PHYTOCHEMICAL INVESTIGATION OF BIOACTIVE CONSTITUENTS FROM ANGELICA SINENSIS

BY

SHIXIN DENG

B.S., Liaoning College of Traditional Chinese Medicine, 1992

DISSERTATION

Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacognosy in the

Graduate College, University of Illinois at Chicago,

2005

CHICAGO, ILLINOIS

DEDICATION

This dissertation is dedicated to my loving family, without

their endless supports and encouragements, I would never have

accomplished it.

ii

ACKNOWLEDGMENTS

First of all, I would like to express my most sincerely appreciation to

Professors Norman R. Farnsworth and Guido F. Pauli. I feel so fortunate and

pleasant to have them as my advisors. Dr. Farnsworth impressed me not only

by his profound and intelligent knowledge in Pharmacognosy and its related

fields, but also by his generosity, wisdom, patience, and the capability to grasp

the key points from millions of minutiae. I would not have brought this

dissertation to reality without his countless encouragement, support and kind

direction. Dr. Pauli brought me into an amazing world where I have learned

many advanced techniques. His strict and serious attitudes on my work,

extensive knowledge, eagerness to teach me updated techniques, as well as

organization habits inspired me to dedicate me to the interesting field of

Pharmacognosy. I am very grateful to Dr. Harry H.S. Fong for his helpful

suggestions in my project. I would like to thank Dr. Judy L. Bolton, Dr.

Richard B. van Breemen and Dr. Jim Wang for reviewing this dissertation and

serving as members of my dissertation defense committee. I also thank Dr.

Jimmy Orjala for serving on my preliminary examination. I really appreciate

Dr. Bolton and her group members, Dr. Birgit Dietz, Dr. Rachel Ruhlen, Ms.

Ping Yao and Ms. Cassia Overk in project 2 of the Botanical Center for their

wonderful job in conducting large numbers of bioassays. I am very grateful to

Dr. Richard van Breemen and Dr. Dejan Nikolic in project 3 and Core C for

providing instant and accurate MS results and analyses. I am very thankful to

Dr. Jim Wang and his student Mr. Jian Lu for their very helpful bioassay

support. I would like to thank the entire faculty, students and post-doctoral

follows in the Botanical Center and Department of Medicinal Chemistry and

Pharmacognosy. Dr. Doel Soejarto afforded kind discussions on botanical

identification. Dr. Geoffrey Cordell and Dr. Steve Swanson provided much help

as the directors of graduate studies. Dr. Shaonong Chen generously offered

ACKNOWLEDGMENTS (continued) iii

numerous assistance whenever I confronted problems during these years, and

his practical knowledge aided in my work. Drs. Hongjie Zhang, Baoning Su,

David Lankin and Robert Kleps provided kind suggestions on NMR

experiments. Dr. Lucas R. Chadwick gave me enormous support based on his

versatile capacity. Drs. Colleen Pierson, Nancy Booth, Allison Turner, Daniel

Fabricant and Elizabeth Krause also provided their enthusiastic help in my

daily work. Dr. Bernie Santarsiero provided the X-ray support for my

experiments.

I would like to thank National Institutes of Health (NIH) (grant P50

AT00155), the Office of Dietary Supplements (ODS), the National Center for

Complementary and Alternative Medicine (NCCAM), National Institutes of

General Medical Sciences (NIGMS) and the Office for Research on Women’s

Health (ORWH) for financially supporting this work. I am grateful to the

University of Illinois at Chicago, College of Pharmacy, and the Department of

Medicinal Chemistry and Pharmacognosy for providing me with a tuition

waiver and stipend to make my studies possible, and the Dean of the College of

Pharmacy, Professor Rosalie Sagraves for offering me a Dean’s Scholarship.

Finally, I would like to express my deepest gratitude to my loving family,

Baoci Deng, Yaxian Zhang, Liqing Deng, Lijuan Deng, Wenzhan Wang,

Xueliang Yuan, Ling Wang, Yue Yuan, Wenfei Hu, and Jinshui Zhao, and

particularly my lovely and clever daughter, Anqi Deng, they never stoped giving

their love and support to me over these years, I really feel happy to have them

in my family.

Without all of the help mentioned above, this dissertation would not

have become reality.

SD

iv

TABLE OF CONTENTS

ACKNOWLEDGMENTS................................................................................ii LIST OF SYMBOLS AND ABBREVIATIONS............................................... viii LIST OF TABLES.......................................................................................xii LIST OF FIGURES....................................................................................xiii

SUMMARY ............................................................................................... xix

1. INTRODUCTION ................................................................................1 1.1. INTRODUCTION AND SCOPE OF STUDY..........................................1 1.2. SIGNIFICANCE OF PREMENSTRUAL SYNDROME ............................3 1.3. PLANTS AS POTENTIAL ALTERNATIVE THERAPIES .........................5 1.4. STATEMENT OF PROBLEM...............................................................7 1.5. PHARMACOGNOSY OF ANGELICA SINENSIS ...................................8 1.5.1. BOTANY, TAXONOMY AND IDENTIFICATION.............................8 1.5.1.1. The family Apiaceae .................................................................8 1.5.1.2. The genus Angelica ................................................................10 1.5.1.3. The species Angelica sinensis (Oliv.) Diels ..............................10 1.5.1.4. Identification of A. sinensis plant material .............................12

1.5.2. PHYTOMEDICINE .....................................................................14 1.5.2.1. Gynecological Uses ................................................................16 1.5.2.2. Other Uses.............................................................................18

1.5.3. PHARMACOLOGY .....................................................................19 1.5.3.1. In vivo activities .....................................................................19 1.5.3.2. In vitro activities.....................................................................22

1.5.4. PHYTOCHEMISTRY...................................................................25

2. EXPERIMENTAL..............................................................................28 2.1. Plant materials ................................................................................28 2.2. Identification of Plant Materials .......................................................28 2.2.1. Macroscopic examination..........................................................29 2.2.2. Microscopic identification..........................................................29 2.2.3. High-performance thin-layer chromatography (HPTLC) .............29 2.2.4. High-performance liquid chromatography (HPLC) .....................30

2.3. Bioassays ........................................................................................30 2.3.1. 5-HT7 receptor binding assay ....................................................30 2.3.2. 5-HT5A receptor binding assay ...................................................32 2.3.3. GABAa receptor binding assay ..................................................32

TABLE OF CONTENTS (continued) v

2.3.4. ERα and ERβ receptor binding assays ......................................33 2.4. Extraction and partitioning .............................................................34 2.4.1. Extraction .................................................................................34 2.4.2. Partitioning ...............................................................................35

2.5. Chromatographic fractionation and isolation...................................35 2.5.1. Solvents ....................................................................................35 2.5.2. Thin-layer chromatography (TLC)..............................................35 2.5.3. Vacuum liquid chromatography (VLC).......................................36 2.5.4. Droplet countercurrent chromatography (DCCC) ......................36 2.5.5. Medium pressure liquid chromatography (MPLC)......................38 2.5.6. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ...38

2.6. Spectroscopic examination of isolates..............................................38 2.6.1. Nuclear magnetic resonance spectroscopy (NMR)......................39 2.6.2. Mass spectrometry (MS) ............................................................39 2.6.3. Ultraviolet spectroscopy (UV).....................................................39 2.6.4. Infrared spectroscopy (IR)..........................................................40 2.6.5. Optical Rotation ([α]D)................................................................40

3. RESULTS.........................................................................................41 3.1. Identification of Plant Materials .......................................................41 3.1.1. Macroscopic examination..........................................................41 3.1.2. Microscopic characteristics .......................................................42 3.1.3. HPTLC.......................................................................................43 3.1.4. HPLC ........................................................................................45 3.1.5. Summary of plant identification................................................46

3.2. Extraction and partitioning .............................................................47 3.3. Biological activities of extract and partitions....................................47 3.4. Fractionation and isolation of pure compounds...............................49 3.4.1. Isolation of 1 (Z-ligustilide) ........................................................52 3.4.2. Isolation of 2 (Z-butylidenephthalide) ........................................52 3.4.3. Isolation of 3 (senkyunolide-I) ...................................................52 3.4.4. Isolation of 4 (riligustilide).........................................................52 3.4.5. Isolation of 5 (gelispirolide)........................................................53 3.4.6. Isolation of 6 (10-angeloylbutylphthalide)..................................53 3.4.7. Isolation of 7 (sinaspirolide) ......................................................53 3.4.8. Isolation of 8 (ansaspirolide)......................................................53 3.4.9. Isolation of 9 (angediligustilide) .................................................53 3.4.10. Isolation of 10 (p-hydroxyphenethyl trans-ferulate) ...................53 3.4.11. Isolation of 11 (angeliferulate) ...................................................54 3.4.12. Isolation of 12 (N-butylbenzenesulphonamide)..........................54

TABLE OF CONTENTS (continued) vi

3.4.13. Isolation of 12 and 13 (Z-6-hydroxy-7-methoxy- dihydroligustilide & N-butylbenzenesulphonamide)...................54 3.4.14. Isolation of 14 (imperatorin)......................................................54 3.4.15. Isolation of 15 (ferulic acid) .......................................................55 3.4.16. Isolation of 16 (vanillin).............................................................55 3.4.17. Isolation of 17 (stigmasterol) .....................................................55 3.4.18. Isolation of 18 (sucrose) ............................................................55 3.4.19. Isolation of 19 (falcarindiol).......................................................55 3.4.20. Isolation of 20 (9Z,17-octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate) ..................................................................................55 3.4.21. Isolation of 21 (heptadeca-1-ene-9,10-epoxy-4,6-diyne-3,8-diol) ................................................................................................56 3.4.22. Isolation of 22 (oplopandiol) ......................................................56 3.4.23. Isolation of 23 (8-hydroxy-1-methoxy-9-heptadecene-4,6-diyn- 3-one)........................................................................................56 3.4.24. Summary of fractionation and isolation ....................................70

3.5. Chemical characterization of isolated compounds ...........................70 3.5.1. Identification of 1 (Z-ligustilide).................................................70 3.5.2. Identification of 2 (Z-butylidenephthalide) .................................72 3.5.3. Identification of 3 (senkyunolide-I) ............................................73 3.5.4. Identification of 4 (riligustilide)..................................................74 3.5.5. Identification of 5 (gelispirolide).................................................76 3.5.6. Identification of 6 (10-angeloylbutylphthalide) ..........................85 3.5.7. Identification of 7 (sinaspirolide) ...............................................92 3.5.8. Identification of 8 (ansaspirolide) ............................................102 3.5.9. Identification of 9 (angediligustilide)........................................111 3.5.10. Identification of 10 (p-hydroxyphenethyl trans-ferulate)..........119 3.5.11. Identification of 11 (angeliferulate) ..........................................120 3.5.12. Identification of 12 (N-butylbenzenesulphonamide).................128 3.5.13. Identification of 12 and 13 (Z-6-hydroxyl-7-methoxyl- dihydroligustilide & N-butylbenzenesulphonamide).................129 3.5.14. Identification of 14 (imperatorin).............................................130 3.5.15. Identification of 15 (ferulic acid)..............................................131 3.5.16. Identification of 16 (vanillin)....................................................132 3.5.17. Identification of 17 (stigmasterol) ............................................132 3.5.18. Identification of 18 (sucrose) ...................................................133 3.5.19. Identification of 19 (falcarindiol)..............................................133 3.5.20. Identification of 20 (9Z,17-octadecadiene-12,14-diyne-1,11,16- triol, 1-acetate) ........................................................................135 3.5.21. Identification of 21 (heptadeca-1-ene-9,10-epoxy-4,6-diyne-3,8- diol).........................................................................................136

TABLE OF CONTENTS (continued) vii

3.5.22. Identification of 22 (oplopandiol) .............................................137 3.5.23. Identification of 23 (8-hydroxy-1-methoxy-9-heptadecene-4,6- diyn-3-one)..............................................................................138 3.5.24. Summary of identification of pure compounds........................139

3.6. Biological characterization of the isolated compounds ...................142 3.6.1. 5-HT7 receptor binding activity ................................................142 3.6.2. GABAa receptor binding activity..............................................143 3.6.3. Anti-TB activity .......................................................................144 3.6.4. Summary of biological activities of the active isolates..............146

3.7. Discussion.....................................................................................147 3.7.1. Stability ..................................................................................147 3.7.2. Full assignments of 1H spectra of known compounds .............147 3.7.3. Major constituents of A. sinenesis ...........................................148 3.7.4. Standardization.......................................................................150

4. CONCLUSIONS AND FUTURE DIRECTIONS .................................152 4.1. Conclusions...................................................................................152 4.2. Future Directions ..........................................................................155 4.2.1. Stability testing of major components and active isolates........155 4.2.2. LC-MS fingerprint identification of constituents in the methanol extract.....................................................................................155 4.2.3. Fractionation of bioactive components from other active fractions ..............................................................................................155 4.2.4. Biological evaluation of the methanol extract in other in vitro assays .....................................................................................156 4.2.5. The biological mechanism of the active isolates.......................156 4.2.6. Toxicity ...................................................................................156

LITERATURE CITED ...............................................................................157 APPENDIX A A summary of distribution and traditional uses of plants in the genus Angelica .............................................................172 APPENDIX B Tabulated NMR data for phthalides isolated from A. sinensis...............................................................................................................178 APPENDIX C MS and NMR spectra of known compounds isolated from A. sinensis .............................................................................183 APPENDIX D Structures of the isolates from A. sinensis .........................221 APPENDIX E Phthalide dimers isolated from plants ................................223 APPENXID F Biological activities of isolates from A. sinensis...................225

TABLE OF CONTENTS (continued) viii

APPENDIX G Simulated and iterated 1H spectra of Z-ligustilide (1) and Z- butylidenephthalide (2) by PERCH NMR software ..............226 APPENDIX H Biological activities of methanol extracts of samples 3 (BC080) and 4 (BC 158)...................................................................228 VITA ........................................................................................................229

ix

LIST OF SYMBOLS AND ABBREVIATIONS

# any Latin numeral in bold font represents isolated compound [α]D specific optical rotation APT attached proton test BuOH butanol ºC degree centigrade δc carbon-13 chemical shift c concentration calcd calculated CCC countercurrent chromatography CHCl3 chloroform CID collision-induced dissociation cm-1 reciprocal centimeters 13C NMR carbon-13 nuclear magnetic resonance COSY COrrelative SpectroscopY 1D, 2D one- or two-dimentional δ (ppm) chemical shift (in parts per million) DEPT distortionless enhancement by polarization transfer DMSO dimethyl sulfoxide DSHEA Dietary Supplement Health and Education Act molar absorptivity E# (in bold font) represents extract after solvent removal ERα alpha estrogen receptor ERβ beta estrogen receptor ESI-MS electrospray ionization - mass spectrometry EtOAc ethyl acetate EtOH ethanol eV electron volt F# (in bold font) represents fraction after solvent removal FDA Food and Drug Administration g gram(s) h hour δH proton chemical shift HMBC heteronuclear multiple-bond connectivity spectroscopy HMQC heteronuclear multiple-bond quantum coherence spectroscopy 1H NMR proton nuclear magnetic resonance HPLC high-performance liquid chromatography HR high resolution 5-HT serotonin 5-HT1A serotonin 1A receptor

SYMBOLS AND ABBREVIATIONS (continued) x

5-HT5A serotonin 5A receptor 5-HT7 serotonin 7 receptor Hz hertz IC50 concentration that inhibits a response by 50% relative to a

positive control Ј coupling constant L liter(s) λ (nm) wavelength (in nanometers) LP lower phase of two-phase solvent mixture M molar concentration [M-H]- deprotonated molecule [M+H]- protonated molecule [M]+ · molecular ion MARC plant material remaining after solvent extraction and filtration max maximum MeOH methanol MeOH-d4 deuterated methanol (NMR solvent) M 10-6 mol/liter min minute(s) mg 10-3 gram(s) or milligram(s) MPLC medium-pressure liquid chromatography µg 10-6 gram(s) or microgram(s) µM 10-6 mol/liter MHz 106 Hertz or megahertz mL 10-3 liter(s) or milliliter(s) MP mobile phase in countercurrent chromatography mp melting point MS mass spectrometry or mass spectrum MS2 tandem mass spectrometry (= MSMS) m/z mass-to-charge ratio ND not determined ν infrared absorption frequency nm nanometers or 10-9 meters nM 10-9 mol/liter NMR Nuclear Magnetic Resonance NOE Nuclear Overhauser effect NOESY Nuclear Overhauser effect correlation spectroscopy NUTS NMR simulation and processing tool used in the present work ORD optical rotatory dispersion P# (in bold font) represents partition after solvent removal PDA Photo Diode Array pet ether petroleum ether

SYMBOLS AND ABBREVIATIONS (continued) xi

PMS premenstrual syndrome ppm parts per million (for MS, represents [experimental mass –

theoretical mass] / [theoretical mass]) Rf retention factor; migration distance of analyte as a fraction of

distance to solvent front in thin-layer chromatography Si silica SP stationary phase in countercurrent chromatography SSRIs selective serotonin reuptake inhibitors t R retention time TCM Traditional Chinese Medicine TLC thin-layer chromatography TMS tetramethylsilane UP upper phase of two-phase solvent mixture UV ultraviolet VLC Vacuum Liquid Chromatography v volumn w weight

xii

LIST OF TABLES

Table 1. Angelica plant materials from different sources................................................28

Table 2. Gradient solvent for system in HPLC fingerprints experiments.........................30

Table 3. Macroscopic characteristics of different Angelica plant materials.....................42

Table 4. Microscopic characteristics of different samples in the genus Angelica ............43

Table 5. Biological activities of extracts and partitions of Angelica sinensis (sample 1) ..48

Table 6. Competitive binding activities to 5-HT7 receptors of the isolates. ....................143

Table 7. Competitive binding activities to GABAa receptors of the isolates. .................144

Table 8. Anti-TB activities of the isolates from Angelica sinensis..................................145

Table 9. Distribution and traditional uses of plants in the genus Angelica.41...............173

Table 10. 1H and 13C NMR spectral data for 1-3 in CDCl3..............................................179

Table 11. 1H and 13C NMR spectral data for 4 and 5 in CDCl3.......................................180

Table 12. 1H and 13C NMR spectral data for 7 and 8 in CDCl3 .......................................181

Table 13. 1D (1H and 13C NMR) and 2D (HMBC) spectral data for 10 and 11 in CDCl3. 182

Table 14. Phthalide dimers isolated from plants ............................................................223

Table 15. Biological activities of isolates from A. sinensis ..............................................225

Table 16. Biological activities of methanol extracts of samples 3 and 4 (A. dehurca).....228

xiii

LIST OF FIGURES

Figure 1. Angelica sinensis (Oliv.) Diels ............................................................................11

Figure 2. The dried roots of Angelica samples 1-4............................................................41

Figure 3. Comparison of TLC fingerprints of different Angelica samples ..........................44

Figure 4. Comparison of HPLC fingerprints of different Angelica samples........................46

Figure 5. Extraction and Partitioning of A. sinensis (Sample 1, BC 165) ..........................47

Figure 6. Fractionation and isolation of compounds 1-23 from A. sinensis. ....................49

Figure 7. VLC fractionation of the PE and CHCl3 partitions of A. sinensis. ......................57

Figure 8. VLC fractination of F3.......................................................................................58

Figure 9. VLC fractionation of F5. ....................................................................................58

Figure 10. VLC fractionation of F7 ...................................................................................59

Figure 11. VLC fractionation of F10. ................................................................................59

Figure 12. VLC fractionation of F3-7 and F3-8.................................................................60

Figure 13. VLC fractionation of F5-4. ...............................................................................60

Figure 14. VLC fractionation of F5-5................................................................................61

Figure 15. DCCC fractionation of F7-5.............................................................................61

Figure 16. MPLC (1) fractionation of F7-7. .......................................................................62

Figure 17. MPLC (2) fractionation of F7-8. .......................................................................62

Figure 18. Preparative TLC fractionation of F7-8-8. .........................................................63

Figure 19. Preparative HPLC (1) fractionation of F3-7-10.................................................64

Figure 20. Preparative HPLC (2) fractionation of F5-5-2...................................................65

Figure 21. Preparative HPLC (3) fractionation of F5-5-4...................................................66

Figure 22. Preparative HPLC (4) fractionation of F7-5-8...................................................67

Figure 23. Preparative HPLC (5) fractionation of F7-7-5...................................................68

LIST OF FIGURES (continued) xiv

Figure 24. Preparative HPLC (6) fractionation of F7-8-5, F7-8-6 and F7-8-7. ..................68

Figure 25. Preparative HPLC (7) fractionation of F10-11, F10-12 and F10-13. ................69

Figure 26. Preparative HPLC (8) fractionation of F3-7-10-5 and F3-7-10-6......................69

Figure 27. Key HMBC correlations of riligustilide (4)........................................................75

Figure 28. X-ray crystal structure of riligustilide (4) ........................................................75

Figure 29. Positive ion electrospray (A) mass spectrum and (B) tandem mass spectrum with collision-induced dissociation of gelispirolide (5) ..........................................78

Figure 30. Gelispirolide (5) 400 MHz 1H NMR spectrum taken in CDCl3 ..........................79

Figure 31. Gelispirolide (5) 400 MHz APT NMR spectrum taken in CDCl3........................80

Figure 32. Gelispirolide (5) 400 MHz COSY NMR spectrum taken in CDCl3.....................81

Figure 33. Gelispirolide (5) 400 MHz HSQC NMR spectrum taken in CDCl3 ....................82

Figure 34. Gelispirolide (5) 400 MHz HMBC NMR spectrum taken in CDCl3 ...................83

Figure 35. Key NOESY correlations of gelispirolide (5) .....................................................84

Figure 36. Positive ion electrospray (A) Mass spectrum and (B) tandem mass spectrum with collision-induced dissociation of of 10-angeloylbutylphthalide (6). ...............87

Figure 37. 10-Angeloylbutylphthalide (6) 360 MHz 1H NMR spectrum taken in CDCl3 ....88

Figure 38. 10-Angeloylbutylphthalide (6) 360 MHz APT NMR spectrum taken in CDCl3..89

Figure 39. 10-Angeloylbutylphthalide (6) 400 MHz COSY spectrum taken in CDCl3........90

Figure 40. 10-Angeloylbutylphthalide (6) 400 MHz HMBC spectrum taken in CDCl3 ......91

Figure 41. Positive ion electrospray (A) mass spectrum and (B) tandem mass spectrum with collision-induced dissociation of sinaspirolide (7).........................................95

Figure 42. Sinaspirolide (7) 400 MHz 1H NMR spectrum taken in CDCl3 .........................96

Figure 43. Sinaspirolide (7) 400 MHz DEPT-135 and 13C NMR spectra taken in CDCl3 ...97

Figure 44. Sinaspirolide (7) 400 MHz COSY spectrum taken in CDCl3 ............................98

Figure 45. Sinaspirolide (7) 400 MHz HSQC spectrum taken in CDCl3............................99

Figure 46. Sinaspirolide (7) 400 MHz HMBC spectrum taken in CDCl3 .........................100

LIST OF FIGURES (continued) xv

Figure 47. Key HMBC correlations of sinaspirolide (7) ...................................................100

Figure 48. Key NOESY correlations of sinapirolide (7)....................................................101

Figure 49. Postulated biosynthetic pathway of sinaspirolide (7).....................................102

Figure 50. Positive ion electrospray (A) mass spectrum and (B) tandem mass spectrum with collision-induced dissociation of ansaspirolide (8). .....................................104

Figure 51. Ansaspirolide (8) 500 MHz 1H spectrum taken in CDCl3...............................105

Figure 52. Ansaspirolide (8) 500 MHz 13C spectrum taken in CDCl3..............................106

Figure 53. Ansaspirolide (8) 500 MHz COSY spectrum taken in CDCl3..........................107

Figure 54. Ansaspirolide (8) 500 MHz HMQC spectrum taken in CDCl3 ........................108

Figure 55. Ansaspirolide (8) 500 MHz HMBC spectrum taken in CDCl3 ........................109

Figure 56. Key NOESY correlations of ansapirolide (8)...................................................110

Figure 57. Postulated biosynthetic pathway of ansapirolide (8)......................................110

Figure 58. Positive ion electrospray (A) mass spectrum and (B) tandem mass spectrum with collision-induced dissociation of angediligustilide(9). .................................112

Figure 59. Angediligustilide(9) 500 MHz 1H NMR spectrum taken in CD3OD.................113

Figure 60. Angediligustilide(9) 500 MHz 13C NMR spectrum taken in CD3OD................114

Figure 61. Angediligustilide(9) 500 MHz COSY spectrum taken in CD3OD ....................114

Figure 62. Angediligustilide(9) 500 MHz HMQC spectrum taken in CD3OD...................115

Figure 63. Angediligustilide(9) 500 MHz HMBC spectrum taken in CD3OD...................116

Figure 64. The structure candidates of angediligustilide (9): head-to-tail linkage (A). ....117

Figure 65. The structure candidates of angediligustilide (9): head-to-head linkage (B). .118

Figure 66. Positive ion electrospray (A) mass spectrum and (B) tandem mass spectrum with collision-induced dissociation of angeferulate (11) .....................................122

Figure 67. Angeferulate (11) 360 MHz 1H NMR spectrum taken in CDCl3......................123

Figure 68. Angeferulate (11) 360 MHz DEPT-135 and 13C spectrum taken in CDCl3 .....124

Figure 69. Angeferulate (11) 360 MHz COSY spectrum taken in CDCl3 .........................125

LIST OF FIGURES (continued) xvi

Figure 70. Angeferulate (11) 360 MHz HMQC spectrum taken in CDCl3.......................126

Figure 71. Angeferulate (11) 360 MHz HMBC spectrum taken in CDCl3........................127

Figure 72. Chemical structures of isolates from A. sinensis (to be continued)................140

Figure 73. Chemical structures of isolates from A. sinensis (continued). .......................141

Figure 74. Z-Ligustilide (1) 360 MHz 1H NMR spectrum taken in CDCl3 ........................184

Figure 75. Z-Ligustilide (1) 360 MHz DEPT-135 and 13C NMR spectra taken in CDCl3 ..185

Figure 76. Z-Ligustilide (1) 360 MHz 1H-1H COSY NMR spectrum taken in CDCl3.........186

Figure 77. Z-Ligustilide (1) 360 MHz 1H-13C HMQC NMR spectrum taken in CDCl3 ......187

Figure 78. Z-Ligustilide (1) 360 MHz HMBC NMR spectrum taken in CDCl3..................188

Figure 79. Z-Butylidenephthalide (2) 360 MHz 1H NMR spectrum taken in CHCl3.........189

Figure 80. Z-Butylidenephthalide (2) 360 MHz DEPT and 13C spectra taken in CDCl3...190

Figure 81. Z-Butylidenephthalide (2) 360 MHz COSY NMR spectrum taken in CDCl3 ...191

Figure 82. Z-Butylidenephthalide (2) 360 MHz HMBC NMR spectrum taken in CDCl3 ..192

Figure 83. Senkyunolide-I (3) 360 MHz 1H NMR spectrum taken in CDCl3....................193

Figure 84. Senkyunolide-I (3) 360 MHz DEPT-135 and 13C spectra taken in CDCl3.......193

Figure 85. Senkyunolide-I (3) 360 MHz COSY spectrum taken in CDCl3 .......................194

Figure 86. Senkyunolide-I (3) 360 MHz HMQC NMR spectrum taken in CDCl3 .............195

Figure 87. Senkyunolide-I (3) 360 MHz HMBC NMR spectrum taken in CDCl3 .............196

Figure 88. (A) Positive ion electrospray exact mass measurement of riligustilide (4). The measured mass was 381.2076 compared to a theoretical mass of 381.2066 for C24H29O4. (B) Positive ion tandem mass spectrum with collision-induced dissociation of the protonated molecule of riligustilide (4) at m/z 381. ..............197

Figure 89. Riligustilide (4) 360 MHz 1H NMR NMR spectrum taken in CDCl3 ................198

Figure 90. Riligustilide (4) 360 MHz DEPT-135 and 13C NMR spectra taken in CDCl3 ...199

Figure 91. Riligustilide (4) 360 MHz COSY NMR spectrum taken in CDCl3....................200

Figure 92. Riligustilide (4) 360 MHz HMQC spectrum taken in CDCl3 ...........................201

LIST OF FIGURES (continued) xvii

Figure 93. Riligustilide (4) 360 MHz HMBC NMR spectrum taken in CDCl3...................202

Figure 94. p-Hydroxyphenethyl trans-ferulate (10) 300 MHz 1H spectrum taken in CDCl3 .................................................................................................................203

Figure 95. p-Hydroxyphenethyl trans-ferulate (10) 300 MHz DEPT-135 and 13C spectra taken in CDCl3 ...................................................................................................204

Figure 96. (A) Positive ion electrospray exact mass measurement of N-butylbenzenesulphonamide (12). The measured mass was 214.0913 compared to a theoretical mass of 214.0902 for C10H17NO2S. (B) Positive ion tandem mass spectrum with collision-induced dissociation of the protonated molecule of N-butylbenzenesulphonamide (12) at m/z 214......................................................204

Figure 97. N-Butylbenzenesulphonamide (12) 500 MHz 1H spectrum taken in CDCl3...205

Figure 98. Positive ion electrospray exact mass measurement of a mixture of N-butylbenzenesulphonamide (12) (A) and Z-6-hydroxyl-7-methoxyl-dihydroligustlide (13) (B). ...................................................................................206

Figure 99. Comparison of the 1H NMR spectra of the isolates 13 and 12 (in CDCl3)......207

Figure 100. (A) Positive ion electrospray exact mass measurement of imperatorin (14). The measured mass was 271.0974 compared to a theoretical mass of 271.0971 for C16H15O4. (B) Positive ion tandem mass spectrum with collision-induced dissociation of the protonated molecule of imperatorin (14) at m/z 271. ...........208

Figure 101. Imperatorin (14) 360 MHz 1H NMR spectrum taken in CDCl3.....................209

Figure 102. Ferulic acid (15) 400 MHz 1H and 13C NMR spectra taken in CDCl3 ...........210

Figure 103. Vanillin (16) 360 MHz 1H and APT 13C NMR spectra taken in CDCl3 ..........211

Figure 104. Sucrose (18) 400 MHz 1H and 13C NMR spectra taken in D2O ....................212

Figure 105. Falcarindiol (19) 400 MHz 1H NMR spectrum taken in CDCl3.....................213

Figure 106. Falcarindiol (19) 400 MHz 13C and DEPT-135 spectra taken in CDCl3........214

Figure 107. Positive ion electrospray exact mass measurement 9Z,17-octadecadiene-12,14-diyne-1,11,16-triol, 1- acetate (20). The measured mass was 355.1924 compared to a theoretical mass of 355.1885 for C20H28O4Na. ............................214

Figure 108. 9Z,17-Octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate (19) 400 MHz 1H NMR spectrum taken in CDCl3...........................................................................215

Figure 109. 9Z,17-Octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate (20) 400 MHz 13C NMR and DEPT-135 spectra taken in CDCl3 ................................................216

LIST OF FIGURES (continued) xviii

Figure 110. Positive ion electrospray exact mass measurement of heptadeca-1-ene-9,10-epoxy-4,6-diyne-3,8-diol (21). The measured mass was 299.1672 compared to a theoretical mass of 299.1623 for C17H24O3Na. ............................217

Figure 111. Heptadeca-1-ene-9,10-epoxy-4,6-diyne-3,8-diol (21) 360 MHz 1H NMR spectrum taken in CDCl3 ...................................................................................218

Figure 112. Oplopandiol (22) 400 MHz 1H NMR spectrum taken in CDCl3 ....................219

Figure 113. Heptadeca-hydroxy-1-methoxy-, Z-9-heptadecene-4,6-diyn-3-one (23) 400 MHz 1H and 13C NMR spectra taken in CDCl3 ....................................................220

Figure 114. Structures of phathalide dimers from plants ..............................................224

Figure 115. Simulation and iteration of 1H NMR spectrum of Z-ligustilide (1) by PERCH NMR software. ....................................................................................................226

Figure 116. Simulation and iteration of 1H NMR spectrum of Z-butylidenephthalide (2) by PERCH NMR software....................................................................................227

xix

SUMMARY

This project was performed in Project 1 in collaboration with Projects 2

and 3 of the UIC/NIH Center for Botanical Dietary Supplements Research. The

work is intended to facilitate the standardization of extracts and products of A.

sinensis, which may be used for evaluating safety and efficacy in human

clinical trials. The following specific aims were addressed in this study. 1. A

series of approaches were established in order to botanically verify raw

materials of A. sinensis. 2. The phytochemical investigation of bioactive

principles from A. sinensis was conducted guided by a 5-HT7 receptor binding

assay. As a result, two botanical samples were verified as Angelica sinensis,

and six new compounds of gelispirolide (5), 10-angeloylbutylphthalide (6), sinaspirolide (7), ansaspirolide (8), angediligustilide (9), angeliferulate (11),

together with 17 known compounds including five phthalides (1-4, 13), a coumarin (14), five polyynes (19-23), and others (10, 12, 15-18) were isolated

and identified. Some of these compounds exhibited serotonergic and

GABAergic activities in the 5-HT7 and GABAa competitive receptor binding

assays, which might contribute to the pharmacological effects of A. sinensis

related to premenstrual symptoms. At the same time, the spectral data

included in the text might serve as reference data for future dereplication and

identification of isolates from this plant or from other species.

1. INTRODUCTION

1.1. INTRODUCTION AND SCOPE OF STUDY

In Asia, the dried root of Angelica sinensis (Oliv.) Diels (Dong Quai or

Dang Gui) has been used for centuries as a women's tonic for alleviating PMS,

menstrual cramps, and symptoms related to menopause, and is regarded as

the second most important herb (Panax ginseng being the first) among

thousands of herbs. It has been used broadly as a dietary supplement for

perimenopausal symptoms in the West. Although suspected by some of having

estrogenic activities based on its use for women’s diseases, A. sinensis has not

been reported to display remarkable estrogenicity pharmacologically or

clinically. This suggests an alternative mechanism for its potential efficacy for

its treatment of gynecological disorders. As interest increases in A. sinensis

products, the development of botanically, chemically and biologically

standardized extracts is becoming necessary and important for the acquisition

of safe and efficacious products, as well as for use in well-controlled clinical

trials.

Our preliminary data (Table 5) showed that a methanol extract of A.

sinensis roots exhibited serotonergic activity with a 59 % inhibition of [3H] LSD

binding to the 5-HT7 receptor at a concentration of 100 µg/mL, and a 97%

inhibition of the binding of [3H] diazepam to the GABAa (γ-aminobutyric acid

neurotransmitter receptor) at a concentration of 50 µg/mL.

The hypothesis proposed herein is that Angelica sinensis contains

active constituents that elicit serotonergic and GABAergic effects. The following

two specific aims will be addressed in this project in accordance with the

above stated hypothesis. Aim 1. Establishment of analytical methods used for verification of plant

raw materials.

2

Acquisition and verification of plant materials was the first step in this

project and was critical for the acquisition of reproducible samples as well as

for future evaluation of the safety and efficacy of the extract. To botanically

verify the plant materials, a series of analytical techniques was validated and

used including the examination of macroscopic and microscopic

characteristics, thin layer chromatography (TLC) and high-performance liquid

chromatography (HPLC) fingerprints.

Aim 2. Isolation and identification of bioactive markers by conducting bioassay-guided fractionation and isolation.

Plant materials authenticated in Aim 1 were extracted with methanol,

and then subjected to partitioning with pet ether (PE), chloroform (CHCl3),

butanol (BuOH) and water (H2O).

The active partitions were subjected to chromatographic fractionation

using a 5-HT7 receptor binding assay in collaboration with personnel in Project

2. A series of chromatographic techniques such as open column, vacuum

liquid chromatography (VLC), medium-pressure liquid chromatography

(MPLC), droplet countercurrent chromatography (DCCC), preparative TLC, and

HPLC with different types of packing materials (normal-phase and reverse-

phase silica gel) and various solvents were used for the separation and

purification of bioactive constituents. The isolates were chemically

characterized using a variety of spectroscopic methods including UV, IR, mass

spectrometry (MS), one-dimensional (1D) and two-dimensional (2D) NMR, and

X-ray diffraction. The determination of biological activities for the active

isolates were expressed in terms of IC50 values for the 5-HT7 and GABAa

receptor binding assays.

3

1.2. SIGNIFICANCE OF PREMENSTRUAL SYNDROME

Epidemiological surveys have indicated that up to 75% of women with

regular menstrual cycles suffer some symptoms of premenstrual syndrome

(PMS), which negatively affects the quality of life.1,2 PMS is a periodic

phenomenon of physical, psychological, or behavioral symptoms. It begins

during the second half of the menstrual cycle and stops at menses.1,2 The

symptoms relating to PMS may include mood swings, depression, tension,

anger, anxiety, irritability, difficulty concentrating, headache, bloating,

mastalgia, and increased appetite with food cravings.3-5 Behavioral irritability

(either anxiety or depression) and fatigue are two basic symptoms.6 PMS can

badly impact personal relationships, social activities, or job performance.7

Premenstrual syndrome significantly decreases with age in the

transition to menopause, and ends at menopause with the overlap of certain

symptoms such as depressed mood, irritability, and anxiety. Women

experiencing PMS are more likely to suffer menopausal hot flushes, depressed

mood, and poor sleep than those without PMS.8,9 Women with a history of PMS

are more likely to suffer mood symptoms during menopause.10 Therefore, PMS

may act as a predictor of menopause to some extent.8

Although the etiology of PMS remains unclear,11 it is generally regarded

as a psycho-neuro-endocrine disorder and is believed to involve reproductive

hormones, and neurotransmitters, as well as other brain processes. 2,12 It has

been proposed that PMS is closely related to a deficiency in central

serotonergic activity,13-15 which involves common symptoms such as

depression, and irritability during the menstrual cycle.6,16,17 Leonard et al.18

suggested that serotonin is a critical factor in regulating mood and might be

involved in pathophysiology based on the phenomenon that psychiatric

disorders (anxiety and depression) are closely related to changes in

serotonergic neurotransmission. Studies by Halbreich et al.19 found low

4

serotonin and melatonin metabolism in women with severe PMS compared

with healthy controls. Serum serotonin concentrations in postmenopausal

women are related to the severity of climacteric symptoms.20 This evidence

suggests a rationale for some herbs with serotonergic activities for the

treatment of PMS.

Serotonin (5-HT) is distributed in many tissues in the central and

peripheral nervous systems where it modulates a variety of physiological and

pathological processes, including anxiety, sleep regulation, feeding, depression,

cardiovascular function, thermoregulation, sexual behavior, circadian rhythms

and aggression by activating different families of receptors.18,21-23 Serotonin

receptors are classified on the basis of operational, structural, and

transductional criteria, and are related by amino acid sequence, pharmacology,

and intracellular mechanisms.24 Since serotonin was discovered in the late

1940s, 14 different types of serotonin receptors from seven families 5-HT1 to 5-

HT7 have been identified.25,26

In additional to serotonin, the neurotransmitter GABA might also be

involved in the symptoms of PMS and menopause. The GABAa receptor is

regarded as one of the most intriguing drug targets to mediate anxiolytic,

sedative, anticovulsant, muscle relaxant and amnesic activities,27 and

modulators of GABAa receptors relate to improvement of depression and

anxiety-related symptoms observed in PMS and menopausal women.28

Current treatment strategies for PMS include both non-pharmacologic

and pharmacologic approaches. Non-pharmacologic approaches such as

dietary modifications (a diet low in salt, fat, caffeine, and sugar), dietary

supplements, aerobic exercise, and stress management have been

recommended to most women as first-line treatments for mild PMS

symptoms.1,3,29 Pharmacologic interventions are usually used for severe

symptoms and include therapy with antidepressants such as selective

5

serotonin reuptake inhibitors (SSRIs), anxiolytics and agents to suppress

ovulation.30 A combination of the novel antipsychotic olanzapine and the

serotonin-selective re-uptake inhibitor (SSRI) fluoxetine were shown to be

effective for depression overlapping with mania in a trial including 28

patients.31 However, an increased risk of breast and endometrial cancer,

random vaginal bleeding, as well as other adverse effects when taking these

medicines hamper their broad clinical application.32-34 A review based on 7

retrospective studies and 24 case reports of bleeding in 43 different people

indicated a risk of upper gastrointestinal (GI) bleeding and perioperative

bleeding during SSRI use.35 To evaluate the safety profile of sertraline

versus other SSRIs immediately following the introduction of sertraline to

the Dutch market, a study of this and other SSRIs was carried out in the

Netherlands to evaluate their safety. This study included 1251 patients

(659 used sertraline and 592 used other SSRIs, i.e. paroxetine, fluoxetine or

fluoxamine) and revealed that nearly three out of four patients reported an

adverse event, such as headache, diarrhea, sweating, or dizziness.36

1.3. PLANTS AS POTENTIAL ALTERNATIVE THERAPIES

Botanical medicines have been applied for the treatment of various

women’s diseases with thousands of years of history in Asia and are sharing a

large market in the form of drugs, dietary supplements, and foods. In the West,

botanical medicines are categorized as complementary/ alternative medicines

(CAM), dietary supplements, or foods. In 1994, the Dietary Supplement Health

and Education Act (DSHEA) defined a dietary supplement as “a product, other

than tobacco, intended to supplement the diet that contains one or more of (a)

vitamin, (b) mineral, (c) herb/botanical, (d) amino acid, (e) supplement that

increases total dietary intake, (f) a concentrate, metabolite, constituent, extract

or combination of (a, b, c, d and/or e)… that must either be in the form of a

6

tablet, capsule , powder, soft gel, gel cap, liquid droplet, or be in some other

forms that is not presented as a conventional food”. FDA also requires a label

disclaimer that “This product has not been evaluated by the FDA. This product

is not intended to diagnose, mitigate, treat, cure or prevent any disease” on all

products, and manufacturers must submit samples of each new product label

to the FDA within a month after marketing.37

In a survey including 223 questionnaires regarding the topic of “which

complementary and alternative therapies benefit which conditions”,

stress/anxiety symptoms were listed as being most important for patients

seeking complementary and alternative therapies.38 There is a recent increase

of interest in complementary and alternative medicines for relief of

premenstrual and menopausal symptoms because of perceived side effects

caused by chemical drugs. Treatment with herbs has shown efficacy for some

patients with PMS.39,40 Hardy40 identified several popular herbal medicines

claimed useful for PMS and menopause. Evening primrose (Oenothera biennis

L.) was one of the plant medicines used for the treatment of PMS, particularly

for the improvement of mastalgia. Efficacy was proposed due to its rich

content of fixed oil, i.e. about 65% linoleic acid and up to 10% of gamma-

linoleic acid. This was based on an observation that abnormally low levels of

essential fatty acids are found in PMS women. Chaste tree berry (Vitex agnus-

castus L.) was indicated to alleviate PMS via its dopaminergic effects. A

standardized product of black cohosh (Cimicifuga racemosa L. Nutt.),

Remifemin, showed relief for menopausal symptoms, as an alternative to

estrogen replacement therapy, although its mechanism of action remains

unclear. Many other herbs with different mechanism of action have been

used for women’s, such as red clover (Trifolium pratense L.) as a

phytoestrogen, dandelion leaf (Taraxacum officinale G.H. Weber ex Wigg.), and

valerian (Valeriana officinalis L.) with sedative and antispasmodic effects for

7

dysmenorrhea. Angelica sinensis is one of the most important and popular

herbs in Asia for the treatment of a variety of gynecological disorders and will

be addressed in more detail in section 1.5.

1.4. STATEMENT OF PROBLEM

There are numerous A. sinensis products on the market. Given the

reality of botanical confusion of plant materials and unclear mechanisms of

action, it is difficult for manufacturers to guarantee the reproducibility, and

further to ensure safety and efficacy of the products. As a result, this might

adversely affect the health of consumers. On the basis of these problems, this

project was designed to address the following specific aims:

A. Establishment of analytical methods for botanical identification of plant materials of A. sinensis.

B. Chemical and biological characterization of active principles from the roots of A. sinensis.

After completion of specific aims A and B, this research will be helpful

for standardization of extracts of A. sinensis and will contribute to the

acquisition of reproducible, safe and efficacious marketed products.

8

1.5. PHARMACOGNOSY OF ANGELICA SINENSIS

1.5.1. BOTANY, TAXONOMY AND IDENTIFICATION

The word Angelica refers to the genus name, derived from the Latin word

angelus, meaning angelic. In European folklore, this genus name is associated

with a mythological story that an angel came to a monk to save an almost

destroyed Europe by offering an herb, and from then on, it was named

angelica. The species name sinensis also originated from a Latin word, Sina,

meaning China.41,42

The following lists the botanical name, synonyms, and the classification

of Angelica sinensis.

Botanical Nomenclature: Angelica sinensis (Oliv.) Diels

Synonym: Angelica polymorpha Maxim. var. sinensis Oliv.

Plant classification:

Kingdom: Plantae-Plant

Subkingdom: Tracheobionta-Vascular plants

Superdivision: Spermatophyta-Seed plants

Division: Magnoliophyta-Flowering plants

Class: Magnoliopsida-Dicotyledons

Subclass: Rosidae

Order: Apiales

Family: Apiaceae-carrot family (formerly Umbelliferae)

Genus: Angelica

Species: sinensis (Oliv.) DIELS

1.5.1.1. The family Apiaceae

As the largest family of the Apiales, Apiaceae (the carrot family) consists

of ca. 430 genera and 3000 species. Most plants are aromatic with hollow

9

stems. The Apiaceae have been recognized since ancient times, both as a

morphologically distinct group of plants and as a source for food, medicines,

poisons, etc., and are widely distributed in most parts of the world, particularly

in the north-temperate zone.43

Umbelliferae is an alternative name for Apiaceae, implicating “bearer of

umbels”. A series of features can be applied to distinguish it from other

families morphologically. Members of the family have alternate leaves,

widening at the base into a sheath that clasps the stem. The stems are often

furrowed. Some parts of the plants usually have a strong aroma, primarily due

to the presence of various essential oils.

The small and compound flowers are always concentrated in umbels;

the flowers have 5 petals, usually uneven, and 5 stamens. The seeds and fruit

form below where the petals and stamen originate. Seeds are in tight pairs,

often conspicuously ribbed, and sometime "winged".

Walters et al.43 described this family with the following morphological

characteristics:

Herbs (rarely shrubs or trees). Leaves alternate (occasionally opposite) or all

basal, simple to variously lobed or compound, estipulate (rarely stipulate) with

petioles sheathing, with internal oil tubules abd often strongly scented. Plant

synoecious (occasionally polygamous, rarely dioecious). Inflorescence of

compound umbels (rarely of simple umbels, heads, or axillary flowers). Flowers

perfect (rarely imperfect) regular or irregular. Sepals 5, small, often very reduced

and apparently absent, distinct. Petals 5 (0), distinct. Stamens 5, distinct,

alternate with petals. Carpels 2, connate; ovary inferior with 2 locules and 1

apical-axile ovule per locule; styles 2, often subtended by bulging stylopodia

(rarely elongated into a beak in fruit). Fruit a schizocarp with 2 mericarps, often

strongly ribbed, sometimes winged and samaroid or covered with tubercles or

prickles.

10

Plants in the Apiaceae have significant economic value. Some

members are edible, such as vegetable crops of Daucus carota L. (carrot),

Apium graveolens L. (celery), and a culinary herb of Petroselinum crispum (P.

Mill.) Nyman ex A.W. Hill (parsley). However, some members are poisonous,

such as Conium maculatum L. (poison hemlock) and Cicuta spp. (water-

hemlock). A. archangelica L. (garden Angelica) which is famous of its green,

crystallized decorative strips.

1.5.1.2. The genus Angelica

Angelica is a genus consisting more than 60 species of tall biennial and

perennial herbs, native to temperate and subarctic regions of the Northern

Hemisphere, such as Asia, Europe, and North America.41 They grow up to 8

feet, with large bipinnate leaves and large compound umbels of white or

greenish-white flowers.

A summary of the distribution and traditional application of plants in

the genus Angelica is listed in APPENDIX A (Table 9 ).41 Many plants in this

genus are important medicinal herbs with the following activities: anti-

inflammatory, diuretic, expectorant and diaphoretic for the treatments of colds,

flu, influenza, hepatitis, arthritis, indigestion, coughs, chronic bronchitis,

bacterial and fungal infections, and for diseases of the urinary tract.

Phytochemicals such as coumarins, polyacetylenes, chalcones, sesquiterpenes

and polysaccharides have been characterized as active principles in this genus,

and exhibit antimicrobial, anticancer, antitumor, analgesic, anti-inflammatory,

hepatoprotective, and nephroprotective biological activities.41

1.5.1.3. The species Angelica sinensis (Oliv.) Diels

Angelica sinensis (Oliv.) Diels is a fragrant, herbaceous perennial plant,

0.5 to 1 m tall.44 It flowers from August to September, and the seeds ripen from

September to October. Indigenous to the west of China, such as Hubei,

11

Sichuan, Yunnan, Guizhou, Shanxi and Gansu provinces, with Gansu being

famous for its superior quality plants,45 Angelica sinensis is rarely available

from wild sources. Currently it is cultivated and harvested in late autumn after

three years.

The American Herbal Pharmacopoeia (2003) describes the

morphological characteristics of A. sinensis as follows:

Stem: Erect, 4-10 dm tall, stout, hollow, fluted, glabrous, greenish-white with purple striations. Leaves: Lowermost leaves 2-3-pinnate, 8-11 cm long, 15-20 cm wide; upper leaves 1-pinnate; petiole 3-11 cm long, with base inflated and

sheathing the stem; leaflets ovate or ovate-lanceolate, margin dentate, fine

white hairs on leaf veins and margin. Inflorescence: Compound umbel, 10-14 per plant, peduncle 4-7 cm, densely covered with fine wooly hairs; 9-36 rays

per umbel, bracts 2 (0), narrow; bracteoles 2-4, narrow. Flowers: Perfect, radially symmetric, with nectariferous disc at apex of the ovary; calyx 5-

toothed; petals 5, white, apices inflexed; stamens 5; ovary inferior, styles 2,

short, basally connate and adnate to the disc, forming a conical stylopodia.

Fruit: Schizocarp splitting into 2 one-seeded carpels compressed against each other and separately attached by the apex to a carpophore; each carpel

ovoid or ellipsoid, the tip rounded or slightly notched, 4-6 mm long, 3-4 mm

wide, with 5 dorsal ribs, the marginal

ridges winged, wings pale purple; oil

glands solitary in each vitta (sinus

between the ribs), with 2 on the

commissure (inner side).”

Figure 1. Angelica sinensis (Oliv.) Diels

Family: Apiaceae-carrot family Medicinal parts: the dried roots Nicknames: Female ginseng or Lady’s ginseng

12

Common names for A. sinensis as it is popularly used in different

areas include Dang Gui, Dong Quai, Tang Kuei, Ming-gui, Chinese

Angelica, Toki (Korea), Tanggi (Japan), etc.45

1.5.1.4. Identification of A. sinensis plant material

Many species other than A. sinensis have been used as its substitutes in

various areas, such as Angelica acutiloba (Sieb. & Zucc.) Kitag in Japan,

Angelica gigas Nak. in Korea, and Levisticum officinale W.D.J. Koch in

Europe.45-47 In addition, there are numerous A. sinensis adulterants, i.e.

Angelica megaphylla Diels, Angelica uchiyamana Yabe, and Ligusticum

glaucescens Franch. The pharmacological effects of these substitutes and

adulterants might be different from A. sinensis (Oliv.) Diels based on

differences in their phytochemical constituents, especially in the essential

oils.46,48 In A. sinensis, the contents of major constituents in its essential oil,

such as ligustilide, butylidenphthalide, and butylphthalide are reported to be

higher than in Angelica acutiloba Kitagawa and in Levisticum officinale W.D.J.

Koch,49,50 especially ligustilide, which is 10-fold higher than in Angelica

acutiloba Kitagawa and Angelica gigas Nak..47,51 The essential oils of six

different species of Angelica were analyzed by HPLC, and the results showed

differences with respect to butylidenephthalide, ligustilide, butylphthalide, and

the carboxylic acids, ferulic acid and nicotinic acid.49 Therefore, it is necessary

to botanically verify plant materials of A. sinensis before use, which could be

conducted on the basis of the macroscopic, microscopic characteristics, as well

as their chemical constituents.

Macroscopic characteristics: The dried root is cylindrical in shape, with 2-10 main branches and more fine branches at the lower part, and

approximately 15-25 cm long. Externally it appears yellowish-brown to brown,

with longitudinally wrinkled and transversely lenticellate. The root is divided

13

into three parts, the root stock, the main root, and the branching root. The

root stock is 1.5-4 cm in diameter long, annulated, apex obtuse and rounded,

possessing purple or yellowish-green remains of stems and leaf sheaths; the

main root is uneven and crude on the surface; the branching root is usually

0.3-1 cm in diameter, with the upper portion thick and the lower portion thin,

mostly twisted and showing a few rootlet scars. Other characteristics include

flexible texture, yellowish-white or yellow-brown fraction, brown and thick bark

with some clefts and many brown dotted secretory cavities, paler wood, and

yellowish-brown cambium ring. It has a strongly aromatic odor, and is sweet

and pungent with a slightly bitter taste.44,45 The highest quality plant materials

are claimed to be strongly aromatic and oily. Woody, withered and not oily

roots, or those with greenish-brown on the fracture are not suggested to be

used for medicinal purposes.49,52

Microscopic characteristics: According to the Pharmacopoeia of the People’s Republic of China (PPRC 2000), transverse sections of the dried roots

of A. sinensis exhibit the following characteristics: “Cork cells in several layers.

Cortex narrow, scattered with a few oil cavities. Phloem broad, more cleft, oil

cavities and oil tubes subrounded, 25-160 µm in diameter, relatively large on

the outer side, gradually becoming small inwards, surrounded by 6-9 secretory

cells, cambium in a ring. Xylem rays grouped, arranged radially,

parenchymatous cells containing starch granules.”

Powdered roots of A. sinensis are yellow-brown, usually exhibiting the

following microscopic features: “parenchymatous cells show a fusiform shape

with slightly thickened walls and very fine oblique crisscross striations,

sometimes thin transverse septa are visible. Vessels are scalariform and

reticulate, up to 80 µm in diameter. Oil cavities or its fragments may be visible.

Cork cells are yellowish. Starch grains are rarely found, which are in ovate,

spherical or ellipical and in 3-8 µm in length, if visible.”52

14

As a traditional technique for botanical identification, examination of

microscopic characteristics individually may not be sufficient to identify plant

materials of A. sinensis in most cases. However, microscopic analysis may be

used to quickly differentiate adulterants from A. sinensis. This is possible since

the abovementioned microscopic features are absent from some common

adulterants.

Phytochemical characteristics: The identification of plant materials on

the basis of different phytochemical constituents focuses on either the

qualitative analysis of characteristic constituents, or the quantitative assay of

major constituents by means of TLC, HPLC, GC-MS and LC-MS. TLC has been

widely used as a rapid method to distinguish A. sinensis from other plants,

including A. pubescens (characterized by osthol and various angelol type

coumarins), A. dahurica (characterized by linear furanocoumarins), L.

chuanxiong (no falcarindiol and coniferyl ferulate), L. porteri (no Z-

butylidenephthalide), and L. officinale (less quantity of ligustilide and

falcarindiol).53,54

Qualitative and/or quantitative HPLC analyses of the constituents in the

roots of A. sinensis have been used for quality control.53,55-58 Ferulic acid is one

of most commonly used markers for the quantitative analysis of A. sinensis

and its products by using reverse-phase HPLC techniques.56,59-62 As a major

bioactive constituent in the essential oil of A. sinensis, Z-ligustilide is another

popular marker for the quality control of A. sinensis and its products.47 GC-MS

and LC-MS have been applied for the chemical analysis of A. sinensis.53,63.

1.5.2. PHYTOMEDICINE

In Asia, the dried root of A. sinensis has been widely used for the

treatment of many diseases, such as dysmenorrhea, amenorrhea,

premenstrual and menopausal syndromes, anemia, abdominal pain, injuries,

15

migraine headaches, and arthritis for thousands of years.45,64,65 Angelica

sinensis is also called “lady’s ginseng” or “female ginseng” to indicate its

importance and popularity for various women’s ailments. The earliest record of

its use is in the Divine Husbandman's Classic of the Materia Medica (Shen Nong

Ben Cao Jing) that was published during the Han Dynasty (AD 25-225). After

being introduced to the West in 1899 by Merck in the form of a liquid extract

named “Eumenol”,66 A. sinensis has been gradually used by westerners, its

many products are included in the Swiss, the Austrian and the German

Pharmacopoeias.41 Currently there are countless A. sinensis products on the

market all over the world.

The common name of A. sinensis in Chinese is Dang Gui, which literally

means “recovery to normal conditions”, indicating its ability to help the body

return to a normal condition of well-being from weakness or illness. A. sinensis

is claimed to have the functions of “Nourish blood and promote its circulation,

regulate the menstruation and relieve its pain……” in the theory of Traditional

Chinese Medicine. In the Chinese Pharmacopoeia (2000), A. sinensis is officially

applied to the symptoms of “menstrual disorders, amenorrhea, dysmenorrhea,

anemia with dizziness and palpitation, constipation, rheumatic arthralgia,

traumatic injuries, carbuncles, boils, and sores.”52 In addition to be used alone,

A. sinensis is often used in combination with other herbs, such as Rhizoma

Ligustici Chuanxiong (Ligusticum chuanxiong), Radix Paeoniae alba (Paeoniae

lactiflorae), and Radix Notoginseng (Panax notoginseng) in various formulas in

Traditional Chinese Medicine (TCM).67,68 Over 80 formulas in the

Pharmacopoeia of the People’s Republic of China (2000 version) and 56

formulas in the Japanese Pharmacopoeia contain A. sinensis root as one of

their components.69,70

16

1.5.2.1. Gynecological Uses

Although A. sinensis has been used for gynecological disorders for many

years, there are few clinical reports in the literature on A. sinensis publishes

dealing with these effects. Three problems exist that hinder its broader use in

modern medicine.

1. Most clinical studies have been performed with formulated herbal

products, i.e., mixtures of different plants, rather than A. sinensis only;

2. Clinical trials have been conducted by using the principles of TCM

theory, which are difficult to convert accurately into the corresponding

terms of western medicine;

3. Only a few randomized placebo-controlled clinical trials have been

carried out.

These problems are grounds for concerns regarding the safety and efficacy of

A. sinensis dietary supplements.

It is generally believed that A. sinensis is estrogenic based on its broad

applications to female ailments. However, in vitro experiments in our UIC/NIH

Botanical Center showed that a methanol extract of A. sinensis has very weak

affinity to estrogen receptors (IC50 > 50µg/ml).71 In a 24-week, double-blind,

placebo-controlled clinical trial involving 71 postmenopausal women, a daily

dose of 4.5 g of a dried aqueous extract of A. sinensis was not found to exhibit

estrogenic activity.72 This indicates that A. sinensis does not have estrogen-like

activities in vivo or in vitro and does not qualify as a phytoestrogen.

A medicinal preparation of herbal extracts (Climex) consisting of

Angelica sinensis and Matricaria chamomilla was evaluated for the treatment of

menopausal symptoms in a placebo-controlled experiment including 55

postmenopausal women.73 Significant improvement of menopausal symptoms

including hot flushes, sleep disturbances and fatigue was observed in the

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study group in comparison with the control group (90-96% vs 15-25%, p <

0.001). It was concluded that hormone-free Climex with A. sinensis as a major

principle was effective in treatment of menopausal symptoms without adverse

effects.

Hardy40 rigorously reviewed the efficacy and safety of some herbs that

have been traditionally used to treat gynecological complaints such as

premenstrual syndrome (PMS) and menopausal symptoms in controlled

studies. He concluded that A. sinensis was effective for PMS when used in

traditional Chinese formulas with good safety profiles.

Approximately 30% of women afflicted with migraine have menstrually

associated attacks. These migraines are often refractory to treatment. Johns

declared that A. sinensis has tranquilizing and sedative effects that can relieve

mood swings and irritability in premenstrual women and recommended it for

the treatment of migraine headaches caused by PMS.74 A liquid extract of A.

sinensis named “Eumenol” was also recommended for the treatment of

menstrual disorders in the West as early as 1927.66

In a clinical study, both the aqueous extract and ligustilide, one of the

major constituents in the essential oil of A. sinensis were administered for 3-7

days for the treatment of dysmenorrhea. As a result, ligustilide exhibited an

efficacy of 77%, much higher than the 38% efficacy observed in the group

receiving the aqueous extract. This indicated that ligustilide is an active

principle for dysmenorrhea, and its mechanism of action might be associated

with its uterine spasmolytic effect.75 Successful treatment on amenorrhea and

dysmenorrhea by using a fluid extract of A. sinensis was also reported.44,76 By

administrating 5 ml of the fluid extract of the roots of A. sinensis to female

patients prior to menstruation three times a day before meals for 7 days, a

significant relief of premenstrual pain and chronic endometritis, as well as

induced menstrual flow in patients, was observed.

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The root of Angelica sinensis (Oliv.) Diels is a major ingredient of 'Four

Things Soup', the most widely used formula of a woman's blood tonic (for blood

vacuity) in China. The other three herbs included in this formula are

Rehmannia glutinosa Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. &

C.A. Mey, Ligusticum wallichii Franch and Paeonia lactiflora Pall. In a case

study, Angelica sinensis was observed to significantly improve anemia of

chronic renal failure.77 Polysaccharides of A. sinensis were demonstrated to

increase hematopoiesis in the bone marrow in an in vivo study.78

1.5.2.2. Other Uses

In addition to gynecological disorders, A. sinensis has also been widely

applied to other diseases due to perceived cardiovascular, hepatoprotective,

hematopoietic, antioxidant, antispasmodic and immunomodulatory activities.

In a clinical study on the treatment of acute cerebral infarcts with an injection

of A. sinensis, 1404 patients were divided in three groups: 692 patients in

Group A were treated with Angelica injection; 390 patients in Group B were

treated with Salvia; and 322 patients in Group C were administered dextran.79

The results showed that the total effective rate in Group A (78.8%) was

markedly higher than the other two groups (B-63.6% and C-59.3%) (P < 0.05).

As an active constituent of A. sinensis, ferulic acid (as sodium ferulate)

has been widely used as a drug in China to treat cardiovascular and

cerebrovascular diseases and prevent thrombosis. Ferulic acid has

antithrombotic, platelet aggregation inhibitory and antioxidant activities. Its

safety and efficacy in coronary heart disease, atherosclerosis, pulmonary heart

disease, and thrombosis have been demonstrated clinically.80 An extract of A.

sinensis roots was evaluated for therapeutic effect on peripheral circulation

and whole blood viscosity after oral administration by humans. The results

showed that it decreased blood viscosity ca. 180 min after oral

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administration.51 Significant therapeutic effects of the roots of A. sinensis on

hemorrheology in patients with acute ischemic stroke was also observed.81

1.5.3. PHARMACOLOGY

1.5.3.1. In vivo activities

Gynecological effects

The anxiolytic effects of essential oil of A. sinensis (EOAS) were studied

in three in vivo experiments with diazepam as a positive control. In the elevated

plus-maze experiment, EOAS at a dose of 30.0 mg/kg increased the percentage

of open-arm time, reduced the percent of protected head dips, and exhibited a

modest anxiolytic effect compared with diazepam. In the light/dark test, EOAS

at a dose of 30.0 mg/kg prolonged the time staying in the light area while

maintaining locomotor activity of the animals. In the stress-induced

hyperthermia experiment, rectal temperature was measured twice after 60 and

70 min of drug administration, EOAS (30.0 mg/kg) inhibited stress-induced

hyperthermia. As a conclusion, these results showed that essential oil of A.

sinensis has an anxiolytic-like effect.82

Ligustilide (5-20 mg/kg, i.p.) and butylidenephthalide (10-30 mg/kg,

i.p.), two major constituents in essential oil of A. sinensis, were found to

reverse the pentobarbital (PB) induced sleeping time in mice. Both compounds

(20 mg/kg, i.p.) reversed the inhibitory effects of yohimbine and methoxamine

(two stimulants of central noradrenergic system) and FG7142 (a

benzodiazepine inverse agonist) on PB sleeping time in mice.83 These results

demonstrated the effects of two phthalide components in A. sinensis on central

noradrenergic and/or GABAa systems.

A polysaccharide from A. sinensis has been shown to have anti-anemic

and immunofunction regulating activity. Its biological mechanism of action

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might involve direct or indirect stimulation of cells in the hematopoietic

inductive microenvironments such as lymphocytes, bone marrow stromal cells

and macrophages.84 As an analgesic, the polysaccharide markedly decreased

the frequency of the writhing reaction induced by estradiol and oxytocin as well

as prolonging the latent period and the elevated pain threshold of mice on the

hot plate after administering polysaccharides (0.25 g kg-1).85

One of the primary benefits of A. sinensis is its ability to relieve

cramps by helping relaxation of the uterus.86 Angelica sinensis has an

adaptive action on the uterus, and its pharmacologically active ingredients

in this aspect can be grouped into two distinctive components: non-volatile

components (water-soluble) and the alcohol-soluble components containing

the essential oil. The water-soluble non-volatile elements of the roots were

observed to be able to increase the contraction of the uterus, while the

volatile elements act as an antispasmodic to relax the muscles of the uterus

in vivo.87,88 The essential oils containing over 70% ligustilide significantly

inhibited the contractile function of isolated uterine smooth muscle of mice

in a concentration-dependent manner whether the uterus was isolated or in

mice treated with oxytocin.89 Ex vivo experiments showed that ligustilide

and butylidenephthalide contained in the volatile oils have a strong

spasmolytic effect on isolated uteri.90,91 They can also inhibit prostaglandin

F2α- (PGF2α-), oxytocin-, or acetylcholine-induced contraction of non-

pregnant rat uteri.92 While the in vitro mechanism of relaxant activity

remains incompletely elucidated, it might involve both histamine receptor

blocking activity and calcium ion channel effects.89,93,94 The muscle

relaxation induced by vasodilating and antispasmodic effects of A. sinensis

are regarded as the mechanism underlying its effectiveness in treating

dysmenorrhea.74

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Other effects

In addition to gynecological effects, A. sinensis exhibited many other in

vivo pharmacological effects, i.e., effects on cardiovascular and blood systems,

as well as immunological, antioxidant, antitumor, pulmonary, and

antiarrhythmic functions.91 In a cardiac hemodynamic experiment, aqueous

extracts of A. sinensis significantly increased coronary blood flow, and reduced

coronary and peripheral vascular resistance and myocardial oxygen

consumption, as well as arterial blood pressure after i.v. administration to

anesthetized dogs (2g/kg).45

Some phthalides such as butylphthalide from Angelica sinensis roots

exhibited a significant relaxant effect on animal tracheal smooth muscle, which

is a direct action on the tracheal smooth muscle suggested by a rapid and

potent anti-BaCl2 action on tracheal smooth muscle. Phthalides displayed a

potent antagonistic effect on acetylcholine- and histamine-induced tracheal

muscle constriction, and histaminergic or cholinergic receptors were not

involved in this action.95 Ligustilide and senkyunolide were found to cause

muscle relaxation in an in vivo study on the crossed extensor reflex in

anesthetized rats.96

The hepato-protective action of ferulic acid (as sodium ferulate) from A.

sinensis was demonstrated in an in vivo study. Sodium ferulate can inhibit the

activity of serum alanine aminotransferase, prevent the depletion of liver

glycogen and glutathione, increase the liver homogenate and microsomal

glutathione S-transferase activities, and reduce the malondialdehyde content,

the membrane fluidity of liver microsomes and the mitochondria in

paracetamol induced liver toxicity in mice.97 Sodium ferulate was also shown

to significantly reduce the increase of medium lipoperoxide (LPO) and inhibit

22

3H-thymidine incorporation dose-dependently, indicating its inhibitory effect

on the proliferation of aortic smooth muscle cells.98

In an in vivo study, a chloroform extract of A. sinensis suppressed the

growth of malignant brain tumors of rat and human origin, and also decreased

the volume of in situ glioblastoma multiforme (GBM).99 Polysaccharides of A.

sinensis exhibited anti-tumor effects in vivo in experimental tumor models

(sarcoma 180, leukemia and Ehrlich ascitic carcinoma),100 they were also

found to activate the immunologic function of rat Kupffer cells.101 An acetone

extract of A. sinensis showed dose-dependent antiproliferative effect on A549,

HT29, DBTRG-05MG and J5 human cancer cells.102

Sodium L-malate from the water extract of A. sinensis was found to

exhibit protective effects against both nephrotoxicity (ED50: 0.4 mg/kg,

perorally) and bone marrow toxicity in rats (ED50: 1.8 mg/kg, perorally).103 A

n-hexane fraction of A. sinensis attenuated various drug-induced amnesia

effects in rats, which are related to memory processes.104 Polysaccharides from

A. sinensis are also suggested to be useful to in the prevention of neutrophil-

dependent mucosal injury in the gastrointestinal tract.105

1.5.3.2. In vitro activities

In vitro evaluation of estrogenic activity of several plants was performed

by Liu et al..71 A methanol extract of A. sinensis (MEAS) was assayed for

competition with estrodiol for binding to ERα and ERβ and for induction of

alkaline phosphatase (AP) activity in cultured Ishikawa (endometrial) cells.

MEAS showed weak ER binding activities (IC50 > 50 µg/mL), as well as weak

induction of the progesterone receptor (PR) and presenelin-2 (pS2). These

findings were consistent with a clinical study which showed non-estrogenicity

of A. sinensis.72 These studies indicated that any activity of A. sinensis against

gynecological disorders must be through non-estrogen pathways.

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Liao et al.106 established receptor binding assays to evaluate the

bioactivities of water extracts of A. sinensis in vitro. These assays were used to

explain these effects on the central nervous system (CNS) at the receptor level.

The results showed that a water extract of A. sinensis acted on 5-HT1A and

GABAa receptors with strong binding affinity (Ki values) of 6 ± 3 µg/mL and

0.6 ± 3 µg/mL, respectively.

An aqueous extract of A. sinensis and its constituent ferulic acid were

found to inhibit platelet aggregation via inhibition of platelet serotonin release,

which was associated with its pharmacological effects on cerebrovascular

disorders.107 Ferulic acid also exhibited DPPH radical scavenging activities

with EC50 of 63.9 µM and acted as an antioxidant by protecting cells from

oxidative stress.108 The mechanism of antioxidant activity was attributable

to its phenolic nucleus and conjugated side chain, which could easily form

a resonance stabilized phenoxy radical.109 Ferulic acid has been used in

many cosmetics due to its photoprotective function and in foods because of

its inhibitory effect on lipid peroxidation. In addition, sodium ferulate (0.8

µM and 3.2 µM) exhibited a dose-dependent inhibition on the synthesis of

thromboxane B2 (TXB2), prostaglandin E2 (PGE2), and F2α (PGF2α) with

inhibitory rates of 93.8%, 90.3% and 47.4% at a concentration of 3.2 µM,

respectively.110,111 Ferulic acid is currently not permitted to be used as a

food additive, cosmetic, or pharmaceutical in the USA.

Butylidenephthalide in A. sinensis showed dose-dependent inhibitory

effects on the aggregation and release reaction of washed rabbit platelets

induced by arachidonic acid, collagen, platelet aggregation factor (PAF), and

adenosine diphosphate (ADP). Anti-aggregation and release activity of

butylidenephthalide were associated with the inhibition of the enzyme COX

and a calcium-antagonizing effect.112 In another study, researchers found that

butylidenephthalide strongly inhibited rat uterine contractions induced by

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prostaglandin F2α, oxytocin, and acetylcholine. The mechanism of its

nonspecific antispasmodic action was different from that of papaverine, which

inhibited tonic contractions more selectively than phasic contractions.113

Polysaccharides of A. sinensis have been studied for different activities.

These have inhibitory effects on the invasion and metastasis of hepatocellular

carcinoma cells.100 In addition, these polysaccharides produce a direct