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Anxiolytic Activity of Apocynum venetum L. and Its Proposed Mechanisms of Action

Permanent Link: http://ufdc.ufl.edu/UFE0021588/00001

Material Information

Title: Anxiolytic Activity of Apocynum venetum L. and Its Proposed Mechanisms of Action
Physical Description: 1 online resource (150 p.)
Language: english
Creator: Grundmann, Oliver
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anxiolytic, apocynum, bioactivation, bioguided, epm, flavonoid, gaba
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Anxiety affects one-eighth of the total population world-wide and has become an important area of research interest in psychopharmacology during this decade. Current treatment options for anxiety disorders present with either a narrow safety margin between the anxiolytic effect and those causing unwanted side effects or a delayed onset of anxiolytic action, that has prompted many researchers to evaluate new compounds in the hope that other anxiolytic drugs will have less undesirable effects. Thus, there is a need of robust anxiolytic compounds that have lesser side effects and a more immediate onset of anxiolytic action than the currently available therapeutics. On the basis of these considerations, it was the purpose of this dissertation to characterize the anxiolytic-like activity of an ethanolic extract prepared from the leaves of Apocynum venetum L. (AV, Apocynaceae). AV is a wild shrub widely distributed throughout mid and northwestern China. An ethanolic extract of AV showed significant antidepressant-like activity in the forced swimming test in rats. However, until now there are no reports on the anxiolytic effects of AV. Thus, the present research focuses on the qualitative analysis and the anxiolytic activity of an ethanolic extract and fractions prepared from the leaves of AV. The extract as well as fractions and subfractions were analyzed using HPLC-UV-MS analysis techniques. The main substance classes in the extract were polyphenolic compounds such as caffeic acid derivatives, flavonoids, and proanthocyanidins. The in vitro oxygen radical absorbance capacity assay revealed a strong antioxidant effect for the extract and for some pure compounds. AV showed a strong anxiolytic effect in vivo which was partially antagonized by a gamma-aminobutyric acid (GABA)-ergic and serotonergic antagonist. Fractionation and further antagonism revealed kaempferol as a contributor to the anxiolytic action of AV. In addition it seems likely that the flavonoid kaempferol, following microbial degradation to para-hydroxyphenylacetic acid in the intestines, contributes to the anxiolytic activity of AV via action on GABA receptors.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Oliver Grundmann.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Butterweck, Veronika.
Local: Co-adviser: Hochhaus, Guenther.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021588:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021588/00001

Material Information

Title: Anxiolytic Activity of Apocynum venetum L. and Its Proposed Mechanisms of Action
Physical Description: 1 online resource (150 p.)
Language: english
Creator: Grundmann, Oliver
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anxiolytic, apocynum, bioactivation, bioguided, epm, flavonoid, gaba
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Anxiety affects one-eighth of the total population world-wide and has become an important area of research interest in psychopharmacology during this decade. Current treatment options for anxiety disorders present with either a narrow safety margin between the anxiolytic effect and those causing unwanted side effects or a delayed onset of anxiolytic action, that has prompted many researchers to evaluate new compounds in the hope that other anxiolytic drugs will have less undesirable effects. Thus, there is a need of robust anxiolytic compounds that have lesser side effects and a more immediate onset of anxiolytic action than the currently available therapeutics. On the basis of these considerations, it was the purpose of this dissertation to characterize the anxiolytic-like activity of an ethanolic extract prepared from the leaves of Apocynum venetum L. (AV, Apocynaceae). AV is a wild shrub widely distributed throughout mid and northwestern China. An ethanolic extract of AV showed significant antidepressant-like activity in the forced swimming test in rats. However, until now there are no reports on the anxiolytic effects of AV. Thus, the present research focuses on the qualitative analysis and the anxiolytic activity of an ethanolic extract and fractions prepared from the leaves of AV. The extract as well as fractions and subfractions were analyzed using HPLC-UV-MS analysis techniques. The main substance classes in the extract were polyphenolic compounds such as caffeic acid derivatives, flavonoids, and proanthocyanidins. The in vitro oxygen radical absorbance capacity assay revealed a strong antioxidant effect for the extract and for some pure compounds. AV showed a strong anxiolytic effect in vivo which was partially antagonized by a gamma-aminobutyric acid (GABA)-ergic and serotonergic antagonist. Fractionation and further antagonism revealed kaempferol as a contributor to the anxiolytic action of AV. In addition it seems likely that the flavonoid kaempferol, following microbial degradation to para-hydroxyphenylacetic acid in the intestines, contributes to the anxiolytic activity of AV via action on GABA receptors.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Oliver Grundmann.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Butterweck, Veronika.
Local: Co-adviser: Hochhaus, Guenther.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021588:00001


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1 ANXIOLYTIC ACTIVITY OF Apocynum venetum L. AND ITS PROPOSED MECHANISMS OF ACTION By OLIVER GRUNDMANN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Oliver Grundmann

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3 To all the people who shaped me in so many ways particular my partner Bob, my father, my aunt, my parents-in-law, and my grandparents. Thank you from the bottom of my heart for all your support and encouragement

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4 ACKNOWLEDGMENTS Many people helped in the completion of this thesis in many different ways. I thank my advisor, Dr. Veronika Butterweck, for providing me with the greatest opportunity and challenge of my life coming over to America. It has profusely changed my life in many ways. Also, I would like to thank Dr. Butterweck for her continued support, providing me with great opportunities, and involving me in research projects besides my dissertation work I have learned so many things during my studies. I would like to thank my dissertation committee, namely Dr. Hartmut Derendorf, Dr. Guenther Hochhaus, Dr. Cary Mobley, and Dr. Saunjoo Yoon, for their advice and providing me with new input and equipment. Special thanks go to the research groups of Dr. David Rossi, Neurological Science Institute, Oregon Health & Science University, and Dr. Junji Terao, Department of Nutrition, University of Tokushima, for letting me join their research group to conduct research on my project. Especially the group members of Dr. Rossi (David, Claudia, James, and Adrianna) were very welcoming and are now valuable friends as is Dr. Hiroyuki Sakakibara, who helped me in so many ways during my stay in Tokushima. Thanks to Kevin Spelman from the University of North Carolina for analyzing the NMR samples and helping with the identification as well. For their continued financial and personal support I would like to thank Tokiwa Phytochemical Co. LTD and especially Dr. Jun-Ichiro Nakajima, who has provided me with every possible advice and information as well as his personal support, and Mr. Takashi Tatsuzaki, and Dr. Sujiro Seo. All of my lab members and friends were the biggest support throughout my years as a graduate student. Immo, Matt, Yui, Mew, Prajakta, Elanor, Ella, Anna-Maria, Stephan, and Victor have been a great help by being there for professional and personal advice. Without them, this dissertation would probably not have turned out the way it did. Summer students, namely

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5 Claire, Andrea, Faryda, and Tanya, have contributed to the success of smaller parts of this work. I thank them for their dedication and hard work. Interns have for sure been a big support for my research. Many thanks go to Katrin, Carmen, Antje, Chris, and Michael for their help with many animal studies and their dedication to the work. Last, but at no means least, I would like to thank my family and friends for their understanding, support, and taking me the way I am. Thank you from the bottom of my heart to my partner Bob for waiting so patiently and endure the separation that followed each of our unities. Thank you to my dad for supporting me and giving me advice if things got rough. Thanks to Michele and Mike for being the best parents-in-law one can wish for. Thanks to my aunt Vera and my grandparents you made me the person I am today and I am proud to be your child. Thanks to Christel and her parents you played a major part and shaped my understanding of the world. Friends, who have always been supportive of me and keep in touch no matter what: Arnfried and Alexandra, Dorys, Asha, Roger, Martina, Marisol, Sam and Brian, David, Arielle, Daniela and Nikki, and my brother Mirko and his wife Claudia.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................................... 4 LIST OF TABLES................................................................................................................................ 8 LIST OF FIGURES ............................................................................................................................ 10 LIST OF ABBREVIATIONS ............................................................................................................ 13 ABSTRACT ........................................................................................................................................ 14 CHAPTER 1 INTRODUCTION ....................................................................................................... 16 Anxiety Disorders ....................................................................................................................... 16 Epidemiology ....................................................................................................................... 16 Physiology, Pathophysiology, and Pharmacology............................................................. 17 Apocynum venetum ................................ ................................ ................................ ...................... 20 Apocynaceae ................................ ................................ ................................ ........................ 20 Apocynum venetum .............................................................................................................. 20 Historic development and taxonomy .......................................................................... 20 Traditional uses and modern indications .................................................................... 23 Known substance classes from Apocynum venetum .................................................. 25 Hypothesis and Specific Aims ................................................................................................... 27 Specific Aim 1 ..................................................................................................................... 28 Specific Aim 2 ..................................................................................................................... 28 Specific Aim 3 ..................................................................................................................... 28 CHAPTER 2 ANALYSIS OF Apocynum venetum EXTRACT AND FRACTIONS ................... 34 Extract Preparation ...................................................................................................................... 34 Extract Analysis .......................................................................................................................... 34 Analytical Equipment .......................................................................................................... 34 Identified compounds from extract..................................................................................... 35 Bioguided Fractionation ............................................................................................................. 35 Preparation of Fractions from Apocynum venetum Extract............................................... 36 Fraction Analysis ................................................................................................................. 37 Preparation of Subfractions of Fraction C from Apocynum venetum ............................... 39 Subfraction Analysis............................................................................................................ 40 Evaluation of Antioxidant Activity ............................................................................................ 42 Conclusions ................................................................................................................................. 45 CHAPTER 3 PHARMACOLOGICAL EVALUATION OF Apocynum venetum AND ITS FRACTIONS ............................................................................................................................... 66 Animal Models ............................................................................................................................ 66

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7 Elevated Plus Maze ............................................................................................................. 66 Light-Dark Transition Test ................................................................................................. 68 Stress-Induced Hyperthermia .............................................................................................. 68 Open Field ............................................................................................................................ 69 Evaluation of Anxiolytic-Like Activity of Apocynum venetum ............................................... 70 Elevated Plus Maze ............................................................................................................. 70 Acute anxiolytic effect ................................................................................................. 70 Anxiolytic activity after chronic treatment ................................................................. 73 Light-Dark Transition Test ................................................................................................. 74 Stress-Induced Hyperthermia .............................................................................................. 75 Evaluation of Anxiolytic-like Activity of Fractions prepared from Apocynum venetum ....... 75 Elevated Plus Maze ............................................................................................................. 75 Anxiolytic profile of fractions and antagonism studies ............................................. 75 Antagonism of diazepam and buspirone ..................................................................... 78 Light-Dark Transition Test ................................................................................................. 79 Evaluation of Anxiolytic-like Activity of Subfractions prepared from Fraction C ................ 80 Evaluation of Locomotor Activity of Apocynum venetum ....................................................... 81 Conclusions ................................................................................................................................. 81 CHAPTER 4 PHARMACOLOGICAL EVALUATION OF KAEMPFEROL AND ITS METABOLITE ........................................................................................................................... 95 Anxiolytic Action of Kaempferol .............................................................................................. 95 Elevated Plus Maze ............................................................................................................. 95 Dose-Response Profile for Kaempferol and Structure-Activity Relationship ................. 95 Antagonism Studies for Kaempferol and Quercetin.......................................................... 96 Evaluation of Locomotor Activity of Kaempferol and Quercetin .................................... 98 Metabolism Theory for Kaempferol and Quercetin in the Intestines ...................................... 98 Anxiolytic Action of Para-Hydroxyphenylacetic Acid........................................................... 101 Elevated Plus Maze ........................................................................................................... 101 Dose-Response Profile for Para-Hydroxyphenylacetic Acid.......................................... 101 Antagonism Studies for Para-Hydroxyphenylacetic Acid .............................................. 102 Evaluation of Locomotor Activity of Para-Hydroxyphenylacetic Acid ........................ 102 Conclusions ............................................................................................................................... 103 CHAPTER 5 DISCUSSION ............................................................................................................ 116 APPENDIX: ADDITIONAL TABLES ....................................................................................... 122 Chapter 2 .................................................................................................................................... 122 Chapter 3 .................................................................................................................................... 123 Chapter 4 .................................................................................................................................... 133 LIST OF REFERENCES ................................................................................................................. 138 BIOGRAPHICAL SKETCH ........................................................................................................... 150

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8 LIST OF TABLES Table page 2-1 Analytical conditions for AV extract and fraction analysis. ................................ ............... 48 2-2 Identified compounds from AV extract. ................................ ................................ ............... 48 2-3 Distribution and amount for fractions derived from AV extract. ................................ ........ 49 2-4 Subfractions of Fraction C from AV. ................................ ................................ .................... 49 2-5 Pooling scheme for subfractions of fraction C from AV. ................................ .................... 49 2-6 Analytical conditions for subfraction A and B from AV fraction C. ................................ .. 50 2-7 Analytical conditions for subfraction C from AV fraction C. ................................ ............. 50 A-1 Results of ORAC assay for relative total antioxidant activity of AV extract and tannin extracts. ................................ ................................ ................................ ...................... 122 A-2 Results of ORAC assay for relative total antioxidant activ ity of caffeic acid derivatives. ................................ ................................ ................................ ............................ 122 A-3 Results of ORAC assay for relative total antioxidant activity of flavonoid aglycones. .. 122 A-4 Results of ORAC assay for relative total antioxidant activity of glycosylated flavonoids. ................................ ................................ ................................ ............................ 122 A-5 Results of ORAC assay for absolute antioxidant activity of AV extract and tannin extrac ts. ................................ ................................ ................................ ................................ 123 A-6 Results of EPM for AV extract dose response profile. ................................ ...................... 123 A-7 Results of EPM for GABA antagonism of AV extract. ................................ ..................... 123 A-8 Results of EPM for 5 HT 1A antagonism of AV extract. ................................ .................... 124 A-9a Results of EPM for detailed dose response profile of AV extract. ................................ ... 124 A-9b continued from table A9a ................................ ................................ ................................ .... 124 A-10 Results of LDT for AV extract and fractions. ................................ ................................ .... 125 A-11 Results of SIH for AV extract. ................................ ................................ ............................ 125 A-12 Chr onic treatment I of AV extract. ................................ ................................ ..................... 126 A-13 Chronic tr eatment II of AV extract. ................................ ................................ .................... 127

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9 A-14 Results of EPM for fractions of AV extract. ................................ ................................ ...... 128 A-15 Results of EPM for GABA antagonism of AV fractions. ................................ ................. 129 A-16 Results of EPM for 5 HT 1A antagonism of AV fractions. ................................ ................. 130 A-17 Results of EPM for concomitant antagonism of fraction C and AV. ............................... 131 A-18 Results of EPM for diazepam and buspirone antagonism. ................................ ................ 131 A-19 Results of EPM for fractions of AV e xtract. ................................ ................................ ...... 132 A-20 Results of Open Field Test for AV extract and antagonists. ................................ ............. 132 A-21 Results of EPM for kaempferol (p.o.) dose r esponse profile. ................................ ........... 133 A-22 Results of EPM for kaempferol (i.p.) dose response profile. ................................ ............ 133 A-23 Results of EPM for quercetin and myricetin (p.o.). ................................ ........................... 133 A-24 Results of EPM for GABA antagonism of kaempferol (p.o.). ................................ .......... 134 A-25 Results of EPM for antagonism of qu ercetin (p.o.). ................................ .......................... 134 A-26 Results of EPM for 5 HT 1A antagonism of kaempferol (p.o.). ................................ ......... 135 A-27 Results of Open Field test for kaem pferol (p.o.). ................................ ............................... 135 A-28 Results of Open Field test for quercetin (p.o.). ................................ ................................ .. 135 A-29 Results of EPM for pHPAA (i.p.) dose response profile. ................................ ................. 136 A-30 Results of EPM for pHPAA (p.o.) dose response profile. ................................ ................ 136 A-31 Results of EPM for antagonism of pHPAA (i.p.). ................................ ............................. 137 A-32 Results of Open Field test for pHPAA (i.p.). ................................ ................................ ..... 137

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10 LIST OF FIGURES Figure page 1-1 Location of the amygdaloid complex in the brain ................................ .............................. 29 1-2 Anxiety circuits of the amygdala after an auditory stimulus. ................................ .............. 29 1-3 Schematic of mechanism of action for anxiolytic action of serotonin agonists. ................ 30 1-4 Apocynum venetum L. ................................ ................................ ................................ ............ 31 1-5 Basic structu re of flavonol s and their respective glycosides or metabolites from AV ..... 31 1-6 Structure of proanthocyanidins present in AV L.. ................................ ............................... 32 1-7 Structure of the caffeic acid derivative chlorogenic acid. ................................ ................... 32 1-8 Structure of apocynins from AV. ................................ ................................ .......................... 33 2-1 UV spectrum of A V extract.. ................................ ................................ ................................ 50 2-3 Mass analysis and distribution of mass to charge ratio for fractions prepared from AV extract. ................................ ................................ ................................ .............................. 53 2-4 Extract ion scheme for tannin reduced and tannin gelatin extract from AV extract. ......... 53 2-5 Acidic TLC of AV extract, tannin extracts, and fractions. ................................ .................. 54 2-6 Hydroethanolic TLC of AV extract, tannin extracts, and fractions. ................................ ... 54 2-7a LC UV spectrum and MS for AV extract. ................................ ................................ ............ 55 2-7b LC UV spectrum and MS for AV fraction A. ................................ ................................ ...... 55 2-7c LC UV spectrum and MS for AV fraction B. ................................ ................................ ...... 56 2-7d LC UV spectrum and M S for AV fraction C. ................................ ................................ ...... 56 2-7e LC UV spectrum and MS for AV fraction D. ................................ ................................ ...... 57 2-7f LC UV spectrum and MS for AV fraction E. ................................ ................................ ...... 57 2-8 TLCs of subfractions from fraction C of AV. ................................ ................................ ...... 58 2-9 Chromatograms and UV spectra of subfractions sA, sB, and sC. ................................ ....... 59 2-10 HPLC chromatogram, UV spectra, and mass spectra of fraction sD from AV fraction C. ................................ ................................ ................................ ................................ ............. 60

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11 2-11 HPLC chromatogram, UV spectra, and mass spectra of fraction sE from AV fraction C. ................................ ................................ ................................ ................................ ............. 61 2-12 1 H NMR spectrum of subfraction sE.. ................................ ................................ .................. 62 2-13 Scheme of the ORAC reaction.. ................................ ................................ ............................ 63 2-14 Antioxidant activity of AV extract and tannin containing and tannin reduced extracts. ................................ ................................ ................................ ................................ ... 64 2-15 Antioxidant activity of caffeic acid derivatives. ................................ ................................ .. 64 2-16 Antioxidant activity of flavonoid aglycones. ................................ ................................ ....... 65 2-17 Antioxidant activity of flavonoid glycosides. ................................ ................................ ...... 65 3-1 Sketch of the Elevated Plus Maze (EPM). ................................ ................................ ............ 84 3-2 Sketch of the Light Dark Transition (LDT) test.. ................................ ................................ 84 3-3 Dose response profile for AV. ................................ ................................ .............................. 85 3-4 GABA antagonism of active AV doses. ................................ ................................ ............... 85 3-5 Serotonin antagonism of active AV doses. ................................ ................................ .......... 86 3-6 Detailed dose response profile for AV. ................................ ................................ ................ 86 3-7 Evaluation of AV extract in the LDT test. ................................ ................................ ............ 87 3-8 Stress Induced Hyperthermia (SIH) test of AV extract. ................................ ...................... 87 3-9 Chronic treatment I with AV extract over 16 days. ................................ ............................. 88 3-10 Chronic treatment II with AV extract over 22 days. ................................ ............................ 88 3-11 Dose response profile for fractions, reconstituted and whole extract of AV in 30 mg/kg. ................................ ................................ ................................ ................................ ..... 89 3-12 Dose response profile for fractions, reconstituted and whole extract of AV in 125 mg/kg. ................................ ................................ ................................ ................................ ..... 89 3-13 GABA an tagonism of active fractions equivalent to 30 mg/kg of whole extract. ............. 90 3-14 GABA Antagonism of active fractions equivalent to 125 mg/kg of whole extract. .......... 90 3-15 5 HT 1A Antagonism of active fractions equivalent to 30 mg/kg of whole extract. .......... 91 3-16 5 HT 1A Antagonism of active fractions equivalent to 125 mg/kg of who le extract. ......... 91

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12 3-17 Concomitant antagonism of diazepam, buspirone, fraction C, and AV with flumazenil and WAY 100635 ................................ ................................ ............................... 92 3-18 Ant agonism of buspirone with flumazenil. ................................ ................................ .......... 92 3-19 Antagonism of diazepam with WAY 100635. ................................ ................................ ..... 93 3-20 Dose response profile for subfract ions of fraction C from AV in 30 mg/kg. .................... 93 3-21 Dose response profile for subfractions of fraction C from AV in 125 mg/kg. .................. 94 3-22 Open Field Test for AV, diazepam, buspirone, and antagonists. ................................ ........ 94 4-1 Structure of the flavonol kaempferol ................................ ................................ .................. 105 4-2 Dose respon se profile for kaempferol after oral administration. ................................ ...... 105 4-3 Dose response profile for kaempferol after intraperitoneal administration. .................... 106 4-4 Dose response profile for quercetin and myricetin after oral administration. .................. 106 4-5 GABA antagonism of kaempferol. ................................ ................................ ..................... 107 4-6 Serotonin antagonism of kaempferol. ................................ ................................ ................. 107 4-7 Antagonism of quercetin. ................................ ................................ ................................ ..... 108 4-8 Open Field Test for kaempferol and diaz epam. ................................ ................................ 108 4-9 Open Field Test for quercetin and diazepam. ................................ ................................ ..... 109 4-10 Intestinal metabolism and absorption of the flavonol kaempfer ol and its metabolites.. 110 4-11 Intestinal metabolism and absorption of the flavonol quercetin and its metabolites.. ..... 111 4-12 Intestinal metabolism and absorption of the flavonol myricetin and its metabolites.. .... 112 4-13 Main routes of trace amine metabolism.. ................................ ................................ ............ 113 4-14 Dose response profile for para hydroxyphenylacetic acid after intraperitoneal administration. ................................ ................................ ................................ ...................... 114 4-15 Dose response profile for para hydroxyphenylacetic acid after oral adminis tration. ...... 114 4-16 Antagonism of para hydroxyphenylacetic acid. ................................ ................................ 115 4-17 Open Field Test for para hydroxyphenylacetic acid. ................................ ......................... 115

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13 LIST OF ABBREVIATIONS AV Apocynum venetum L., Apocynaceae GABA Gamma-aminobutyric acid FST Forced Swimming Test HPLC High pressure liquid chromatography UV Ultraviolet MS Mass spectrometry EPM Elevated plus maze LDT Light-dark transition test SIH Stress-induced hyperthermia OFT Open Field Test 5-HT 5-hydroxytryptamine, serotonin Diaz. Diazepam Busp. Buspirone i.p. Intraperitoneal p.o. Per os, given orally Sal. Saline pHPAA Para-Hydroxyphenylacetic acid ORAC Oxygen Radical Absorbance Capacity assay HPA Hypothalamus-Pituitary-Adrenal

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANXIOLYTIC A CTIVITY OF A pocynum venetum L. AND ITS PROPOSED MECHANISMS OF ACTION By Oliver Grundmann December 2007 Chair: Veronika Butterweck Cochair: Guenther Hochhaus Major: Pharmaceutical Sciences Anxiety affects one eight h of the total population world wide and h as become an important area of research interest in psychopharmacology during this decade. Current treatment options for anxiety disorders present with either a narrow safety margin between the anxiolytic effect and those causing unwanted side effects or a delayed onset of anxiolytic action, that has prompted many researchers to evaluate new compounds in the hope that other anxiolytic drugs will have less undesirable effects. Thus, there is a need of robust anxiolytic compounds that have lesser side effects and a more immediate onset of anxiolytic action than the currently available therapeutics. On the basis of these considerations, it was the purpose of this dissertation to characterize the anxiolytic-like activity of an ethanolic extract prepared from the leaves of Apocynum venetum L. (AV, Apocynaceae). AV is a wild shrub widely distributed throughout mid and northwestern China. An ethanolic extract of AV showed significant antidepressant-like activity in the forced swimming test in rats. However, until now there are no reports on the anxiolytic effects of AV. Thus, the present research focuses on the qualitative analysis and the anxiolytic activity of an ethanolic extract and fractions prepared from the leaves of AV. The extract as well

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15 as fractions and subfractions were analyzed using HPLC-UV-MS analytical techniques. The main substance classes in the extract were polyphenolic compounds such as caffeic acid derivatives, flavonoids, and proanthocyanidins. The in vitro oxygen radical absorbance capacity assay revealed a strong antioxidant effect for the extract and for some pure compounds. AV showed a strong anxiolytic effect in vivo which was partially antagonized by a gamma-aminobutyric acid (GABA)-ergic and serotonergic antagonist. Fractionation and further antagonism revealed kaempferol as a contributor to the anxiolytic action of AV. In addition it seems likely that the flavonoid kaempferol, following microbial degradation to para-hydroxyphenylacetic acid in the intestines, contributes to the anxiolytic activity of AV via action on GABA receptors.

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16 CHAPTER 1 INTRODUCTION Anxiety Disorders Epidemiology Anxiety disorders affect more than one-eighth of the total population world-wide and has become an important area of research interest in psychopharmacology during this decade [1]. Approximately 18% of the US adult population will suffer from any anxiety disorder during their life, making it the most common mental disorder. Fewer than half of these people receive appropriate treatment due to reasons such as lack of appropriate screening, lack of access to medication, being non-responders to medication, and exhibiting subclinical symptoms. However, of those 18% diagnosed with an anxiety disorder, 22.8% are classified as severe, 33.7% as moderate, and 43.5% as mild (N=9282) [2]. The highest incidences occurred for special phobias, social phobias, and generalized anxiety disorder. Anxiety disorders show a high comorbidity with major depressive disorders (ranging from 37% for separation anxiety disorder to 62% for generalized anxiety disorder, then also called anxious depression). Interestingly, the odds ratio for women to develop a mental disorder is 1.6 times compared to men. The population with a concurrent major depression and anxiety disorder especially shows a higher degree of treatment non-responders compared to those suffering from either one of the mental disorders alone [3]. Anxiety disorders can be classified according to their respective disruptive action on a persons social interaction and behavior. Phobias for example are related to certain objects or situations that cause irrational fear and cause an avoidance behavior (e.g. arachnophobia, ophidiophobia). In contrast, a generalized anxiety disorder is a chronic state of constant, undefined, and unexplainable fear. Common chronic symptoms include muscle tensions, headaches, heart palpitations, insomnia, and dizziness [4].

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17 Physiology, Pathophysiology, and Pharmacology In general, anxiety and fear are emotional responses to environmental stimuli, usually inherent as a reflex in human behavior to avoid a threatening or unpleasant situation. It is therefore a basic instinct and causes the body to react in a certain way to avoid the threat or flee. A person suffering from a chronic anxiety disorder presents with an inappropriate, irrational, or illogical response to an otherwise normal situation. Research on emotions has been stimulated mainly by the search for the neuropharmacological basis for fear and which brain regions might contribute or be involved with the development of an anxiety disorder. One brain region has frequently been involved in anxiety research and is probably linked to the development of the disorder in a profound way the amygdala (from Greek translating to due to its almond shaped appearance). The amygdaloid complex is located within the medial temporal lobes of the brain (figure 1 1) and comprises different nuclei including the basolateral, medial, and central n ucleus [5] The exact purpose of each of the nuclei is not yet known, but the complex has multiple connections with almost all ot her regions of the brain. The amygdala has been linked to emotional states and conditioned learning and memory. In the case of an auditory stimulus, the amygdala receives input from the thalamic nuclei and the auditory cortex which reaches the lateral nucleus and are passed on and amplified either directly to the central nucleus or by intra-amygdaloid pathways through the basolateral and other nuclei. The central nucleus then passes the information to higher brain regions for processing, but also triggers an immediate reaction causing behavioral, autonomic nervous system, and hormonal (through activation of the hypothalamic-pituitary-adrenal axis) responses (figure 1-2). The basolateral nucleus is therefore the main integrator for incoming stimuli since its removal renders rodents less affected by a threatening stimulus [6]. The central nucleus seems to be the efferent site of the amygdala in eliciting conditioned fear responses [7]. Due to its complex interaction

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18 with other parts of the brain, it has been a major challenge to differentiate the circuits involved in emotional states like anxiety from other emotional states like joy or depression. The general receptor hypothesis for anxiety is a major part of the pharmacology of anxiolytics currently used in treatment, but it seems likely that other factors play important roles as well. -aminobutyric acid (GABA), seems to contribute to the anxiolytic effect of benzodiazepines in different nuclei of the amygdala, predominantly the basolateral and the central nuclei [8] as well as a band of GABA receptors between the central and medial nuclei of the amygdala [9]. While GABA and especially the widely distributed GABAA receptors play therefore a pivotal role in serving as major output for the central nucleus, the basolateral nucleus is linked to the main excitatory neurotransmitter glutamate [10]. Since both nuclei are interconnected and specific lesions to the central and basolateral nuclei have been linked to a loss of fear in animals [11], it seems likely that feedback projections from the central to the basolateral nucleus serve as inhibitors for a fear stimulus that then projects back to the central nucleus via the basal and accessory basal nuclei. The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) has also been linked to anxiolysis and treatment options (e.g. buspirone) target a specific subreceptor, 5-HT1A, to induce anti-anxiety effects [12]. Although less pronounced, there are serotonergic projections in the amygdaloid complex, mainly presynaptical on GABA-ergic nerve terminals, inhibiting the synaptic GABA release, leading to a disinhibition of a specialized group of neurons (called ovoid-shaped non-pyramidal neurons) in the central nucleus, which then extend to pyramidal neuron projections outside the amygdala, thus causing an inhibition of excitatory neurons in the prefrontal cortex [13], which leads to an anti-anxiety effect (figure 1-3).

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19 The development of anxiety disorders might partially be related to a dysfunctional feedback mechanism in the amygdala or a dysregulation of response to the HPA (hypothalamus-pituitary-adrenal) axis, which causes the increased secretion of the stress hormone cortisol [8]. The main therapeutic approaches for the treatment of anxiety disorders are benzodiazepines and selective serotonin reuptake inhibitors (SSRIs), which act on the GABAA receptor or the serotonergic neurotransmission, respectively [14]. Use of SSRIs has increased over the last decade due to lesser side effects and a higher effectiveness especially for obsessive-compulsive disorders compared to the long-established benzodiazepines, which have been linked to hepatotoxicity [15]. One disadvantage of SSRIs and many other anti-anxiety drugs are the delayed onset of action and limitation of use based on interaction with comedication or other side effects. Another treatment option are the azapirones, mainly represented by buspirone, which have a benefit-over-risk advantage in the treatment of specific anxiety disorders over benzodiazepines [16]. However, the current non-responder rate to pharmacotherapy is as high as 40% [17] and necessitates the search for new therapeutic approaches. Although there is limited clinical data on the efficacy of herbal medicines for the treatment of anxiety disorders [18], there is strong evidence from traditional uses, case reports, and numerous animal behavioral experiments that showed promising results [19]. Extracts from Hypericum perforatum [20], Passiflora incarnata [21], Tilia tomentosa [22], and Piper methysticum [23] show significant anxiolytic activity in various animal models. In addition isolated compounds like kaempferol [22], quercetin, chrysin, and apigenin [24] show binding affinity or modulation of GABAA and GABAC receptors in vitro.

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20 Apocynum venetum Apocynaceae Taxonomic classification The family of Apocynaceae has a rich diversity, which consists of various habits such as trees, shrubs, lianas, and herbs mainly found in temperate and subtropical to tropical regions across the world [25]. The presence of a milky juice, which has been used as latex both traditionally and recently, is nearly ubiquitous throughout the family and is an important family characteristic. Many species produce substantial amounts of both medicinal and toxic alkaloids (Nerium, Strychnos, Rauwolfia, Pachypodium) and saponins (Acokanthera, Apocynum, Cerbera, Nerium, Strophanthus). While the alkaloids are mainly found in underground parts like roots and rhizomes, the cardio-active saponins and cardiac glycosides can be present in leaves and all aerial parts as well. The family is rich in polyphenolic compounds (proanthocyanidins, flavonols) and fibers. Traditional uses of several species are therefore mainly for medicinal purposes (anti-arrhythmic, aphrodisiac, nerve calming), hunting (poisonous arrow tips), or economic applications (rubber, fiber). Several alkaloids are still used as drugs, including reserpine from Rauwolfia serpentina and the semisynthetic derivatives vincristine and vinblastine from Vinca minor. Apocynum venetum Historic development and taxonomy The Latinized name Apocynum is derived from the Greek description by Dioscorides for a plant native to Greece that was used to poison dogs [26]. The name consiApocynum was used for any plant producing a milky juice that was latex-like in its behavior until the French botanist Joseph

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21 Pitton de Tournefort (1656-1708) distinguished the genus further between the plant families Euphorbiaceae, Asclepiadaceae, and Apocynaceae. Further revision of the genus was accomplished by the Swedish botanist Carl Linnaeus (1707-1778), who revolutionized the taxonomic system to its modern state. He described Apocynum venetum (AV, figure 1-4), which he originally named Trachomitum venetum, with the following words [27]: engel, und eirundlanzetfrmigen Blttern, ein etwa zwei Schuh hohes Gewchs mit perennirender Wurzel auf den Inseln des Adriatischen Meeres, vorzglich der kleinen Insel Lio einheimisch und in Sibirien, welches in unsern Grten im August bald purpurroth, b ald wei blht. Die groe, ziemlich dicke Wurzel welche einen scharfen, brennenden Milchsaft von sich giebt, ist von den Alten fr ein Schleim abfhrendes Mittel gehalten worden; verdient aber keine Nachahmung ceous stalk and eliptic lancetshaped leaves, an approximately two shoes high shrub with perennial root on the islands of the adriatic sea, predominantly on the small island Lio and in Siberia, which flowers purple red or white in our gardens during August. The big, pretty thick root which secrets a hot, burning milk juice, has been used by the eldest as a mucus purgative remedy, but does not deserve But even the classification by Linnaeus was not to be final since the sexual systematic was not able to summarize all Apocynaceae gena into the family but instead divided them based on the number of petals (monogyna or digyna), which classified the genus Apocynum wrongly to be closer related to the family Asclepiadaceae. This mistake was later correc ted by the British botanist Robert Brown (1773 1858), who is best known for the discovery of the Brownian motion of molecules and the naming of the cell nucleus. The use of the word Apocynaceae as the name for the now correctly classified plant family dates back to 1836, when John Lindley (1799-1865), another British botanist, redefined the was done by Robert E. Woodson (1904-1963) in 1930 and attempted to solidify the use of Trachomitum venetum to be used for AVwas introduced by Augusto

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22 Bguinot (1875-1940) to distinguish the Eurasian species (as it refers to the Italian city of Venice or Venetian) from the North American species including Apocynum cannabinum, Apocynum androsaemifolium, and Apocynum medium. However, the use of AV to describe the species has remained consistently in modern science, but both names can be used equally. Further differentiation was made by assigning subspecies, which resulted in the complete name of AV L. subsp. sarmatiense [26], which confusingly can also be used as a synonym for the species, then being called Apocynum sarmatiense or Trachomitum sarmatiense. Apocynum venetum has also been mentioned in several botanical books as being native to the Mediterranean area, namely Italy, Greece and Turkey, and most of the temperate zones of Russia, China, and Japan. An [28]: -branched perennial, 40-240 cm, glabrous (except for minutely pubescent pedicels, calyx and corolla), with creeping rhizome. Leaves very variable in size and shape, from narrowly ovate and oblong-lanceolate o narrowly lanceolate, 2-7 cm 6-25 mm, margin denticulate; petiole 2-6 mm. Inflorescence loose, usually much-branched, many flowered, rising well above uppermost leaves. Calyx 1.5-2 mm, lobes acute. Corolla pink, 5-7 mm, pubescent; lobes oblong, rounded, 2.5-3.5 1.2-1.5 mm. Pollen mostly fertile (40-95%). Follicles 7While the North American species Apocynum cannabinum has white flowers and is a source of fiber), the species Apocynum venetum flowers with a pink florescence and is therefore called AV are which translates to Luobuma. A tea prepared from the leaves is called Luobumaye. The tea has also been used and recently marketed as an -written as [29]. The taste of the tea preparation from the roasted leaves has been compared to that of green tea with the distinction that it lacks the stimulating effects of caffeine but instead has a calming effect due to the content of supposedly sedative substances [30].

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23 Traditional uses and modern indications Medicinal uses of AV date back to the first century anno domini where the leaves were used to prepare a tea in China and other Asian countries [31]. The tea was used to [32] The roots of Apocynum cannabinum and Apocynum androsaemifolium have also been used in the first half of 20th century in Europe for the treatment of heart diseases and it has been suggested to use the roots of AV instead due to their ten times higher lethal dose [33]. The leaves of AV are not linked to toxic effects since the aerial parts are devoid of alkaloids and the cardiac glycoside cymarin, which is present in underground parts [33]. Many of the traditional uses for AV leaves are still found as indications in the 2000 scientifically investigated in the last three decades: n and insomnia due to hyperactivity of the liver, edema with [32]. The effect of AV on the cardiovascular system has been the best investigated indication to date. Various research groups have found a cholesterol-lowering effect [34], prevention of the oxidation of low density lipoprotein (LDL) and fatty acids [29,35], and general antioxidant, radical--[36] of an aqueous extract both in vitro and in vivo. It has also been shown that AV inhibits the formation of advanced glycation end products that have been shown to contribute to diabetic vascular complications in the kidneys and the retina [31]. The antihypertensive effect of AV has been investigated in isolated vasculature [37] and in vivo using sodium chloride induced hypertensive, spontaneous hypertensive, and renal (after removal of one kidney) hypertensive rats [38]. The extract seems to be both acting directly on the smooth vascular cell and on the kidney, thereby reducing the afterload and the resistance.

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24 In addition, a direct diuretic effect of has been shown in an in vivo animal study using rats [39]. One human study summed the effects of AV on the cardiovascular system under the general term -patients treated with the extract had a significantly lower blood pressure and elevated high density lipoprotein (HDL) concentrations compared to a control group [40]. The traditional use of AV includes its calming effects on the nerves, which can be caused by a wide variety of pharmacological activities including sedative, antidepressant, anti manic, and anxiolytic mechanisms. In recent years, the antidepressant activity has been investigated in animal models of depression, namely the forced swimming test (FST) in rats and the tail suspension test (TST) in mice [41,42]. After chronic treatment for 8 weeks the extract influences the concentration of the neurotransmitters norepinephrine and dopamine in the hypothalamus and the striatum, two regions of the brain commonly associated with depression, and causes a down regulation of beta-adrenergic receptors, which is a classical sign for an antidepressant effect also seen after treatment with therapeutically used antidepressants like imipramine [43]. Further studies investigated pure compounds isolated from AV in concentrations present in the extract. The flavonol glycosides hyperoside (Quercetin-3-O--D-galactoside) and isoquercitrin (Quercetin-3-O--D-glucoside) showed a significant antidepressant effect in the TST when given alone and in combination as sub-active concentrations [42]. It has been shown that these flavonols are able to reach the brain after oral administration and metabolism in the intestines as the respective glucuronide miquelianin (Quercetin-3-O--D-glucuronic acid), which is able to cross the blood-brain barrier [44]. In addition, the extract was able to inhibit lipid-peroxidation in vitro using the brain cell line PC-12, which is also an indicator for the prevention of depressive symptoms since oxidative stress seems to play an important role in the

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25 development of many CNS disorders [45]. A major advantage of AV as a potential new treatment option for depression is its lack of interaction with Cytochrome P450 (CYP) 3A enzymes [46], which account for many drug-herb interactions. St. Johns Wort (Hypericum perforatum, Clusiaceae) is a popular case of such an interaction, although it has a long application history for the treatment of mild to moderate depression [47]. Known substance classes from Apocynum venetum The leaves of AV which are used to prepare the tea and the extract used in this research investigation contain a variety of substance classes, of which the majority are closely related and can be summarized as belonging to the class of polyphenolic compounds derived from either the isopentenyl synthesis pathway or the shikimic acid pathway with some less common derivatives from the polyketide synthesis pathway [48]. An important class of compounds is the flavonoids, which have been linked to a variety of pharmacological activities. The leaves of AV mainly contain glycosides of the flavonols quercetin (hyperoside, isoquercitrin) and kaempferol (astragalin, trifolin) and also small quantities of the aglycones (figure 1-5). Various pharmacological activities have been shown for both quercetin and kaempferol. Quercetin is present in many fruits and vegetables and in general aerial parts of plants, especially high in onions (Allium cepa, Alliaceae) and has been linked to many pharmacological activities such as anticarcinogenic, antiasthmatic, antihyperlipidemic, antithrombotic, antiviral, and antibiotic effects due to its high antioxidant activity [49]. Kaempferol is also a wide-spread flavonol, present in fruits and vegetables with high concentrations found in strawberries (Fragaria x ananassa, Rosaceae) and has also been linked to health benefits including anti-inflammatory [50], anti-HIV [51], antiviral [52], antiestrogenic and antiproliferative [53], as well as muscle relaxant effects [54]. The CNS activity of flavonoids has been an important research issue since the discovery of an anxiolytic and anticonvulsant effects of the flavone chrysine in

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26 1990 [55] both in vivo in animals and in vitro by GABA-receptor binding experiments. These findings suggest the possibility for these compounds or their metabolites to reach the respective brain areas. Few studies have been investigating the penetration of flavonoids into the brain, but it has been shown in vitro that miquelianin is able to cross the blood-brain barrier [44] and quercetin concentrations have been measured in vivo [56]. Another important class of compounds is the proanthocyanidins, which consist of monomeric flavan-3-ole compounds which assemble to form diand oligomeric proanthocyanidins via a new carbon-carbon bound, which can present in a variety of colors, often resembling the dark red or blue color of a fruit and are found in high amounts in vegetables, nuts, and seeds as well [57]. They have been linked to a variety of pharmacological activities including antibacterial, antiviral, anticarcinogenic, anti-inflammatory, anti-allergic, and smooth-muscle relaxing actions [58]. The main monomeric flavanoles present in the leaves of AV are catechin, epicatechin, gallocatechin, and epigallocatechin, which are then assembled to form the dimers (figure 1-6) epicatechin--gallocatechin, epigallocatechin--epicatechin, and procyanidin B2 (epicatechin--epicatechin). Recently, additional dimers and trimers consisting of the monomers epigallocatechin, epicatechin, and gallocatechin have been identified [42]. The proanthocyanidins present in AV leaves have been investigated for their antioxidant activity using in vitro systems and showed a potent effect against peroxynitrite-induced damage as well as prevention of protein damage by formation of advanced glycation end products [31]. One important substance found in high concentrations in the leaves from AV is chlorogenic acid [30], which occurs in many fruits and vegetables and is especially high in apples [59]. This caffeic acid derivative (figure 1-7) has been linked to many health benefits

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27 including prevention of cancer, weight loss and reduction of risk to develop diabetes [60], antioxidant, and recently, anxiolytic activities [61]. Another class of compounds present in the leaves of AV is the apocynins, which are special in that they consist of a flavanole and a caffeic acid derivative that form a new carbon-carbon bound [62]. Although the four to date identified apocynins (figure 1-8) are present in very low concentrations, they have been linked to pharmacological activities and have shown to possess hepatoprotective effects [63]. These substances are, however, not phylogenetically related to the catechol derivative apocynin [64] present in the roots of Picrorhiza kurroa (Scrophulariaceae). Hypothesis and Specific Aims The use of natural medicines for the treatment of diseases has been practiced in almost all cultures for thousands of years. Many modern treatment options are either structurally or mechanistically based on natural products (e.g. cocaine, morphine, vincristine, simvastatin, captopril). The need for new approaches to treat especially chronic diseases like depression, anxiety, schizophrenia, cancer, metabolic diseases, heart diseases, and alike that have a tremendous social and economic impact has never been stronger due to side effects, treatment resistance, or healthcare cost of current medications to mention a few. Especially central-nervous system (CNS) diseases are increasing and need constant medical and pharmaceutical attention. It has been shown that AV possesses CNS effects and might be a treatment option for depression. The goal of this research was to chemically characterize major constituents from an ethanolic extract prepared from AV, investigate possible anxiolytic activities of the extract, and use bio-guided fractionation as well as antagonism studies to get further insight into its mechanism of action.

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28 Specific Aim 1 The analytical evaluation of the ethanolic leaf extract from AV using an LC-MS method including qualitative composition. This was followed by bio-guided fractionation of the whole extract which included qualitative identification of fraction and subfraction composition using the same methods as for the whole extract and identification of putative anxiolytic compounds by 1H-NMR analysis for structure elucidation. Finally, the extract and pure substances identified from AV were evaluated for antioxidant activity. Specific Aim 2 The evaluation of putative anxiolytic effects of the whole extract using animal behavior models of anxiety including the Elevated Plus Maze, Light-Dark Transition test, and Stress-Induced Hyperthermia. Bio-guided fractionation and antagonism studies were used to narrow down possible active compounds in the extract and evaluate the anxiolytic mechanisms of action for AV. Specific Aim 3 Following bio-guided fractionation, isolated compounds from AV were tested for anxiolytic activity using the same pharmacological models as used before. The mechanism of action for these compounds was further investigated by antagonism studies. A possible model for the bioactivation of compounds linked to the route of administration was presented.

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29 Figure 1-1 Location of the amygdaloid complex in the brain [65]. Figure 1-2 Anxiety circuits of the amygdala after an auditory stimulus [5]. Body Auditory Cortex Thalamus Stimulus Lateral nucleus Basal nucleus Accessory basal nucleus Central nucleus Higher brain regions Behavior Autonomic Nervous System Hypothalamic Pituitary Adrenal Axis Amygdala

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30 Figure 1-3 Schematic of mechanism of action for anxiolytic action of serotonin agonists (3A: physiological condition, no serotonergic agonist, excretion of neurotransmitter GABA into synaptic cleft, inhibition of postsynaptic GABA transmission; 3B: binding of serotonergic agonist inhibits GABA release from presynaptic neuron into synaptic cleft, disinhibition of postsynaptic neuron leads to inhibition of excitatory neurons on pyramidal cells outside the amygdaloid complex). Presynaptical neuron Posts ynaptical neuron Presynaptic 5 HT 1A receptor Presynaptic GABA A receptor 5 HT1A agonist binds GABA Inhibition Activation A B

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31 Figure 1-4 Apocynum venetum L. [66], taken in the autonomous Xinjiang region of China. Figure 1-5 Basic structure of flavonols and their respective glycosides or metabolites from AV. (R1 = OH, R2 = R3 = H Kaempfe rol ; R1 = OH, R2 = OH, R3 = H Quercetin ; R1 = D galactose, R2 = OH, R3 = H Hyperoside R1 = D glucose, R2 = OH, R3 = H Isoquercitrin ; R1 = D glucuronopyranoside, R2 = OH, R3 = H Miquelianin ; R1 = D glucose, R2 = R3 = H Astragalin ; R1 = D galactos e, R2 = R3 = H Trifolin ).

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32 Figure 1-6 Structure of proanthocyanidins present in AV L. (6a, epicatechin-(4 ) gallocatechin; 6 b, epigallocatechin (4 ) epicatechin; 6 c, epicatechin (4 ) epicatechin). Figure 1-7 Structure of the caffeic acid derivative chlorogenic acid. 6a 6b 6c

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33 Figure 1-8 Structure of apocynins from AV (8a, Apocynin A, 8b, Apocynin B, 8c, Apocynin C, 8d, Apocynin D). 8a 8 b 8c 8d

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34 CHAPTER 2 ANALYSIS OF Apocynum venetum EXTRACT AND FRACTIONS Extract Preparation The extract was prepared and analyzed at the research and quality control laboratory of Tokiwa Phytochemical Co. LTD, Chiba, Japan. Leaves of Apoynum venetum (500 g) were refluxed for 1 h in aqueous ethanol (70% v/v, 300 ml) twice and the combined alcoholic extract was evaporated to dryness (140 g). The extract (67.5 g) was dissolved in hot water (1000 ml), and adjusted to pH 3.0 with sulfuric acid, then filtered. The filtrate was chromatographed on DIAION HP-20 (3.6 cm i.d.318 cm) and eluted with water (1000 ml) and then aqueous ethanol (70% v/v, 1000 ml). The aqueous ethanol fraction was collected and evaporated to dryness to obtain commercial AV extract (21 g). The extract was standardized on an amount of 2.1% hyperoside and 2.7% isoquercitrin, respectively. This procedure was repeated to obtain a total of 50 g AV extract (batch no. 79750101). Extract Analysis Analytical Equipment The extract was analyzed on a Waters Alliance 2695 High Pressure Liquid Chromatography (HPLC) system coupled with a Waters 996 photodiode-array (PDA) detector and MicroMass Quattro Micro atmospheric pressure ionization (API) mass spectrometer (HPLC-MS). The analytical conditions were as follows: Sample: AV extract, 4 mg/mL in 50% Ethanol Column: Waters Sunfire C-18, 3.5 m, 2.1 x 100 mm Mobile Phase: Gradient, solvent A: deionized water, solvent B: Acetonitrile, solvent C: 1% formic acid in deionized water (see table 2-1 for gradient time program) Flow rate: 0.2 mL/min. Oven temperature: 40 C Injection volume: 5 L UV detection: PDA start wavelength: 190 nm, stop wavelength: 400 nm

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35 Mass spectrometer: ionization mode: positive electron spray ionization (ESI), mass range: 200-2000 Identified compounds from extract A total of 16 compounds, that have been mentioned in the literature, have been identified according to the ultraviolet (UV) and the respective mass spectra (figures 2-1 and 2-2). Relative amounts of flavone glycosides were calculated to be 17.4% for quercetin glycosides and 2.7% for kaempferol glycosides of the total extract mass. Table 2-2 lists all identified compounds with their respective retention time, ions, and predominant daughter ion. Since the scanned mass range was between 200 and 2000 dalton, lower mass fragments from monomeric compounds could not be detected. The hydroethanolic extract used for all further analysis is rich in polyphenolic compounds, mainly the caffeic acid derivative chlorogenic acid, the flavonols quercetin and kaempferol and their respective glycosides, and oligomeric proanthocyanidins. This is in well agreement with literature sources [29,42]. Bioguided Fractionation Bioguided fractionation is commonly used to elucidate possible pharmacological active compounds from a complex mixture of an extract. One assumption for the usefulness of bioguided fractionation is that the extract actually exhibits the pharmacological activity in question. The other approach would be to start with a single compound that is known from an extract and evaluate its pharmacological activity. However, if the compound alone does not show any activity, this does not exclude a possible contribution of this substance to the activity seen with the whole extract since many extracts resemble the pharmacological response as a synergistic effect of active compounds and facilitating compounds, that enhance absorption or bioactivation of the active substances [67]. Thus, bioguided fractionation provides a powerful

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36 tool to identify active compounds from pharmacological active extracts with smaller chances of loosing a possible active substance when testing single compounds. Preparation of Fractions from Apocynum venetum Extract Fractionation of AV extract (batch no. 79750101) was achieved on a Sephadex LH-20 column (lot. no. 051201) with a flow rate of 2.0 mL/min. over a period of 28 hours. Overall, 160 tubes with each 18g were collected (figure 2-3 and table 2-3). The tubes were pooled into five fractions according to their mass to charge (m/z) ratio with the exception of the fifth fraction being the washing fraction. The total recovery for the fractions was 94.16% or 47.08 g of AV extract. The detailed conditions of the fractionation were as follows: Sample: AV extract, 50 g in 50% Ethanol Column: Sephadex LH 20, total volume: 1,000 mL Mobile Phase: 75% Ethanol in deionized water for fractions A-D, 70% acetone in deionized water for fraction E Flow rate: 2.0 mL/min. for fractions A-D, 0.5 mL/min. for fraction E An additional sub-extraction was conducted to derive a tannin-free and a tannin-containing extract of AV, respectively. These extracts were evaluated for their antioxidant potential in order to confirm the hypothesis of a major antioxidant effect being linked to oligoand polymeric macromolecular polyphenols like tannins. The extracts were prepared according to the scheme shown in figure 2-4. Basically, 100.0 g of AV extract was dissolved in a hydroethanolic solution and stirred for homogenization. Insoluble parts were separated and protease digested gelatine added. This procedure has been used for many years to separate high molecular tannins from smaller molecules by protein precipitation [68]. Following centrifugation, the solution was classified as the tannin-reduced extract and the precipitate as the tannin-gelatin extract. The extracts were analyzed by TLC under the same conditions as the fractions.

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37 Fraction Analysis Analysis of the five fractions derived from AV extract was conducted using the same HPLC-MS system as for the analysis of the whole extract and a thin-layer chromatography of the whole extract, the fractions, and pure compounds. Analytical conditions for the LC-MS analysis of the fractions were the same as with the whole extract. Two thin-layer chromatography systems were used for analysis of the fractions with the first being alcoholic-acidic for detection and separation of flavonoids (figure 2-5) and the second being more hydrophilic for detection of monomeric proanthocyanidins and caffeic acid derivatives (figure 2-6). With both TLCs, the whole extract, the tannin-reduced and tannin-containing extracts, as well as the pure compounds hyperoside, isoquercitrin, and chlorogenic acid were evaluated. The conditions for the acidic TLC were as follows: Stationary phase: Silica Gel 60 F254 (Merck) Mobile phase: ethyl acetate:formic acid:glacial acetic acid:water (100:11:11:26) Detection agent: phosphomolybdic acid in ethanol Analytical conditions for the second, hydroethanolic TLC are listed below: Stationary phase: RP-18 254S (Merck) Mobile phase: 40% ethanol in deionized water Detection agent: phosphomolybdic acid in ethanol The TLC analysis used chlorogenic acid, hyperoside, and isoquercitrin as reference standards since these substances are present in the highest amounts in AV extract. From the chromatogram it can be seen, that mainly fraction B contains the quercetin glycosides hyperoside and isoquercitrin as well as chlorogenic acid, while the other fractions do not contain any significant amounts of these compounds. All three compounds can be considered marker compounds for the extract. Analysis of the tannin-reduced extract from AV revealed that

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38 hyperoside, isoquercitrin, and chlorogenic acid are present in this extract, while the tannin-containing extract did not have any significant amounts of these compounds. Since both TLCs did show a band at the very bottom just above the start line for the tannin-containing extract, this might be an indication for higher molecular weight compounds. HPLC-MS analysis of the whole extract and fractions was performed under the same analytical conditions as for the whole extract alone. Mass fractionation patterns were split into low mass (m/z 150-650), medium mass (m/z 600-1050), and high mass (m/z 1000-2000) range. Both the intensity of ions as well as the area under the curve for the UV chromatogram were used to evaluate the composition of each of the fractions (figure 2-7 a-f). Based on these findings it can be concluded, that fraction A mainly consists of lower molecular weight compounds (m/z 291) as well as some oligomeric proanthocyanidins (m/z 1000-1400) with a higher hydrophilicity. Fraction B is rich in the flavonol glycosides hyperoside and isoquercitrin (m/z 465) as well as the caffeic acid derivative chlorogenic acid (m/z 355). Both fractions A and B account for about 44% of the total extract due to the high amounts of chlorogenic acid and quercetin glycosides. Fraction C mainly consists of the flavonol aglycone kaempferol (m/z 287) and the flavone gallocatechin/epigallocatechin (m/z 307) as well as oligomeric proanthocyanidins (m/z 1000-1500). The main constituent of fraction D is the flavonol aglycone quercetin (m/z 303) as well as a gallocatechin dimer (m/z 611) and possibly catechin (m/z 291), which could also be a daughter ion from the dimer. Fraction E mainly consisted of higher molecular compounds (m/z 600-2000) which could be diand oligomeric proanthocyanidins as well as higher molecular polymers that were not covered by the mass range scan (above 2000 dalton). The fragmentation pattern shows a higher ion density for heavier fragments (600-2000), which is an indicator for compounds with

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39 higher molecular masses. Table 2-2 attempts to categorize most of the identified compounds from AV extract to their respective fractions. However, especially the proanthocyanidins are difficult to be classified in a specific fraction solely based on the mass fragmentation pattern and UV spectrum since these compounds show a very similar fragmentation and daughter ions. In the case of Hyperoside-malonate, Trifolin-malonate, Astragalin-malonate, and Trifolin/Astragalin-acetate, either the UV spectra presented no clear indication of the presence of the compound or the mass fragmentation pattern was not conclusive. Preparation of Subfractions of Fraction C from Apocynum venetum Pharmacological evaluation of fractions A through E in animal behavior tests revealed a strong anxiolytic effect for fraction C of AV extract (see chapter 3). Through further fractionation, it was the goal to isolate single compounds from the extract for evaluation in vivo using the same animal model as has been used for the whole extract and the fractions. The fractionation was conducted on an octadecylsilica column (ODS column) using 4.0 g of fraction C prepared from AV extract as stated above. Following are the separation conditions: Sample: AV Fraction C, 4.0 g 50% Ethanol Column: ODS, total volume: 360 mL Mobile Phase: Gradient with 10% to 70% acetonitrile in deionized water (table 2-4) Flow rate: 5.0 mL/min. A total of 12 subfractions were obtained. Total recovery of all subfractions from fraction C was 98.975% or 3.959 g of fraction C. In order for reasonable amounts to be used in the in vivo animal experiments, these 12 subfractions were pooled together to derive 5 subfractions (sA-sE, table 2-5). Percent amount of all subfractions from fraction C accounted for 2.67% of AV extract compared to 3.96% of fraction C. This constitutes an overall recovery of 67.42%. The loss of approximately 33% might be due to the fractionation process, since a multiple step approach was

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40 necessary to derive the subfractions. During this process, residues remain on the fractionation columns that can only be completed removed by reconstituting the column even when multiple wash runs have been collected. Subfraction Analysis Similar to the analysis of the initial fractions derived from AV extract, the subfractions were analyzed using both TLC and HPLC-MS. TLC conditions were as follows: Stationary phase: RP-18 254S (Merck) Mobile phase: 20% acetonitrile for hydrophilic compounds, 50% acetonitrile for lipophilic compounds Detection agent: phosphomolybdic acid in ethanol The TLC was conducted using the flavanols catechin, gallocatechin, and epicatechin as well as the flavonol aglycone kaempferol as reference substances since these substances have been identified in fraction C (figure 2-8). The hydrophilic TLC of fractions C-1 to C-4 identified gallocatechin and epigallocatechin as the major compounds. Fractions C-6 through C-9 were rich in catechin and another flavonol not identified by reference. Both the 20% acetonitrile and 50% acetonitrile TLCs identified fraction C-12 as containing mainly kaempferol, but there were other substances not identified by reference, that may be flavanols (purple) as well as flavonols (brown). All fractions definitely contained at least two substances or more. After pooling of fractions C-1 through C-12 into five subfractions sA to sE, subfractions sA, sB, and sC were evaluated by HPLC-MS under the following analytical conditions: Sample: subfraction A, subfraction B, and subfraction C in acetonitrile Column: Develosil C-30-UG-5, 5 m, 4.6 x 250 mm Mobile Phase: Gradient, solvent A: acetonitrile, solvent B: deionized water, solvent C: 1% formic acid in deionized water (see tables 2-6 and 2-7 for gradient time program) UV detection: 280 nm Mass spectrometer: ionization mode: positive electron spray ionization (ESI), mass range: 130-1000

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41 The chromatograms and mass spectra (figure 2-9) identify some of the known compounds, but especially in subfraction sC, most of the compounds are yet unidentified. Major constituents in subfraction sA are epigallocatechin and gallocatechin, which is in well agreement with the results from the TLC. Subfraction sB is rich in epicatechin, but also contains catechin among at least three unidentified compounds. Subfraction sC mainly consists of the flavonol glycosides hyperoside and isoquercitrin and their respective malonates as well as the aglycone quercetin. The complexity of sC is higher compared to the other two fractions with about half of the total mass being yet unidentified compounds. Subfractions sD and sE were analyzed under similar analytical conditions (figure 2-10 and 2-11), but an isocratic mobile phase was used instead of a gradient method: Sample: subfraction sD and subfraction sE in acetonitrile Column: Develosil C-30-UG-5, 5 m, 4.6 x 250 mm Mobile Phase: isocratic (subfraction D: acetonitrile, deionized water, 10% formic acid in deionized water(15:75:10), subfraction E: acetonitrile, deionized water, 10% formic acid in deionized water(40:50:10) UV detection: 280 nm Mass spectrometer: ionization mode: positive electron spray ionization (ESI), mass range: 150-1000 The identification of compounds from subfraction sD was not possible due to the low amounts present. Subfraction sE mainly consisted of kaempferol and small amounts of afzelechin. The kaempferol content was determined by an external standard curve under the same analytical conditions, standard concentration range 1.125 g/mL to 18 g/mL, retention time for kaempferol 16.46 minutes, injection volume for sE 5.5 g/mL, correlation coefficient r=0.9998, calibration curve equation: y=2126.6 x 490.475. An absolute amount of 3.4252 g/mL kaempferol and a relative content of 62.3% of kaempferol in subfraction sE were calculated.

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42 In order to unequivocally confirm the structure of kaempferol in subfraction sE, an additional 1H-nuclear magnetic resonance (H-NMR) spectrum was recorded. The spectrum was taken by Mr. Kevin Spelman at the University of North Carolina, Greensboro, NC. Subfraction sE was analyzed under the following analytical conditions: Sample: subfraction sE in deuteriated methanol (CD3H 3.33 ppm) H-NMR analyzer: Jeol JNM ECA 500 Frequ ency: 500 MHz, field strength: 11.747 T Acquisition temperature: 411.8 C, acquisition duration: 1.745 s Resolution: 0.57 Hz The H-NMR spectrum presented with clear, but less intense shifts for kaempferol compared to the solvent signal (figure 2-12). Signals for the hydrogen atoms in ring A were --8). The two doublets in ring B are located at -r H---respective hydrogens on the hydroxyl groups (positions C-5, C-7, C--did present with Other shifts from the H-NMR spectrum could not be clearly attributed to afzelechin, which was found from the mass spectrum. This might be due to low sample amounts as well as a low concentration of afzelechin in subfraction sE. Evaluation of Antioxidant Activity Antioxidant Assay The assessment of antioxidant properties of therapeutic compounds and in particular natural products has gained enormous interest in the past two decades [69]. The realization that reactive oxygen species (ROS) have either a major impact on the progression of or play an important role in the development of many degenerative or chronic diseases such as dementia, cancer, inflammation, Alzheimer, atherosclerosis, or depression as well as their role in aging have triggered tremendous interest in the prevention of ROS generation [70]. Many natural

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43 products and food sources have been linked to antioxidant activity due to the high concentrations of polyphenolic compounds [71]. Since the extract prepared from the leaves of AV are high in polyphenolic compounds such as flavonoids, caffeic acid derivatives, and proanthocyanidins, the evaluation of its antioxidant activity comes to mind. Other research groups evaluated the prevenetion of lipid-peroxidation [35] and peroxynitrite radical scavenging effects [30] of the extract. The extract, fractions, and isolated compounds showed a high antioxidant activity. However, there are no reports of the total antioxidant capacity of the extract using the oxygen radical absorbance capacity (ORAC) assay. The ORAC has become a standard assay for the evaluation of antioxidant activity both for food and medicinal purposes [72]. The assay is easy and fast to perform and has the advantage of evaluating both the slow and fast acting antioxidant effects of extracts or isolated compounds. This is accomplished by a kinetic reaction that evaluates the loss of fluorescence of a fluorescent probe (fluorescein, Sigma-Aldrich) over time due to the activity of a radical (2-Azobis(2-amidinopropane) dihydrochloride (AAPH, Sigma-Aldrich)) source (figure 2-13). The parameter evaluated is the area under the fluorescence-time curve (AUC). If a compound or extract has a significant antioxidant or radical-scavenging activity, the loss of fluorescence is delayed and therefore the area under the curve higher. The concentration of the radical has to be high enough for the final fluorescence to be below 5% of the initial fluorescence in order to evaluate the area under the curve with sufficient accuracy. Results are compared to a standard curve of the water-soluble vitamin E derivative Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich).

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44 Evaluation of AV extract, the tannin-containing and tannin-reduced extracts, and pure compounds from the whole extract as well as structurally related substances was conducted under the following conditions: Equipment: Plate reader Biotek Synergy HT, analysis software KC 4, excitation wavelength: 485 nm, emission wavelength: 538 nm, temperature: 37 C, kinetic reading every 2 minutes for 70 minutes (36 readings) Samples: AV extract, tannin-containing extract, tannin-reduced extract, naringenin (Roth, Karlsruhe, Germany), luteolin (Roth), apigenin (Roth), kaempferol (Fisher Scientific), quercetin (Sigma-Aldrich), myricetin (Sigma-Aldrich), rutin (Roth), isoquercitrin (Tokiwa Phytochemical LTD), hyperoside (Tokiwa Phytochemical LTD, chlorogenic acid (Roth), rosmarinic acid (Roth), caffeic acid (Roth), ferulic acid (Roth), isoferulic acid (Roth), cynarin (Roth); all samples were run in triplicates (n=3), trolox standard curve was run with every 96-well plate Sample preparation: 5 mg of extracts were dissolved in 1 mL of dimethyl sulfoxide (DMSO, Fisher Scientific) and diluted 1:500, 1:1,000, 1:2,000 and 1:4,000 with buffer; pure compounds were prepared in concentrations of 3.125 to 25 M in buffer with 0.002% DMSO Standard: Trolox standard was prepared in concentrations of 3.125 to 200 M with 0.002% DMSO Buffer: all samples were diluted in phosphate buffer containing 0.75 M potassium hydrogen phosphate (Fisher Scientific) and 0.75 M natrium dihydrogen phosphate (Fisher Scientific) in deionized water, buffer was used as blank containing 0.002% DMSO Fluorescent probe: Fluorescein was dissolved in methanol to achieve a 12.5 M stock solution, 10 L of the stock solution were dissolved in 50 mL of buffer (final concentration: 2.5 nM) Radical source: 360 mg of AAPH were dissolved in 5 mL buffer and added directly before the start of the kinetic reading (final concentration: 266 mM) Calculation: Microsoft Excel spreadsheet template, AUC calculation by trapezoidal rule, relative Trolox equivalents calculated by: [(AUCsample AUCblank)/(AUCTrolox AUCblank)]x(mol Trolox/mol sample) [molarity fraction for pure compounds only] Statistics: Statistical analysis was performed using GraphPad Prism ver 4.0, One-Way Analysis of Variance (ANOVA) followed by Student-Newman-Keuls post-hoc multiple comparison test. Trolox standard curves were linear in a range between 3.125 and 200 mol (correlation coefficients (r) between 0.9777 and 0.9876). The whole extract as well as the tannin-containing extract showed a high relative total antioxidant activity (TAA) in a concentration of 0.01 mg/mL compared to the trolox standard (figure 2-14). The absolute antioxidant activity for the extracts

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45 was calculated in micromole trolox equivalents (mol TE)/g dry weight (relative total antioxidant activity x dilution factor). Absolute antioxidant activity for the whole extract was 1012 5.8 mol TE/g (mean S.E.M), for the tannin-containing extract 977.2 57.8 mol TE/g, and for the tannin-reduced extract 497.8 27.2 mol TE/g. The tannin-reduced extract did not show an increased relative antioxidant potential compared to trolox. The caffeic acid derivatives showed a structure-related antioxidant effect with caffeic acid, ferulic and isoferulic acid, and rosmarinic acid being stronger antioxidant while chlorogenic acid and cynarin did not possess as high an activity in the ORAC (figure 2-15). The flavanon naringenin and the flavonol quercetin had a strong antioxidant effect compared to trolox, while all other flavonols (kaempferol and myricetin) and flavons (apigenin and luteolin) did not present with a strong effect (figure 2-16). All of the glycosylated flavonoids (apigetrin: apigenin-7-O-glucoside, cynaroside: luteolin-7-O-glucoside, hyperoside: quercetin-3-O-galactoside, isoquercitrin: quercetin-7-O-glucoside, rutin: quercetin-3-O-rutinoside) showed a high antioxidant activity (figure 2-17). Conclusions A hydroethanolic leaf extract prepared from Apocynum venetum L., Apocynaceae and several fractions and subfractions were analyzed by HPLC-MS for qualitative composition. The extract, two derived tannin-reduced and tannin-containing extracts, as well as pure compounds were investigated for their antioxidant activity in vitro. Structure confirmation was mainly based on UV spectra and mass spectra and compared to an internal library. The structures of compounds in subfraction E of fraction C were further analyzed using H-NMR for unequivocal structure elucidation. The extract presents with a typical leaf extract spectrum and is high in polyphenolic compounds, mainly proanthocyanidins and flavonoids. The caffeic acid derivative chlorogenic acid was also found in high amounts, which is common for aerial parts of plants and

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46 herbs [73]. Main flavonoid compounds were the glycosylated quercetin derivatives hyperoside and isoquercitrin. Several oligomeric proanthocyanidins and kaempferol derivatives were also present. Kaempferol was structurally determined by HPLC-MS and 1H-NMR as the main component in subfraction E of fraction C, while the other compound afzelechin was identified based on its UV and mass spectra. Relative total antioxidant activity of the whole extract was evaluated using the ORAC assay and found to be comparably high (2.026 TAA) to foods usually associated with a high antioxidant activity such as grapes (2.75 TAA), cherries (1.65 TAA), and plums (2.65 TAA) [69]. In addition, the absolute antioxidant activity of the extract (1012 mol TE/g) was as high as dark chocolate (1031 mol TE/g) [74]. The antioxidant activities of pure compounds can be attributed to a structural relationship of free hydroxyl-groups and contributing factors like methylated hydroxyl-groups. Since chlorogenic acid is an ester between caffeic acid and quinic acid, it seems likely that quinic acid reduces the antioxidant effect of caffeic acid [75]. The higher the number of free aromatic hydroxyl groups present in the molecule that are not sterically hindered, the higher the antioxidant capacity of the molecule. Rosmarinic acid shows a high antioxidant activity since it is a dimer of caffeic acid. The cinnamic acid derivatives ferulic and isoferulic acid presented with high antioxidant activities since the methylated hydroxylgroup in ortho-position stabilizes the hydroxyl-radical generated. Similar structure-activity relationships were confirmed for other compounds such as quercetin and the glycosylated flavonoids and corresponded well with literature results [61,72]. The limitation of the use of in vitro assays for the evaluation of antioxidant activity in vivo is clearly the neglected metabolism and potential inactivation by the intestines and the liver [76]. Therefore, any reported antioxidant values for foods, nutritional supplements, and herbal extracts from in vitro test systems without consideration of metabolism should be interpreted with caution. Findings of both the analytical

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47 assessment as well as the antioxidant activity of AV extract are in good agreement with previously published results from other research groups [29,30,35,42].

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48 Table 2-1 Analytical conditions for AV extract and fraction analysis. Time Solvent A% Solvent B% Solvent C% Flow Curve 0 75 15 10 0.2 1 10 70 20 10 0.2 6 20 50 40 10 0.2 6 25 0 90 10 0.2 6 40 75 15 10 0.2 11 Table 2-2 Identified compounds from AV extract. For UV and MS spectra, see figures 2-1 and 2-2. Compound number Co mpound name Molecular weight (mol/g) Retention time (min.) Mass ion (m/z) Daughter ion Fraction 1 Chlorogenic acid 354 4.13 355 163 (n.d.) B 2 Hyperoside 464 10.7 0 465 303 B 3 Isoquercitrin 464 11.27 465 303 B 4 Trifolin 448 13.63 449 287 B 5 Astragal in 448 14.92 449 287 B 6 Hyperoside malonate 550 15.35 551 303 (B) 7 Hyperoside acetate 506 16.28 507 303 B 8 Isoquercitrin acetate 506 17.57 507 303 B 9 Trifolin malonate 534 18.05 535 287 (D) 10 Isoquercitrin malonate 550 18.48 551 303 B 11 Astrag alin malonate 534 19.09 535 287 (D) 12 Trifolin /Astragalin acetate 490 19.53 491 287 (B), C 13 Quercetin 302 22.05 303 153 (n.d.) D 14 Kaempferol 287 25.22 287 153 (n.d.) C 15 Catechin/Epicatechin 290 4.53 291 139 (n.d.) A 16 Gallocatechin/Epigalloca techin 307 3.49 307 174 (n.d.) A, C 17 Gallocatechin dimer 611 9.51 611 347 B, D, E n.d.: not detected, but daughter ion mass taken from literature [77], Letters in brackets refer to the compound not being conclusively identified for the respective fraction

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49 Table 2-3 Distribution and amount for fractions derived from AV extract. Fraction Tube no. Weight (g) % of total AV extrac t mg eq. to 30 mg/kg AV extract mg eq. to 125 mg/kg AV extract A 1 42 8.09 g 16.18 % 4.9 mg 20.2 mg B 43 62 13.91 g 27.82 % 8.3 mg 34.8 mg C 63 99 1.98 g 3.96 % 1.2 mg 5.0 mg D 100 160 1.88 g 3.76 % 1.1 mg 4.7 mg E wash 21.22 g 42.44 % 12.7 mg 53.1 mg Total 47.08 g 94.16 % 28.2 mg 117.8 mg Table 2-4 Subfractions of Fraction C from AV. Subfraction Eluent Amount (mg) Amount (%) C 1 10% ACN 476 mg 1 1.9 00 % C 2 10% ACN 294 mg 7. 35 0 % C 3 10% ACN 307 mg 7. 675 % C 4 10% ACN 640 mg 16 .000 % C 5 20% ACN 157 mg 3.9 25 % C 6 20% ACN 109 mg 2.7 25 % C 7 20% ACN 216 mg 5.4 00 % C 8 20% ACN 380 mg 9.5 00 % C 9 20% ACN 238 mg 5 95 0 % C 10 20% 40% ACN 990 mg 24.75 0 % C 11 70% ACN 62 mg 1. 55 0 % C 12 70% ACN 90 mg 2. 25 0 % Total 3,959 mg 98.9 75 % Table 2-5 Pooling scheme for subfractions of fraction C from AV. Pooled sub fraction Composition Amount (mg) % of Fraction C % o f AV extract mg eq. to 30 mg/kg AV extract mg eq. t o 125 mg/kg AV extract sA C 1, C 2, C 3, C 4 1 717 42.925 1.16 0.35 1.45 sB C 5, C 7, C 8 753 18.825 0.51 0.15 0.64 sC C 9, C 10, C 11 1 290 32.25 0 0 .87 0.26 1.09 sD C 6 109 2.725 0. 07 0.02 0.09 sE C 12 90 2.25 0 0.06 0.02 0.08 Total 3,959 98.969 2.67 0.8 0 3.35

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50 Table 2-6 Analytical conditions for subfraction A and B from AV fraction C. Time Solvent A% Solvent B% Solvent C% 0 5 85 10 20 15 75 10 25 15 75 10 30 70 20 10 Table 2-7 Analytical conditions for subfraction C from AV fraction C. Time Solvent A% Solvent B% Solvent C% 0 15 75 10 20 25 65 10 35 35 55 10 40 50 40 10 Figure 2-1 UV spectrum of AV extract. Wavelength: 254 nm, injection volume: 5 L, dilution: 4 mg extract/mL. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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51 Figure 2-2a Mass spectra of compounds from AV. 1 chlorogenic acid 4 Trifolin 5 Astragalin 6 Hyperoside malonate 10 Isoquercitrin malonate 7 Hyperoside acetate 8 Isoquercitrin acetate 2 Hyperoside 3 Isoquercitrin 9 Trifolin malonate 11 Astragalin malonate 12 Trifolin /Astragalin acetate

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52 Figure 2-2b Mass spectra of compounds from AV continued. 1 3 Quercetin 1 4 Kaempferol 1 5 Catechin/Epicatechin 1 6 Gallocatechin/ Epigallocatechin 1 7 Gallocatechin dimer

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53 Figure 2-3 Mass analysis and distribution of mass to charge ratio for fractions prepared from AV extract. Figure 2-4 Extraction scheme for tannin-reduced and tannin-gelatin extract from AV extract. Apocynum venetum extract 100.0 g Aqueous solution 5120 mL Tannin reduced extract 63.64 g Tannin gelatin extract 68.73 g Insoluble parts 0.86 g Add 400 mL of 50% Ethanol and dissolve Add 4,600 mL of hot water, stir 1 h, centrifuge Add 10% protease digested gelatin solution (389 g), stir 30 min, centrifuge Reduced pressure dry at 60 C Concentrate and freeze dry Reduced pressure dry at 60 C

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54 Figure 2-5 Acidic TLC of AV extract, tannin-extracts, and fractions. Figure 2-6 Hydroethanolic TLC of AV extract, tannin extracts, and fractions. 1 2 3 4 5 6 7 8 9 10 11 12 1 Chlorogeni c acid 2 Hyperoside 3 Isoquercitrin 4 Apocynum venetum extract 5 Fraction A 6 Fraction B 7 Fraction C 8 Fraction D 9 Fraction E 10 Apocynum venetum extract 11 Tannin reduced extract 12 Tannin gelatin extract 1 2 3 4 5 6 7 8 9 10 11 12 1 Chlorogenic acid 2 Hyperoside 3 Isoquercitrin 4 Apocynum venetum extract 5 Fract ion A 6 Fraction B 7 Fraction C 8 Fraction D 9 Fraction E 10 Apocynum venetum extract 11 Tannin reduced extract 12 Tannin gelatin extract

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55 Figure 2-7a LC-UV spectrum (254 nm) and MS for AV extract. (1: UV spectrum at 254 nm, 2: mass spectrum range 150-650, 3: mass spectrum range 600-1050, 4: mass spectrum range 1000-2000) Figure 2-7b LC-UV spectrum (254 nm) and MS for AV fraction A. (1: UV spectrum at 254 nm, 2: mass spectrum range 150-650, 3: mass spectrum range 600-1050, 4: mass spectrum range 1000-2000) 1 2 3 4 1 2 3 4

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56 Figure 2-7c LC-UV spectrum (254 nm) and MS for AV fraction B. (1: UV spectrum at 254 nm, 2: mass spectrum range 150-650, 3: mass spectrum range 600-1050, 4: mass spectrum range 1000-2000) Figure 2-7d LC-UV spectrum (254 nm) and MS for AV fraction C. (1: UV spectrum at 254 nm, 2: mass spectrum range 150-650, 3: mass spectrum range 600-1050, 4: mass spectrum range 1000-2000) 1 2 3 4 1 2 3 4

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57 Figure 2-7e LC-UV spectrum (254 nm) and MS for AV fraction D. (1: UV spectrum at 254 nm, 2: mass spectrum range 150-650, 3: mass spectrum range 600-1050, 4: mass spectrum range 1000-2000) Figure 2-7f LC-UV spectrum (254 nm) and MS for AV fraction E. (1: UV spectrum at 254 nm, 2: mass spectrum range 150-650, 3: mass spectrum range 600-1050, 4: mass spectrum range 1000-2000) 1 2 3 4 1 2 3 4

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58 Figure 2-8 TLCs of subfractions from fraction C of AV. 20%MeCN 50%MeCN 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 4 1 4 1 5 1 6 1 7 1 Catechin 2 Gallocatechin 3 Epigallocatechin 4 Fraction C 5 C 1 6 C 2 7 C 3 8 C 4 9 C 5 10 C 6 11 C 7 12 C 8 13 C 9 14 C 10 15 C 11 16 C 12 17 Kaempferol

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59 Figure 2-9 Chromatograms and UV spectra of subfractions sA, sB, and sC. Detection wavelength: 280 nm, A: subfraction sA, (+)-gallocatechin (retention time 11.48 minutes, (-)-epigallocatechin (retention time 17.78 minutes), B: subfraction sB, (+)-catechin (retention time 13.23 minutes), (-)-epicatechin (retention time 21.92 minutes), C: subfraction sC, 4 hyperoside, 5 isoquercitrin, 6 hyperosideor isoquercitrin-malonate, 10 quercetin 1 2 3 4 5 6 7 8 9 10 A B B 1 2 3 4 5 6 7 8 9 10 C A B

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60 Figure 2-10 HPLC chromatogram, UV spectra, and mass spectra of fraction sD from AV fraction C. A and B represent respective UV and mass spectra of peaks A (retention time 7.12 minutes) and B (retention time 7.44 minutes), UV wavelength for chromatogram: 280 nm. A A A B B B

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61 Figure 2-11 HPLC chromatogram, UV spectra, and mass spectra of fraction sE from AV fraction C. A and B represent respective UV and mass spectra of peaks A (retention time 8.18 minutes, afzelechin) and B (retention time 9.70 minutes, kaempferol), UV wavelength for chromatogram: 280 nm. B B A A B A

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62 Figure 2-12 1H-NMR spectrum of subfraction sE. Detailed section for kaempferol shifts between 6 ppm and 8.5 ppm shown in insert.

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63 Figure 2-13 Scheme of the ORAC reaction. The fluorescent probe fluorescein is oxidized and decomposes to a non-fluorescent product when exposed to ROS [72].

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64 Figure 2-14 Antioxidant activity of AV extract and tannin-containing and tannin-reduced extracts. *** p<0.001 against trolox, n=3 for extracts and trolox Figure 2-15 Antioxidant activity of caffeic acid derivatives. *** p<0.001 against trolox, ** p<0.01 against trolox, p<0.05 against trolox, n=3 for compounds and n=9 for trolox

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65 Figure 2-16 Antioxidant activity of flavonoid aglycones. *** p<0.001 against trolox, n=3 for compounds and n=12 for trolox Figure 2-17 Antioxidant activity of flavonoid glycosides. *** p<0.001 against trolox, n=3 for compounds and n=9 for trolox

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66 CHAPTER 3 PHARMACOLOGICAL EVALUATION OF Apocynum venetum AND ITS FRACTIONS Animal Models Elevated Plus Maze The elevated plus maze (EPM) is considered to be an etiologically valid animal model of anxiety because it uses natural stimuli (fear of a novel open space and fear of balancing on a relatively narrow, raised platform) that can induce anxiety in humans [78]. The model was introduced about 20 years ago and has been used extensively for the evaluation of natural products as well as synthetic compounds for their potential use as anxiolytics [79]. An anxiolytic agent increases the frequency of entries into the open arms and increases the time spent in open arms of the EPM (figure 3-1). Known anxiolytic agents such as the benzodiazepine diazepam and the azapirone buspirone hydrochloride, which are used clinically for the treatment of anxiety disorders, show reliable anxiolytic effects in the EPM [80-83]. In addition, antidepressants have become a major treatment option for generalized anxiety disorders [4,84] and other anxiety disorders, especially if a comorbid depression is present. A major disadvantage of the EPM is its sensitivity to external noise and interruptions [85,86], which have to be closely monitored. The conditions under which the EPM is conducted have to be kept as consistent as possible, including parameters such as light, time of the day, and environment. Therefore, the EPM was conducted in the same room between the hours of 7 AM and 1 PM. The animals were allowed to adapt to the environment at least for one hour prior to the experiment, in most cases cages were brought into the experimental room the evening before the experiment. Light conditions were kept consistent and observers were handling the animals a week before beginning of the experiments. The maze consisted of two open (31 cm 5 cm 1 cm) and two closed (31 cm 5 cm 15 cm) arms, extending from a central platform (5 cm 5

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67 cm) and elevated to a height of 40 cm above the floor. On the wooden base frame, the closed arms were made of opaque plastic, while the open arms were made of the same material with a slight elevated edge. Mice were individually placed on the center of the maze facing a closed arm, and the number of entries and the time spent in closed and open arms were recorded during a 6-min observation period. Two mice were recorded simultaneously. Arm entries were defined as entry of all four paws into an arm [79]. The percentage time spent on open arms (100 open/total time) was calculated for each animal. On the day of behavioral evaluation applicable to all animal models used, observers did not use deodorants or other fragrances that would distract or influence the behavior of the animals. Animals were assigned randomly to the control or treatment groups and used repeatedly with a wash out period of at least 4 days before the next evaluation. All animals used were between the ages of 6 to 18 weeks weighing between 18 and 32 g. Animals with a higher weight were excluded since fat distribution might change the distribution of compounds with a high volume of distribution (e.g. diazepam, lipophilic, uncharged molecules) and therefore influence the pharmacological response. Animals with 0% time spent on open arms or presenting with clear symptoms of abnormal behavior (e.g. no movement at all in home cage, incorrect application to animal) were excluded from the experiment prior to statistical evaluation and replaced by a new, randomly chosen animal [87]. All animals were housed and all experiments performed according to the policies and guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Florida, Gainesville, USA [88] It has been shown that there is considerable difference between the anxiolytic response of different mice strains in the EPM [89]. Therefore, it was important to select a strain that provides sufficient sensitivity to the EPM. Male C57BL/6 (black six) mice were selected since this strain

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68 showed an intermediate to good anxiolytic response in the EPM as well as a good response in the Light-Dark Transition test [90] as both models have a high predictive (ability to predict drugs effective in animal models to be effective in humans) as well as constructive (similar pathological mechanism in animals and humans) validity [91]. Light-Dark Transition Test The light-dark transition test was introduced in 1985 and frequently has been used to identify novel anxiolytic compounds [92]. The model uses the fear of a novel, brightly-lighted area and the avoidance behavior to enter the lighted area by hiding in a dark compartment (figure 3-2). Anxiolytic agents increase the percent time spent in the lighted area without affecting the number of transitions between the two compartments, which would be an indicator for a stimulant effect [93]. Grey, opaque plastic boxes (33 cm 45 cm 30 cm) were used of which one third was covered by a lid extending down to the bottom of the box with a pass through at the bottom which allowed transition between the lighted and dark compartments. The box was equally lighted with six 40 watt light bulbs. Mice were individually placed in the center of the lighted area facing the entrance to the dark compartment. Three animals were evaluated simultaneously. Animals were handled in the same manner as for the EPM. It has been proposed that the EPM and the LDT represent a different set of behaviors, therefore possibly reflecting two different types of anxiety. While the EPM is based on a factor of exploration and elevation, the LDT uses mainly neophobia and locomotion [94]. This might be an important factor to consider when evaluating the anxiolytic response of novel substances and comparing the results of both models. Stress-Induced Hyperthermia A method of inducing a stressful condition in rodents is exposing them to an open field box (33 cm 45 cm 30 cm) similar to that used for the Light-Dark transition test but without a

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69 covered compartment which leads to a rise in core body temperature [95]. This elevated body temperature has been shown across species upon emotional stress [96,97] and is accompanied by a rise in heart rate and an overactive autonomic system as is common for many anxiety disorders [98]. The open field was equally lighted with six 40 watt light bulbs. Basal temperature was measured before the oral treatment by means of a temperature probe (RET-3, Physitemp, Clifton, NJ) connected to a digital thermometer (Thermalert TH-5, Physitemp, reading accuracy of 0.1 C). The probe was dipped into vegetable oil and inserted into the rectum until a stable temperature was achieved for 20 sec. Animals were treated and returned to their home cages. Diazepam in a dose of 5 mg/kg was used as a known stress-reducing and anxiolytic agent. Exposure to the open field was 60 min. after treatment for a period of 10 minutes followed by a temperature measurement. The parameter evaluated is the difference in basal temperature and temperature after open field stress. If the difference is significantly lower compared to a control group, it can be concluded that the substance or extract does reduce stress and lowers anxiety if it does not affect body temperature without the stress factor since some compounds have a hypothermic effect [99]. There are indicators that SIH presents with a different set of behaviors compared to both the EPM and the LDT [100]. Since SIH evaluated exposure to a stressful environment, both the LDT and EPM allow for the animal to avoid the stressful condition. When comparing SIH, EPM, and LDT, these considerations should be kept in mind when interpreting the results. Open Field The Open Field evaluates mainly locomotor activity in rodents, but has also been used in a modified version for anxiolytic activities [101]. The Open Field consisted of either a round grey plastic arena measuring 70 cm in diameter that was divided into several concentric units by black painted lines, dividing the arena into 19 fields and was surrounded by 34 cm high walls or a grey,

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70 opaque plastic box (33 cm 45 cm 30 cm) that was later divided into six equally sized compartments by the evaluation software. The arena or box was equally lighted by six 40 watt light bulbs. Two animals were evaluated at the same time in the round arena and three animals were evaluated simultaneously in the grey boxes under the same experimental conditions as used for the Light-Dark transition test. The animal was set in the center of the arena or box and videotaped for a 5 minute period. Parameters evaluated were the number of field transitions and the total distance run during the evaluation. Substances that are known stimulants (e.g. caffeine, cocaine, methamphetamine) cause the animal to run a longer distance and increase the number of field transitions [102]. Sedatives and muscle relaxants (e.g. diazepam in higher doses) on the other hand reduce these parameters [103,104]. Evaluation of Anxiolytic-Like Activity of Apocynum venetum Elevated Plus Maze Acute anxiolytic effect The first evaluation of anxiolytic activity of the whole extract prepared according to chapter 2 evaluated a dose range of 15 to 250 mg/kg. In order to evaluate the involvement of the GABA and serotonin system in the anxiolytic action of the extract, the active doses of AV were antagonized with the GABAA receptor antagonist flumazenil and the 5-HT1A antagonist WAY-100635. The known anxiolytics diazepam (benzodiazepine acting via GABA-ergic mechanism) and buspirone (azapirone acting via the serotonergic 5-HT1A receptor) were also antagonized using the respective antagonist. The conditions for the EPM were as follows: Control p.o.: 0.5% propylene glycol (Sigma-Aldrich, St. Louis, MO) in deionized water Diazepam: Ampoules (10 mg/2 mL, Hoffmann-La Roche, Basel, Switzerland) were diluted to 1.5 mg/kg (300 L/10 mL) with deionized water containing 0.5% propylene glycol Buspirone hydrochloride: 10 mg/kg (Sigma-Aldrich), 10 mg/10 mL deionized water with 0.5% propylene glycol

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71 AV extract: 15, 30, 60, 125, and 250 mg/kg (respective dose in deionized water with 0.5% propylene glycol) Flumazenil: 3 mg/kg (Hoffmann-La Roche, Nutley, NJ) were dissolved in 0.9% saline solution with 0.5% propylene glycol WAY-100635: 0.5 mg/kg (Sigma-Aldrich) were dissolved in 0.9% saline solution with 0.5% propylene glycol Control i.p.: 0.5% propylene glycol in 0.9% saline solution Control p.o., diazepam, buspirone, and AV extract were given orally 60 min. before evaluation Control i.p., flumazenil, and WAY-100635 were given intraperitoneally 75 min. before evaluation Recording of EPM for 2 mice consecutively with the high-resolution video camera WV-CP244 (Panasonic, Secaucus, NJ, U.S.A) Computerized analysis of videos by TopScan, Top View Animal Behaviour Analyzing System (version 1.00, Clever Sys Inc. Preston, VA, U.S.A) conducted by an unbiased and treatment-blinded person Statistical analysis: Calculation of the percentage time and number of entries on the open arms with 95% confidence limits and comparisons of the results were performed using GraphPad Prism (version 4.00, GraphPad Software Inc., San Diego, CA) with one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison test The extract showed a clear anxiolytic effect at doses of 30 and 125 mg/kg by increasing both the percentage time spent on the open arms and the number of entries in the open arms, but was not active at doses of 15, 60, and 250 mg/kg (figure 3-3). The observation of an inverted U-shaped activity has been observed before and is a common, yet not completely explained phenomenon [105]. Interestingly, the same dose-response profile was observed for the antidepressant effect of the same extract of AV in the Forced Swimming Test (FST) in rats [41]. The two active doses were further investigated by antagonism studies using flumazenil and WAY-100635. AV in a dose of 125 mg/kg was completely antagonized by flumazenil (3 mg/kg, i.p.) as was diazepam (figure 3-4). The anxiolytic activity of the extract in a dose of 30 mg/kg was reduced with concomitant treatment of flumazenil, but not significantly lower compared to just the extract alone. This might be an indicator of a partial influence of the GABA system in the anxiolytic action at a dose of 30 mg/kg. Flumazenil itself did not present with an anxiolytic

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72 nor anxiogenic effect in the EPM compared to control. The anxiolytic activity of the extract in a dose of 125 mg/kg was not antagonized or lowered by WAY-100635, but at a dose of 30 mg/kg there was a reduced percentage time spent on open arms as well as number of entries in the open arms, although not significantly lower compared to the extract alone in this dose (figure 3-5). Like with flumazenil this might be an indicator of a partial involvement of the serotonergic system in the anxiolytic action for the extract, while the higher dose of 125 mg/kg seems to be predominantly acting via the GABA-ergic system. Similar observations of an inactive dose between two distinct active doses have been observed before and one explanation might be the interaction between different receptor systems [106,107]. Indeed, if both antagonists were given concomitantly, the anxiolytic effects of the extract were effectively antagonized in both concentrations of 30 and 125 mg/kg (figure 3-17). Another explanation for this phenomenon might be an absorption and/or metabolism issue of active compounds in the complex extract matrix. One component, acting on both the GABA-ergic and serotonergic system, is soluble in lower concentration, but competes for absorption from the gut in higher concentrations, while the other compound is absorbed but needs a higher concentration to exerts its anxiolytic action through the GABA-ergic system. Metabolism might also play an important role as will be explained later. A more detailed dose-response profile for AV extract was conducted in order to evaluate the range of active doses and inactive doses between the two distinct anxiolytic concentrations of 30 and 125 mg/kg. Doses of 15, 22.5, 30, 37.5, 45, and 60 mg/kg as well as 75, 100, 125, 150, and 200 mg/kg were evaluated and compared to diazepam (1.5 mg/kg) and control (figure 3-6). treatments to control since the number of treatments and animals used per treatment did not

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73 allow for a multiple comparison as used by the Student-Newman-Keuls test. Comparisons were made between control, diazepam, and the extract in a dose from 15 to 60 mg/kg and in a second experiment between another control and diazepam group as well as AV extract in a dose range from 60 to 200 mg/kg. The detailed dose-response profile revealed two active concentration ranges between 22.5 to 30 mg/kg and 100 to 125 mg/kg. The least active dose was 45 mg/kg. The sudden drop in activity after 30 and 125 mg/kg might be attributed to solubility or absorption issues. Whether this is related to absorption from the intestines or transfer into the brain due to transporters is not clear. Anxiolytic activity after chronic treatment AV extract has been evaluated after chronic treatment in two separate experiments. Mice were assigned to a treatment group in a circulating fashion (first cage: control, diazepam, AV 30, AV 60, and AV 125; second cage: diazepam, AV 30, AV 60, AV 125, and control, etc.). The first experiment covered a range of 16 days (chronic treatment I), with evaluations in the EPM after 8 and 16 days, respectively. In this study, animals were administered control (0.5% propylene glycol), diazepam (1.5 mg/kg with 0.5% propylene glycol), and AV extract (30, 60, and 125 mg/kg containing 0.5% propylene glycol) on a daily basis between 8 AM and 1 PM. On the 8th day, all animals were treated with control one hour before exposure to the EPM. The purpose of this design was to evaluate a possible anxiolytic effect assuming that concentrations of active compounds or metabolites have reached a steady-state. On day 16, animals were treated with the assigned treatment (figure 3-9). The second experiment was conducted over a period of 22 days (chronic treatment II) and animals were evaluated in the EPM after 14 and 22 days of treatment with control, diazepam, and AV extract in the same manner as in the first chronic study. However, on days 14 and 22, animals were treated with the assigned treatment 60 min. before evaluation in the maze (figure 3-

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74 10). Statistical analysis was conducted using a One-Way ANOVA design followed by Dunnetts post-hoc comparison. The results did not show an as strong or as significant an anxiolytic effect as has been observed after acute treatment for diazepam as well as for the three doses of AV in both chronic treatments. Diazepam as well as the extract in 30 mg/kg did show an increase in percentage time spent in the open arms, as well as in the number of entries on the open arms after the first evaluation in the EPM (day 8 for chronic treatment I and day 14 for chronic treatment II). However, these effects diminished over time and did not present during the second evaluation. Similar observations for diazepam and other herbal extracts have been made by other research groups [108-110]. It has been proposed that adaptive mechanisms in brain receptor density or responsiveness are responsible for the loss of anxiolytic and sedative activity in animal behavior models while antidepressant effects can be observed consistently after chronic administration. Light-Dark Transition Test The extract was tested in the two active doses of 30 and 125 mg/kg and compared to control and diazepam (1.5 mg/kg). All treatments were given 60 minutes before evaluation in the LDT. The protocol was otherwise similar to the EPM. In addition, fraction C from AV was evaluated according to the equivalent dose of the whole extract (see figure 2-3 for dose). Results were compared using One-Way ANOVA followed by Student-Newman-Keuls multiple comparison test. Interestingly, only the extract in a dose of 30 mg/kg showed a significant anxiolytic effect stronger than diazepam, whereas the higher 125 mg/kg dose was not significantly different from control (figure 3-7). Fraction C showed an activity in both equivalent doses of 30 and 125 mg/kg of whole extract. This dose dependent effect might be due to different paradigms evaluated in the LDT compared to the EPM as mentioned earlier in this chapter. The number of transitions

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75 between the light and the dark compartments of the LDT were not significantly different between all groups, which excludes a possible stimulant effect and specifies the increased time spent in the lighted area as an anxiolytic effect. Stress-Induced Hyperthermia A third paradigm was used to evaluate a putative stress-lowering and anxiolytic effect of AV. Animals were treated orally with control (0.5% propylene glycol), diazepam (5 mg/kg in 0.5 % propylene glycol), and AV extract (30, 60, and 125 mg/kg in 0.5% propylene glycol) after basal temperature was measured. Sixty minutes afterwards, animals were exposed to the open field box for 10 minutes and rectal temperature was measured again. The temperature difference was compared between groups using a One-Way ANOVA followed by Student-Newman-Keuls multiple comparison test. Diazepam had a strong hypothermic effect on the animals, reducing the body temperature even slightly below the initial basal temperature (figure 3-7). AV in 30 mg/kg did also reduce the temperature increase compared to control, while both 60 and 125 mg/kg did not have a significant effect. As discussed above, the stress paradigm evaluates a different behavior in the SIH model compared to the EPM and LDT. Therefore, the indication of different mechanisms of action for the lower (30 mg/kg) and higher (125 mg/kg) anxiolytic dose of AV are actually reflected in a differential outcome in these animal models of behavior. Evaluation of Anxiolytic-like Activity of Fractions prepared from Apocynum venetum Elevated Plus Maze Anxiolytic profile of fractions and antagonism studies After evaluation of the whole extract prepared from AV, five fractions were derived as mentioned in chapter 2. Since the whole extract presented with a distinct anxiolytic profile of two separate active doses, all five fractions were evaluated according to their weight contribution

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76 to the whole extract in their equivalent doses of 30 and 125 mg/kg, respectively (see doses used in table 2-3). The fractions were tested in the EPM according to the same protocol used for the whole extract. Antagonism studies were conducted in the same manner using flumazenil for the GABA-ergic system and WAY-100635 for antagonism of the 5-HT1A receptor. An additional group was given the reconstituted extract, which consisted of all five fractions added together to resemble the whole extract composition. In addition to the fractions and reconstituted extract, the whole extract was tested again to compare the activity of the fractions to the whole AV extract. Fractions C, D, and E were active in a dose equivalent to 30 mg/kg of whole extract as did the whole extract (figure 3-11). However, the reconstituted extract did not show a significant anxiolytic effect in 30 mg/kg. The whole extract showed an anxiolytic activity in dose of 125 mg/kg, as did fractions A, C, and E in an equivalent dose (figure 3-12). The reconstituted extract did again not present with a significant anxiolytic activity in this dose. Diazepam did show a significant anxiolytic effect in both experiments. The lack of activity for the reconstituted extracts might be due to a pooling of substances from the fractions that were pulled apart from the whole extract and now interact in a different manner not resembling the original interaction. Such behavior is not uncommon with pooled fractions and in bio-guided fractionations [111,112]. GABA-antagonism with flumazenil for the active fractions revealed an interesting profile. While fractions C and E were not antagonized by flumazenil in an equivalent dose of 30 mg/kg (figure 3-13) but fraction D was, all active fractions, namely A, C, and E, were antagonized in the equivalent dose of 125 mg/kg of whole extract (figure 3-14).

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77 Antagonism by WAY-100635 on the serotonin 1A receptor presented with opposite profiles. All fractions C, D, and E were antagonized by WAY-100635 in a dose equivalent to 30 mg/kg of whole extract (figure 3-15), while the effects of fractions A and C were significantly different from control with concomitant administration of the antagonist (figure 3-16). Buspirone was used as a known anxiolytic acting via the serotonergic system and did present with significant anxiolytic effects in both experiments. Since the anxiolytic activity of the whole extract was mainly linked to the GABA-ergic system in the higher dose of 125 mg/kg, the activity of the extract in 30 mg/kg was reduced by both flumazenil and WAY-100635, but not significantly from the extract alone. The fractionation and antagonism of the fractions revealed the anxiolytic mechanism of action for AV. While the fractions in the lower active dose of 30 mg/kg were mainly inhibited by WAY-100635 and therefore indicate the main mechanism of action to be mediated via the serotonergic 1A receptor, the fractions according to higher dose of 125 mg/kg were inhibited by the GABA antagonist flumazenil and therefore make the anxiolytic action via this system likely. Fraction E however did not entirely follow this proposed activity scheme since it was inhibited by both flumazenil and WAY-100635 with the exception of flumazenil antagonism in the dose equivalent to 30 mg/kg of whole extract. This might be due to oligomeric substances like proanthocyanidins that are metabolized in the intestines or liver to monomeric compounds that then show activity. The oligomeric molecules would then serve as precursors or prodrugs for active substances. Concomitant administration of both antagonists flumazenil and WAY-100635 with the most active fraction C in both equivalent doses of 30 and 125 mg/kg of whole extract blocked the anxiolytic activity effectively in both concentrations (figure 3-17). This clearly shows that the

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78 observed anxiolytic effects are mediated through either GABA or serotonin or both receptor systems. In addition, both diazepam and buspirone were antagonized by joined administration of the antagonists (figure 3-17). Antagonism of diazepam and buspirone The contribution and the neural network between the GABA-ergic and serotonergic systems related to the anxiolytic actions of drugs are complex and yet not fully understood (see chapter 1). There seems to be some evidence that GABA neurons serve mainly as presynaptic neurons and serotonergic neurons as postsynaptic neurons, although this is only true for certain brain regions such as the amygdala and the hypothalamus. In general, GABA serves as the main inhibitory neurotransmitter in the brain and depending on the location of the GABA receptor, it can have different effects on the release and action of other neurotransmitters. Since diazepam acts specifically on the benzodiazepine binding site of GABAA receptors and buspirone is an agonist on 5-HT1A receptors, the interaction of both systems plays a major contribution to the knowledge of anxiolytic activity. Therefore, it was of interest to evaluate potential inhibitions of diazepam using WAY-100635 as an antagonist and flumazenil for buspirone. Since experiments with the whole extract showed that flumazenil antagonizes the anxiolytic action of diazepam and WAY-100635 inhibits the anxiolytic effect of buspirone, a cross-over antagonism study was conducted using the same concentrations for both the agonists (diazepam in 1.5 mg/kg and buspirone in 10 mg/kg) and antagonists (flumazenil in 3 mg/kg and WAY-100635 in 0.5 mg/kg) and evaluate the anxiolytic action in the EPM under the same conditions as described before. Antagonism of buspirone using flumazenil significantly reduced the percent time spent on the open arms, whereas diazepam was still active (figure 3-18). The number of entries in the open arms did not present with a significant difference between control, diazepam and buspirone plus flumazenil. On the other hand, coadministration of WAY-100635

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79 with diazepam did not lower the anxiolytic activity of the benzodiazepine as well as buspirone alone. Buspirone and diazepam plus WAY-100635 showed a significant anxiolytic activity compared to control (figure 3-19), whereas only the number of entries for diazepam plus WAY-100635 was significantly higher compared to control. The observation of an antagonistic action of flumazenil on the anxiolytic effect of buspirone has been shown before and has been attributed to the position of the serotonergic interneuron that is activated by presynaptic GABA-ergic neurons [106]. Interestingly, there has been no literature source that investigated the potential inhibition of the anxiolytic action of diazepam by WAY-100635 so far. Indeed, the current hypothesis that GABA receptors are mainly located presynaptical and therefore are not influenced by inhibition of the serotonergic system has been confirmed with these results. The anxiolytic activity of diazepam was not affected by coadministration of WAY-100635, since diazepam alone did show the same percent spent on the open arms. Light-Dark Transition Test The LDT was used to evaluate the most active fraction C in both doses equivalent to 30 and 125 mg/kg whole extract since the LDT uses a different paradigm than the EPM. The LDT was conducted with both active concentrations of fraction C as well as the whole extract in 30 and 125 mg/kg (figure 3-7). Results for the whole extract have been discussed earlier in this chapter. Fraction C showed a significant anxiolytic activity by an increase in time spent in the lighted area of the LDT compared to control. The number of transitions between the lighted and dark compartments was not affected by the treatment, which makes the observed increase spent in the lighted area specific to an anxiolytic effect rather than a stimulant or sedative activity. It seems therefore be likely that compounds from fraction C, which mainly contained the flavonol kaempferol and proanthocyanidins, contribute to the anxiolytic activity of AV.

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80 Evaluation of Anxiolytic-like Activity of Subfractions prepared from Fraction C Elevated Plus Maze Subfractions of fraction C, namely sA, sB, sC, sD, and sE, were evaluated in the EPM. Qualitative analysis of subfractions revealed kaempferol as the main ingredient and small amounts of afzelechin in sE while other subfractions mainly consisted of proanthocyanidins like catechin, epicatechin, and gallocatechin. Subfractions were given according to their amount equivalent to the whole extract in 30 or 125 mg/kg (see table 2-5). Subfractions sA and sC in a dose equivalent to 30 mg/kg of whole extract showed anxiolytic activity in the EPM by increasing the percentage time spent on open arms as did diazepam compared to control (figure 3-20). The number of entries on the open arms was only significant higher for diazepam and sC. The subfractions in an equivalent dose of 125 mg/kg of whole extract did present with a different anxiolytic profile with only subfraction sA being significantly different from control in both parameters of percent time spent on and number of entries in the open arms (figure 3-21). Subfraction sA mainly consisted of (+)-gallocatechin and (-)epigallocatechin (see chapter 2). It has been shown by other research groups that epigallocatechin gallate modulates GABA binding by enhancing the binding of diazepam to the benzodiazepine site of specific GABAA receptors [113]. In addition, epigallocatechin gallate shows a bimodal action at GABAA receptors, enhancing GABA binding at low doses and inhibiting it at high doses as has been shown with apigenin. In addition, epigallocatechin gallate did not alter GABA binding to cultured hippocampal neurons, but did reverse the action of the negative GABA modulator -carboline in a dose-dependent manner. Recently, it has been shown that epigallocatechin gallate after intraperitoneal injection presented with anxiolytic effects in the EPM [114].

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81 Evaluation of Locomotor Activity of Apocynum venetum Open Field Test The Open Field used for the evaluation of AV consisted of the round plastic arenas as stated earlier in this chapter. 6-8 animals were treated either with control, diazepam (1.5 mg/kg), buspirone (10 mg/kg), or AV extract (30 and 125 mg/kg). Flumazenil (3 mg/kg) and WAY-100635 (0.5 mg/kg) were given intraperitoneal. All oral solutions were also given 0.9% sodium chloride and all i.p. treatments were also administered control orally. All solutions contained 0.5% propylene glycol as a solubilizer. There were no significant differences between the treatment groups in number of field transitions and total distance run (figure 3-22). The slightly lower number of field transitions and distance run of buspirone has been reported before in the literature and has been attributed to the receptor blocking effects of buspirone on dopamine receptors [115]. However, this reduced locomotor activity was not significant compared to the other groups. This excludes a stimulant or sedative effect that would interfere with the anxiolytic activity observed in the EPM, LDT, and SIH. AV therefore has a clear anxiolytic-like effect in these animal models. Conclusions An ethanolic extract prepared from the leaves of AV was evaluated for anxiolytic activity in several animal behavior models of anxiety. The extract showed a clear anxiolytic-like effect at two distinct dose ranges of 22.5-30 and 100-125 mg/kg comparable to the anxiolytic activity of the benzodiazepine diazepam and the azapirone buspirone. The dose-response profile presented with a biphasic U-shaped activity, which has been observed before but has not been fully understood to date. Antagonism studies of the whole extract revealed the GABA-ergic system as the main mediator of anxiolytic effect in the extract dose of 125 mg/kg, but no significant reduction for both the GABA-ergic and serotonergic antagonist in the lower active dose of 30

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82 mg/kg. The Open Field test was used to exclude any stimulant or sedative effects of AV. Both active concentrations of the extract as well as diazepam, buspirone, and the antagonists flumazenil and WAY-100635 in the same concentrations used in the behavioral test did not alter locomotor activity. Chronic treatment with AV over a twoand three-week period in three doses (30, 60, and 125 mg/kg) did not show significant anxiolytic effects, but diazepam also failed to increase the observed parameters significant from control. The observation of loss of anxiolytic activity after chronic treatment has been observed before and has been attributed to adaptive mechanisms of the respective receptor systems. Since the antidepressant effects of AV persist after chronic treatment, it is likely that different receptor systems are involved in the anxiolytic action of the extract. Bio-guided fractionation of the extract lead to the discovery of three fractions presenting with anxiolytic effects in an equivalent dose of 30 mg/kg as well as three different fractions showing an anti-anxiety activity in the equivalent dose of 125 mg/kg. This is an indicator for the contribution of at least two compounds from the extract to the anxiolytic effect seen in the EPM, LDT, and SIH. Antagonism of the active fractions in their respective equivalent doses showed that all fractions in a concentration equal to 30 mg/kg of whole extract were antagonized by WAY-100635, indicating a major involvement of the 5-HT1A receptor in the anxiolytic action of AV in this dose. Flumazenil failed to antagonize two fractions in a dose equal to 30 mg/kg of whole extract, but effectively reduced the anxiolytic action of all fractions in the higher dose equivalent to 125 mg/kg of whole extract. This confirms prior observations of the main involvement of the GABA-ergic system in the anxiolytic action of the extract at the higher dose of 125 mg/kg.

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83 Since fraction C showed the highest anxiolytic activity in both doses equivalent to 30 and 125 mg/kg whole extract, it was further evaluated in the LDT and by preparation of subfractions. Fraction C showed a significant anxiolytic activity in the LDT in both dose ranges, whereas the whole extract did not present with a significant anxiolytic activity in the higher dose of 125 mg/kg but with significant anxiolytic effects in the lower dose of 30 mg/kg. Since the LDT uses a different set of parameters to evaluate anti-anxiety effects than the EPM, the different activity profiles for the extract might be indicative of different receptor systems targeted and specific treatment options for different subclasses of anxiety disorders. The SIH paradigm is used both for the evaluation of stress-reducing and anxiolytic agents. The results of the SIH were similar to the findings in the LDT, where AV in the lower dose of 30 mg/kg did show an activity (significantly lower difference between basal and stress body temperature), but higher doses of 60 and 125 mg/kg failed to reduce body temperature after stress exposure. Subfractionation of fraction C lead to two different active subfractions, namely sA and sC. While sA was active in both equivalent doses of 30 and 125 mg/kg of whole extract, sC only showed significant anxiolytic activity in the lower dose. Main constituents of subfraction sA were epigallocatechin and gallocatechin. Epigallocatechin gallate has been shown to produce anxiolytic-like effects in the EPM before following intraperitoneal administration. The metabolism of epigallocatechin gallate to epigallocatechin and various other metabolites has been shown in humans [116]. Another major constituent of fraction C was the flavonol kaempferol, which was mainly present in subfraction sE. Although this subfraction did not show a significant anxiolytic effect, which might be attributed to the concomitant administration of afzelechin, kaempferol was evaluated further for its anxiolytic properties in the EPM (see chapter 4).

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84 Figure 3-1 Sketch of the Elevated Plus Maze (EPM). The dark areas represent the walls surrounding the closed arms Figure 3-2 Sketch of the Light-Dark Transition (LDT) test. The dark-shaded area is covered by a lid with a pass through at the bottom that allows transition between the dark and light compartments.

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85 Figure 3-3 Dose-response profile for AV. ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-4 GABA-antagonism of active AV doses. ** p<0.01 against control, *** p<0.001 against control, + p<0.05 against treatment alone, +++ p<0.001 against treatment alone, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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86 Figure 3-5 Serotonin-antagonism of active AV doses. p<0.05 against control, *** p<0.001 against control, + p<0.05 against treatment alone, +++ p<0.001 against treatment alone, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-6 Detailed dose-response profile for AV. p<0.05 against control, ** p<0.01 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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87 Figure 3-7 Evaluation of AV extract in the LDT test. p<0.05 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent in Lighted area, b: Number of Transitions between dark and lighted compartment Figure 3-8 Stress-Induced Hyperthermia (SIH) test of AV extract. p<0.05 against control, *** p<0.001 against control, n=6-10 per treatment group

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88 Figure 3-9 Chronic treatment I with AV extract over 16 days. p<0.05 against control, mean S.E.M, n=8-10 per treatment group, a and c: % Time spent on Open Arms, b and d: Number of Entries in Open Arms Figure 3-10 Chronic treatment II with AV extract over 22 days. p<0.05 against control, ** p<0.01 against control, mean S.E.M, n=7-9 per treatment group, a and c: % Time spent on Open Arms, b and d: Number of Entries in Open Arms

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89 Figure 3-11 Dose-response profile for fractions, reconstituted and whole extract of AV in 30 mg/kg. p<0.05 against control, ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-12 Dose-response profile for fractions, reconstituted and whole extract of AV in 125 mg/kg. p<0.05 against control, ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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90 Figure 3-13 GABA antagonism of active fractions equivalent to 30 mg/kg of whole extract. p<0.05 against control, ** p<0.01 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-14 GABA Antagonism of active fractions equivalent to 125 mg/kg of whole extract. p<0.05 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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91 Figure 3-15 5-HT1A Antagonism of active fractions equivalent to 30 mg/kg of whole extract. p<0.05 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-16 5-HT1A Antagonism of active fractions equivalent to 125 mg/kg of whole extract. p<0.05 against control, ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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92 Figure 3-17 Concomitant antagonism of diazepam, buspirone, fraction C, and AV with flumazenil and WAY-100635, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-18 Antagonism of buspirone with flumazenil. *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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93 Figure 3-19 Antagonism of diazepam with WAY-100635. ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-20 Dose-response profile for subfractions of fraction C from AV in 30 mg/kg. p<0.05 against control, ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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94 Figure 3-21 Dose-response profile for subfractions of fraction C from AV in 125 mg/kg. p<0.05 against control, ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 3-22 Open Field Test for AV, diazepam, buspirone, and antagonists. Box and whiskers with median and range, n=7 per treatment group, a: number of field transitions, b: total distance in mm

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95 CHAPTER 4 PHARMACOLOGICAL EVALUATION OF KAEMPFEROL AND ITS METABOLITE Anxiolytic Action of Kaempferol Elevated Plus Maze After evaluation of the whole extract and fractions prepared from the leaves of AV and confirmation of anxiolytic activity of the whole extract and certain fractions, further investigation of pure compounds found in AV and in active fractions seemed obvious. One such compound was kaempferol (figure 4-1), which was present as one of the main constituents in fraction C. Kaempferol is a weak agonist at GABAA receptors, but did not show an anxiolytic effect after i.p. administration in the EPM [22]. Since bioactivation might be an important step in the putative anxiolytic effects, kaempferol was administered orally in doses equivalent to those present in AV and a dose-response profile was derived after oral as well as intraperitoneal administration of kaempferol (Fisher Scientific, Pittsburgh, PA) using the EPM in mice. Dose-Response Profile for Kaempferol and Structure-Activity Relationship Following oral administration of kaempferol (in a dose range from 0.01 to 1.0 mg/kg dissolved (for the lower concentrations) or suspended (for higher concentrations) in 0.5% propylene glycol in deionized water and compared to control and diazepam (1.5 mg/kg, p.o.). The dose range was chosen based on the concentration of kaempferol present in AV (0.02 mg in 30 mg of whole extract and 0.08 mg in 125 mg of whole extract). Kaempferol showed a significant anxiolytic effect in doses above 0.02 mg/kg by increasing the percentage time spent on the open arms and at least at the concentrations of 0.02 and 0.08 mg/kg by increasing the number of entries in the open arms (figure 4-2). Kaempferol in a dose of 0.08 mg/kg showed the highest anxiolytic effect. The plateau at higher doses might be an indicator of saturation of absorption or due to the solubility issues and application as a suspension at these concentrations.

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96 Following intraperitoneal injection of kaempferol in isotonic saline solution (0.9% sodium chloride), kaempferol did not show an anxiolytic effect in doses of 0.02, 0.5, and 1.0 mg/kg, respectively (figure 4-3) in both parameters evaluated. Diazepam presented with clear anxiolytic effects in both experiments compared to control. The lack of anxiolytic activity after i.p. administration confirms prior findings [22] while it supports the theory of possible bioactivation in the intestines either by the microbial flora or the enterocytes. This will be discussed later in this chapter. The degree of hydroxylation in the B ring of the flavonol structure has profound implications on both the metabolism and absorption of the respective flavonol [117,118]. While kaempferol has one hydroxyl group in para-position, quercetin presents with two hydroxyl -substituted flavonol with hydroxyl groups in Since the dose range for kaempferol covered 0.01 to 1.0 mg/kg, both quercetin and myricetin were given orally to mice in concentrations of 0.02 and 0.5 mg/kg and were evaluated in the EPM under the same experimental conditions like kaempferol (figure 4-4). Quercetin presented with clear anxiolytic-like effects comparable to diazepam in both concentrations evaluated, while myricetin did not show an increase in percentage time spent on open arms. Further explanation and a theory for a structure-activity relationship is discussed later in this chapter. Antagonism Studies for Kaempferol and Quercetin Since both kaempferol and quercetin presented with an acute anxiolytic effect after oral administration, further insight into their mechanism of action was studied by antagonizing possible mechanisms of action of the flavonols with flumazenil on the GABA-ergic system and WAY-100635 on the serotonergic system. Kaempferol concentrations studied were equivalent to the total AV extract in 30 mg/kg (0.02 mg/kg kaempferol) and 125 mg/kg (0.08 mg/kg

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97 kaempferol). The higher active dose of 0.5 mg/kg was chosen for quercetin. After coadministration of flumazenil, the lower concentration of kaempferol did not show a significant reduction in percentage time spent in or entries on open arms, but the higher concentration of 0.08 mg/kg was significantly reduced in these parameters to oral kaempferol treatment alone (figure 4-5). Following WAY-100635 antagonism on the serotonergic system, both concentrations of kaempferol were not significantly lowered in their percentage time spent on open arms, but the lower dose showed significantly lower entries on the open arms compared to kaempferol treatment alone (figure 4-6). Both diazepam and buspirone showed clear anxiolytic-like effects in the experiments. The lack of a clear reduction in percentage time spent on open arms for the serotonin antagonism of the lower kaempferol dose and the clear reduction in both parameters for the GABA-antagonism of the higher dose might be an indicator for a diverse action of kaempferol on a variety of neurotransmitter systems and/or enzymes. As has been shown previously, kaempferol is a potent inhibitor of monoamine oxidase [119] and protects cerebellar granule cells from stress-induced apoptosis [120], although these studies have been conducted in vitro. Quercetin in a concentration of 0.5 mg/kg was neither inhibited by flumazenil nor WAY-100635 (figure 4-7). Flumazenil had no influence on the percentage time spent on open arms while WAY-100635 lead to an increase in this parameter. However, the number of entries for quercetin with concomitant administration of the antagonists was not significantly different from control whereas diazepam presented with increased values in these parameters. The anxiolytic mechanism of action for quercetin can therefore not be explained via interaction with GABA-ergic or serotonergic neurons following oral administration to mice.

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98 Evaluation of Locomotor Activity of Kaempferol and Quercetin The Open Field test is commonly used to investigate possible stimulant or sedative effects of drugs by exposing the animal to an open arena divided into equal compartments by lines on the ground and videotaping the distance and number of field transitions between the compartments. Kaempferol and quercetin in their respective concentrations used for the antagonism studies as well as diazepam did not present with any differences in distance run and number of field transitions compared to control (figures 4-8 and 4-9). This excludes a stimulant or sedative effect of kaempferol or quercetin which might influence the anxiolytic behavior observed in the EPM. Metabolism Theory for Kaempferol and Quercetin in the Intestines Extensive research has been conducted on flavonoid absorption and metabolism in the past decades, especially since these compounds have been linked to many health benefits [121,122]. The main attention has been put on quercetin and anthocyanidins, since these compounds have the highest antioxidant activity and therefore seemed most promising to promote health [123,124]. The complex intestinal conversion of flavonoids by both the bacterial microflora and the enterocyte make it hard to predict overall absorption of flavonoids and their metabolites. Recently, the metabolic degradation of kaempferol, quercetin, and related substances by intestinal bacteria has been studied in isolated pig [117,125] or rat caecum [126]. The flavonols kaempferol, quercetin, and myricetin were metabolized to phloroglucinol derived from ring A, while kaempferol was transformed to 4-hydroxyphenylacetic acid (para-hydroxyphenylacetic acid, pHPAA), quercetin to 3,4-dihydroxyphenylacetic acid (DOPAC) and further to 3-hydroxyphenylacetic acid (meta-hydroxyphenylacetic acid, mHPAA), and myricetin to 3,5-dihydroxyphenylacetic acid derived from ring B (figures 4-10, 4-11, and 4-12). This degradation occurred within the first hour (10% of original amount converted) for kaempferol

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99 and quercetin and reached completeness after 8 hours of incubation time. It has been shown before that the bacterium Clostridium orbiscindens degrades kaempferol to pHPAA and quercetin to mHPAA and DOPAC both in humans [127] and in rats [128]. The respective intermediate was further metabolized to 4-methylphenol (pHM) for kaempferol and 3,4-dihydroxymethylbenzoic acid or 3-methylphenol for quercetin, respectively. Absorption studies of these compounds in vivo have not been conducted yet, but in vitro cell culture assays using CACO-2 cells (human colon adenocarcinoma cell) have shown, that both mHPAA and DOPAC are absorbed to some degree (2.7 and 0.3% of initial concentration, respectively) [129]. Another in vivo study compared the absorption and splanchnic metabolism of various flavonoids using a cannulated perfused rat intestine from jejunum to ileum and a biliary duct [118]. This study concluded that kaempferol is absorbed in higher amounts (58% net absorption) as either the aglycone or glucuronide than quercetin and its metabolites (15% net absorption). In addition, kaempferol reaches the peripheral tissues in much higher amounts (49% of total perfused concentration) compared to quercetin (9% of total perfused concentration). Since the degradation products of kaempferol as well as the aglycone and the glucuronide are absorbed, it is likely that these compounds can reach both the systemic circulation and may be able to penetrate into the brain. It has been shown, that rat cortical neurons are able to accumulate kaempferol, but not quercetin [130]. This might be indicative of a selective mechanism for kaempferol in the brain. Quercetin on the other hand has been shown to inhibit ionic currents of GABAA, GABAC, nicotinic acetylcholine, 5-HT3A, and glutamate receptors [24], although these experiments have been conducted in vitro using electrophysiological methods. If quercetin itself is able to reach the CNS has not clearly been shown to date using in

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100 vivo models, but some in vitro studies showed that quercetin [131] and its glucuronic acid metabolite miquelianin [44] are able to cross the blood-brain barrier, although in very low amounts. Trace amines are precursors or degradation products of neuroactive biogenic amines in the brain (figure 4-13). They play an important role in homeostasis by acting either as neuromodulators or neurotransmitters on specific receptors themselves [132]. Endogenous ligands are largely unknown for these receptors to date. Older studies indicate an involvement of the catabolic trace amines meta-hydroxyphenylacetic acid and para-hydroxyphenylacetic acid in several psychological disorders like agoraphobia [133] schizophrenia [134] and aggression [135] For schizophrenia and agoraphobia, para hydroxyphenylacetic acid plasma levels were significantly reduced, whereas for isolated aggressive mice, this parameter was increased compared to control groups. Another study investigated the uptake of para hydroxyphenylacetic acid into the brain and found specifically high concentrations [136] In addition, these trace amines show an active transport mechanism out of the brain, which is sensitive to probenecid [137]. Subcutaneous injections of para-hydroxyphenylacetic acid lead to rapid increases in brain concentrations, which could not be inhibited by probenecid [138]. Certain anxiolytic and antipsychotic drugs like chlorpromazine or sulpiride increase the levels of para-hydroxyphenylacetic acid and meta-hydroxyphenylacetic acid in certain brain regions [139]. Although these studies are older, newer research is supporting the hypothesis of a complex involvement of the trace amines in several CNS diseases [140]. Another recent development investigated the inhibitory action of para-hydroxylated benzaldehyde derivatives on GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) [141]. The two enzymes GABA-T and SSADH are mainly responsible for the degr-aminobutyric

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101 acid (GABA), which is the main inhibitory neurotransmitter in the brain. Increased levels of GABA have been linked to anxiolysis in both humans and animals [142]. The structure-activity relationship includes the necessity of the benzene ring and a hydroxyl group in para-position in the aromatic system. Since AV contains kaempferol and its glucosides in sufficient amounts, a possible metabolic activation by the intestinal microflora to form para-hydroxyphenylacetic acid and absorption of this compound were investigated. Since kaempferol itself did not present with an anxiolytic activity after intraperitoneal administration, bioactivation in the gut seems to be reasonable. As drugs applied intraperitoneal are still subjected to liver metabolism [143] and glucuronidation, the glucuronic acid metabolite of kaempferol did not appear to be a likely candidate for the anxiolytic activity. Anxiolytic Action of Para-Hydroxyphenylacetic Acid Elevated Plus Maze Since the before-mentioned theory of bioactivation of kaempferol by intestinal microflora to para-hydroxyphenylacetic acid (pHPAA), it was of interest to study the possible influence of its action on trace amine levels in the brain using the EPM in mice. Para-hydroxyphenylacetic acid was administered both intraperitoneal and oral in a dose range of 0.001 to 1.0 mg/kg. Active doses were antagonized using flumazenil (GABA antagonist) and WAY-100635 (5-HT1A antagonist). The EPM was conducted under the same conditions mentioned in chapter 2 for the whole extract and all subsequent EPMs. Dose-Response Profile for Para-Hydroxyphenylacetic Acid Following intraperitoneal injection of pHPAA (Sigma-Aldrich) in a dose-range between 0.001 and 1.0 mg/kg, only two concentrations, namely 0.01 and 0.02 mg/kg exerted an anxiolytic effect in the EPM (figure 4-14). This sharp dose-response curve might be indicative of an active

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102 transporter that is saturable above a certain concentration reached in the plasma. In addition, the active transporter for efflux of para-hydroxyphenylacetic acid might become activated above certain levels reached in the brain and actively secret pHPAA back out of the brain. After oral administration in three different doses, pHPAA did not show anxiolytic effects (figure 4-15). It has been shown before that pHPAA is nitrated both in the oral cavity and the stomach to 3-nitro-4-hydroxyphenylacetic acid, which is excreted through the urine [144]. If this nitrated metabolite still exerts anxiolytic activity or can cross the blood-brain barrier is unknown. Since para-hydroxyphenylacetic acid after oral administration lacked the anxiolytic effect seen after intraperitoneal injection, it seems likely that the nitrated metabolite is not able to induce anxiolytic-like effects. Antagonism Studies for Para-Hydroxyphenylacetic Acid Antagonism of the active doses of pHPAA was conducted using flumazenil as a GABA antagonist and WAY-100635 as a serotonin antagonist. Flumazenil did reduce the percentage time spent on open arms for pHPAA in a dose of 0.01 mg/kg, but the reduction in this parameter was not significant for pHPAA in a dose of 0.02 mg/kg, although there was a marked decrease (figure 4-16). Both doses were not inhibited by WAY-100635, clearly stating the involvement of the GABA-ergic system for the anxiolytic-like effects of pHPAA. Both diazepam and buspirone did exert anti-anxiety effects. Although the activity of pHPAA in the higher dose was not significantly reduced with concomitant administration of flumazenil, it can be concluded that the pharmacological mechanism of action for pHPAA is mainly exerted through the GABA-ergic system. Evaluation of Locomotor Activity of Para-Hydroxyphenylacetic Acid The Open Field Test was conducted under the same conditions as mentioned in chapter 2 with three animals being evaluated simultaneously in opaque, grey plastic boxes as used for the

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103 Light-Dark Transition test and the Open Field for kaempferol. All treatments, namely control, diazepam (1.5 mg/kg), buspirone (10 mg/kg), and pHPAA in a concentration of 0.02 mg/kg were not significantly different from each other in the number of field transitions and the total distance run (figure 4-17). This excludes a possible sedative or stimulant effect of pHPAA. Conclusions Pharmacological activities of flavonoids for the treatment of CNS diseases such as depression an anxiety has been studied extensively and certain flavonoids such as chrysin and quercetin have been shown to exhibit central nervous system activities both in vitro by receptor binding studies and in vivo using animal behavior models. The putative anxiolytic effects of kaempferol however have not been evaluated in vivo to date. One publication showed a moderate binding affinity of kaempferol to GABAA receptors, but failed to observe anxiolytic effects after intraperitoneal treatment [22]. The findings from our group suggest that both kaempferol and quercetin might exert part of their anxiolytic activity by bioactivation to phenylacetic acid derivatives in the intestines. Oral application of kaempferol seems to play an important role in its bioactivation since kaempferol did not show an anxiolytic effect after intraperitoneal administration in mice. A fast degradation to para-hydroxyphenylacetic acid has been shown ex vivo and it is suggested from structurally similar compounds, that para-hydroxyphenylacetic acid is well absorbed from the gut into the systemic circulation. After oral application of para-hydroxyphenylacetic acid, no anxiolytic activity was observed, which is in well agreement with metabolism to a nitro-compound in the stomach and therefore inactivation of para-hydroxyphenylacetic acid as has been reported earlier. Both kaempferol and para-hydroxyphenylacetic acid were antagonized by coadministration of flumazenil following oral or intraperitoneal application, respectively. This strongly suggests the involvement of GABA-ergic

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104 neurons in the anxiolytic activity of these compounds. Quercetin was neither antagonized by flumazenil nor WAY-100635, which might be indicative of a different receptor or enzyme system involved in its anxiolytic mechanism of action. Older publications suggest the involvement of trace amines and their degradation products in the pathophysiology of various CNS diseases. It has been shown that para-hydroxyphenylacetic acid is transported into the brain rapidly and that a probenecid-sensitive transporter controls the efflux of this compound from the brain.

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105 Figure 4-1 Structure of the flavonol kaempferol Figure 4-2 Dose-response profile for kaempferol after oral administration. p<0.05 against control, ** p<0.01 against control, mean S.E.M, n=10-14 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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106 Figure 4-3 Dose-response profile for kaempferol after intraperitoneal administration. *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 4-4 Dose-response profile for quercetin and myricetin after oral administration. p<0.05 against control, ** p<0.01 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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107 Figure 4-5 GABA-antagonism of kaempferol. p<0.05 against control, ** p<0.01 against control, + p<0.05 against treatment alone, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 4-6 Serotonin-antagonism of kaempferol. p<0.05 against control, ** p<0.01 against control, *** p<0.001 against control, + p<0.05 against treatment alone, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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108 Figure 4-7 Antagonism of quercetin. p<0.05 against control, ** p<0.01 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 4-8 Open Field Test for kaempferol and diazepam. Box and whiskers with median and range, n=8 per treatment group, a: number of field transitions, b: total distance in mm

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109 Figure 4-9 Open Field Test for quercetin and diazepam. Box and whiskers with median and range, n=8 per treatment group, a: number of field transitions, b: total distance in mm

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110 Figure 4-10 Intestinal metabolism and absorption of the flavonol kaempferol and its metabolites. UGT: UDP-glucuronosyl-transferase, MRP: multi drug resistance related protein, active transport, transmembrane or proposed active transport, metabolism by intestinal microflora, pHPAA: para-hydroxyphenylacetic acid, pMH: para-methylphenol. Ring A metabolite in blue, ring B metabolites in red.

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111 Figure 4-11 Intestinal metabolism and absorption of the flavonol quercetin and its metabolites. UGT: UDP-glucuronosyl-transferase, MRP: multi drug resistance related protein, active transport, transmembrane or proposed active transport, metabolism by intestinal microflora, DOPAC: 3,4-dihydroxyphenylacetic acid, mHPAA: meta-hydroxyphenylacetic acid. Ring A metabolite in blue, ring B metabolites in red.

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112 Figure 4-12 Intestinal metabolism and absorption of the flavonol myricetin and its metabolites. UGT: UDP-glucuronosyl-transferase, MRP: multi drug resistance related protein, active transport, transmembrane or proposed active transport, metabolism by intestinal microflora, 3,4,5-PAA: 3,4,5-trihydroxyphenylacetic acid, 3,5-PAA: 3,5-dihydroxyphenylacetic acid. Ring A metabolite in blue, ring B metabolites in red.

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113 Figure 4-13 Main routes of trace amine metabolism. AADC: amino acid decarboxylase, TH: tyrosine hydroxylase, NMT: N-methyltransferase, MAO: monoamine oxidase, PNMT: phenylethanolamine N--hydroxylase, COMT: catechol-O-methyltransferase [132]. Kaempferol metabolite pHPAA and quercetin metabolite DOPAC are in red.

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114 Figure 4-14 Dose-response profile for para-hydroxyphenylacetic acid after intraperitoneal administration. ** p<0.01 against control, *** p<0.001 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 4-15 Dose-response profile for para-hydroxyphenylacetic acid after oral administration. p<0.05 against control, ** p<0.01 against control, mean S.E.M, n=10 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms

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115 Figure 4-16 Antagonism of para-hydroxyphenylacetic acid. p<0.05 against control, ** p<0.01 against control, *** p<0.001 against control, ++ p<0.01 against treatment alone, mean S.E.M, n=10-20 per treatment group, a: % Time spent on Open Arms, b: Number of Entries in Open Arms Figure 4-17 Open Field Test for para-hydroxyphenylacetic acid. Box and whiskers with median and range, n=10 per treatment group, a: number of field transitions, b: total distance in mm

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116 CHAPTER 5 DISCUSSION The purpose of this dissertation was to chemically characterize an extract prepared from the leaves of Apocynum venetum L. (Apocynaceae) and evaluate the extract and prepared fractions for a putative anxiolytic activity. In addition, pure compounds from AV have been evaluated for their mechanism of anxiolytic action using antagonists for the two most common receptor systems involved in anxiety. Many natural products derived from plants have been used for centuries to cure or treat diseases. Based on their traditional uses or their chemical profile, extracts have been evaluated for a wide variety of pharmacological activities. AV has been used as a tea in traditional Asian medicine for the treatment of liver diseases, for relieve of tension, as an antiphlogistic, and for the induction of diuresis [32]. Most of these activities have been scientifically confirmed to some degree. Based on prior studies that showed an antidepressant effect of an ethanolic AV extract [41], it was therefore of interest to investigate other treatment options for CNS diseases such as anxiety disorders, since this pathological symptom complex is one of the most common mental illnesses world-wide [2]. The first aim was to qualitatively characterize the composition of an ethanolic leave extract from AV using a chromatographic system coupled with a mass spectrometer to achieve high sensitivity. The extract contained mainly flavonoids, caffeic acid derivatives, and oligomeric anthocyanidins, which is in well agreement with prior findings [42]. Since the antidepressant effects of the extract have been linked to the quercetin glycosides hyperoside and isoquercitrin, the extract was standardized to contain 2.1% hyperoside and 2.7% isoquercitrin. All other compounds were identified based on their respective retention times, ultraviolet, and mass spectra. The extract profile was typical for a plant leaf extract with the main constituents being

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117 the caffeic acid derivative chlorogenic acid, the proanthocyanidins catechin, epicatechin, gallocatechin, and epigallocatechin, and the flavonol aglycones quercetin and kaempferol and their respective galactosides and glucosides. The flavonol aglycones in the extract did most likely result from acid hydrolysis of the respective glycosides as is commonly seen with plant extracts [145]. The monomeric proanthocyanidins are also present in the form of oligomeric proanthocyanidins, combining up to four monomers as has been reported to date [42]. The appearance of monomers like catechin or epigallocatechin results both from naturally occurring monomers and hydrolytic processes during extract preparation. Following fractionation, all of the major compounds from the whole extract were recovered in appropriate fractions, although it is not clear if this was achieved quantitatively (recovery rates were 94% for fractionation and 98% for subfractionation). One possible explanation for the loss of anxiolytic activity of the reconstituted fractions might be a change in interaction between certain compounds from the extract due to the fractionation. The structure of kaempferol was confirmed by 1H-NMR analysis. The antioxidant properties of the extract and some compounds in comparison to structurally related substances showed a high antioxidant effect of the whole extract as well as a tannin-rich extract evaluated by the oxygen radical absorbance capacity (ORAC) assay. This is in well agreement with literature sources that evaluated a variety of radical-scavenging activities of AV [29,30]. Caffeic acid derivatives, quercetin, and the glycosylated flavonoids presented with a high antioxidant activity based on structure-activity relationships as has been reported before [69,74]. Pharmacological evaluation in 3 different animal models of anxiety of the whole extract revealed a significant anxiolytic activity in two distinct dose ranges of 22.5-30 and 100-125 mg/kg comparable to the known anxiolytics diazepam and buspirone. This unique double U-

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118 shaped activity is an extension to the occurrence of U-shaped activities, which are not yet fully understood, but may be due to hormesis. Hormesis is a concept first described in toxicology where a low-dose stimulation occurred followed by a high-dose inhibition [105]. This phenomenon has been observed in a variety of receptor systems and hormonal responses including the dopamine and serotonin receptors as well as corticosterone and estrogen, which all play a role in stress and anxiety disorders. Another explanation of the specific occurrence of a double U-shaped anxiolytic effect observed in AV might be the involvement of two receptor systems and a receptor selective dose-response profile. This is supported by fractionation and antagonism studies on the GABA-ergic and serotonergic receptors, where the extract and fractions were antagonized by flumazenil (GABAA antagonist) in a dose equivalent to 125 mg/kg and the fractions equivalent to 30 mg/kg of whole extract were significantly antagonized by WAY-100635 (5-HT1A antagonist). The involvement and interconnection of GABA and serotonin as neurotransmitters for anxiety responses in neuronal circuits has been shown both in vitro [13] and in vivo [146]. Another possibility for the distinct activity of the two doses might be related to solubility limitations of active compounds, biotransformation to active or inactive metabolites, or enzyme saturation. At this point, it is not clear which of these mechanisms is responsible for the observed anxiolytic dose-response profile keeping in mind that it might even be the contribution of more than one discussed or yet not known mechanism. The activity of AV in a dose of 30 mg/kg was observed in all three animal models, namely the Elevated Plus Maze (EPM), the Light-Dark Transition (LDT) test, and the Stress-Induced Hyperthermia (SIH) while the higher concentration of 125 mg/kg only exerted anxiolytic-like effects in the EPM. All three animal models use distinct paradigms for the induction of anxiety

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119 and evaluate different parameters related to the respective condition. In fact, the differential response of the two AV doses supports the theory of different receptor systems being targeted at the two doses. It has been suggested in the literature that the EPM and the LDT can distinguish between specific anxiety disorders [94]. In addition, the SIH uses a stress-related paradigm for the induction of an anxiety behavior where the animal is not able to avoid exposure to the stressful situation, which is not the case for both the EPM and the LDT [100]. Chronic treatment over 16 or 22 days with AV did mostly not present with a sustained significant anxiolytic effect and neither did the known anxiolytic diazepam, which was also administered. This lack of anxiolytic effect after chronic administration has been observed before and attributed to adaptive mechanisms of receptor systems, neuronal circuits, or hormonal feedback mechanisms [108,109]. Fraction C derived from AV showed the strongest anxiolytic effects in both doses equivalent to 30 and 125 mg/kg in both the EPM and the LDT and was subjected to subfractionation and further pharmacological evaluation. The main constituents in fraction C are kaempferol and the proanthocyanidins gallocatechin and epigallocatechin. Subfractionation of fraction C and evaluation in the EPM showed an anxiolytic effect for subfractions A and C in an equivalent dose of 30 mg/kg whole extract and subfraction A for a dose of 125 mg/kg whole extract. Subfraction A mainly consisted of the proanthocyanidins gallocatechin and epigallocatechin. There have been reports of an anxiolytic activity of epigallocatechin gallate after intraperitoneal injection in the EPM [114] as well as a biphasic action on GABAA receptors [113]. So far, there have been no reports of metabolism of epigallocatechin gallate to epigallocatechin after oral administration, but the results from the EPM would suggest such a mechanism for bioactivation. In addition to the activity of subfraction

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120 A, there was no anxiolytic effect observed for the other subfractions, including subfraction sE, which consisted mainly of kaempferol and afzelechin. Since a major aim and outcome of this research included a putative anxiolytic activity of kaempferol following bioactivation, a possible interaction between afzelechin and kaempferol might account for a lack of anxiolytic activity of subfraction sE. An interesting aspect and theory of this research included the mechanism by which flavonols and specifically kaempferol are metabolized to an active metabolite by bacterial degradation in the intestines. Studies in pig caecum have shown that flavonols like quercetin and kaempferol are degraded by intestinal bacteria to phenylacetic acid metabolites that are also degradation products of trace amines in the brain [125,132]. It has been shown, that these degradation products are readily absorbed through the enterocytes in the intestines [129]. In addition, metabolism of kaempferol and net absorption into the systemic circulation of the aglycone was significantly higher compared to quercetin [118]. About three decades ago the interest in trace amines and their physiological activities resulted in a series of papers that found a connection between altered trace amine levels and a number of pathophysiological CNS conditions like anxiety disorders [133], pathological aggression [135], migraines [140], or schizophrenia [134]. They also found that elevated plasma levels of para-hydroxyphenylacetic acid lead to an increase in brain levels of this trace amine degradation product [136,138]. In addition, certain antipsychotic drugs showed a pronounced effect on brain levels of paraand meta-hydroxyphenylacetic acid [139]. This field of research now receives a renaissance with the discovery of specific trace amino acid receptors (TAARs) in the brain, although endogenous ligands have not been identified yet [132].

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121 In conclusion, an ethanolic extract from the leaves of AV presented with a unique anxiolytic activity in various animal models. The pharmacological mechanisms of action for this diverse effect are mediated through the GABA-ergic as well as serotonergic neurotransmitter systems, which suggest the involvement of at least two active substances in the extract. Following fractionation, the flavonol kaempferol was identified as a contributor to the anxiolytic effects observed with the extract. Kaempferol and its metabolite para-hydroxyphenylacetic acid mediate their anxiolytic activity mainly through GABA, while quercetin and yet not identified compounds are responsible for either a direct action on inhibitory neurotransmission in the brain or anxiolytic action through the serotonergic system as has been shown by antagonism studies. The successful approach of using bio-guided fractionation for evaluation and elucidation of pharmacological activity has been used before to elucidate the mechanism of action for complex biological matrices like plant extracts [147]. This research is an example for the effective utilization of bio-guided fractionation and antagonism experiments of a plant extract in vivo to derive active fractions and single compounds.

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122 APPENDIX ADDITIONAL TABLES Chapter 2 Table A-1 Results of ORAC assay for relative total antioxidant activity of AV extract and tannin extracts. Trolox AV extract Tannin containing extract Tannin reduced extract # of values 3 3 3 3 Mean 1.00 00 0 2.024 00 1.954 0 0.9957 0 Std. Dev 0.08974 0.02007 0.2004 0.09411 Std. Error 0.05181 0.01159 0.1157 0.05433 Table A-2 Results of ORAC assay for relative total antioxidant activity of caffeic acid derivatives. Trolox Cynarin Chlorogenic acid Ferulic acid Isoferulic acid Rosmarinic acid Caffeic acid # of values 9 3 3 3 3 3 3 Mean 0.9999 1.614 00 1.081 00 1.945 00 2.173 00 2.605 000 1.882 00 Std. Dev 0.5931 0.02346 0.1283 0 0.02883 0.02211 0.004582 0.07351 Std. Error 0.1977 0.01354 0.07405 0.01664 0.01277 0.002646 0.04244 Table A-3 Results of ORAC assay for relative total antioxidant activity of flavonoid aglycones. Trolox Luteolin Apigenin Myricetin Quercetin Kaempferol Naringenin # of values 12 3 3 3 3 3 3 Mean 1.000 0 1.266 00 1.540 00 0.7757 0 2.099 000 1.268 00 2.376 00 Std. Dev 0.5191 0.08156 0.04771 0.02098 0.008327 0.06035 0.1323 0 Std. Error 0.149 9 0.04709 0.02755 0.01212 0.004807 0.03484 0.07639 Table A-4 Results of ORAC assay for relative total antioxidant activity of glycosylated flavonoids. Trolox Rutin Hyperoside Isoquercitrin Apigetrin Cynaroside # of values 9 3 3 3 3 3 Mean 1.000 00 1.6 53 00 1.519 00 1.558 00 1.858 00 1.589 00 Std. Dev 0.1522 0 0.06609 0.06855 0.03412 0.05050 0.1595 0 Std. Error 0.05073 0.03816 0.03958 0.01970 0.02916 0.09212

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123 Table A-5 Results of ORAC assay for absolute antioxidant activity of AV extract and tannin extracts in mol trolox equivalents/g dry weight. AV extract Tannin containing extract Tannin reduced extract # of values 3 3 3 Mean 1012 .000 977.2 0 497.8 0 Std. Dev 10.04 0 100.2 0 47.05 Std. Error 5.795 57.84 27.17 Chapter 3 Table A-6 Results of EPM for AV extract dose-response profile. Cont.: Control, Diaz.: Diazepam, Busp.: Buspirone, AV: Apocynum venetum Cont Diaz Busp AV 15 AV 30 AV 60 AV 125 AV 250 # of values 10 10 10 10 10 10 10 10 % Time OA Mean 3.27 00 17.12 0 15.64 0 2.75 00 13.5 00 5.82 0 17. 54 0 3.19 0 Std. Dev 2.365 0 8.303 6.519 2.808 0 7.662 5.170 7.288 3.540 Std. Error 0.7479 2.626 2.062 0.8879 2.423 1.635 2.305 1.120 # of Entries OA Mean 1.900 0 12.30 00 6.800 0 2.100 0 5.500 0 3.100 0 7.300 0 1.900 0 Std. Dev 1.101 0 2.111 0 1.932 0 1.524 0 2.12 1 0 1.595 0 2.983 0 1.663 0 Std. Error 0.3480 0.6675 0.6110 0.4819 0.6708 0.5044 0.9434 0.5260 Table A-7 Results of EPM for GABA antagonism of AV extract. Cont.: Control, Sal.: Saline, Diaz.: Diazepam, Flum.: Flumazenil, AV: Apocynum venetum Cont./ Sal. C ont./ Flum. Diaz./ Sal. Diaz./ Flum. AV 30/ Sal. AV 30/ Flum. AV 125/ Sal. AV 125/ Flum. # of values 10 10 10 10 10 10 10 10 % Time OA Mean 2.710 0 1.630 0 15.31 0 2.160 0 13.50 0 8.360 19.45 0 0.7100 Std. Dev. 3.046 0 1.500 0 7.394 2.092 0 7.662 4.135 13.33 0 1 .006 0 Std. Error 0.9633 0.4742 2.338 0.6617 2.423 1.308 4.217 0.3181 # Entries OA Mean 2.200 0 1.500 0 7.500 1.500 0 6.200 0 3.600 0 6.800 1.500 0 Std. Dev. 2.860 0 1.080 0 1.581 1.269 0 2.486 0 2.633 0 4.315 1.434 0 Std. Error 0.9043 0.3416 0.500 0.4014 0.7860 0 .8327 1.365 0.4534

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124 Table A-8 Results of EPM for 5-HT1A antagonism of AV extract. Cont.: Control, Sal.: Saline, Busp.: Buspirone, WAY: WAY-100635, AV: Apocynum venetum Cont./ Sal. Cont./ WAY Busp ./ Sal. Busp ./ WAY AV 30/ Sal. AV 30/ WAY AV 125/ Sal. AV 125/ WAY # of values 10 10 10 10 10 10 10 10 % Time OA Mean 2.720 4.120 12.90 1.120 13.50 7.550 15.99 12.38 Std. Dev. 1.659 3.380 7.025 1.133 7.662 4.049 4.578 6.661 Std. Error 0.5245 1.069 2.221 1.069 2.423 1.281 1.448 2.106 # Entries OA Mean 2.30 0 2.300 0 4.200 0.9000 6.200 3.300 6.100 5.800 Std. Dev. 1.567 1.494 0 3.259 0.7379 2.486 1.418 3.784 3.706 Std. Error 0.4955 0.4726 1.031 0.2333 0.7860 0.4485 1.197 1.172 Table A-9a Results of EPM for detailed dose-response profile of AV extract. Cont.: Control, Sal.: Saline, Diaz.: Diazepam, AV: Apocynum venetum Cont. Diaz. AV 15 AV 22.5 AV 30 AV 37.5 AV 45 AV 60 # of values 10 10 10 10 10 10 10 10 % Time OA Mean 6.800 15.74 9.870 14.89 17.28 8.350 5.980 7.030 Std. Dev. 2.740 6.984 3.478 8.130 7.28 8 3.532 3.936 5.693 Std. Error 0.8664 2.209 1.100 2.571 2.305 1.117 1.245 1.800 # Entries OA Mean 5.700 0 8.800 0 7.000 8.100 0 8.300 0 6.800 0 3.500 0 4.600 Std. Dev. 2.312 0 2.530 0 3.197 3.035 0 2.908 0 2.098 0 1.581 0 3.596 Std. Error 0.7311 0.8000 1.011 0.95 97 0.9195 0.6633 0.5000 1.137 Table A-9b continued from table A9a Cont. Diaz. AV 75 AV 100 AV 125 AV 150 AV 200 # of values 10 10 10 10 10 10 10 % Time OA Mean 3.310 0 10.70 8.320 9.320 10.41 6.440 6.100 Std. Dev. 1.446 0 3.994 4.793 6.286 4.985 4.950 2.487 Std. Error 0.4572 1.263 1.516 1.988 1.577 1.565 0.7866 # Entries OA Mean 3.000 0 6.200 0 3.700 4.400 5.800 3.600 4.000 Std. Dev. 0.7071 2.440 0 1.767 2.413 2.974 2.366 1.247 Std. Error 0.2357 0.7717 0.5588 0.7630 0.9404 0.7483 0.3944

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125 Table A-10 Results of LDT for AV extract and fractions. Cont.: Control, Diaz.: Diazepam, AV: Apocynum venetum, C: Fraction C Cont. Diaz. AV 30 AV 125 C 30 C 125 # of values 10 10 10 10 10 10 % Time spent in lighted area Mean 30.94 0 43.31 0 53.46 0 39.23 0 43.53 0 42 .87 0 Std. Dev. 7.463 5.300 9.641 4.889 12.65 0 15.04 0 Std. Error 2.360 1.676 3.049 1.546 3.999 4.757 # of Transitions between lighted and dark compartments Mean 20.00 0 21.30 0 21.10 0 21.90 0 20.90 0 18.10 0 Std. Dev. 7.196 5.100 6.208 9.146 9.267 6.173 St d. Error 2.275 1.613 1.963 2.892 2.930 1.952 Table A-11 Results of SIH for AV extract. Cont.: Control, Diaz.: Diazepam, AV: Apocynum venetum Cont. Diaz. AV 30 AV 60 AV 125 # of values 10 6 9 9 9 stress temperature) Mean 1.810 0 0.1167 0 1.078 0 1.589 0 1.767 0 Std. Dev. 0.7385 0.1329 0 0.4438 0.5754 0.6892 Std. Error 0.2335 0.05426 0.1479 0.1918 0.2297

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126 Table A-12 Chronic treatment I of AV extract. Cont.: Control, Diaz.: Diazepam, AV: Apocynum venetum Cont. Diaz. AV 30 AV 60 AV 125 Day 8, treatment with water # of values 8 9 10 9 8 % Time OA Mean 13.30 23.79 33.89 18.66 22.56 Std. Dev. 14.54 19.42 22.53 11.38 11.42 Std. Error 5.1 39 6.473 7.125 3.792 4.039 # Entries OA Mean 5.125 7.778 7.800 6.778 9.125 Std. Dev 2.696 2.224 2.781 2.682 3.091 Std. Error 0.9531 0.7412 0.8794 0.8941 1.093 Day 16, assigned treatment # of values 8 8 7 9 6 % Time OA Mean 7.650 14.81 9.957 11.22 1 1.90 Std. Dev. 5.124 9.173 4.971 6.098 4.297 Std. Error 1.812 3.243 1.879 2.033 1.754 # Entries OA Mean 4.500 6.375 5.286 5.333 6.500 Std. Dev. 2.619 2.875 1.976 2.121 1.517 Std. Error 0.9258 1.017 0.7469 0.7071 0.6191

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127 Table A-13 Chronic treatment II of AV extract. Cont.: Control, Diaz.: Diazepam, AV: Apocynum venetum Cont. Diaz. AV 30 AV 60 AV 125 Day 14, assigned treatment # of values 7 8 9 9 9 % Time OA Mean 9.786 19.11 15.30 12.56 13.83 Std. Dev. 5.296 5.250 12.50 6.488 5.749 Std. Error 2.002 1.856 4.167 2.163 1.916 # Entries OA Mean 7.571 11.00 7.889 7.333 8.778 Std. Dev 3.259 4.036 3.790 2.291 3.993 Std. Error 1.232 1.427 1.263 0.7638 1.331 Day 22, assigned treatment # of values 7 8 7 9 7 % Time OA Mean 5.100 13.24 3.629 9.767 6 .957 Std. Dev. 4.523 6.245 2.396 7.384 7.177 Std. Error 1.709 2.208 0.9057 2.461 2.713 # Entries OA Mean 3.143 8.500 2.714 5.333 4.857 Std. Dev. 1.069 3.338 1.604 2.693 3.716 Std. Error 0.4041 1.180 0.6061 0.8975 1.405

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128 Table A-14 Results of EPM for fractions of AV extract. Cont.: Control, Diaz.: Diazepam, A-E: fractions A-E, AV(R): reconstituted Apocynum venetum extract, AV: Apocynum venetum Cont. Diaz. A B C D E AV(R) AV AV extract, reconstituted extract, and fractions equivalent to 30 mg/kg # of values 10 10 10 10 10 10 10 10 10 % Time OA Mean 2.250 11.79 6.010 0 5.750 11.15 9.570 9.240 6.890 10.16 Std. Dev. 1.403 5.191 3.119 0 1.880 5.788 6.036 6.977 2.872 5.776 Std. Error 0.4438 1.641 0.9863 0.5947 1.830 1.909 2.206 0.9083 1.827 # Entries OA Mean 2.700 6.900 3.400 0 2.500 5.800 4.900 0 3.000 0 3.300 4.300 Std. Dev. 1.567 2.601 2.171 0 0.8498 2.936 2.846 0 1.826 0 1.636 2.214 Std. Error 0.4955 0.822 0.6864 0.2687 0.9286 0.9000 0.5774 0.5175 0.700 AV extract, reconstituted extract, and fraction s equivalent to 125 mg/kg # of values 10 10 10 10 10 10 10 10 10 % Time OA Mean 2.520 12.04 8.410 2.990 14.64 6.220 7.950 6.010 8.350 Std. Dev. 1.057 3.664 3.301 1.688 7.311 3.988 4.097 2.658 3.941 Std. Error 0.3343 1.159 1.044 0.5336 2.312 1.261 1.29 6 0.8406 1.246 # Entries OA Mean 2.600 8.900 4.200 2.500 6.400 3.800 4.900 0 3.300 4.500 Std. Dev. 1.350 3.071 1.932 0.9718 2.221 2.530 2.025 0 1.703 2.014 Std. Error 0.4269 0.971 0.6110 0.3073 0.7024 0.800 0.6403 0.5385 0.636

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129 Table A-15 Results of EPM for GABA antagonism of AV fractions. Cont.: Control, Diaz.: Diazepam, A-E: fractions A-E Cont. Diaz. A C D E GABA antagonism of fractions equivalent to 30 mg/kg whole extract # of values 10 10 N .a. 10 10 10 % Time OA Mean 3.480 9.520 N .a. 9.000 3.85 0 8.290 Std. Dev. 2.753 4.776 N .a. 3.960 2.508 3.795 Std. Error 0.8705 1.510 N .a. 1.252 0.7931 1.200 # Entries OA Mean 3.400 6.000 N .a. 4.700 2.700 3.500 Std. Dev. 2.675 3.197 N .a. 2.111 0.9487 2.068 Std. Error 0.8459 1.011 N .a. 0.6675 0.3000 0.6540 GABA antagonism of fractions equivalent to 125 mg/kg whole extract # of values 10 10 10 10 N .a. 10 % Time OA Mean 3.480 9.520 4.620 7.520 N .a. 6.470 Std. Dev. 2.753 4.776 2.523 3.852 N .a. 6.314 Std. Error 0.8705 1.510 0.7979 1.218 N .a. 1.997 # Entri es OA Mean 3.400 6.000 2.900 4.250 N .a. 4.100 Std. Dev. 2.675 3.197 1.370 2.188 N .a. 3.178 Std. Error 0.8459 1.011 0.4333 0.7734 N .a. 1.005

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130 Table A-16 Results of EPM for 5-HT1A antagonism of AV fractions. Cont.: Control, Busp.: Buspirone, A-E: fractions A-E Cont. Busp A C D E 5 HT 1A antagonism of fractions equivalent to 30 mg/kg whole extract # of values 10 10 N .a. 10 10 10 % Time OA Mean 3.360 7.760 N.a. 3.640 2.280 1.550 Std. Dev. 2.131 1.930 N.a. 1.750 2.212 1.870 Std. Error 0.6740 0.6103 N .a. 0.5534 0.6995 0.5913 # Entries OA Mean 2.300 3.600 N.a. 2.200 1.500 0.9000 Std. Dev. 1.160 1.506 N.a. 1.398 1.354 0.8756 Std. Error 0.3667 0.4761 N.a. 0.4422 0.4282 0.2769 5 HT 1A antagonism of fractions equivalent to 125 mg/kg whole extract # of values 10 10 10 10 N.a. 10 % Time OA Mean 3.360 7.760 8.690 10.65 N.a. 3.580 Std. Dev. 2.131 1.930 3.562 4.643 N.a. 3.467 Std. Error 0.6740 0.6103 1.126 1.468 N.a. 1.096 # Entries OA Mean 2.300 3.600 4.400 4.800 N.a. 2.700 Std. Dev. 1.160 1.506 1.43 0 1.751 N.a. 1.567 Std. Error 0.3667 0.4761 0.4522 0.5538 N.a. 0.4955

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131 Table A-17 Results of EPM for concomitant antagonism of fraction C and AV. Cont.: Control, Sal.: Saline, Diaz.: Diazepam, Busp.: Buspirone, Fract. C: fraction C, AV: Apocynum venetum, Flum.: Flumazenil, WAY: WAY-100635 Cont./ Sal. Flum./WAY (i.p.) Diaz. Busp. Fract. C Fract. C Cont. AV AV Dose (mg/kg) 1.5 10 30 125 0 30 125 # of values 10 10 10 10 10 10 10 10 % Time OA Mean 5.970 6.940 5.380 4.880 5.010 2.760 4.820 4.960 Std. Dev. 3.352 3.262 2.453 2.778 4.909 3.089 1.711 3.762 Std. Error 1.060 1.032 0.7757 0.8784 1.552 0.9770 0.5412 1.190 # Entries OA Mean 3.100 4.000 2.900 2.500 3.100 1.300 3.000 3.000 Std. Dev. 1.370 1.944 1.197 1.841 1.853 1.337 3.266 2.211 Std. Error 0.4333 0.6146 0.3786 0.5821 0.5859 0.4230 1.033 0.6992 Table A 18 Results of EPM for diazepam and buspirone antagonism. Cont.: Control, Diaz.: Diazepam, Busp.: Buspirone Cont. Diaz. Busp. Diazepam antagonism by WAY 100635 # of values 10 10 10 % Time OA Mean 3.360 7.760 9.790 Std. Dev. 2.131 1.930 5.037 Std. Error 0.6740 0.6103 1.593 # Entries OA Mean 2.300 3.600 5.500 Std. Dev. 1.160 1.506 2.415 Std. Error 0.3667 0.4761 0.7638 Buspirone antagonism by flumazenil # of values 10 10 10 % Time O A Mean 3.480 9.520 5.260 Std. Dev. 2.753 4.776 2.432 Std. Error 0.8705 1.510 0.7690 # Entries OA Mean 3.400 6.000 3.400 Std. Dev. 2.675 3.197 1.075 Std. Error 0.8459 1.011 0.3399

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132 Table A-19 Results of EPM for fractions of AV extract. Cont.: Control, Diaz.: Diazepam, sA-sE: subfractions sA-sE of fraction C derived from Apocynum venetum extract Cont. Diaz. sA sB sC sD sE Subfractions of fraction C equivalent to 30 mg/kg # of values 10 10 10 10 10 10 10 % Time OA Mean 5.530 11.88 12.56 8.330 15. 11 7.050 7.480 Std. Dev. 3.077 5.835 5.275 5.440 4.690 4.630 4.612 Std. Error 0.9730 1.845 1.668 1.720 1.483 1.464 1.459 # Entries OA Mean 4.000 0 8.000 7.300 0 4.700 0 8.300 4.000 0 4.200 0 Std. Dev. 2.108 0 3.367 2.584 0 2.312 0 2.406 2.708 0 2.741 0 Std. Er ror 0.6667 1.065 0.8172 0.7311 0.7608 0.8563 0.8667 Subfractions of fraction C equivalent to 125 mg/kg # of values 10 10 10 10 10 10 10 % Time OA Mean 7.140 19.41 0 17.58 10.90 9.930 12.07 8.830 Std. Dev. 3.204 8.830 10.03 4.988 4.201 6.525 4.988 Std. Error 1.013 2.792 3.171 1.577 1.328 2.063 1.577 # Entries OA Mean 4.500 0 10.70 0 8.700 0 7.200 5.400 6.700 4.500 0 Std. Dev. 2.369 0 4.877 2.452 0 3.190 2.366 3.234 2.461 0 Std. Error 0.7491 1.542 0.7753 1.009 0.7483 1.023 0.7782 Table A-20 Results of Open Field Test for AV extract and antagonists. Cont.: Control, Sal.: Saline, Diaz.: Diazepam, Busp.: Buspirone, AV: Apocynum venetum, Flum.: Flumazenil, WAY: WAY-100635 Cont./ Sal. Diaz./ Sal. Busp./ Sal. AV 30/ Sal. AV 125/ Sal. Sal./ Flum. Sal./ WAY # of values 7 7 7 7 7 7 7 # of Field Transitions Mean 138.5 141.3 113.0 125.7 151.6 133.0 134.1 Std. Dev. 45.04 28.41 43.67 36.03 45.46 46.08 40.05 Std. Error 15.93 10.04 16.51 14.71 17.18 16.29 14.16 Total Distance (mm) Mean 4198 4478 3267 3931 4216 390 8 3972 Std. Dev. 1387 672.9 992.0 1009 1232 1182 1229 Std. Error 490.3 237.9 374.9 411.8 465.6 417.9 434.6

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133 Chapter 4 Table A-21 Results of EPM for kaempferol (p.o.) dose-response profile. Cont.: Control, Diaz.: Diazepam, K.: Kaempferol Cont. Diaz. K. 0.01 K. 0.02 K. 0.08 K. 0.5 K. 1.0 # of values 11 13 13 14 10 10 11 % Time OA Mean 2.400 7.946 5.462 9.121 10.93 9.680 9.209 Std. Dev. 1.288 4.400 3.746 4.929 7.040 6.814 4.191 Std. Error 0.3885 1.220 1.039 1.317 2.226 2.155 1.264 # Entries OA Mean 2.182 5.077 3.769 5.357 5.500 5.100 3.909 Std. Dev. 1.779 2.565 2.166 2.023 3.274 3.143 2.119 Std. Error 0.5363 0.7113 0.6008 0.5407 1.035 0.9939 0.6390 Table A-22 Results of EPM for kaempferol (i.p.) dose-response profile. Cont.: Control, Sal.: Saline, Diaz.: Diazepam, K.: Kaempferol Cont./Sal. Diaz./Sal. Cont./K. 0.02 Cont./K. 0.5 Cont./K. 1.0 # of values 10 10 10 10 10 % Time OA Mean 2.550 9.090 5.080 4.940 4.380 Std. Dev. 1.597 4.795 2.995 2.904 3.189 Std. Error 0.5051 1.516 0.9473 0.9182 1.0 08 # Entries OA Mean 2.100 6.100 3.200 3. 400 2.700 Std. Dev. 0.8756 2.283 1.317 1.430 1.947 Std. Error 0.2769 0.7219 0.4163 0.4522 0.6155 Table A-23 Results of EPM for quercetin and myricetin (p.o.). Cont.: Control, Diaz.: Diazepam, Q.: Quercetin, M.: Myricetin Cont. Diaz. Q 0.02 Q 0.5 M. 0.02 M. 0.5 # of values 10 10 10 10 10 10 % Time OA Mean 2.580 7.670 8.060 9.990 4.060 6.230 Std. Dev. 1.802 3.356 4.293 6.803 2.432 3.790 Std. Error 0.5698 1.061 1.358 2.151 0.7692 1.198 # Entries OA Mean 2.600 6.000 5.500 5.900 3.800 5.200 Std. Dev. 1.506 2.582 2.173 2.234 1.989 2.530 Std. Error 0.4761 0.8165 0.6872 0.7063 0.6289 0.8000

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134 Table A-24 Results of EPM for GABA antagonism of kaempferol (p.o.). Cont.: Control, Sal.: Saline, Diaz.: Diazepam, K.: Kaempferol, Flum.: Flumazenil Cont./ Sal. Diaz./ Sal. K. 0.02/ Sal.. K. 0.02/ Flum. K. 0.08/ Sal. K. 0.08/ Flum. # of values 10 10 10 10 10 10 % Time OA Mean 3.870 13.38 15.42 12.73 15.02 5.540 Std. Dev. 2.037 7.074 7.630 6.516 9.584 3.935 Std. Er ror 0.6441 2.237 2.413 2.061 3.031 1.244 # Entries OA Mean 2.900 8.200 7.800 6.400 6.900 3.500 Std. Dev. 1.197 3.765 2.741 3.836 3.071 1.780 Std. Error 0.3786 1.191 0.8667 1.213 0.9713 0.5627 Table A-25 Results of EPM for antagonism of quercetin (p.o.). Cont.: Control, Sal.: Saline, Diaz.: Diazepam, Q.: Quercetin, Flum.: Flumazenil, WAY: WAY-100635 Cont./ Sal. Diaz./ Sal. Q. 0.5/ Sal.. Q. 0.5/ Flum. Q. 0.5/ WAY # of values 10 10 10 10 10 % Time OA Mean 3.170 8.080 6.330 6.420 7.260 Std. Dev. 2.17 9 2.312 2.234 2.657 3.523 Std. Error 0.6890 0.7312 0.7065 0.8402 1.114 # Entries OA Mean 2.800 5.400 4.100 3.800 4.100 Std. Dev. 1.751 1.506 1.370 1.229 1.287 Std. Error 0.5538 0.4761 0.4333 0.3887 0.4069

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135 Table A-26 Results of EPM for 5-HT1A antagonism of kaempferol (p.o.). Cont.: Control, Sal.: Saline, Busp.: Buspirone, K.: Kaempferol, WAY: WAY-100635 Cont./ Sal. Busp. / Sal. K. 0.02/ Sal.. K. 0.02/ WAY K. 0.08/ Sal. K. 0.08/ WAY # of values 10 10 10 10 10 10 % Time OA Mean 3.730 10.89 15.42 11. 22 15.02 9.640 Std. Dev. 1.885 4.281 7.630 6.380 9.584 4.002 Std. Error 0.5961 1.354 2.413 2.018 3.031 1.266 # Entries OA Mean 2.700 5.400 7.800 4.600 6.900 5.600 Std. Dev. 1.337 1.838 2.741 1.955 3.071 2.011 Std. Error 0.4230 0.5812 0.8667 0.6182 0. 9713 0.6360 Table A-27 Results of Open Field test for kaempferol (p.o.). Cont.: Control, Diaz.: Diazepam, K.: Kaempferol Cont. Diaz. K. 0.02 K. 0.08 # of values 8 8 8 8 # Field Transitions Mean 64.63 55.63 62.13 61.88 Std. Dev. 22.73 21.25 18.84 21. 30 Std. Error 8.038 7.514 6.661 7.532 Total Distance (mm) Mean 2511 2227 2295 2302 Std. Dev. 707.2 782.9 592.5 622.0 Std. Error 250.0 276.8 209.5 219.9 Table A-28 Results of Open Field test for quercetin (p.o.). Cont.: Control, Diaz.: Diazepam, Q.: Quercetin Cont. Diaz. Q. 0.5 # of values 8 8 8 # Field Transitions Mean 81.75 65.75 75.00 Std. Dev. 15.03 15.40 24.08 Std. Error 5.314 5.444 8.513 Total Distance (mm) Mean 2704 2478 2385 Std. Dev. 374.9 526.1 545.8 Std. Error 132.5 186.0 193.0

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136 Table A-29 Results of EPM for pHPAA (i.p.) dose-response profile. Cont.: Control, Sal.: Saline, Diaz.: Diazepam, pHPAA: para-hydroxyphenylacetic acid Cont./Sal. Diaz./Sal. Cont./pHPAA Dose (mg/kg) 1.5 0.001 0.005 0.01 0.02 0.08 0.5 1.0 # of values 10 1 0 10 10 10 10 10 10 10 % Time OA Mean 5.970 13.33 4.180 6.660 14.35 14.64 8.780 9.060 9.120 Std. Dev. 3.857 7.625 3.387 4.285 5.780 4.446 3.535 4.920 4.214 Std. Error 1.220 2.411 1.071 1.355 1.828 1.406 1.118 1.556 1.332 # Entries OA Mean 3.400 8.000 2.700 3.900 8.700 9.300 4.500 3.700 4.300 Std. Dev. 2.221 5.011 1.636 3.348 3.622 2.312 2.173 2.111 1.947 Std. Error 0.7024 1.585 0.5175 1.059 1.146 0.7311 0.6872 0.6675 0.6155 Table A-30 Results of EPM for pHPAA (p.o.) dose-response profile. Cont.: Control, Diaz.: Diazepam, pHPAA: para-hydroxyphenylacetic acid Cont. Diaz. pHPAA 0.02 pHPAA 0.5 pHPAA 1.0 # of values 10 10 10 10 10 % Time OA Mean 3.330 9.190 4.790 7.230 5.070 Std. Dev. 1.875 5.655 2.629 3.908 3.360 Std. Error 0.5929 1.788 0.8313 1. 236 1.062 # Entries OA Mean 2.400 5.100 2.800 3.600 3.400 Std. Dev. 0.9661 2.807 1.874 1.838 2.066 Std. Error 0.3055 0.8876 0.5925 0.5812 0.6532

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137 Table A-31 Results of EPM for antagonism of pHPAA (i.p.). Cont.: Control, Sal.: Saline, Diaz.: Diazepam, Busp.: Buspirone, pHPAA: para-hydroxyphenylacetic acid, Flum.: Flumazenil, WAY: WAY-100635 Cont./ Sal. Diaz./ Sal. Busp./ Sal. Cont./pHPAA 0.01 Cont./pHPAA 0.02 Sal. Sal. Sal. Sal. Flum. WAY Sal. Flum. WAY # of values 20 17 10 14 13 11 10 13 12 % Tim e OA Mean 4.130 0 8.782 7.870 9.550 4.600 0 9.264 8.250 4.400 0 9.658 Std. Dev. 1.795 0 4.537 2.744 4.139 2.339 0 4.406 3.986 2.609 0 5.434 Std. Error 0.4014 1.100 0.8678 1.106 0.6488 1.328 1.260 0.7235 1.569 # Entries OA Mean 2.350 0 6.118 3.900 5.071 0 2.69 2 0 4.727 0 5.100 0 2.308 0 4.500 0 Std. Dev. 0.9881 4.372 2.283 3.245 0 1.437 0 2.867 0 2.767 0 1.182 0 2.468 0 Std. Error 0.2209 1.060 0.7219 0.8674 0.3985 0.8644 0.8750 0.3279 0.7124 Table A-32 Results of Open Field test for pHPAA (i.p.). Cont.: Control, Sal.: Saline, Diaz.: Diazepam, Busp.: Buspirone, pHPAA: para-hydroxyphenylacetic acid Cont./Sal. Diaz./Sal. Busp./Sal. Cont./pHPAA 0.02 # of values 10 10 10 10 # Field Transitions Mean 62.90 57.10 63.70 51.00 Std. Dev. 24.43 10.06 17.56 17.28 Std. Error 7 .727 3.181 5.554 5.465 Total Distance (mm) Mean 2198 2281 2257 1904 Std. Dev. 594.5 360.7 557.3 444.5 Std. Error 188.0 114.1 176.2 140.6

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150 BIOGRAPHICAL SKETCH Oliver Grundmann, born in 1978 in Osnabrueck, Germany, received his high school diploma in 1997 from Hannah-Arendt Gymnasium in Lengerich, Germany. He entered college to start his pharmacy studies in 1998 after completion of a one-year civil service obligation with the regional hospital in Lengerich, Germany as a nurse assistant. Upon completion of his pharmacy studies at the Westfaelische-Wilhelms-Universitaet in Muenster, Germany and receiving his European pharmacist license in spring 2004, he was offered a position as a graduate student with the research group of Dr. Veronika Butterweck at the University of Florida. He entered the program in August 2004 and started work as a teaching assistant in the College of Pharmacy. In 2005, he was granted a JSPS summer fellowship to visit the research group of Dr. Junji Terao at the University of Tokushima, Japan. In the same year, he pursued a Master of Science in forensic toxicology with a minor in statistics, which he completed in spring 2007. He started an additional teaching position with the distance education program in forensic sciences in summer 2007. He received a poster award at the Annual Meeting of the American Society of Pharmacognosy in summer 2007. Currently, he has published four peer-reviewed original reseach papers and one review article. His doctoral studies focused on the anxiolytic and antidepressant activity of natural products and their pharmacology. Oliver Grundmann graduated with a Doctor of Philosophy in pharmaceutical sciences in fall 2007.