m, H-11a), 2.06 (s, OAc), 1.88 (dd, 4.4, 12.4, H-8), 1.80 (2H, (m, H-12), 1.77 (m, H-22b), 1.62 (overlapped, H-1a), 1.60 (m, H-6a), 1.40 (s, H-27), 1.39 (overlapped, H-5), 1.38 (s, H-18), 1.35 (m, H- 2b), 1.31 (s, H-29), 1.30 (overlapped, H-7a), 1.28 (overlapped, H-1b), 1.27 (d, 6.9, H-21), 1.26 (s, H-26), 1.22 (s, H-28), 1.15 (m, H-11b), 1.14 (overlapped, H- 7b), 1.06 (s, H-30), 0.76 (m, H-6b), 0.59 (d, 3.7, H-19a), 0.32 (d, 3.7, H-19b), 13C-
NMR spectral data (500 MHz, pyridine-d5) δ 220.0 (s, C-16), 170.9 (s, OAc), 170.7 (s, OAc), 107.6 (d, C-1'), 88.8 (d, C-3), 83.0 (d, C-15), 73.2 (d, C-2'), 72.6 (d, C-3'), 72.1 (d, C-23), 72.1 (d, C-4'), 65.2 (d, C-24), 64.4 (t, C-5'), 60.0 (d, C- 17), 58.6 (s, C-25), 48.3 (d, C-8), 47.5 (d, C-5), 46.1 (s, C-14), 41.6 (s, C-13), 41.4 (s, C-4), 37.0 (t, C-22), 33.1 (t, C-12), 32.2 (t, C-1), 30.5 (t, C-19), 30.1 (t, C- 2), 28.0 (d, C-20), 26.8 (s, C-10), 26.7 (t, C-7), 26.0 (t, C-11), 25.7 (q, C-29), 24.7 (q, C-26), 21.2 (q, OAc), 21.0 (t, C-6), 21.0 (q, OAc), 20.4 (q, C-21), 20.1 (s, C-9), −1 19.8 (q, C-18), 19.4 (q, C-27), 15.5 (q, C-30), 12.0 (q, C-28); IR νmaxNaCl (cm ): 3466, 2937, 2871, 1737, 1455, 1376, 1241, 1089, 757; HRESIMS m/z 705.4205
(calc 705.4214 for C39H61O11).
20 Cimiracemoside M: white powder, mp 107-109 °C, [α] D -19.00 (c 0.30, 1 CHCl3); H-NMR data (500 MHz, pyridine-d5) δ 5.41 (overlapped, H-4'), 5.39 (brt,
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8.4, H-23), 4.90 (d, 7.2, H-1'), 4.39 (s, H-15), 4.38 (overlapped, H-5'a), 4.29 (t, 8.6, H-2'), 4.08 (t, 6.7, H-3'), 3.63 (t, 10.5, H-5'b), 3.55 (dd, 4.0, 12.8, H-3), 3.05 (d, 8.4, H-24), 2.69 (brt, 12.8, H-22a), 2.39 (d, 6.5, H-17), 2.31 (m, H-2a), 2.13 (overlapped, H-20), 2.10 (overlapped, H-7a), 2.10 (m, H-11a), 2.07 (s, OAc), 1.99 (s, OAc), 1.95 (m, H-2b), 1.88 (dd, 4.6, 11.5, H-8), 1.82 (2H, (m, H-12), 1.78 (m, H-22b), 1.60 (overlapped, H-1a), 1.59 (m, H-6a), 1.41 (s, H-27), 1.39 (overlapped, H-5), 1.38 (s, H-18), 1.34 (s, H-29), 1.28 (overlapped, H-1b), 1.27 (d, 6.8, H-21), 1.26 (s, H-26), 1.22 (s, H-28), 1.18 (overlapped, H-7b), 1.15 (m, H- 11b), 1.07 (s, H-30), 0.79 (m, H-6b), 0.60 (d, 3.6, H-19a), 0.33 (d, 3.6, H-19b), 13 C-NMR data (500 MHz, pyridine-d5) δ 220.0 (s, C-16), 170.5 (s, OAc), 170.5 (s, OAc), 107.4 (d, C-1'), 88.5 (d, C-3), 83.0 (d, C-15), 75.8 (d, C-3'), 75.0 (d, C- 2'), 73.2 (d, C-4'), 72.1 (d, C-23), 65.2 (d, C-24), 63.2 (t, C-5'), 60.0 (d, C-17), 58.6 (s, C-25), 48.3 (d, C-8), 47.4 (d, C-5), 46.1 (s, C-14), 41.6 (s, C-13), 41.4 (s, C-4), 37.0 (t, C-22), 33.1 (t, C-12), 32.2 (t, C-1), 30.0 (t, C-2), 30.0 (t, C-19), 28.0 (d, C-20), 26.8 (s, C-10), 26.7 (t, C-11), 26.4 (t, C-7), 25.7 (q, C-29), 24.7 (q, C- 26), 21.1 (t, C-6), 21.0 (q, OAc), 20.9 (q, OAc), 20.4 (q, C-21), 20.1 (s, C-9), 19.8 −1 (q, C-18), 19.4 (q, C-27), 15.4 (q, C-30), 12.0 (q, C-28); IR νmaxNaCl (cm ): 3465, 2933, 2866, 1732, 1370, 1235, 1041, 750; HRESIMS m/z 727.4041 (calc
727.4033 for C39 H60O11Na).
20 Cimiracemoside N: pale yellow powder, mp 172-174 °C, [α] D -70.36 (c 0.367, 1 CHCl3); H-NMR data (500 MHz, pyridine-d5) δ 5.12 (overlapped, H-12), 4.78 (d, 6.8, H-1'), 4.45 (t, 14.5, H-2'), 4.33 (brs, H-4'), 4.31 (d, 11.0.2, H-5'a), 4.24 (d, 6.9, H-3'), 4.24 (brt, 6.9, H-16), 4.07 (d, 10.5, H-26a), 3.80 (d, 11.0.2, H-5'b), 3.68 (s, H-24), 3.63 (d, 10.5, H-26b), 3.44 (dd, 4.2, 13.0, H-3), 2.71 (dd, 8.8, 15.4, H- 11a), 2.28 (m, H-2a), 2.23 (overlapped, H-20), 2.14 (s, H-OAc), 1.88 (m, H-15a), 1.83 (m, H-2b), 1.79 (overlapped, H-17), 1.76 (m, H-15b), 1.60 (m, H-8), 1.58 (overlapped, H-22a), 1.49 (overlapped, H-1a), 1.48 (s, H-27), 1.45 (overlapped, H-22b), 1.42 (s, H-18), 1.38 (m, H-6a), 1.29 (overlapped, H-7a), 1.27 (s, H-29), 1.22 (overlapped, H-5), 1.16 (overlapped, H-11b), 1.12 (overlapped, H-1b), 1.02 (d, 6.3, H-21), 0.96 (s, H-30), 0.89 (overlapped, H-7b), 0.85 (s, H-28), 0.63 (m, H-
179
6b), 0.54 (d, 3.6, H-19a), 0.19 (d, 3.6, H-19b), 13C-NMR data (500 MHz, pyridine- d5) δ 170.7 (s, OAc), 107.5 (d, C-1'), 105.9 (s, C-23), 88.1 (d, C-3), 77.1 (d, C- 12), 74.7 (d, C-16), 74.5 (d, C-3'), 72.9 (d, C-2'), 69.6 (d, C-4'), 68.1 (t, C-26), 66.8 (t, C-5'), 62.5 (d, C-24), 62.2 (s, C-25), 56.2 (d, C-17), 48.8 (s, C-13), 47.8 (s, C-14), 47.0 (d, C-5), 45.6 (d, C-8), 44.1 (t, C-15), 41.2 (s, C-4), 37.5 (t, C-22), 36.6 (t, C-11), 31.9 (t, C-1), 29.8 (t, C-2), 29.5 (t, C-19), 26.7 (s, C-10), 25.7 (q, C-29), 25.6 (t, C-7), 23.3 (d, C-20), 21.7 (q, OAc), 21.3 (q, C-21), 20.3 (t, C-6), 20.1 (s, C-9), 19.6 (q, C-28), 15.3 (q, C-30), 14.3 (q, C-27), 13.5 (q, C-18), IR −1 νmaxNaCl (cm ): 3454, 2935, 2871, 1729, 1455, 1372, 1244, 1070, 1030, 754;
HRESIMS 661.3962 (calc 661.3952 for C37H57O11).
20 Cimiracemoside O: pale yellow powder, mp 143-145 °C, [α] D -60.00 (c 0.160, 1 CHCl3); H-NMR data (500 MHz, pyridine-d5) δ 5.76 (s, H-26), 5.41 (ddd, 5.4, 9.7, 9.7, H-4'), 5.10 (brd, 6.0, H-12), 4.85 (d, 7.3, H-1'), 4.62 (dd, 7.1, 14.3, H-16), 4.34 (dd, 5.5,11.4, H-5'b), 4.28 (dd, 9.1,9.2, H-3'), 4.04 (dd, 8.5, 8.3, H-2'), 3.95 (s, H-24), 3.61 (dd, 10.9, 11.4, H-5'a), 3.43 (dd, 4.0, 11.4, H-3), 2.71 (m, H-11a), 2.24 (overlapped, H-22a), 2.24 (m, H-2a), 2.16 (s, OAc), 1.99 (s, OAc), 1.85 (m, H-2b), 1.80 (overlapped, H-20), 1.79 (s, H-27), 1.79 (s, H-29), 1.78 (overlapped, H-17), 1.75 (m, H-15a), 1.70 (dd, 6.5, 18.0, H-22b), 1.62 (m, H-8), 1.55 (m, H- 15b), 1.51 (overlapped, H-1a), 1.37 (s, H-18), 1.27 (m, H-6a), 1.25 (overlapped, H-7a), 1.21 (overlapped, H-5), 1.21 (overlapped, H-11b), 1.12 (overlapped, H- 1b), 0.98 (s, H-30), 0.98 (d, 6.5, H-21), 0.89 (overlapped, H-7b), 0.80 (s, H-28), 0.67 (m, H-6b), 0.57 (d, 4.5, H-19a), 0.23 (d, 4.5, H-19b), 13C-NMR data (500
MHz, pyridine-d5) δ 170.6 (s, OAc), 170.6 (s, OAc), 107.3 (d, C-1'), 105.8 (s, C- 23), 98.4 (d, C-26), 88.2 (d, C-3), 77.0 (d, C-12), 75.7 (d, C-2'), 75.0 (d, C-3'), 73.1 (d, C-4'), 73.0 (d, C-16), 65.8 (s, C-25), 63.4 (d, C-24), 63.2 (t, C-5'), 56.4 (d, C-17), 48.7 (s, C-13), 47.8 (s, C-14), 46.9 (d, C-5), 45.7 (d, C-8), 43.5 (t, C-15), 41.2 (s, C-4), 37.6 (t, C-22), 36.7 (t, C-11), 31.9 (t, C-1), 29.8 (t, C-19), 29.5 (t, C- 2), 26.7 (s, C-10), 26.0 (d, C-20), 25.6 (t, C-7), 25.6 (q, C-29), 21.6 (q, OAc), 21.0 (q, C-21), 20.9 (q, OAc), 20.4 (t, C-6), 20.1 (s, C-9), 19.5 (q, C-28), 15.3 (q, C- -1 30), 13.5 (q, C-18), 13.1 (q, C-27). IR νmax NaCl (cm ) 3449, 2940, 2869, 1732,
180
1455, 1371, 1241, 1048, 983, 755; HRESIMS m/z 701.3889 (calc 701.3901 for
C39H57O11 [M - H2O + H]+).
20 Cimiracemoside P: pale yellow powder, mp 151-153 °C, [α] D -69.77 (c 0.440, 1 CHCl3); H-NMR data (500 MHz, pyridine-d5) δ 5.11 (brd 6.8, H-12), 4.87 (d, 7.1, H-1'), 4.41 (s, H-24), 4.38 (m, H-5'a), 4.22 (m, H-4'), 4.20 (t, 8.6, H-3'), 4.05 (m, H-2'), 4.05 (brt, 7.7, H-16), 3.76 (9.8, 10.1, H-5'b), 3.48 (brd, 7.0, H-3), 2.75 (m, H-11a), 2.32 (m, H-2a), 2.21 (overlapped, H-22a), 2.16 (s, OAc), 1.90 (overlapped, H-15a), 1.88 (m, H-2b), 1.86 (overlapped, H-17), 1.85 (overlapped, H-20), 1.67 (overlapped, H-22b), 1.65 (s, H-27), 1.64 (overlapped, H-15b), 1.63 (m, H-8), 1.57 (overlapped, H-1a), 1.43 (overlapped, H-6a), 1.33 (s, H-18), 1.33 (s, H-29), 1.27 (overlapped, H-5), 1.25 (overlapped, H-7a), 1.21 (overlapped, H- 11b), 1.14 (overlapped, H-1b), 1.02 (s, H-30), 0.95 (overlapped, H-7b), 0.93 (d, 5.0, H-21), 0.85 (s, H-28), 0.70 (m, H-6b), 0.57 (d, 3.8, H-19a), 0.26 (d, 3.8, H- 13 19b), C-NMR data (500 MHz, pyridine-d5) δ 172.4 (s, C-26), 170.5 (s, OAc), 107.6 (d, C-1'), 106.2 (s, C-23), 88.1 (d, C-3), 78.7 (d, C-3'), 76.8 (d, C-12), 75.6 (d, C-16), 75.4 (d, C-2'), 71.3 (d, C-4'), 67.2 (t, C-5'), 62.7 (d, C-24), 58.6 (s, C- 25), 55.6 (d, C-17), 48.8 (s, C-13), 48.0 (s, C-14), 47.0 (d, C-8), 45.7 (d, C-5), 43.2 (t, C-15), 41.2 (s, C-4), 36.6 (t, C-11), 35.6 (t, C-22), 31.9 (t, C-1), 29.9 (t, C- 2), 29.6 (t, C-19), 26.8 (s, C-10), 25.7 (t, C-7), 25.7 (q, C-29), 25.3 (d, C-20), 21.6 (q, OAc), 20.7 (q, C-21), 20.4 (t, C-6), 20.1 (s, C-9), 19.5 (q, C-28), 15.3 (q, C- -1 30), 13.5 (q, C-18), 11.1 (q, C-27). IR νmax NaCl (cm ) 3423, 2956, 2934, 2870, 1787, 1731, 1454, 1362, 1243, 1041, 968, 753; HRESIMS m/z 675.3758 (calc
675.3744 for C37H55O11).
20 23-epi-26-deoxyactein: colorless needles, mp 251-253 °C, [α] D -61.18 (c 1 0.255, CHCl3-MeOH, 1:1); H-NMR data (500 MHz, pyridine-d5) δ 5.10 (brd, 5.3, H-12), 4.83 (d, 7.2, H-1'), 4.35 (dd, 10.5, 4.2, H-5'a), 4.23 (brd, 6.7, H-16), 4.21 (m, H-4'), 4.14 (t, 8.6, H-3'), 4.05 (d, 10.4, H-26a), 4.04 (s, H-24), 4.01 (t, 7.8, H- 2'), 3.73 (t, 10.2, H-5'b), 3.62 (d, 10.4, H-26b), 3.44 (dd, 3.4, 10.8, H-3), 2.70 (dd, 8.6, 15.5, H-11a), 2.28 H-2a), 2.23 H-20), 2.12 (s, OAc), 1.89 (dd, 6.7, 11.8, H-
181
15a), 1.84 H-2b), 1.78 H-15b), 1.76 (overlapped, H-17), 1.59 (brd, 12.4, H-8), 1.59 (brd, 12.4, H-22a), 1.52 H-1a), 1.45 (s, H-27), 1.44 (brd, 12.4, H-22b), 1.40 (s, H-18), 1.4 H-6a), 1.29 (s, H-29), 1.22 (brd, 10.1, H-5), 1.21 H-7a), 1.17 (brd, 15.5, H-11b), 1.11 H-1b), 1.00 (d, 6.7, H-21), 0.99 (s, H-30), 0.91 H-7b), 0.83 (s, H-28), 0.60 (brd, 13.6, 11.1, H-6b), 0.52 (d, 3.8, H-19a), 0.19 (d, 3.8, H-19b),, 13 C-NMR data (500 MHz, pyridine-d5) δ 170.7 (s, OAc), 107.5 (d, C-1'), 105.9 (s, C-23), 88.1 (d, C-3), 78.7 (d, C-3'), 77.1 (d, C-12), 75.6 (d, C-2'), 74.5 (d, C-16), 71.3 (d, C-4'), 67.2 (t, C-5'), 67.1 (t, C-26), 62.5 (d, C-24), 62.3 (s, C-25), 56.2 (d, C-17), 48.8 (s, C-13), 47.9 (s, C-14), 47.0 (d, C-5), 45.6 (d, C-8), 44.2 (t, C-15), 41.2 (s, C-4), 37.6 (t, C-22), 36.7 (t, C-11), 32.0 (t, C-1), 30.0 (t, C-2), 29.5 (t, C- 19), 26.8 (s, C-10), 25.7 (t, C-7), 25.7 (q, C-29), 23.3 (d, C-20), 21.7 (q, C-21), 21.4 (q, OAc), 20.4 (t, C-6), 20. 2 (s, C-9), 19.7 (q, C-28), 15.3 (q, C-30), 14.3 (q, C-27), 13.5 (q, C-18), FABMS m/z (relative intensity %) 661.4 (0.75), 613.2 (2.0), 460.1 (11), 307.1 (100), 289.1 (43), 219.2 (9), 154.1 (100), 136.1 (100), 107.9
(75), 78.9 (35), 66.3 (27); HRFABMS m/z 683.3779 (calcd 683.3771 for C37
H56O10Na).
20 26-deoxyactein: colorless needles, mp 253-254 °C, [α] D -52.17 (c 0.025, 1 CHCl3); H-NMR data (500 MHz, pyridine-d5) δ 5.11 (dd, 3.6, 8.8, H-12), 4.86 (d, 7.6, H-1'), 4.53 (brdd, 7.2, 14.2, H-16), 4.38 (dd, 11.2, 5.1, H-5'b), 4.24 (m, H-4'), 4.18 (t, 8.7, H-3'), 4.05 (t, 7.8, H-2'), 3.95 (d, 10.0, H-26a), 3.88 (d, 10.0, H-26b), 3.77 (t, 10.0, H-5'a), 3.71 (s, H-24), 3.48 (dd, 4.3, 11.7, H-3), 2.74 (dd, 7.1, 15.9, H-11a), 2.28 (H-2a), 2.19 (brd, 12.4, H-22a), 2.15 (s,OAc), 1.91 (dd, 8.0, 12.6, H- 15a), 1.88 (H-2b), 1.80 (overlapped, H-17), 1.79 (H-20), 1.68 (dd, 6.6, 12.6, H- 15b), 1.64 (dd, 4.7, 14.4, H-8), 1.6 (H-22b), 1.52 (H-1a), 1.47 (s, H-27), 1.44 (H- 6a), 1.37 (s,H-29), 1.35 (s,H-18), 1.3 (H-7a), 1.26 (brd, 12.4, H-5), 1.20 (dd, 3.7, 15.9, H-11b), 1.15 (H-1b), 1.02 (s, H-30), 0.96 (d, 5.7, H-21), 0.95 (H-7b), 0.86 (s, H-28), 0.69 (brd, 12.7, H-6b), 0.60 (d, 4.0, H-19a), 0.25 (d, 4.0, H-19b) 13C-
NMR data (500 MHz, pyridine-d5) δ 170.6 (s, OAc), 107.5 (d, C-1'), 105.9 (s, C- 23), 88.1 (d, C-3), 78.7 (d, C-3'), 77.1 (d, C-12), 75.6 (d, C-2'), 73.0 (d, C-16), 71.3 (d, C-4'), 68.8 (t, C-26), 67.1 (t, C-5'), 63.3 (s, C-25), 63.3 (d, C-24), 56.5 (d,
182
C-17), 48.8 (s, C-13), 48.0 (s, C-14), 47.0 (d, C-5), 45.8 (d, C-8), 43.7 (t, C-15), 41.2 (s, C-4), 36.7 (t, C-11), 36.7 (t, C-22), 32.0 (t, C-1), 29.9 (t, C-2), 29.6 (t, C- 19), 26.8 (s, C-10), 26.0 (d, C-20), 25.7 (t, C-7), 25.7 (q, C-29), 21.0 (q, C-21), 20.7 (q, OAc), 20.5 (t, C-6), 20. 2 (s, C-9), 19.6 (q, C-28), 15.4 (q, C-30), 13.8 (q, C-27), 13.5 (q, C-18), FABMS m/z (relative intensity %) 661.4 (6), 451.3 (25), 307.1 (100), 289.1 (58), 219 (15), 154.1 (100), 136.1 (80), 107.9 (60), 78.9 (60);
HRFABMS m/z 683.3775 (calcd 683.3771 for C37 H56O10Na).
183
10.2 Mass spectral data supporting the presence of additional nitrogenous constituents in Black Cohosh.
Samp le g11 on amide column crjuly 150403 TOF MS ES+ 15.19 100 180 180.1 7.42e3
%
5.64 180.1 3.92 163.1
0 crjuly150403 TOF MS ES+ 27.24 100 342 342.2 1.82e3
%
4.97 342.3
8.25 6.28 342.2 11.81 3.33 137.17.24 20.57 344.3 163.1 208.2 270.2 0 Time 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Appendix Figure 1. Additional LC-MS/MS spectral analysis supporting the presence of compounds in Cimicifuga racemosa fraction g11 with an odd number of nitrogens (n=1,3,5,..etc.).
184
Sample g11 on amide column ms/msSample g11 on amide column ms/ms 15-JUL-2004 crjuly150404 119 (16.523) Sm (SG, 1x3.00); Cm (112:127) 3: TOF MSMS 172.10ES+ 70.1025 100 501
%
112.1264
0 crjuly150404 72 (13.992) Sm (SG, 1x3.00); Cm (62:81) 3: TOF MSMS 172.10ES+ 70.1059 100 564
%
172.1561 130.1292
94.1059 112.1177 154.1461 71.0933 113.1096 137.1125
0 m/z 70 80 90 100 110 120 130 140 150 160 170
Appendix Figure 2. High resolution positive-ion electrospray tandem mass spectrum of the protonated molecule of m/z 172.1561, in fraction g11, obtained utilizing a quadrupole time-of-flight mass spectrometer. This mass and fragmentation pattern are similar to cimipronidine.
185
Sample g11 on amide column ms/msSample g11 on amide column ms/ms 15-JUL-2004 crjuly150404 48 (15.528) Sm (SG, 1x3.00); Cm (41:55) 5: TOF MSMS 154.10ES+ 154.1411 100 99.0
70.1059
%
94.0980
95.0904
112.1177
77.0875
0 m/z 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160
Appendix Figure 3. High resolution positive-ion electrospray tandem mass spectrum of the protonated molecule of m/z 154.1411 from fraction g11 obtained utilizing a quadrupole time- of-flight mass spectrometer. The fragments are similar to cimipronidine.
186
Sample g11 on amide column ms/msSample g11 on amide column ms/ms 15-JUL-2004 crjuly150404 177 (27.079) Sm (SG, 1x3.00); Cm (148:217) 7: TOF MSMS 342.10ES+ 180.1536 100 255
163.1179
%
342.2438
145.0986
85.0697 137.0982 117.1167 301.2376 0 crjuly150404 63 (15.034) Sm (SG, 1x3.00); Cm (54:68) 4: TOF MSMS 180.10ES+ 145.1084 109 100 117.1123
%
163.1179
115.0979
91.0898 180.1426
0 m/z 80 100 120 140 160 180 200 220 240 260 280 300 320 340
Appendix Figure 4. High resolution positive-ion electrospray tandem mass spectrum of the protonated molecules of m/z 342.2438 and 180.1426 obtained utilizing a quadrupole time-of- flight mass spectrometer. The fragmentation pattern varies from the pattern seen with cimipronidine.
187
10.3 Additional Black Cohosh Documentation
Appendix Figure 5. Permit to Collect Cimicifuga species in Great Smoky Mountain National Park (GSMNP). Permits were held by the UIC/NIH Center from 1999 to 2002 to collect in the GSMNP.
188
Appendix Figure 6. Permit to Collect Cimicifuga species in the Blue Ridge Parkway (BRP). Permits were held by the UIC/NIH Center from 1999 to 2002 to collect in the BRP.
189
Appendix Figure 7. Results for the clinical black cohosh extract, screen for heavy metals by USP <561> using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The result of 13 ppm is within acceptable limits for total heavy metals set forth by USP of 20 ppm.
190
Appendix Figure 8. Results for the clinical black cohosh extract screen for Lead (Pb) and Cadmium (Cd) using a graphite furnace. The results were below level of quantitation. This indicated no potential toxic concentration of either heavy metal was present in the extract. The lead level at <0.1 ppm is below the California Proposition 65 level of 0.5 ppm of lead daily.
191
Appendix Figure 9. USP pesticide screen of black cohosh extract. No detectable pesticides in the extract were reported.
192
Appendix Figure 9 (continued). USP pesticide screen of black cohosh extract. No detectable pesticides in the extract were reported.
193
Appendix Figure 10. Ames Test screening for mutagenicity of TA98 without metabolic activation (-S9) of Black Cohosh Phase I extract. The result was no mutagenic effect.
194
Appendix Figure 11. Ames Test screening for mutagenicity of TA100 without metabolic acitivation (-S9) of Black Cohosh Phase I extract. The result was no mutagenic effect.
195
Appendix Figure 12. Ames Test screening for mutagenicity of TA98 with metabolic acitivation of Black Cohosh Phase I extract. The result was no mutagenic effect.
196
Appendix Figure 13. Ames Test screening for mutagenicity of TA100 with metabolic activation of Black Cohosh Phase I extract. The result was no mutagenic effect.
197
Appendix Figure 14. Ames Test screening methodology and positive control data.
198
Appendix Figure 15. Material data safety sheet from Pure World Botanicals on clinical black cohosh extract.
199
Appendix Figure 16. Pure World Botanicals certificate of analysis of Clinical Black Cohosh extract.
200
Appendix Figure 17. Batch record of Phase I clinical encapsulation from Pharmavite corporation.
201
Appendix Figure 18. Additional batch record report from Pharmavite corporation for the Phase I clinical capsule.
202
Appendix Figure 19. Mixing formulation record of Phase I clinical capsule from Pharmavite Corporation.
203
Appendix Figure 20. Clinical capsule specifications summary from Pharmavite corporation.
204
11.0 Vita
1.0 Personal Data
Daniel S. Fabricant Program for Collaborative Research in the Pharmaceutical Sciences, UIC/ NIH Center for Botanical Dietary Supplement Research, College of Pharmacy, University of Illinois at Chicago 833 S. Wood Street (M/C 877), Chicago, Illinois 60612 Phone: 312.996.7253 Fax: 312.413.5894 E-mail: [email protected]
2.0 Education
1998-2005 Doctoral Candidate, Pharmacognosy, UIC/NIH Center for Botanical Dietary Supplements Research, Program for Collaborative Research in the Pharmaceutical Sciences (PCRPS), University of Illinois at Chicago, Chicago, Illinois.
1997 B.S., Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
3.0 Positions Held and Responsibilities
2005 Assistant Director, Research, ConsumerLab.com, Pasadena, Maryland
2000-2005 Research Assistant, NIH/ODS Center for Botanical Dietary Supplement Research. University of Illinois at Chicago. Development of a chemically, biologically and botanically standardized dosage form of Cimicifuga racemosa for use in Clinical Trials. Isolation and structure elucidation of bioactive constituents from polar fractions. HPLC-ELSD and RAPD-PCR method development for identification of botanicals. Procurement and identification of plant samples. Organic natural product synthesis. Consulted with clinical arm on DSHEA for IRB approval. Advisors: Norman R. Farnsworth and Guido F. Pauli
1999- 2001 Production Associate, NAPRALERT database, University of Illinois at Chicago. Evaluation of scientific literature relating to the chemistry, biological activity and traditional uses of natural products, coding of pharmacological activity data into database.
1998 Teaching Assistant, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at
205
Chicago. Taught over 30 lecture and recitation sections for professional pharmacy medicinal/organic chemistry courses.
1998 Editorial Assistant, WHO Collaborating Centre for Research in Traditional Medicine, University of Illinois at Chicago. Research and editing of WHO monographs on Selected Medicinal Plants, Volume 1.
1997 Research and Development Scientist, Viragen Inc., Hialeah, FL. HPLC and SEC method development for quantification and purification of interferons. ELISA techniques for identification of interferons. Performed cGMP HPLC stability studies of interferons. Supervisor: Duy Nguyen, Ph.D.
1997 Undergraduate Teaching Assistant, University of North Carolina- Chapel Hill. Chemistry 11-Freshman Chemistry Laboratory. Proctored lectures, laboratory experiments and examinations. Supervisor: Don C. Jicha, Ph.D.
1996 Undergraduate Research Assistant, University of North Carolina- Chapel Hill. Protein synthesis and purification. Mentor: Gary J. Pielak, Ph.D.
1994-1995 Summer Intern, Dade County Medical Examiners’ Office, Toxicology Lab, Miami, FL. HPLC, GC method development for detection and quantification of parent compounds and drug metabolites. Mentor: W. Lee Hearn, Ph.D.
4.0 Society Memberships
2003-2005 American Society of Pharmacognosy (ASP)
1998-2002 American Association for the Advancement of Science (AAAS)
1995-2002 American Chemical Society (ACS)
5.0 Consulting Activities
2003 Bio-equivalence studies on generic Misoprostol. Development of HPLC-ELSD, -MS methodology.
2003 United States Pharmacopoeia. Monograph evaluation of Cimicifuga racemosa extracts.
2002 American Herbal Pharmacopoeia Monographs on Cimicifuga racemosa.
206
1998 Tom’s of Maine, Research and development work. Performed stability studies on a variety botanical extracts. Developed formulation and extraction protocols of bulk plant material used for commercial products.
6.0 Additional Training
2003 Food and Drug Law Institute (FDLI)- Dietary Supplements at a Crossroads, January 15-16, Washington D.C.
2002 Food and Drug Law Institute (FDLI)-Introduction to Food Law, January 14-15, Washington D.C., Sponsored by ODS
2000 NCCAM Workshop on RT-PCR of Botanicals, November 2-3, NIEHS, RTP, North Carolina. Sponsored by ODS
7.0 Peer Reviewed Publications
2005 Cimipronidine a novel alkaloid from C. racemosa J Nat Prod (Re- Submitted)
2005 Synthesis of cimiracemate B, a phenylpropanoid found in Cimicifuga racemosa. Chen SN, Fabricant DS, Pauli GF, Fong HH, Farnsworth NR. Nat Prod Res. 19(3):287-90
2005 Marcel Dekker/Office of Dietary Supplements Encyclopedia of Dietary Supplements, New York, Paul Coates, Editor, Black Cohosh by Fabricant DS, Farnsworth NR, pp.41-54
2003 Black cohosh acts as a mixed competitive ligand and partial agonist of the serotonin receptor. Burdette JE, Liu J, Chen SN, Fabricant DS, Piersen CE, Barker EL, Pezzuto JM, Mesecar A, van Breemen RB, Farnsworth NR, Bolton JL. J. Agric. Food Chem. 51(19): 5661- 5670.
2002 A preliminary RAPD-PCR analysis of Cimicifuga species and other botanicals used for women’s health. Xu H, Fabricant DS, Piersen CE, Bolton JL, Pezzuto JM, Fong H, Totura S, Farnsworth NR, Constantinou AI. Phytomedicine 9(8): 757-62.
2002 Black Cohosh: an alternative therapy for menopause? Mahady, GB, Fabricant DS, Chadwick LR, Dietz B. Nutr. Clin. Care 5(6): 283-9.
2002 High-performance liquid chromatographic analysis of Black Cohosh (Cimicifuga racemosa) constituents with in-line evaporative light
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scattering and photodiode array detection. Li WK, Chen SN, Fabricant D, Angerhofer CK, Fong HHS, Farnsworth NR, Fitzloff JF. Anal. Chim. Acta 471(1): 61-75.
2002 Black Cohosh (Cimicifuga racemosa) protects against menadione- induced DNA damage through scavenging of reactive-oxygen species: Bioassay-directed isolation and characterization of active principles. Burdette JE, Chen S, Lu ZZ, Xu H, White B, Fabricant DS, Liu J, Fong HHS, Farnsworth NR, Constantinou AI, van Breemen RB, Pezzuto JM, Bolton JL. J Agric Food Chem. 50(24): 7022-8.
2002 Cimiracemates A-D, novel phenylpropanoid esters from the rhizomes of Cimicifuga racemosa Chen SN, Fabricant DS, Lu ZZ, Zhang HJ, Fong HHS, Farnsworth NR. Phytochemistry 61(4): 409- 13.
2002 Cimiracemosides I-P, new 9,19-cycloartane triterpene glycosides from C. racemosa Chen SN, Fabricant DS, Lu ZZ, Fong HHS, Farnsworth NR. J. Nat. Prod. 65(10): 1391-7.
2002 The absolute configurations of 26-deoxyactein and 23-epi-26- deoxyactein. Chen SN, Li WK, Fabricant DS, Santarsiero BD, Mesecar A, Fitzloff JF, Fong HHS, Farnsworth NR. J. Nat. Prod. 65(4):601-5.
2001 The Value of Plants Used in Traditional Medicine for Drug Discovery. Fabricant DS, Farnsworth NR. Environ. Health Perspect. 109(S1): 69-75.
2000 Plants Used Against Cancer: An Extension of the Work of Jonathan Hartwell. Graham JG, Fabricant DS, Quinn ML, Farnsworth NR, J. Ethnopharmacology 73 (3): 347-377.
8.0 Poster Presentations
2004 A novel guanidine alkaloid from C.racemosa. Fabricant DS, Chen SN, Nikolic D, Jaki B, Lankin DC, van Breemen RB, Pauli GF, Farnsworth NR, In: 45th Annual ASP meeting, July 23-25; Tuscon, Arizona.
2002 Development of a chemical, biologically and botanically standardized dosage form of Cimicifuga racemosa for use in clinical trials. Fabricant DS, Chen SN, Nikolic D, Burdette JE, Li WK, Fitzloff JF, van Breemen RB, Bolton JL, Fong HHS, Farnsworth, In:
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NCCAM/ODS Center Directors Meeting 2002, July 8-10; Bethesda, Maryland.
2001 Geographical and seasonal variation of major chemical constituents of Cimicifuga racemosa. Fabricant DS, Chen SN, Li WK, Fitzloff JF, Fong HHS, Farnsworth NR, In: Proceedings ASP/CRN meeting: Botanical Dietary Supplements: Natural Products at a Crossroads, November 8-11; Asilomar, California.
2001 Using RAPD-PCR markers to identify plant species and variants. Xu H, Fabricant DS, Johnson HE, Piersen CE, Bolton JL, Farnsworth NR, Constantinou A, In: Proceedings ASP/CRN meeting: Botanical Dietary Supplements: Natural Products at a Crossroads, November 8-11; Asilomar, California.
2001 High performance liquid chromatographic analysis of Black Cohosh (Cimicifuga racemosa L.) with in-line photodiode array and evaporative light scattering detection. Li W, Chen SN, Fabricant DS, Fong HHS, Farnsworth NR, Fitzloff JF, In: Abstracts 222nd ACS National Meeting, August 26-30, Chicago, Illinois.
9.0 Lectures Presented
2003 UIC/NIH Center for Botanical Dietary Supplements Research Brainstorming Sessions. Food and Drug Law: Dietary Supplements. February 24th .
2002 UIC/NIH Center for Botanical Dietary Supplements Research Brainstorming Sessions. Cimicifuga racemosa: Research Update. October 21st
10.0 Meetings attended
2002 NCCAM/ODS 2nd Annual Center Directors Meeting 2002, July 8-10; Bethesda, Maryland.
2001 ASP/CRN meeting: Botanical Dietary Supplements: Natural Products at a Crossroads, November 8-11; Asilomar, California.
1999 ASP midterm meeting: Botanical Dietary Supplements: May 1-3; Tunica, Mississippi.
1998 39th Annual ASP meeting: July 25-28; Orlando, Florida.
11.0 Honors and Awards
2004 Myron Goldsmith Scholarship
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2001-2005 College of Pharmacy Dean’s Scholarship Recipient
1994 Dean’s List, Ithaca College
13.0 Volunteer and Other Activities
2000 Co-Coordinator. Norman R. Farnsworth’s 70th birthday celebration at the Drake Hotel, Chicago, Illinois. Raised over $ 25,000 in funds from ticket sales and industrial contributions to cover expenses. Responsible for site-selection, student participation and coordination of international participation.
1998-1999 Graduate Student Council Representative, University of Illinois at Chicago. Participated as a representative for the National Graduate Student Meeting (NAGPS). Co-Chairperson on subcommittee for incorporation of optical and dental health care plans for graduate student health insurance at UIC.
1996-1997 Americorps, University of North Carolina-Chapel Hill. Literacy tutor for migrant workers learning English as a second language (ESOL). Organized Carr Court summer camp for educationally challenged children.
1995-1997 Varsity Letterman, Football, University of North Carolina-Chapel Hill.
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