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Electronic Theses and Dissertations Graduate School

2010

Science Based Authentication of Dietary Supplements: Studies on the Chemical Constituents, Analytical Method and Biological Activities of Scutellaria Spp and Pfaffiaaniculata P

Jing Li

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This Dissertation is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. SCIENCE BASED AUTHENTICATION OF DIETARY SUPPLEMENTS

---STUDIES ON THE CHEMICAL CONSTITUENTS, ANALYTICAL METHOD AND

BIOLOGICAL ACTIVITIES OF SCUTELLARIA SPP AND PFAFFIA PANICULATA

KUNTZE

A Dissertation

presented in partial fulfillment of requirements for the degree of Doctor of Philosophy in the Department of Pharmacognosy The University of Mississippi

JING LI

Nov. 2010

Copyright © 2010 by Jing Li

All rights reserved

ii ABSTRACT

During the last decade, the use of herbal medicine has expanded globally and gained popularity. With the tremendous expansion in the use of herbal medicine worldwide, safety and efficacy as well as quality control of herbal medicines have become more and more issues of concern for both health authorities and the public. Although herbal medicine has been in use for hundreds to thousands years, very limited science-based data exist to explicit its chemical constituents, pharmacological activities and toxicity. Therefore, lack of scientific evidence for herbal medicine usage elicits the need for systematic studies of herbal medicine, including phytochemical, analytical, and biological investigation

Two projects are included in the dissertation. The first one is the “Science Based

Authentication of Dietary Supplements - Studies of Scutellaria Species on the Chemical

Constituents, Analytical Method, and Biological Activities”. The second project is the

“Phytochemical Investigation of Pfaffia paniculata Kuntze”. American skullcap (Scutellaria lateriflora L.), belonging to the family Labiatae, is native to North America and was used as a tonic, tranquilizing, and antispasmodic remedies. Since 1989 safety concerns about American skullcap were raised due to the reports of hepatotoxic reactions after skullcap-containing preparations were ingested. Therefore, this plant was listed in the FDA Poisonous Plant

Database and has been classified as an “Herb of Undefined Safety”. Meanwhile, it was reported to be adulterated with germander, e.g. Teucrium chamaedry, and T. canadense, which is a group of plants with hepatotoxicity. Very few studies have been previously reported associated with

iii the quality control, toxicity investigation, and pharmacological activities of this herbal medicine as well as compounds isolated from it. The primary objective of this project is to explore the chemical constituents of American skullcap, which will be used as reference markers to develop a validated chemical fingerprinting method for the differentiation of it from its adulterant, germander. From the MeOH extract of the aerial parts of this plant sixteen compounds, including three minor coumarins in which two are new, six flavonoids, four triterpenes, two steroids, and one fatty acid were isolated and characterized. In order to compare the similarity of chemical components between American skullcap and Chinese skullcap (S. baicalensis), also to obtain more marker compounds, the chemical components of Chinese skullcap, which is the most often used herb medicine in this genus, were also studied and seventeen flavonoids have been isolated. Based on the chemical constituents, a simple HPLC-PDA fingerprinting method was developed, which can be used to authenticate S. lateriflora species, to distinguish it from its potential adulterants, to compare the chemical profiles of related species in this genus, and to assess the quality of commercial products claiming to contain S. lateriflora. In the present study three classes of compounds, including eleven flavonoids isolated from two Scutellaria species mentioned above and two phenylpropanoids, as well as one diterpene isolated from T. canadense, were used as markers simultaneously to provide comprehensive analysis for the above mentioned purposes. As part of the efforts of this project, a HPLC/ESI-MSn method was established which allowed a fast characterization of thirteen flavonoids and one stilbene derivative from American skullcap without time-consuming isolation of compounds even if

iv reference compounds are not available. The biological activities of the two skullcap extracts and thirteen flavonoids isolated from them were evaluated on a panel of bioassays, including cytotoxicity, antioxidant, anti-inflammatory, estrogenic activity and activations on peroxisome proliferator-activated receptor-alpha and -gamma (PARP and PPARγ). Neither skullcap extracts nor pure compounds tested showed cytotoxicity against a panel of mammalian normal and solid tumor cell lines at the tested concentrations. During the investigation of chemical constituents of American skullcap no neoclerodane diterpenes, which are responsible for the hepatotoxicity in germander (Kouzi et al., 1994), were found. Taken together, these evidences suggest that the reports of hepatotoxic reactions on American skullcap might very likely be due to its adulterant germander. In the present bioassay study, the most significant finding was the identification of PPARs activation of skullcap extracts as well as several characteristic flavonoids, such as baicalein, baicalin, wogonin, and chrysin etc. that provides some information on possible new application of skullcap, especially American skullcap, for the treatment of diabetic-related diseases. Also the potent activity of these flavonoids on PPARs suggests the potential of these flavonoids as leads for PPARα and PPARγ agonists.

P. paniculata Kuntze, belonging to the family Amaranthaceae, is a large, rambling, shrubby ground vine with an intricate, deep, and extensive root system. It is the most employed species in commercial preparations in Brazil as “Brazilian ginseng” and has been commonly used for three centuries with the same indications as American and Asian ginseng. More than fifty preparations, which contained or were produced from plant P. paniculata are available on

v USA market, but no analytical method(s) existed to assess the quality of P. paniculata raw materials, and the products claiming to contain P. paniculata. In order to develop an appropriate validated quantitative analytical method for the quality control of this plant material and dosage form the detailed phytochemical investigation of P. paniculata was carried out. Two new nortriterpenoids pfaffine A and B, one new compound pfaffine C, along with eleven known compounds were isolated and identified from the roots of this plant. These compounds will be used as markers for quality control of raw materials of P. paniculata as well as products claiming to contain this species.

vi DEDICATION

This dissertation is dedicated to my family who loved and supported me; and to prof.

Ikhlas A. Khan who encouraged and assisted me during the years of my studies.

vii LIST OF ABBREVIATION

12-LOX 12-lipoxygenase ADME absorption, distribution, metabolism, and excretion AFB1 aflatoxin B1 AHP American Herbal Pharmacopoeia AHPA American Herbal Products Association AIDS acquired immune deficiency syndrome ALT Alanine transaminase AP acetaminophen AP-1 activator protein-1 APCI atmospheric pressure chemical ionisation API atmospheric pressure ionisation ATCC American Type Culture Collection AZT 3‟-Azido-2‟,3‟-dideoxythymidine BDS botanical dietary supplement CAM complementary/alternative medicine CC column chromatography CD circular dichroism CE capillary electrophoresis CEs cotton effects Cf-FAB continuous flow-FAB CHO Chinese hamster ovary CID collision induced dissociation CIP Cahn–Ingold–Prelog COSY correlation spectroscopy COX-2 cyclooxygenase-2 DCFH-DA 2′,7′-dichlorodihydrofluorescein diacetate DEPT distortionless enhancement by polarization transfer DNA deoxyribonucleic acid DPPH 2,2-diphenyl-l-picrylhydrazyl DS dietary supplement DSCG disodium cromoglycate DSHEA Dietary Supplement and Health Education Act dTTP thymidine triphosphate E2 17 β -estradiol ECD electronic circular dichroism EI electron impact ELSD evaporative light scattering detector

viii EMEA European Medicines Evaluation Agency EPC electroplanar chromatography ESI electrospray ionisation ESR electron spin resonance FAB fast atom bombardment FBS fetal bovine serum FDA U.S. Food and Drug Administration GC gas chromatography HNSCC head and neck squamous cell carcinoma HBV hepatitis B virus HIV-1 human immunodeficiency virus -1 HMBC heteronuclear multiple-bond connectivity HPLC high performance liquid chromatography HSCCC high-speed counter-current chromatography HSQC heteronuclear single quantum coherence HTLV-1 human T-lymphotropic virus type-1 HX-XOD hypoxanthine-xanthine oxidase IC50 inhibition concentration at 50% ICAM intercellular adhesion molecule IL interleukin IR infrared i.p. intraperitoneal injection iNOS inducible NO synthase LPS lipopolysaccharide LTs leukotrienes MALDI matrix-assisted laser desorption ionisation MECC micellar electrokinetic capillary chromatography MIC minimum inhibitory concentration MLV murine leukemia viruses mRNA messenger RNA MRSA methicillin-resistant Staphylococcus aureus MS mass spectrometry NCNPR National Center for Natural Products Research NCS newborn calf serum NF-ƙ B nuclear factor-ƙ B NMR nuclear magnetic resonance NOESY nuclear overhauser effect spectroscopy ODS octadecyl silane OPLC overpressured-layer chromatography

ix PAC passive cutaneous anaphylaxis PBMC peripheral blood mononuclear cells PCR polymerase chain reaction PDGF-A platelet-derived growth factor-A PGE2 prostaglandin E2 PHA phytohemagglutinin PKC protein kinase C PMA phorbol 12-myristate-13-acetate PMS phenazine methosulfate PPAR peroxisome proliferator-activated receptor PSA prostate-specific antigen RLU relative luciferase units RNA ribonucleic acid ROS reactive oxygen species RPC rotation planar chromatography RP-IP-HPLC reversed-phase ion-pairing HPLC RSD relative standard deviation RSV respiratory syncytial virus RT reverse transcriptases SAX-HPLC strong anion-exchange HPLC SBE extract of S. baicalensis SEC size-exclusion chromatography SFDA State Food and Drug Administration in China SLE extract of S. lateriflora TGF-β1 transforming growth factor-β1 TIC total ion current TLC thin-layer chromatography TNF tumor necrosis factor TPA 12-O-tetradecanoylphorbol-13-acetate tR retention time TSP thermospray ionisation UV ultraviolet absorbance VLC vacuum liquid chromatography WHO World Health Organization XTT 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]- 2H-tetrazolium hydroxide YES yeast estrogen screen

x ACKNOWLEDGMENTS

Throughout my academic experiences, there have been many people who deserve credit and appreciation for their roles in making this dissertation possible. First and foremost is my advisor, Dr. Ikhlas A. Khan. It would be impossible for me to have this exceptional journey that I had dreamed of without the support and guidance of Dr. Khan. I would like to thank Dr. Khan for giving me the freedom to pursue the things that I found most interesting. I also truly appreciate his understanding, patience and inspiring through this period of my life and career.

I would like to give my heartfelt appreciation to my committee members: Dr. Larry A.

Walker, Dr. Mahmoud El Sohly, Dr. Jordan K. Zjawiony, and Dr. Marc Slattery for their very helpful insights, constructive comments and timely suggestions. I would also like to thank all the faculty members of our department for sharing their valuable knowledge, and rigorous attitude toward scientific research. Sincere thanks are given to Dr. Daneel Ferreira for his help in writing manuscript.

I would also like to thank Dr. Shabana Khan for a series of biological studies of the crude extracts as well as pure compounds isolated during my Ph. D. work. I am grateful for her help and contributions to my dissertation.

I am fortunate in having the opportunity to work with the friendly people in Dr. Khan‟s research group at NCNPR, who have always given me a hand whenever I needed. I really appreciate all of their friendship and encouragement to finish this dissertation. Special thanks are given to Dr. Yanhong Wang for his help in LC-MSn and HPLC experiments; to Dr. Troy

xi Smillie for constant support; to Dr. Vaishali Joshi for the authentication of the plant material, and to Dr. Yuanqing Ding for the calculation of CD spectra of the new compounds.

This dissertation is funded in part by the Food and Drug Administration (FDA) “Science

Based Authentication of Dietary Supplements” under grant number 2U01 FD 002071-07 and the

United States Department of Agriculture, Agriculture Research Service Specific Cooperative

Agreement under No. 58-6408-06-067. I would like to thank the financial support by NCNPR and Dr. Khan for providing graduate research assistantship. I would also like to thank the Center for Research Excellence in Natural Products Neuroscience (CORE-NPN) for providing the neuroscience fellowship during the year 2009.

I would like to acknowledge the Department of Pharmacognosy, School of Pharmacy for providing all requested assistance. I would also like to thank my fellow graduate students, past and present members, for their friendship that I have enjoyably experienced.

I would like to give my deepest expression of love and appreciation to my wonderful family. Particularly to my understanding and patient husband, Dr. Jiangnan Peng, who has been proud and supportive of my work and who has shared the many uncertainties, challenges and sacrifices for completing this dissertation. His love and support provided me the strength and perseverance I needed to achieve my goals. To my precious son, Peter and daughter Lily, whose innocence and happiness taught me the beauty of life and the patience to live it. To my parents

Fuqiang Li, and Lanqin Gu, who has been my role-model for hard work, persistence and personal sacrifices, and who instilled in me the inspiration to set high goals and the confidence to

xii achieve them.

Each and everyone listed above and others who have not been named directly, but whose friendship and encouragement remains important to me, deserve my gratitude and my admiration for supporting me throughout this journey.

xiii TABLE OF CONTENTS

CHAPTER PAGE

Chapter 1. Introduction

1.1. General background 1

1.2. Overview of the Scutellaria genus 2

1.3. Objectives of the dissertation 21

Chapter 2. Extraction, Isolation, and Characterization of Chemical Constituents of Scutellaria

lateriflora L. and S. baicalensis Georgi

2.1. Introduction 24

2.2. Experimental sections 25

2.2.1. General experimental procedures 25

2.2.2. Plant material 25

2.2.3. Extraction and isolation 26

2.3. Results and discussion 32

2.4. Conclusion 48

Chapter 3. Chemical Fingerprint Analysis of Scutellaria Species, Germander and Dietary

Supplements Claiming to Contain Skullcap Using HPLC-PDA

3.1. Introduction 49

xiv 3.2. Background and objectives 52

3.3. Experimental sections 54

3.3.1. Reagents and chemicals 54

3.3.2. Standard solutions 55

3.3.3. Sample preparation 55

3.3.4 Instrumentation and HPLC conditions 56

3.3.5. Method development 58

3.4. Results and discussion 65

3. 5. Conclusion 72

Chapter 4. Identification of Flavonoids in Scutellaria lateriflora Using High-Performance Liquid

Chromatography-Electrospray Tandem Mass Spectrometry-Ultraviolet Photodiode

Array

4.1. Introduction 73

4.2. Experimental sections 75

4.2.1. Reagents and chemicals 75

4.2.2. Standard solutions 76

4.2.3. Sample preparation 77

4.2.4. LC/ESI-MSn conditions 77

4.3. Results and discussion 78

4.3.1 Fragmentation pathways of twelve standard flavonoids 79

xv 4.3.2 Characterization of phenolic compounds from S. lateriflora using HPLC-DAD/ESI-MSn 91

4.4. Conclusion 99

Chapter 5. Evaluation of Bioactivities of Methanol Extracts of S. baicalensis L. and S. lateriflora

Georgi and Flavonoids Isolated from Them

5.1. Introduction 101

5.2. Materials and methods 102

5.2.1. Materials 102

5.2.2. Methods 105

5.2.2.1. Assays for in vitro cytotoxicity 105

5.2.2.2. Assay for antioxidant activity-inhibition of intracellular ROS generation 105

5.2.2.3. Assay for anti-inflammatory activity (inhibition of NF-kB) 106

5.2.2.4. Assay for estrogenic activity 107

5.2.2.5. Assay for the activity of PPARα and PPARγ in CHO cells 108

5.3. Results and Discussion 108

5.3.1. In vitro cytotoxicity 108

5.3.2. Antioxidant activity 109

5.3.3. Anti-inflammatory activity 111

5.3.4. Estrogenic activity 113

5.3.5. Activation of PPARα and PPARγ 115

xvi 5.4. Conclusion 119

Chapter 6. Introduction of Pfaffia paniculata Kuntze 125

Chapter 7. Phytochemical investigation of Pfaffia paniculata (Martius) Kuntze

7.1. Introduction 131

7.2. Experimental sections 135

7.2.1. General experimental procedures 135

7.2.2. Plant material 135

7.2.3. Extraction and isolation 135

7.3. Results and discussion 140

7.4. Conclusion 164

Chapter 8. Concluding remarks

8.1. Contributions of this dissertation 165

8.2. Future work 168

8.3. Related publications 169

Bibliography 171

Vita 294

xvii LIST OF TABLES

1-1. The Species Studied and the Number of Phenolic Compounds Reported 5

1-2. The Number of Neoclerodane Diterpenoid Reported in Scutellaria Species 6

1-3. Ten Commonly Used BDS and Their Potential Adulterants (AHPA) 23

2-1. NMR Data for Compounds 1 and 2 in MeOH-d4 35

2-2. Conformational Analysis of Compound 1 41

2-3. Key Transitions and Oscillator and Rotatory Strengths of Conformer 1a of Compound 1 at

the B3LYP/6-31G** Level in the Gas Phase 42

3-1. Species Analyzed in Present Study 58

3-2. Products Claiming to Contain S. lateriflora 58

3-3. HPLC Gradient Condition I for Fingerprint Analysis 61

3-4. HPLC Gradient Condition II for Fingerprint Analysis 61

3-5. HPLC Gradient Condition III for Fingerprint Analysis 62

3-6. HPLC Gradient Condition IV for Fingerprint Analysis 63

3-7. Retention Times and UV Absorptions of Standard Flavonoids Obtained Under Optimized

HPLC Conditions 64

4-1. LC-UV/MSn Characteristics of of Twelve Standard Flavonoids 81

4-2. ESI-MSn Product Ions of Three Methoxylated flavonoids 86

4-3. Relative Intensity of Ions in ESI-MS/MS of Chrysin and Daidzein 89

xviii 4-4. LC-UV/ESI-MSn Identification of Phenolic Compounds in S. lateriflora 93-94

5-1. Cytotoxicities of Skullcap Extracts and Flavonoids Isolated from Them 110

5-2. Antioxidant Activities of Skullcap Extracts and Flavoniods Isolated from Them 111

5-3. Anti-inflammatory Activity of Skullcap Extracts and Flavonoids Isolated from Them: Effect

on NF- κB Mediated Transcription 113

5-4. Estrogenic Response of Skullcap Extracts and Flavonoids Isolated from Them 115

5-5. Fold Induction of Tested Samples on PPARs at Different Concentrations 121

7-1. NMR Data of Compound 40 in Pyridine-d5 143

7-2. NMR Data of Compound 41 in Pridine-d5 149-150

7-3. 1H-NMR Data of Compound 42a and 42b 160

7-4. 13C-NMR Data of Compound 42a and 42b 161

xix LIST OF FIGURES

2-1. Structures of compounds isolated from S. lateriflora 33

2-2. Key HMBC correlations of compound 1 35

1 2-3. H-NMR spectrum of compound 1 in acetone-d6 36

13 2-4. C-NMR spectrum of compound 1 in acetone-d6 36

1 1 2-5. H- H COSY spectrum of compound 1 in acetone-d6 37

2-6. HSQC spectrum of compound 1 in acetone-d6 37

2-7. HMBC spectrum of compound 1 in acetone-d6 38

2-8. Optimized geometries of conformers of compound 1 at B3LYP/6-31G** level

in the gas phase 40

2-9. Calculated ECD spectra of conformer 1a and the experimental ECD of

compound 1 41

2-10. Electron density surfaces of some orbitals involved in key transitions in the ECD

spectra of 1 at the B3LYP/6-31G** level in the gas phase 42

1 2-11. H-NMR spectrum of compound 2 in acetone-d6 43

13 2-12. C-NMR spectrum of compound 2 in acetone-d6 44

1 1 2-13. H- H COSY spectrum of compound 2 in acetone-d6 44

2-14. HSQC spectrum of compound 2 in acetone-d6 45

2-15. HMBC spectrum of compound 2 in acetone-d6 45

2-16. Structures of compounds isolated from S. baicalensis 47

xx 3-1. Chemical structures of fourteen standard compounds investigated in this study 57

3-2. HPLC chromatogram of S. lateriflora chromatographed using a gradient elution system

shown in Table 4-3 61

3-3. HPLC chromatogram of S. lateriflora chromatographed using a gradient elution system

shown in Table 4-4 62

3-4. HPLC chromatogram of S. lateriflora chromatographed using a gradient elution system

shown in Table 4-5 62

3-5. HPLC chromatogram of S. lateriflora chromatographed using a gradient elution system

shown in Table 4-6 63

3-6. HPLC chromatogram of a mixture of eleven standard flavonoids under optimized gradient

system 64

3-7. HPLC chromatogram of a mixture of three standard compounds isolated from T. candense

under optimized gradient system 65

3-8. HPLC chromatographic fingerprinting profile of S. lateriflora 63

3-9. Comparison of HPLC profiles of S. lateriflora with its potential adulterants detected at

wavelength 250 nm 67

3-10. Comparison of HPLC profiles of Scutellaria related species detected at wavelength

280 nm 69

3-11. Comparison of HPLC profiles of S. lateriflora obtained from different sources detected at

wavelength 280 nm 70

xxi 3-12. Comparison of HPLC profiles of S. barabata from different sources detected at wavelength

280 nm 70

3-13. Comparison of HPLC profiles of different part of S. baicalensis detected at wavelength 280

nm 71

3-14. Comparison of HPLC profiles of commercial products claiming to contain S. lateriflora

detected at wavelength 280 nm 71

4-1. Basic ring structure of flavonoids with labelling convention 74

4-2. Basic structures of the major flavonoid classes 74

4-3. Chemical structures of twelve standard flavonoids investigated 76

4-4. ESI-MS3 spectra of three methoxylated flavone isomers: (a) wogonin; (b) oroxylin A; (c)

gankwanin 84

4-5. HPLC/ESI-MSn analysis of the extract of skullcap 91

4-6. Chemical structures of fourteen compounds identified from skullcap 92

5-1. Structures of flavonoids tested 104

5-2. Effect of tested samples on PPARα activation 122

5-3. Effect of tested samples on PPARγ activation 122

5-4. Dual effects of tested samples on PPARs activation at 10 µM 123

5-5. Dual effects of tested samples on PPARs activation at 30 µM 123

7-1. Structures of compounds isolated from Pfaffia paniculata 134

7-2. Key HMBC and NOE correlations of compound 40 144

xxii 1 7-3. H-NMR spectrum of compound 40 in pyridine-d5 144

13 7-4. C-NMR spectrum of compound 40 in pyridine-d5 145

7-5. DEPT spectrum of compound 40 in pyridine-d5 145

1 1 7-6. H- H COSY spectrum of compound 40 in pyridine-d5 146

7-7. HSQC spectrum of compound 40 in pyridine-d5 146

7-8. HMBC spectrum of compound 40 in pyridine-d5 147

7-9. ROESY spectrum of compound 40 in pyridine-d5 147

1 7-10. H-NMR spectrum of compound 41 in pyridine-d5 151

13 7-11. C-NMR spectrum of compound 41 in pyridine-d5 151

7-12. DEPT spectrum of compound 41 in pyridine-d5 152

1 1 7-13. H- H COSY spectrum of compound 41 in pyridine-d5 152

7-14. HSQC spectrum of compound 41 in pyridine-d5 153

7-15. HMBC spectrum of compound 41 in pyridine-d5 153

7-16. ROESY spectrum of compound 41 in pyridine-d5 154

7-17. Z-E isomerization of compound 42 in aqueous MeOH 156

1 7-18. H-NMR spectrum of compound 42 in MeOH-d4 157

13 7-19. C-NMR spectrum of compound 42 in MeOH-d4 157

1 1 7-20. H- H COSY spectrum of compound 42 in MeOH-d4 158

7-21. HSQC spectrum of compound 42 in MeOH-d4 158

7-22. HMBC spectrum of compound 42 in MeOH-d4 159

xxiii 7-23. Key HMBC correlations of compound 42a 159

7-24. Z-E isomerization test of compound 54 in aqueous MeOH 163

xxiv LIST OF CHARTS

2-1. Scheme for isolation of chemical constitunets from S. lateriflora ...... 30

2-2. Scheme for isolation of chemical constituents from S. baicalensis 31

4-1. Nomenclature adopted for the different retrocyclization cleavage fragments of deprotonated

flavones, flavonols and flavanones observed in this study 79

4-2. Proposed key fragmentation pathways of the anion baicalein ...... 82

4-3. Proposed key fragmentation pathways of the anion wogonin ...... 85

4-4. Proposed key fragmentation pathways of the anion naringenin ...... 87

4-5. Proposed key fragmentation pathways of the anion hesperitin ...... 88

4-6. Proposed key fragmentation pathways of the anion quercetin ...... 90

4-7. Proposed key fragmentation pathways from the anion daidzein ...... 90

4-8. Proposed key fragmentation pathways of compound 32 agylcone ...... 95

7-1. Scheme for isolation of chemical constituents from Pfaffia paniculata ...... 139

xxv

First Project

Studies on Scutellaria Species: Chemical Constituents,

Analytical Methods and Biological Activities

0 Chapter 1

Introduction

1.1. General background

Traditional medicine is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement or treatment of physical and mental illnesses (WHO definition). It involves the use of herbal medicines, animal parts and minerals, among which herbal medicines are the most widely used. The terms complementary/alternative medicine (CAM), non-conventional medicine are used interchangeably with traditional medicine in some countries. During the last decade, use of traditional medicine has expanded globally and has gained popularity. According to the World

Health Organization (WHO), 80% of the population depends on traditional medicine for primary health care in some Asian and African countries. In many developed countries, 70% to 80% of the population has used some form of alternative or complementary medicine (e.g. acupuncture).

With the tremendous expansion in the use of herbal medicine worldwide, safety and efficacy as well as quality control of herbal medicines have become issues of concern for both health authorities and the public.

The safety problems associated with herbal medicines may result from adverse effects of the herbs themselves and/or potential toxicity related to misidentification or mislabeling of the plant species used in herbal medicines; the use of incorrect parts of plants; adulteration and contamination with other plant species, undisclosed prescription or over-the-count drugs or

1 heavy metals when the necessary naturally occurring plant substances are in short supply or expensive or when the supplier intends to intensify a specific pharmacologic effect. In these cases science-based research is needed to provide solid evidence of questionable herbal medicines in order to ensure that traditional medicine is used properly.

Several methods can be used to be certain that the correct plant species has been acquired. These methods include taxonomic examination (macroscopic and/or microscopic) or biochemical [deoxyribonucleic acid (DNA) analysis using polymerase chain reaction (PCR) techniques] or chemical (chromatographic methods) analysis, or metabolomics study. The taxonomic or genetic analysis can only be applied to intact or milled plants and are not possible for them to deal with plant extracts. Under these circumstances chromatographic methods, including, but not limited to, high performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), thin-layer chromatography (TLC), can be used. Among them HPLC coupled with different detectors, such as ultraviolet absorbance (UV) detector, evaporative light scattering detector (ELSD), or mass spectrometry (MS), are the most popularly used methods to identify plants used in the herbal medicines. These methods commonly analyze a group of “active” molecules or arbitrarily chosen “marker” compounds in complex plant extracts. Also, metabolomics can be applied to authenticate the plant raw materials as well as their products. NMR and MS are currently considered to be the most universal approaches for metabolomics study. Acquisition of a complete metabolite profile of a plant through metabolomics allows the identification of the plant species. Some pioneer applications have been presented recently (Chio et al., 2004; Le Gall et al., 2004)

1.2. Overview of the Scutellaria genus

2 Scutelllaria is a unique cosmopolitan genus of the subfamily Scutellarioideae belonging to Labiatae (Lamiaceae) family. About 360 species are found spread throughout the world and in different climate areas (Poton, 1992). There are above 100 species in China and over 90 species in North American.

Essential oils, alcohols (derivatives of phenylethanol) and organic acids (esters and glycosides of vanillic, syringic, and ferulic acids), iridoids, sesquiterpenes, neoclerodane diterpenoids, steroids, cardenolides, tannins, and flavonoids have been isolated from the genus

Scutellaria (Chemesova et al., 1993; Plant Resources of the USSR. 1991). Among them phenolic compounds and clerodane diterpenoids are the main secondary metabolites reported.

Malikov‟s group published a thorough summary of phenolic compounds isolated and identified from about 60 species in this genus up to 2002 (Malikov et al., 2002). Additional new phenolic compounds have been reported from 2002 to 2010. We have updated and summarized these data in Table 1-1. These reported phenolic compounds include: flavones, flavanones, isoflavones, chalcones, biflavones, lignoflavones and phenylpropanoid glycosides. The last one, so-called phenylpropanoids, are biogenetic precursors of flavonoids. These compounds are known to contain reactive phenolic hydroxyls and carbonyls. These groups of compounds are certainly interrelated and undergo mutual transformations (Mabry et al., 1975). Owing to the ability to transform into chalcones and to form quinones, flavonoids are considered to be highly physiologically active compounds with a wide spectrum of pharmacological activities. S. baicalensis is the most commonly used and thoroughly studied species in all aspects in this genus. Sixty-four phenolic compounds have been isolated from this species. Other species have been reported as rich sources of flavonoids including S. rivularis, S. indica, S. prastrata, S.

3 amoena, and S. discolor from which 38, 33, 29, 28, and 27 flavonoids have been isolated, respectively. Neoclerodane diterpenoids are the other characteristic compounds in some species.

Bruno et al. reviewed the neoclerodane diterpenoids isolated from this genus up to June 2001, and reported that 120 natural neoclerodane diterpenoids have been isolated from Scutellaria species. Thereafter 27 additional new neoclerodane diterpenoids have been reported during

2002-2010 and the data are compiled in Table 1-2. Moreover, 22 new neo-clerodane diterpenoid alkaloids were reported from S. Barbata recently. Among them, a few compounds have shown very potent activity against species of Lepidoptera, but whether this activity will be retained under field conditions is not known. The responses of different species of insects to a specific neoclerodane vary and small changes to the structural composition of a molecule can significantly alter its activity (Bruno et al., 2002).

Many species of this genus are extensively used in traditional medical systems of China,

India, Korea, Japan, European countries, and North America. Among them two species, including American skullcap (S. lateriflora L.) and Chinese skullcap (S. baicalensis Georgi) are the most popular ones with different medical uses, respectively. Besides, Extracts of S. orientalis are used as anti-hypertensive agents (Zaprametov et al., 1988).

4 Table 1-1. The Species Studied and the Number of Phenolic Compounds Reported species number of species number of compounds compounds S. adenostegia Briq. 6 (15) S. orientalis L. 12 (6) S. adsurgens M. Pop. 4 (14) S. ovata Hill. 7 (7) S. alpina L. 20 (20) S. oxystegia Juz. 3 (3) S. altaica Fisch. ex. Sweet 1 (1) S. peregrina Ldb. 0 (1) S. altissima L. 11 (11) S. phyllostachya Juz. 16 (7) S. amoena C. H. Wright. 28 (16) S. planipes L. 21 (13) S. araxensis Grossh. 0 (5) S. polyodon Juz. 17 (13) S. baicalensis Georgi. 64 (62) S. prilipkoana Grossh. 0 (1) S. barbata D. Don. 16 (1) S. prostrata Jacq. et Benth. 29 (30) S. columnae L (All) 2 (2) S. przewalskii Juz. 17 (18) S. comosa Juz. 19 (14) S. pycnoclada Juz. 0 (16) S. creticola Juz. 10 (14) S. ramosissima M. Pop. 11 (11) S. discolor Colebr. 27 (23) S. rehderiana Diels. 10 (7) S. epilobiifolia A. Hamilt. 4 (10) S. repens Buch-Ham, ex D. 14 (14) Don S. galericulata I. 4 (23) S. rivularis Wall. 38 (28) S. glabrata Vved. 6 (7) S. scandens Buch-Ham. ex 17 (16) D. Don. S. granulosa Juz. 2 (2) S. scordifolia Fisch. ex 2 (14) Schrank S. grossa Wall. 24 (19) S. sedelmeyeria Juz. 0 (3) S. ikonnikovii Juz. 7 (7) S. sevanensis Sosn. 19 (19) S. immaculata Nevski 25 (8) S. squarrosa Nevski 6 (6) S. incona Spreng. 0 (1) S. strigillosa Hemsl 15 (15) S. indica L. 36 (33) S. supina L. 0 (19) S. iskanderi Juz. 0 (10) S. tenax W. W. Smith. 9 (5) S. karjaginii Grossh 3 (3) S. tournefortii Benth. 0 (4) S. lateriflora L. 17 (1) S. transiliensis Juz. 0 (6) S. litwinowii Bornm. Et. Sint. 7 (10) S. ussuriensis (Regel) Kubo 2 (2) S. nepetoides M. Pop. 4 (4) S. virularis Wall 0 (2) S. ocellata Juz. 7 (7) S. viscidulla Bunge 13 (7) S. oreophila Grossh. 5 (5) 57 species 635 The number in parenthesis indicates the data reported by Malikov‟s group.

5 Table 1-2. The Number of Neoclerodane Diterpenoid Reported in Scutellaria Species species number of species number of compounds compounds S. albida L. 7 S. hematochlora 1 S. alpine L. 19 S. lateriflora L. 5 S. alpine L.ssp. 8 S. linearis Benth. 4 javalambrensis Pau. S. altissima L. 2 Scutellaria parvula Michx. 1 S. baicalensis Georgi. 1 S. orientalis ssp. pinnatifida 5 S. barbata D. Don. 23 S. orientalis ssp sintenisii 2 Sausskn. ex Bornm. Scutellaria luteo-caerulea 5 S. polyodon Juz. 15 Bornm. & Sint. S. columnae L (All) 8 S. pontica C. Koch 9 S. cypria Rech. fil. var. 3 S. repens Buch-Ham, ex D. 16 cypria Don S. cypria Rech. var. elatior 1 S. rivularis Wall. 11 R. D. Meikle S. discolor Colebr. 13 Scutellaria rubicunda 2 Hornem ssp linneana (Carnel) Rech. S. drummondii Benth. 3 S. seleriana Loesen 1 S. galericulata I. 8 S. violacea Heyne ex Wall. 2 S. grossa Wall. 6 S. woronowii Juz. 2 S. guatemalensis 4

American skullcap is native to North America. It was introduced into American medicine in 1773 by Dr. Lawrence Van Derveer who used it to treat cases of hydrophobia. The name of mad-dog herb stems from this. Subsequently, it came to be utilized primarily for its

6 reputed tonic, tranquilizing, and antispasmodic remedies. It was introduced officially in The

United States Pharmacopia in the edition of period from 1863 to 1916 and then in The National

Formulary until it was dropped in 1947 (Claus, 1956). In America, skullcap is regulated as a dietary supplement (DS). Since 1989 safety concerns of skullcap were raised. A report in the

British Medical Journal summarized the observed hepatotoxic effects of the herb in four women who had been consuming proprietary products supposedly containing skullcap for the relief of stress (MacGregor et al., 1989). Two cases of “skullcap” poisoning, including one fatality, were reported from the Risk Hospital in Oslo, Norway, in 1991 (Huxtable, 1992). This plant was listed in the FDA Poisonous Plant Database (FDA, 2008) and has been classified as an “Herb of

Undefined Safety” (Awad et al., 2003). Studies in Britain showed that many wholesalers there were substituting germander for skullcap (Phillipson et al, 1984). Germander is a group of

Teucrium species, e.g. T. chamaedry, and T. canadense. The capsules containing germander were marketed in France as a weight-control supplement and were associated with thirty cases of hepatotoxicity (Pittler et al., 2003). Germander was banned in France and Italy in 1992. The only approved use for germander in United State is as a natural flavoring in alcoholic beverages.

The toxicological studies have shown that one of the major neoclerodane diterpenes in germander, teucrin A, is responsible for the hepatotoxicity. The furan ring moiety of this diterpene is the key for toxicity. When it was reduced to tetrahydrofuran the hepatotoxicity disappeared (Kouzi et al., 1994). American Herbal Products Association (AHPA) recommends that appropriate steps be taken to assure that skullcap raw materials are free of the noted adulterant, germander.

The root of S. baicalensis is used as a well known traditional Chinese medicine called

7 “Huang Qin”. It was documented in an ancient Chinese herbal, “Shen-Nong-Ben-Cao-Jing”, as a remedy for suppurative dermatitis, diarrhea and inflammatory diseases, and is officially listed in the Chinese Pharmacopeia and Japanese Pharmacopeia JPXIII. Traditional Chinese physicians use it as a remedy for fever, allergic disease and inflammation, especially against bacterial and viral infections of the respiratory and gastrointestinal tract, i. e., chronic bronchitis, bacillary dysentery, viral hepatitis, and acute biliary tract infection. Other species, such as S. amoena C. H. Wright, S. hypericifolia Levl. S. tenax W. W. Smith var patentipillosa (N.-M.) G.

Y. Wu, S. rehderiana Diels. S. viscidula Bge., and S. likiangensis Diels., have been used as substitute as “Huang Qin” in China (Song et al., 1981). Over 60 phenolic compounds were isolated from S. baicalensis. Only one diterpene was reported from it (Hussein et al., 1996). The plant extract as well as some of the phenolic compounds isolated from it demonstrated diverse bioactivities, including antitumor, antiviral, anti-inflammatory, antibacterial, antiallergic, antioxidant, and anti-SARS coronavirus activities. But data associated with these bioactivities are scattered over a huge volume of literature. Here we give a comprehensive review of the diverse bioactivities of S. baicalensis, the phenolic compounds isolated from it, and preparations containing this plant species.

Anticancer activity

The root extract of S. baicalensis (SBE) has a broad spectrum of anticancer activity. It inhibits various cancer cell growths, including hepatocellular carcinoma, cholangiocarcinoma

(Yano et al, 1994), pancreatic carcinoma (Motoo et al, 1994), urothelial carcinoma (Ikemoto et al, 2000) and breast cancer (So et al, 1997). The results of these early investigations were substantiated by a series of studies. Ye et al. demonstrated that SBE exhibited a concentration-

8 dependent growth inhibitory effects on squamous cell carcinoma (KB, SCC-25), prostate cancer

(PC-3, LNCaP), colon cancer (KM-12, HCT-15), breast cancer (MCF-7) and liver cancer

(HepG2). The inhibition concentration at 50% (IC50) for PC-3, LNCaP, MCF-7, KB, HepG2,

KM-12, SCC-25, HCT-15 was 0.52, 0.82, 0.9, 1.0, 1.1, 1.1, 1.2, 1.5 mg/mL, respectively.

Different types of cancers have different sensitivity to the effect of this herb, with prostate and breast cancer being most sensitive (Ye et al., 2002). Because SBE has a strong anti- inflammatory effect, Zhang et al. tested its anticancer activity on head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo and investigated its effect on cyclooxygenase-2 (COX-

2), which converts arachidonic acid to prostaglandin E2 (PGE2) and is highly expressed in

HNSCC. They found the production of PGE2 was strongly inhibited by SBE in a dose- dependent manner. These findings suggested that the anticancer activity of SBE can be attributed, in part, to its inhibitory effect on PGE2 production via suppression of COX-2 expression and arachidonic acid release from cell membrane (Zhang et al., 2003). Further they evaluated SBE as an anti-cancer agent both in vitro (in androgen-dependent and androgen- independent cell lines) and in vivo (in nude mice xengrafted with prostate cancer cells) and to explore its ability to affect both COX-2 and cell cycle regulation pathway. The study, together with others (Bonham et al., 2005), demonstrated that SBE inhibits androgen-independent prostate cancer cells (PC-3) more strongly than it ihibits androgen-dependent cells (LCNaP), which could be viable alternative therapy to prostate cancer patients who fail to respond to conventional hormone-based treatment. Another study showed that SBE inhibited the proliferation of lymphocytic leukemia, lymphoma and myeloma cell lines by induction of apoptosis associated with the modulation of the bc1 family of genes and mitochondrial damage.

9 These results suggested that SBE is a potential anti-cancer agent against these hematological malignancies (Kumagai et al., 2007).

On the other hand, SBE showed potent activity in combined treatment with standard anticancer therapeutics as a biological modulator. SBE pretreatment decreased cisplatin-induced kaolin intake in the rat model of simulated nausea. It may play a therapeutic role in chemotherapy-induced emesis (Aung et al., 2003).

S. baicalensis contains three major flavonoids, baicalein, baicalin and wogonin. The anticancer activity of these compounds have been screened in different cell types (Chang et al.,

2001; Ikemoto et al., 2000; Lee et al., 2005; Ma et al., 2005; Sonoda et al., 2004). Taken together, the differences in the anti-cancer activities of these flavonoids in different cell lines, in part, may be cell type specific. For example, several studies implicated 12-lipoxygenase (12-

LOX) as a regulator of human cancer development. As a 12-LOX inhibitor, baicalein has been shown to induce apoptosis in human gastric, breast, colon, hepatoma, prostate, and pancreatic cancer cells (Chan et al, 2000; Chen et al, 2000; Ding et al, 1999; Kuntz et al, 1999; Po et al,

2002; Wong et al, 2001). One study showed that baicalein exerted a concentration-dependent, growth-inhibitory effect on three cell lines of human hepatocellular carcinoma (HCC). Baicalein strongly inhibited the activity of topoisomerase II and suppressed the proliferation of all three cell lines after administration 6 h (Matsuzaki et al., 1996). The mechanisms of antiproliferative effect of baicalein and related compounds, baicalin, wogonin, esculetin and scoparone, in human

T-lymphoid leukemia cells (CEM cells) were investigated. The results demonstrated that baicalein exhibited the greatest antiproliferative activity with an IC50 value of 4.7 0.5 μM and a maximal suppression of 91.5 1.4 % in CEM cells. The protein tyrosine kinase activity in the

10 CEM cells was significantly reduced by baicalein (10-6-10-4 M). The reverse transcription- polymerase chain reaction analysis of messenger ribonucleic acid (RNA) levels of platelet- derived growth factor-A (PDGF-A) and transforming growth factor-β1 (TGF-β1) demonstrated that baicalein reduced the PDGF-A messenger RNA (mRNA) level, while less affected the TGF-

β1 mRNA. Baicalein exhibited the greater reduction on the expression of PDGF-A mRNA than esculetin did (Huang et al., 1994). So, antiproliferative effect of baicalein may be partly mediated through inhibition of growth-related signal, protein tyrosine kinase, and reduction of expression of growth factor, PDGF-A. Baicalein also inhibited growth by suppressing the COX-

2 expression resulting in decreased levels of PGE2 of two HNSCC cell lines SCC-25 and KB

(Zhang et al., 2003). Others have shown that baicalin stimulated activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) to induce expression of quinone reductase gene; this enzyme may be able to act as a potential cancer chemopreventive agent by induction of phase II detoxification enzyme (Park, H.J. et al., 2004). Besides baicalin, wogonin has shown to have an apoptosis- inducing activity against human monocytic leukemia cell line THP-1, but not in human fetal lung normal cells (Himeji et al., 2007).

Antiviral activities

SBE as well as its flavonoids showed anti-human immunodeficiency virus -1 (HIV-1), anti-hepatitis B (anti-HBV), and anti-respiratory syncytial virus (anti-RSV) activities

(Buimovici-Klein et al., 1990; Ono et al., 1990).

Baicalein was found to be a potent inhibitor of the activities of reverse transcriptases

(RT) of murine leukemia viruses (MLV) and HIV. Under the reaction conditions specified for each of the MLV- and HIV- reverse transcriptases, both enzyme activities were inhibited by

11 more than 90% in the presence of 2 μg/mL baicalein (Ono, K., 1989).

Baicalin produced concentration-dependent inhibition of human T-lymphotropic virus type-1 (HTLV-1) replication in productively infected T and B cells. Moreover, baicalin treatment selectively reduced the detectable levels of HTLV-1 p19 gag protein in infected cells by 70% at concentrations that produced insignificant effects on total cellular protein and DNA synthesis with no loss in cell viability. Resistance to HTLV-1 infection and virus-mediated transformation was noted in uninfected peripheral blood lymphocytes pretreated with baicalin before cocultivation with lethally irradiated chronically infected cells. Baicalin inhibited reverse transcriptase activity in HTLV-1-infected cells as well as the activity of purified reverse transcriptase from Moloney murine leukemia virus and Rous-associated virus type-2 (Baylor et al., 1992).

The inhibitory effect of baicalin against HIV-1 infection and replication has been studied in vitro (Kitamura et al., 1998; Li et al., 1993). The results showed that baicalin markedly inhibited replication of HIV-1 in a concentration-dependent manner in normal peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA). The effect was more pronounced when the cells were pretreated with baicalin. Further, baicalin inhibited HIV-1 replication in PHA-stimulated PBMC from asymptomatic HIV-1-seropositive carries. The 50% inhibitory concentration for HIV-1 replication was approximately 0.5 μg/mL. At a concentration of 2 μg/mL of baicalin, copy numbers of HIV-1 proviral DNA were approximately 50 times less than in untreated controls. In a cell-free infection system, baicalin inhibited the activity of HIV-

1 reverse transcriptase, but not the activity of human DNA polymerases α and γ (DNA polymerase β was slightly inhibited). It also did not inhibit HIV-2 or murine leukemia virus RT.

12 The mechanism of its anti-HIV-1 activity remains to be determined. Another study demonstrated that baicalin, at the noncytotoxic concentrations, inhibited both T cell topic (X4) and monocyte topic (R5) HIV-1 Env protein mediated fusion with cells expressing CD4/CXCR4 or CD4/CCR5. Furthermore, presence of baicalin at the initial stage of HIV-1 viral adsorption blocked the replication of HIV-1 early strong stop DNA in cells. Since baicalin did not inhibit binding of HIV-1 gp 120 to CD4, it was proposed that baicalin may interfere with the interaction of HIV-1 Env with chemokine correceptors and block HIV-1 entry of target cells (Li et al.,

2000). Baicalin coupling with Zn exhibited lower cytotoxicity and higher anti-HIV-1 activity without changing its target on HIV-1 RT and on HIV-1 entry compared with baicalin in vitro

(Wang Q. et al., 2004). Combination therapy for AIDs patients has been studied. Synergistic anti-HIV effects of baicalin with 3′-Azido-2′, 3′-dideoxythymidine (AZT) have been reported

(Inada et al., 1994), suggesting that baicalin might be potentially useful as part of a drug combination regimen for the treatment of HIV-1 infections.

The difference in HIV-1 reverse transcriptase inhibitory activity between baicalein and baicalin has been examined. The results indicated that HIV-1 reverse transcriptase inhibitory activity of baicalein was four times higher than baicalin (Wu et al., 2001). The studies of structure-activity relationship revealed that the presence of both the unsaturated double bond between 2 and 3 of the flavonoid pyrone ring, and the three hydroxyl groups introduced on positions 5, 6 and 7, were a prerequisite for the inhibition of reverse transcriptase activity. In general, the presence of substituents (hydroxyl and halogen) in the B-ring increase toxicity and/or decrease activity (Hu et al., 1994; Ono et al., 1990). Lee et al investigated the in vitro effects of baicalein and baicalin on the three distinctive enzymic activities of the HIV-1 integrase

13 - endonucleolytic, integration, and disintegration activity. They found that both compounds inhibited the three enzymatic activities in a dose-dependent manner. The 50% inhibitory concentrations of baicalein and baicalin for endonucleolytic activities of HIV-1 integrase were

4.4, 3.3 and 25.9, 4.0 μM, respectively. In general, baicalein exhibited nearly 6- to 10-fold stronger inhibition than baicalin for the three enzymic activities. These data demonstrated that baicalein or baicalin can be used as a leading compound to develop anti-acquired immune deficiency syndrome (anti-AIDS) chemotherapeutic agents targeting the HIV-1 integrase (Lee

M.J. et al, 2003).

SBE displayed anti-HBV effects at non-toxic dose, with inhibition percentage of

41.3±2.5 (p<0.001). Further study showed that the three major components (i.e., baicalein, baicalin, and wogonin) extracted from S. baicalensis displayed the most effective anti-HBsAg activities with inhibition percentage of 14.6±2.9, 19.5±3.4, and 45.4±2.7 respectively, at a dose of 20 µg/mL. Only wogonin can suppress HBV surface antigen production (p<0.001) without evidence of cytotoxicity. The latter action was confirmed by measuring an endogenous HBV

DNA polymerase repairing activity, since both the relaxed circular and linear forms of HBV

DNA are significantly reduced in the wogonin-treated group (Huang et al., 2000).

70% EtOH extract of S. baicalensis showed potent antiviral activities against RSV with

IC50 22.8 μg/mL and with selective index (SI=IC50/CC50) 1.7. Further purification of the active extract of S. baicalensis led to the identification of wogonin and oroxylin A as the most potent anti-RSV components. Their IC50 values were 7.4 μg/mL and 14.5 μg/mL, and SI values were

16.1 and 4.0, respectively (Ma et al., 2002). This data offered scientific support and chemical basis for the traditional uses of this herb to relieve viral infection in the upper respiratory tract.

14 Anti-inflammatory activity

The methanol extracts of S. baicalensis, S. rivularis and their major constituents, including baicalein, baicalin and wogonin, possessed anti-inflammatory activity in vivo in several animal models of inflammation, including carrageenan-induced paw edema and adjuvant-induced arthritis in rats (Krakauer et al., 2001; Kubo et al., 1984; Lin et al., 1996; Park

S. et al., 2004). The anti-inflammatory action has been reported to be partly due to inhibition of leukotriene synthesis by lipooxygenase in leukocyte (Butenko et al., 1993; Kimura et al., 1987).

Further investigations showed that these flavonoids possessed the inhibitory effects of cyclooxygenase-2 and inducible NO synthase (iNOS) expression in the lipopolysaccharide

(LPS)-stimulated mouse macrophage cell line, RAW 264.7 (Chi et al., 2001; Kim et al., 1999;

Wakabayshi et al., 2000). Among them, wogonin was found to be one of the most potent suppressors. Wogonin produced dose-dependent inhibitory effects on 12-O- tetradecanoylphorbol-13-acetate (TPA) induced COX-2 expression, and prostaglandin E2 (PGE2) production on the dorsal skin of mice. When applied topically (250-1000 μg/3 days) to the dorsal skin of mice, wogonin inhibited COX-2 expression and PGE2 production induced by multiple treatment with TPA. At a dose of 200 μg/site/treatment, wogonin caused 55.3% reduction of PEG2 production on the dorsal skin compared with an increased production in the

ATP-treated control group. It also significantly inhibited mouse ear edema induced by TPA in both preventive (58.1% inhibition) as well as curative treatment (31.3% inhibition) schedules at

200 μg/ear/treatment. Also wogonin exhibited preventive effect against ethanol-induced gastric mucosal damage in rats by either anti-inflammatory effects through dual actions on arachidonic acid metabolism, i.e., induction of prostaglandin D2 and suppression of 5S-

15 hydroxyeicosatetraenoic acid (5S-HETE), or preventive induction of profuse apoptosis in the stomach (Park, S. et al., 2004). Wogonin was the only flavonoid proved to down-regulate COX-

2 expression in mouse skin induced by TPA treatment (Park et al., 2001). In addition to inhibition of inflammation-associated enzymes, such as cyclooxygenases (COX) and lipoxygenases, these flavonoids have been found to regulate the expression level of several inflammation-associated genes. For instance, baicalin inhibited the production of various cytokines from human peripheral blood mononuclear cells induced by super-antigen treatment

(Krakauer et al., 2001). Wogonin was also found to possess significant suppression on several inflammatory-associated genes expression in intact as well as in the TPA-induced inflamed mouse skin. It potently reduced COX-2 and tumor necrosis factor (TNF)-а gene expression while intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-1β were weakly affected.

Therefore, from these results, it is suggested that the modulation of preinflammatory gene expression may be one of the in vivo action mechanisms of anti-inflammation by wogonin.

Investigations reported, all together, give a scientific basis for using Scutellairae species as a source for anti-inflammatory agents (Chi et al., 2003).

Antioxidant activity

SBE could inhibit lipid peroxidation in rat liver homogenate (Kimura et al., 1981) and in beef heart mitochondria (Hodnick et al., 1994). Antioxidant effects of SBE have been traced to several of its flavones, which include baicalein, wogonin, scullcapflavone I and II (Kimura et al.,

1982; Takaji et al., 1980). Several results suggested that baicalein could inhibit iron-dependent lipid peroxidation in microsomes (Gao et al., 1995). It was suggested that baicalein might be exerting its protective effect through chelation of metals. Baicalein could also scavenge reactive

16 - oxygen species (ROS) such as hydroxyl radicals (·OH), superoxide anion radicals (·O2 )

(Hamada et al., 1993) and singlet oxygens (Criado et al., 1995). When a hypoxanthine (HX)-

- xanthine oxidase (XOD) system was used for generation of O2 , baicalein inhibited XOD

(Hanasaki et al., 1994). This observation is consistent with an early study (Chang et al., 1993), in which baicalein behaved as a non-competitive inhibitor of XOD. Further direct free radical scavenging abilities of baicalein, baicalin, wogonin and wogonin glucuronide were examined by electron spin resonance (ESR) spectrometry method. The results showed that the direct free radical scavenging abilities of the four flavonoids decreased in the order of baicalein > baicalin > wogonin > wogonin glucuronide. Baicalein and baicalin were shown to protect several types of tissues against damage caused by ROS. With an o-di-hydroxyl group in the A ring, baicalein and baicalin are much better free radical scavenger than wogonin and wogonoside. The direct free radical scavenging abilities of baicalein and baicalin on alkyl radical and 2,2-diphenyl-l- picrylhydrazyl (DPPH) radical suggested that flavonoid with o-di-hydroxyl structure in the A ring was an effective radical scavenger (Gao et al., 1998; 1999; 2000). Morimoto et al identified a mechanism capable of consuming large amount of H2O2 via a peroxidase reaction that oxidizes baicalein to 6,7-dehydrobaicalein (Morimoto et al., 1998). Kim et al reported that S. baicalensis prevented neuronal cell death in the hippocampal CA 1 region induced by a four-vessel occlusion (Kim et al., 2001), and attenuated apoptosis by inhibiting protein oxidation in a H2O2- treated neuronal cell line (Choi et al., 2002). Based on the radical quenching and antioxidative effects, baicalein could inhibit hippocampal neuronal death induced by 5 min of cerebral ischemia in gerbils (Hamada et al., 1993), and baicalin, as a biological response modifier, promoted the repair of DNA single strand breakage caused by H2O2 in cultured NIH3T3

17 fibroblasts (Chen X. et al., 2003). Also, wogonin was reported to have a protective effect on neuronal cells damaged by oxygen and glucose deprivation in rat hippocampal slices in culture

(Son et al., 2004), and could inhibit ischemic brain injury in a rat model of permanent middle cerebral artery occlusion (Choi et al., 2002). In addition, baicalein as well as baicalin could reduce cytotoxicity of amyloid β protein in PC12 cells, possibly by reduction of oxidative stress; hence these flavonoids may be useful in the chemoprevention of Alzheimer‟s disease (Heo et al.,

2004).

Antibacterial activity

The water extracts of S. baicalensis were found to have growth-inhibition activity against

Salmonella typhimurium. The mechanism might be due to the fact that SBE could stimulate the transcription of polyphosphate kinase, especially, in the early growth-stage of S. typhimurium

(Hahm et al, 2001).

In 1981, five flavonoids, including baicalein, baicalin and wogonin etc. isolated from S. baicalensis, were tested against several strains of bacteria, including Escherichia coli IFO 3301,

Saracina lutae RIMD 3232, Bacillus subtilis IAM 1521, and Staphylococcus aureus 209P. It was found that compound VIII [2(S)-5,7,2′,6′-tetrahydroxyflavanone] and baicalein had antibacterial activity at a concentration of 250 μg/ mL (Kubo et al., 1981). Although baicalin used alone showed only moderate antibacterial activity against Staphylococcus aureus, significant synergistic activities against both methicillin-resistant Staphylococcus aureus

(MRSA) and penicillin resistant S. aureus in combination with β-lactams were observed. When combined with 16 μg/mL baicalin, minimum inhibitory concentrations (MICs) of benzypenicillin against methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant

18 Staphylococcus aureus were reduced from 125 and 250 μg/mL to 4 and 16 μg/mL, respectively.

This activity of baicalin was dose-dependent. Viable counts showed that the killing of MRSA and β-lactam-resistant S. aureus cells by 10 to 50 μg/mL ampicillin, amoxicillin, benzylpenicillin, methicillin and cefotaxime was potentiated by 25 μg/mL baicalin. Therefore, baicalin has the potential to restore the effectiveness of β-lactam antibiotics against MRSA and other strains of β-lactam-resistant S. aureus (Liu et al., 2000). In view of its limited toxicity, baicalin offers potential for the development of a valuable adjunct to β-lactam treatments against otherwise resistant strains of microorganisms.

Antiallergic activity

Leukotrienes (LTs) were reported to support various cell growths, and basophil as well as its precursor numbers increased in atopic patients (Reilly et al., 1987). Baicalein was found to inhibit the production of LT B4 by human alveolar macrophages (Sakamoto, K. et al., 1980).

The antiallergic effect of baicalein on human basophil growth in vitro was examined using different basophilic cells and shown that two-week cultured numbers of basophilic cell were inhibited by baicalein (1-100 μM) in a dose-dependent manner (Tanno et al., 1989; 1991).

Further study was performed to examine the effects of baicalein on the proliferation and the differentiation of human basophilic leukemia cell line KU812F. The results showed that baicalein suppressed the growth of basophilic leukemia cell at a concentration of approx. 10 μM, and suggested that baicalein may affect allergic diseases by suppressing the growth and the differentiation of basophils/mast cells (Tanno et al., 1994). Koda and his colleagues reported the anti-allergic drug disodium cromoglycate (DSCG) and disodium baicalein 6-phosphate, which suppressed the increase in capillary permeability caused by 48 h homologous passive cutaneous

19 anaphylaxis (PAC) in rats, inhibited not only hyaluronidase activity in vitro but also the activation of hyaluronidase induced by PAC in a dose-related manner. They suggested that potent hyaluronidase inhibitory substances might have anti-allergic effects (Kakegawa et al.,

1992; Sakamoto, K. et al., 1980). The anti-allergic activity of baicalein was also evidenced from the facts that it inhibited the histamine release from rat peritoneal exudate cells induced by antigens, compound 48/80 and calcium ionophore A-23187, and from its inhibitory effect on

Shultz-Dale reaction using sensitized guinea pig ileum (Kakegawa et al., 1992).

Other activities

SBE was observed to have a specific inhibitory effect on CYP1A1/2 among cytochrome

P450 (CYP) enzymes involved in aflatoxin B1 (AFB1) metabolism by rat and human microsomes (Kim et al, 2001). Further studies indicated that baicalein and wogonin might have beneficial effects against benzo(a)pyrene- and AFB1-induced toxicities (Ueng et al, 2003). Also baicalein inhibited hepatic testosterone 6-hydroxylation (CYP3A4) activity with an IC50 of 17.4

μM (Kim et al., 2002). Baicalin had protective effects on acetaminophen (AP)-induced hepatotoxicity. The hepatoprotective effects of baicalin against AP overdose may be due to its ability to block the bioactivation of AP by inhibiting CYP2E1 expression (Jang et al., 2003).

Seki et al reported that the lipogenesis in the hamster sebaceous glands was suppressed 54% by

10-4 M wogonin. Thus, the therapeutic effects of experimentally used SBE for acne vulgaris could be due to inhibition of lipogenesis by its active ingredients such as wogonin (Seki et al.,

1993).

Chinese skullcap and its principal active flavonoids exhibit diverse bioactivities, including anticancer, antiflammatory, antiviral, antioxidation, antiallergic, and antibacteria

20 activities, but the mechanisms of these activities have not been completely elucidated yet.

Beside baicalein and baicalin some minor components might have others special bioactivities and should been investigated in the future.

1.3. Objectives of the dissertation

In America, the marketing of dietary supplement has grown dramatically following the passage of the Dietary Supplement and Health Education Act (DSHEA) in 1994. Based on the

National Health Statistics Reports published in 2008, approximately 38 percent of adults and approximately 12 percent of children in the United States are using some form of DS. The annual sales exceed $23 billion. As use of these products increases, so does the potential for adverse effects. The data provided by National Poison Control Center showed that the number of adverse reaction reports increased steady each year since 1994, and the total number of such reports was 35,400 from 1994 to 1999. In the circumstances, more and more attention has been attracted from the government agencies, academic institutions, as well as industry. Great effects have been made to prevent consumers from unsafe products. For example, the American Herbal

Products Association (AHPA) listed the known potential adulterants that have been encountered in trade (see Table 1-3), and recommends that appropriate steps be taken to assure that the listed raw materials are free of the noted adulterant; FDA set up the Poisonous Plant Database to list all plants which have shown some possible toxicity. Also, FDA supported NCNPR by means of funds appropriated by the U.S. congress to develop authentication, identification tools and evaluate the safety of botanical dietary supplements in a broad range of fields including cultivation, taxonomy, microscopy, chemical analysis, genetic fingerprinting, safety evaluation and absorption, distribution, metabolism, and excretion (ADME) studies. Two projects of my

21 dissertation are part of these efforts.

The objectives of the first project

In America there are many skullcap dietary supplement products on the market. As mentioned in section 1.2., this plant species has been classified as “Herb of Undefined Safety” by the FDA due to the reports of hepatotoxic reactions after skullcap-containing preparations were ingested. Meanwhile, it was reported to be adulterated with germander, which is a group of plants with hepatotoxicity, e.g. Teucrium chamaedry and T. canadense. But only limited data associated with its chemical & pharmacological aspects are available. Therefore, the objectives of this project was to explore the chemical constituents in it, to develop a reliable analytical method(s) for the differentiation of skullcap from germander, to evaluate the toxicity as well as bioactivities of the crude extract and compounds isolated from it. Taken together, all information generated here would be useful to solve the problems encountered with this herb and to provide solid scientific support for the traditional usages of this plant.

22 Table 1-3. Ten Commonly Used BDS and Their Potential Adulterants (AHPA) No. herb in commerce adulterant 1 Eleuthero root (Eleutherococcus Periploca sepium root senticosus) 2 Plantain leaf (Plantago lanceolata) Digitalis lanata leaf 3 Skullcap herb (Scutellaria lateriflora) Germander herb (Teucrium chamaedrys) 4 Stephania root (Stephania tetrandra)a Aristolochia fangchi root 5 Asian species of Cocculus spp., Aristolochia fangchi root Diploclisia spp., Menispermum spp. and Sinomenium spp. root 6 Asian species of Akebia and Clematis Aristolochia mandshuriensis stem stem 7 Costus root (Saussurea costusa) Aristolochia debilis root 8 Vladimiria souliei root Aristolochia debilis root 9 Black cohosh root/rhizome (Actaea Chinese cimicifuga root/rhizomec racemosab) (Actaea spp.) 10 Ginkgo (Ginkgo biloba) leaf extract Ginkgo (Ginkgo biloba) leaf extract standardized to flavonol glycosides and with added flavonol glycosides or terpenes aglycones (e.g., rutin, quercetin, etc.) a. Synonym = Saussurea lappa; b. Synonym = Cimicifuga racemosa c. Also known as sheng ma or Rhizoma Cimicifugae; is Actaea cimicifuga, syn. Cimicifuga foetida; Actaea dahurica, syn. C. dahurica; A. heracleifolia, syn. C. heracleifolia; and possibly other Asian species of Actaea.

23 Chapter 2

Extraction, Isolation and Characterization of

Chemical Constituents of Scutellaria lateriflora L and S. baicalensis Georgi

2.1 Introduction

As mentioned in the previous chapter, the goal of this project was to develop and validate an analytical method to differentiate between S. lateriflora and its potential adulterant germanders, and to evaluate the bioactivities of individual compounds isolated from S. lateriflora and S. baicalensis to provide solid scientific support for the traditional usages of this plant. All of these can be achieved only when qualified reference standards are available.

Literature data pertaining to the chemical constituents of S. lateriflora are limited.

Sesquiterpenes were reported as the main components of the essential oil of S. lateriflora

(Yaghmai, 1988). Five neo-clerodane diterpenoids were isolated from the Me2CO extract of S. lateriflora collected in the „Orto Botanico dell‟Università di Milano‟ at Tuscolano (Brescia,

Italy) (Bruno et al., 1998). Three flavone glucuronides and a flavanone glucuronide were identified along with five flavones by HPLC-UV/MS (Gafner et al., 2003). In order to obtain reference markers the present chemical investigation was undertaken. During the course of this research, all efforts have been made to obtain more chemical markers with enough quantity for structural elucidation and quality control. Flavonoids are rich in many species of genus

Scutellaria, for instance S. baicalensis. On this base, the study of chemical components of S. baicalensis was carried out since it was reported to be rich in flavonoids and more than sixty flavonoids have been identified from this plant (Malikov et al., 2002).

24 2.2 Experimental sections

2.2.1. General experimental procedures

Optical rotations were measured in MeOH using a Rudolph Research Auto Pol IV polarmeter with a sodium lamp (589 nm) and a 1 dm microcell. UV spectra were acquired on a

Varian 50 Bio UV spectrophotometer. Circular dichroism (CD) spectra were measured using an

Olis DSM 20 CD instrument. Infrared (IR) spectra were obtained on a Bruker Tensor 27 spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded in pyridine-d5 or methanol-d4 on either a Varian 600 or Bruker Avance 400 NMR. All chemical shifts (δ) are given in ppm with reference to solvents and coupling constants (Ј) are given in Hz. HRESIMS were acquired on a Bruker MicroTOF mass spectrometer. A highly porous synthetic resin

(Diaion HP-20) was purchased from Mitsubishi Kagaku, Ltd. (Tokyo, Japan). Column chromatography (CC) was carried out on silica gel (40 µm for flash chromatography, J. T.

Baker), 100 C18-reversed phase silica gel (Sigma-Aldrich, 230-400 mesh) and Sephadex LH-20

(Mitsubishi Kagaku, Tokyo, Japan). The fractions were monitored by TLC on normal-phase silica gel 60 F254 plates (Merck, Germany) and reversed-phase C18 F254s plates (Merck,

Darmstadt, Germany). Spots were visualized under UV light or by heating at 105 °C for 1-2 min after spraying with anisaldehyde/H2SO4 reagent. HPLC was performed on an ODS column

(Phenomenex Luna C18, 10x250 mm, 5 μm) as well as a silica NP-column (Phenomenex Luna

Silica, 10x250 mm, 5 μm) and the elution was monitored at 240 nm.

2.2.2. Plant material

The aerial parts of S. lateriflora were purchased from Starwest botanicals

(http://www.starwest-botanicals.com) and authenticated by Dr. Vaishali Joshi at the National

25 Center for Natural Products Research (NCNPR), University of Mississippi, where a voucher specimen (voucher no. 2120) has been deposited.

S. baicalensis ethanol extract was purchased from Chinese Herbs Direct and authenticated by comparing its TLC profile with that of the authentic sample provided by Dr.

Vaishali Joshi. A voucher specimen (Voucher No. 1181) has been deposited in the repository of

NCNPR.

2.2.3. Extraction and isolation

The dried powder of the aerial parts (850 g) of S. lateriflora was extracted by immersing in MeOH (3 x 3 L) at room temperature for two days each time and the MeOH extracts were combined. A brown crude viscous residue (127.6 g) was obtained after evaporation of the solvent in vacuo. This MeOH extract (126.0 g) was subjected to passage over a Diaion HP-20 column (1.65 kg) eluting with 95% EtOH (7 L), MeOH-Me2CO (1:1, 8 L), Me2CO (6 L), EtOAc

(5 L), and CHCl3 (5 L), successively, to give seven fractions (A-G) (see scheme 2-1). Fraction D

(9 g) was subjected to normal-phase silica gel CC (288 g) eluting with cyclohexane-EtOAc-

MeOH mixtures of increasing polarity, to afford scutellaric acid (11, 11.2 mg) and a mixture of

1-triacontanol and 1-dotriacontanol in the ratio of 2:1 (32.1 mg). Fraction E (21.45 g) was chromatographed on silica gel (556 g) and eluted with a gradient of CHCl3-MeOH (99:1 to 7:3) to give 52 fractions. Fraction E-6 (128.1 mg) was subjected to reversed-phase silica gel chromatography (Biotage RP-18, 40 g), eluted with MeOH-H2O 92:8 to afford lupenol (42.1 mg). Fraction E-8 (407.7 mg) afforded β-sitosterol (14, 213.2 mg) after crystallization from

MeOH. Fraction E-9 (103.2 mg) was purified by preparative TLC using hexanes-EtOAc (9:1) to give a mixture of arachidic acid, behenic acid, and lignoceric acid in the ratio of 2:1:0.3 (42.7

26 mg). Fraction E-10 was separated by Sephadex LH-20 CC, eluted with MeOH to afford two main subfractions. Subfraction one was repeatedly purified by HPLC on RP-18 using a MeOH-

H2O gradient as well as on silica NP-column using a hexanes-EtOAc gradient to afford decursin

(3, 0.37 mg), scuteflorin A (1, 0.95 mg), and B (2, 0.51 mg). Fraction F (55 g) was subjected to flash CC on silica gel (761 g), [cyclohexane-EtOAc (83:17 to 4:6), CHCl3-MeOH (94:6 to

87:13), MeOH] to give six main fractions. Subfraction F-1 was purified further by crystallization from MeOH to furnish palmitic acid (16, 21.3 mg). Subfraction F-2 (612.7 mg) was subjected to normal-phase silica gel chromatography with a gradient of cyclohexane-EtOAc

(1:1 to 1:9) to give four fractions F2-1-F2-4. Oroxylin A (5, 13.2 mg) was obtained by crystallization (MeOH) from fraction F2-2. Fraction F2-1 and F2-3 were purified on Sephadex

LH-20, eluted with MeOH to afford dihydrooroxylin A (9, 4.6 mg) and dihydrochrysin (8, 3.2 mg), respectively. The MeOH soluble part of subfraction F-3 (748 mg) was subjected to

Sephadex LH-20 CC eluted with MeOH to afford oroxylin A (5, 21.5 mg), wogonin (6, 27.9 mg), and chrysin (42.1 mg). The MeOH insoluble part of subfraction F-3 was crystallized from

MeOH to furnish ursolic acid (13, 57.9 mg). Subfraction F-4 (1.0223 g) was subjected to

Sephadex LH-20 CC, eluted with MeOH followed by normal-phase silica gel chromatography with a gradient of cyclohexane-EtOAc (93:7 to 4:6) to give oroxylin A (5, 12.9 mg), wogonin (6,

12.7 mg), and pomolic acid (12, 17.9 mg). Subfractions F-5 and F-6 were purified by crystallization from MeOH to afford 5,7-dihydroxy-8,2′-dimethoxyflavone (7, 7.8 mg) and daucosterol (15, 89.9 mg), respectively.

25 Compound 1: white powder; [α] D + 25.2 (c 0.032, MeOH); UV (MeOH) λmax (log ε)

-1 1 260 (1.31), 330 (0.58) nm; IR νmax 1720 (C=O), 1510-1620 cm (aromatic –C=C–); for H

27 13 (aceton-d6, 600MHz) and C NMR (aceton-d6, 125 MHz) data, see Table 1; HR-ESI-MS m/z

365.1013 (M+Na, calcd. for 365.1001) and 707.2011 (2M+Na, calcd. for 707.2105).

25 Compound 2: white powder; [α] D + 5.8 (c 0.017, MeOH); UV λmax (log ε) 255 (3.31),

-1 1 330 (1.17) nm; IR νmax 1730 (C=O), 1520-1610 cm (aromatic –C=C–); for H (aceton-d6,

13 600MHz) and C NMR (aceton-d6, 125 MHz) data, see Table 1; HR-ESI-MS m/z 365.1011

(M+Na, calcd for 365.1001) and 707.2085 (2M+Na, calcd. for 707.2105).

Methods of circular dichroism (CD) calculations: All calculations were performed at

298 K by the Gaussian03 program package (Frisch et al., 2003). The AM1 method was employed to scan the potential energy surface (PES) to identify conformers of compound 1.

Ground-state geometries were optimized at the B3LYP/6-31G** level, total energies of individual conformers were obtained, and vibrational analysis was done to confirm these minima. Single point energies of conformers of compound 1 were calculated at the B3LYP-

SCRF/6-31G**//B3LYP/6-31G** level with the COSMO model in MeOH. Conformational distributions were calculated at B3LYP/6-31G** and B3LYP-SCRF/6-31G**//B3LYP/6-31G** levels. TDDFT was employed to calculate excitation energy (in nm) and rotatory strength R in dipole velocity (Rvel) and dipole length (Rlen) forms, at the B3LYP/6-31G** and B3LYP/AUG- cc-pVDZ//B3LYP/6-31G** levels in the gas phase and at the B3LYP-SCRF/6-

31G**//B3LYP/6-31G** level in MeOH. The calculated rotatory strengths were simulated into an electronic circular dichroism (ECD) curve by using the Gaussian function:

Where ζ is the width of the band at 1/e height and ΔEi and Ri are the excitation energies and rotatory strength for transition i, respectively. ζ= 0.20 eV and Rlen were used.

28 The methanol soluble part of the extract of S. baicalensis was subjected to vacuum liquid chromatography (VLC) on silica gel eluting with gradients of CHCl3/MeOH to afford fifteen fractions. Fraction 3, 6, 11, 13, and 15 were further separated using different chromatographic methods including silica gel column, Sephadex LH-20, and preparative thin layer chromatography as shown in scheme 2-2. The methanol unsolvable part of the extract of S. baicalensis was dissolved in H2O and adjusted to pH 2 using HCl. The acidic solution was treated by adding MeOH to give baicalin crystals.

29 SL ( S. laterifloria, 850.6 g)

MeOH, extract, 3000mlx3

Reside MeOH portion (SL-2) (SL-1, 127.6g, 14%) CHCl3, 3000mlx3 column with Diaion@ HP-20 eluting with differnt solvents CHCl3 portion 20g residue

SLRE-1-3 SLR-CHCl3 SLR-EtOAcSLR-Me CO SLR-14-20 SLRE-12-13 SLRE-10-11 SLRE-9 SLRE-8 SLRE-4-6 2 55g 6.77g (2.38g) (30g) 9g 5.7g 10g 3.47g 2.28g fatty acids VLC silica gel column JLI65 Hex-EtOAc EtOAc-MeOH VLC MeOH SLRE46-22-23 JLI65-61 JLI65-81 SLRE46-10-13 5,7-dihydroxy-2',8- lupenol sitosterol 0.6127g dimethoxyflavone SLRE46-19-21 SLRE46-61-63 SLRE46-1-2-s 1.0223g daucosterol SLRI-9-42-p Palmitic acid arachidic acid:behenic SLRI-43-47 SLRE46-14-18 Sephadex LH-20 acid: lignoceric acid 3a,24-dihydroxy-olean-12-en-28-oic acid 0.748g (2:1:0.3) Biotage SLRI-9-42-p Hex:CHCl3 1:1 to 1:9 SLRE46-19-21-20 1-triacontanol:1-dotriacontanol (2:1) SLRE46-19-21-70-77 Biotage-RP

wogonin SLRE46-10-13-69-77 SLRE46-10-13-81'-87' chrysin SLRE46-10-13-60-68 8.1mg 49.4 mg oroxylin A SLRE46-10-13-1'-53' SLRE46-19-21-70-77-86-91 Sephadex LH-20 19.6 mg 3ß,19a-dihydroxy-urs-12-en-28-oic acid dissolve in MeOH Sephadex LH-20 SLRE46-19-21-70-77-16-61

SLRE46-10-13-60-68-1-13 dihydrooroxylin A SLRE46-10-13-1'-53'-3-4 MeOH residue SLRE46-10-13-1'-53'-8 JL-I-53-2 SL-R-E-4-6-14-1 dihydrochrysin Sephadex LH-20 JL-I-53-1 uroslic acid SLRE46-10-13-60-68-24-25

SL-R-E-4-6-14-18-83-101 SL-R-E-4-6-14-18-109-125 SL-R-E-4-6-14-18-83-101 JL-I-53-2-109-125 PTLC chrysin HPLC

SL-R-E-4-6-14-18-83-101-1 SL-R-E-4-6-14-18-83-101-2 JL-I-53-2-83-101-1 JL-I-53-2-83-101-2 oroxylin A wogonin lateriflorin A lateriflorin B decursin Chart 2-1. Scheme for isolation of chemical constituents from S. lateriflora

30 S. baicalensis extract (184.6 g)

MeOH

SB1-1 (86 g) SB1-2

H2O, adjust pH Silica gel SB1-2-1 VLC baicalin

fr.1 fr.3 fr.6 fr.11 fr.13 fr.15 silica gel column MeOH+CHCl3 Sephadex LH-20 Sephadex LH-20

SB1-6-1 solutionSB1-11-75-81 SB1-3-36 ganhuangenin silica gel cry. skullcapflavone II column SB1-11-110-122 SB1-15-1 2,6,2',4'-tetrahydroxy rutin -6'-methoxychalcone SB1-6-1-1 SB1-6-2 SB1-15-2 SB1-3-41-51 daidzein baicalein wogonoside Sephadex LH-20 Sephadex LH-20 SB1-15-3,4 MW=574

SB1-3-41-51-1 SB1-3-41-51-2 Sephadex LH-20 SB1-13-86 PTLC SB1-13-A-8-11 SB1-13-45 hesperidin ganhuangemin hesperetin SB1-3-41-51-1-1 SB1-3-41-51-1-2 SB1-13-85 wogonin genkwanin naringenin

SB1-13-54 viscidulin I SB1-3-41-51-2-1 SB1-3-41-51-2-2 oroxylin A quercetin Chart 2-2. Scheme for isolation of chemical constituents from S. baicalensis

31 2.3. Results and discussion

From the MeOH extract of the aerial parts of S. lateriflora three minor coumarins, scuteflorins A (1) and B (2), and decursin (3), along with six flavonoids, chrysin (4, Yuldashev et al., 2005), oroxylin A (5, Xiao et al., 2003), wogonin (6, Xiao et al., 2003), 5,7-dihydroxy-2′,8- dimethoxyflavone (7, Tomimori et al., 1988), dihydrochrysin (8, Jung et al., 1990), dihydrooroxylin A (9, Xiao et al., 2003), four triterpenes, lupenol (10, Patra et al., 1988), scutellaric acid (3a,24-dihydroxy-olean-12-en-28-oic acid) (11, Morota et al., 1995), pomolic acid (3ß,19a-dihydroxy-urs-12-en-28-oic acid) (12, An et al., 2005), ursolic acid (13, An et al.,

2005 ), two steroids, β-sitosterol (14) and daucosterol (15, Tang et al., 2002) and palmitic acid

(16, Huang et al., 2007) were obtained (Figure 2-1). Among them two coumarins (1) and (2) are new. Their structures were established by means of 1D and 2D NMR spectra as well as HRMS data. The absolute configuration of them was determined by comparison of the experimental and theoretical calculated CD spectra. The structures of known compounds were elucidated by comparison of their NMR data with those reported. The absolute configuration of the two flavanones, dihydrochrysin and dihydrooroxylin A was determined based on the specific rotation data and comparison with the literature data. All of the known compounds except chrysin and wogonin are reported for the first time from this plant.

32

33 Structural elucidation of two new compounds

Scuteflorin A (1) was obtained as a white powder, [α]D + 25.2 (c 0.032, MeOH). The

HR-ESI-TOF-MS spectrum of compound 1 gave an [M+Na]+ ion at m/z 365.1013 and

+ [2M+Na] at 707.2011 that are consistent with the pseudomolecular formula C19H18O6Na (calcd. for [M+Na]+ 365.1001 and calcd. for [2M+Na]+ 707.2105, respectively). The characteristic bright blue fluorescence under UV 254 light and the UV absorption at λ 255 and 330 nm (α，β- unsaturated C=O) along with an IR absorption band at 1719 cm-1 (C=O of α-pyrone and ester) indicated that this compound might possess a coumarin skeleton. The 1H NMR spectrum of 1

(Table 2-1, Figure 2-3) shows a pair of doublets at δH 8.04 and 6.36 ascribable to H-3 and H-4 of a coumarin moiety. The two singlets at δH 8.11 and 6.90 were assigned to H-5 and H-8 and suggested 6, 7-disubstitution of the aromatic ring. The 6, 7-disubstitution was determined to result from a dihydro-γ-pyranone ring based on the 1H and 13C NMR data (Figure 2-3 and 2-4) and the (heteronuclear multiple-bond connectivity) HMBC correlations as depicted in Figure 2-2.

H-5 (δH 8.11) correlated with a carbonyl carbon at C-4′ (δC 187.8), H-3′ (δH 5.71) with C-2′ (δC

83.6 ), C-5′ (δC 26.2), C-6′ (δC 20.1), and C-4′ (δC 187.8), δH H-5′ (1.58) with C-2′, C-3′, and C-

6′, H-6′ (δH 1.39) with C-2′, C-3′, and C-5′. The 4′-carbonyl substitution explained the downfield shift of H-5. The remaining resonances in the 1H and 13C NMR spectra corresponded to a senecioyloxy group (Erdelmeier et al., 1985) which was located at C-3′ according to the HMBC correlation of H-3′ with C-1″ (δC 165.2 ppm). Thus, the planar structure of compound 1 was deduced as 3′-senecioyloxy-4′-oxo-3′, 4′-dihydroxanthyletin and was given the trivial name scuteflorin A.

34 Table 2-1. NMR Data for Compounds 1 and 2 in Aceton-d6 compound 1 compound 2 no. δC δH (J in Hz) HMBC δC δH (J in Hz)

2 159.9 * 3 115.6 6.36, d (9.6) 115.6 6.35, d (9.6) 4 144.5 8.04, d (9.6) 144.4 8.04, d (9.6) 5 128.7 8.11, s C-4,7,9, 4′ 128.7 8.11, s 6 118.2 118.1 7 162.2 162.1 8 106.0 6.90, s C-6,7,9,10 106.0 6.91, s 9 160.6 160.4 10 115.1 115.1 2′ 83.6 83.5 3′ 76.5 5.71, s C-2′,4′,5′,6′,1″ 77.2 5.78, s 4′ 187.8 187.6 5′ 26.2 1.58, s C-2′,3′,6′ 26.4 1.59, s 6′ 20.1 1.39, s C-2′,3′,5′ 20.1 1.41, s 1″ 165.2 166.6 2″ 115.5 5.85, br C-1″,3″,4″,5″ 127.9 3″ 160.5 140.4 6.27, qq (8.4, 1.5) 4″ 27.5 1.97, d (1.2) C-2″,3″,5″ 20.7 1.95, p (1.5) 5″ 20.5 2.20, d (1.1) C-2″,3″,4″ 16.1 2.00, dq (7.2, 1.5) * Not observed

H O H H H O H

O O O O

H Figure 2-2. Key HMBC correlations of compound 1.

35

1 Figure 2-3. H-NMR spectrum of compound 1 in aceton-d6

13 Figure 2-4. C-NMR spectrum of compound 1 in aceton-d6

36

Figure 2-5. H-H COSY spectrum of compound 1 in aceton-d6

Figure 2-6. HSQC spectrum of compound 1 in aceton-d6

37

Figure 2-7. HMBC spectrum of compound 1 in aceton-d6

Compound 1 has a positive specific rotation (+ 25.2) compared to + 135 of (+)-decursin

(3) with its C-3΄S absolute configuration (Nemoto et al., 2003). However, due to the considerable structural differences between compounds 1 and 3 one may expect significant changes in optical rotation value. We thus took recourse to ECD to determine its absolute configuration by comparing the experimentally observed and theoretically calculated ECD spectra. Using this approach, we have successfully defined the absolute configuration of several natural products (Ding et al., 2007; 2008; 2009). The potential energy surface of compound 1 in the gas phase was scanned at the AM1 level by rotating about the C3′-O (C1″), C1″-O (C3′) and

C1″- C2″ bonds. Six conformers were found and redefined at the B3LYP/6-31G** level.

Conformational analysis indicated that conformer 1a is predominant [95.6% at the B3LYP/6-

31G** level in the gas phase by Gibbs free energies and 95.9% at the B3LYP-SCRF/6-

38 31G**//B3LYP/6-31G** level in MeOH with “COnductor-like continuum Solvent MOdel”

(COSMO) by total energies] (see Figure 8, Table 2-2). Theoretical calculation of the ECD of conformer 1a was performed by the time dependent density functional theory (TDDFT) method at the B3LYP/6-31G** and B3LYP/AUG-cc-pVDZ//B3LYP/6-31G** levels in the gas phase, and at the B3LYP-SCRF/6-31G**//B3LYP/6-31G** level in MeOH. The calculated ECD spectra of 1a in the gas phase and in MeOH, together with the experimental ECD of 1 in MeOH, are shown in Figure 9. The overall patterns of the calculated ECD spectra are consistent with that of the experimental one, i.e., positive Cotton effects (CE) in the 250-300 nm region and negative CEs in the 300-350 nm region. Considering the extended π-system of the dihydro-γ- pyranone containing coumarin chromophore in 1, the positive CE near 260 nm and the shoulder near 290 nm in the experimental ECD would be characteristic for this molecule. This is supported by analysis of the molecular orbitals involved in key transitions generating the ECD spectrum of 1a at the B3LYP/6-31G** level in the gas phase. The calculated positive rotatory strength at 272 nm (Table 2-3), which likely contributes to the positive CE near 260 nm in the experimental ECD, originates via the transitions from MO88 to MO91 (Figure 2-10). Another calculated positive rotatory strength at 294 nm, indicative of the shoulder near 290 nm in the experimental ECD, is derived from the transition from MO90 to MO92. The calculated negative rotatory strength at 317 nm, which may be associated with the negative CE beyond 300 nm in the experimental ECD, is generated by the transition from MO90 to MO91. All four MOs involve the π-electrons in the dihydro-γ-pyranone containing coumarin chromophore. The calculated positive rotatory strength at 257nm (Table 2-3), which originates via the transition from MO86 to MO93, also contributes to the positive CE near 260 nm in the experimental ECD spectrum

39 (Figure 2-10). The strong calculated positive rotatory strength at 210 nm results from the transition from MO87 to MO93. Since these three MOs involve the electrons of the - unsaturated ester system that may rotate along the C3′-O (C1″), C1″-O (C3′) and C1″- C2″ bonds in solution, caution should be exercised in using the CEs near these wavelengths to predict absolute configuration. Based on the above evidence, we conclude that compound 1 retains the same absolute configuration at C-3΄ as in (+)-decursin (3), but is assigned R in (1) due to the reversal of the Cahn–Ingold–Prelog (CIP) priorities.

Figure 2-8. Optimized geometries of conformers of compound 1 at B3LYP/6-31G** level in the gas phase.

40 Table 2-2. Conformational Analysis of Compound 1.

species in gas phase in methanol a b c d e f g h ΔE PE% ΔE′ PE′% ΔG PG% ΔEs PEs% 1a 0.00 93.1 0.00 94.3 0.00 95.6 0.00 95.9 1b 4.83 0.0 4.99 0.0 5.73 0.0 4.95 0.0 1c 8.17 0.0 7.96 0.0 8.08 0.0 6.66 0.0 1d 1.95 3.4 2.08 2.8 2.24 2.2 2.29 2.0 1e 7.27 0.0 7.31 0.0 7.71 0.0 7.14 0.0 1f 1.95 3.4 2.08 2.8 2.24 2.2 2.29 2.0 a,c,e Relative energy, relative energy with ZPE, and relative Gibbs free energy at B3LYP/6- 31G** level in the gas phase, respectively (kcal/mol). b,d,f Conformational distribution calculated by using the respective parameters above at B3LYP/6-31G** level in the gas phase. g,h Relative energy (kcal/mol) and conformational distribution in methanol solution at B3LYP- SCRF/6-31G**//B3LYP/6-31G** level with COSMO model, respectively.

Figure 2-9. Calculated ECD spectra of conformer 1a and the experimental ECD of compound 1 (red ▬ and olive ▬, in the gas phase at the B3LYP/6-31G** level; black ▬, in MeOH solvent at the B3LYP-SCRF/6-31G**//B3LYP/6-31G** level with COSMO; green ▬, in the gas phase at the B3LYP/AUG-cc-pVDZ//B3LYP/6-31G** level; blue ▬, experimental ECD in MeOH).

41 Table 2-3. Key Transitions and Oscillator and Rotatory Strengths of Conformer 1a of Compound 1 at the B3LYP/6-31G** Level in the Gas Phase.

a b c d e Excited State ΔE (eV) (nm) f Rvel Rlen

90→91 3.91 317 0.180 -21.8 -19.8

90→92 4.21 294 0.162 25.8 26.4

88→91 4.55 272 0.039 7.9 7.4

86→93 4.83 257 0.011 18.6 19.2

87→93 5.89 211 0.431 84.8 84.1

a Excitation energy. b Wavelength. c Oscillator strength. d Rotatory strength in velocity form

(10 -40 cgs). e Rotatory strength in length form (10 -40 cgs).

Figure 2-10. Electron density surfaces of some orbitals involved in key transitions in the ECD spectra of 1 at the B3LYP/6-31G** level in the gas phase.

Scuteflorin B (2) was obtained as white powder. [α]D + 5.8 (c=0.017, MeOH). Its molecular formula C19H18O6 was determined to be same as compound 1 by the high resolution

ESI-TOF-MS data 365.1011 (calcd. [M+Na]+ for 365.1001) and 707.2085 (calcd. [2M+Na]+ for

42 707.2105). Analysis of the NMR data [1H, 13C, heteronuclear single quantum coherence

(HSQC), correlation spectroscopy (COSY) and HMBC] (shown in Figure 2-11, 2-12, 2-13, 2-14, and 2-15) of 2 showed that the 1H and 13C NMR data of 2 are similar to those of compound 1 except that of the senecioyloxy group in 1 was replaced by an angeloyl group in 2 by comparison with literature data (Erdelmeier et al., 1985) and 2D NMR data. Thus, compound 2 was assigned as 3′-angeloyloxy-4′-oxo-3′,4′-dihydroxanthyletin and was given the trivial name scuteflorin B.

Since compound 2 is also dextrorotatory, the 3΄ R absolute configuration of 2 should be the same as in compound 1.

1 Figure 2-11. H-NMR spectrum of compound 2 in aceton-d6

43

13 Figure 2-12. C-NMR spectrum of compound 2 in aceton-d6

Figure 2-13. H-H COSY spectrum of compound 2 in aceton-d6

44

Figure 2-14. HSQC spectrum of compound 2 in aceton-d6

Figure 2-15. HMBC spectrum of compound 2 in aceton-d6

45 Seventeen flavonoids have been isolated after repeated chromatographic purification from five fractions of S. baicalensis. Their structures were elucidated as baicalein (17), baicalin

(18), wogonin (6), wogonoside (19) and oroxylin A (5) (Miyaichi et al., 1988b), quercetin (24), rutin (25), naringenin (27), hesperetin (28), hesperidin (29), and daidzein (30) (Kimura et al.,

1981), viscidulin I (23) and 2,6,2',4'-tetrahydroxy-6'-methoxychalcone (31, Iinuma et al., 1989), skullcapflavone II (20, Kimura et al., 1981), ganhuangenin (21, Liu, Y.L. et al., 1984), genkwanin (22, Bosabalidis et al., 1998), ganhuangemin (26, Liu, M.L. et al., 1984) (see Figure

2-16) using 1H-NMR, 13C-NMR, HMQC and HMBC as well as comparison with reported data.

46 R2' R4' R8 R7 O

R6' R6 R5 O R5=R6=R7=OH, R8=R2'=R4'=R6'=H baicalein (17) R5=R6=OH, R7=O-GlcUA, R8=R2'=R4'=R6'=H baicalin (18) R5=R7=OH, R8=OCH3, R6=R2'=R4'=R6'=H wogonin (6) R5=OH, R8=OCH3, R7=O-GlcUA, R6=R2'=R4''=R6'=H wogonoside (19) R5=R7=OH, R6=OCH3, R8=R2'=R4'=R6'=H oroxylin A (5) R5=R2'=OH, R6=R7=R8=R6'=OCH3, R4'=H skullcapflavone II (20) R5=R7=R2'=R4'=OH, R8=R6'=OCH3, R6=H ganhuangenin (21) R5=R4'=OH, R7=OCH3, R6=R8=R2'=R6'=H, genkwanin (22) R3' R2' R4' HO O

R6' R3 OH O

R3=R2'=R6'=OH, R3'=R4'=H viscidulin I (23) R3=R3'=R4'=OH, R2'=R6'=H quercetin (24) R3=O-rutinosyl, R3'=R4'=OH, R2'=R6'=H rutin (25) R3' R2' R4'

R7 O

R6' R3 OH O

R3=R7=R2'=R6'=OH, R3'=R4'=H ganhuangemin (26) R7=R4'=OH, R3=R2'=R3'=R6'=H naringenin (27) R7=R3'=OH, R4'=OCH3, R3=R2'=R6'=H hesperetin (28) R7=O-rutinosyl, R3'=OH, R4'=OCH3, R3=R2'=R6'=H hesperidin (29) HO O OH O OH

O OH OH OH H3CO daidzein (30) 2,6,2',4'-tetrahydroxy-6'-methoxychalcone (31)

Figure 2-16. Structures of compounds isolated from S. baicalensis

47 2.4. Conclusion

Two types of secondary metabolites, i.e. flavonoids and diterpenoids have been reported in many species of the genus Scutellaria, such as S. baicalensis, S. discolor, S. riuvlaris, S. galericulata etc. However; this is the first report on the isolation of coumarins from the genus

Scutellaria. Similar prenylated dihyropyranocoumarins were reported from Chinese traditional medicines (e.g. Angelica gigas and Peucedanum spp) with cytotoxicity (Ahn, et al., 1997), neuroprotective (Kang et al., 2005, Lee J. et al., 2003) and protein kinase C (PKC) (Ahn, et al.,

1997) activating activities. All the compounds except wogonin and chrysin are reported for the first time from S. lateriflora. The major flavonoids isolated from S. baicalensis as well as S. lateriflora will be used as chemical markers for quality control analysis. Meanwhile, these pure compounds will be tested on a panel of bioassays to provide scientific support for their traditional uses as well as to find new applications.

48 Chapter 3

Chemical Fingerprint Analysis of Scutellaria Species, Germander

and Dietary Supplements Claiming to Contain Skullcap Using HPLC-PDA

3.1 Introduction

Quality control when dealing with botanicals is much more difficult than for synthetic drugs because of the high variability of chemical components involved. As botanical dietary supplement (BDS), singular and in combinations, contain a myriad of mostly unique, or species- specific compounds in complex matrices, it is difficult to completely characterize all of these compounds. It is also equally difficult to know precisely which one is responsible for the therapeutic effects of an herbal medicine because these compounds often work synergistically.

In recent years, significant efforts have been made to devise methods for the quality control of herbal materials as well as their products by utilizing quantitative methods and/or qualitative fingerprinting technologies (Lin et al., 2001; Yang et al., 2005). Quantitative analysis methods commonly used by the industry are analyzing the product for the presence of chemical markers known to be present in the specific herbal as indicators or standards to assess quality of the herbal medicine. Even though this has been an acceptable method for quality control, for herbal complex systems, the presence of the chemical markers do not always guarantee an individual is getting the actual herb stated by the product label, especially if the product has been spiked with the chemical markers. So, the quantization method for the chemical markers can confirm the compounds presence, but it may not confirm the presence of the plant material. Qualitative analysis is typically used to demonstrate the general characteristics of herbal materials or their

49 products. Many kinds of chemical fingerprint analysis methods, such as TLC, GC, HPLC-UV, and HPLC-MS, are in rapidly developing that offer great potential for monitoring the quality of herbal materials, particularly for identifying a particular herb and distinguishing it from closely related species (Jiang, et al., 2005; Zschocke et al., 1998). By fingerprinting the entire pattern of components present in one herb or its products, the complete information could be acquired. The quality information acquired from fingerprinting profile makes the chemical composition in a given herb visible and comparable in wholeness to determine not only the presence or absence of desired markers or active constituents but the complete set of ratios of all detectable analytes.

Fingerprinting profile possesses two levels of significance: the „elementary‟ quality control and

„intensive‟ quality control. The former („elementary‟ quality control) serves the routine authentication and rapid overall quantifiable estimation of herbs and their products even if some ingredients in the fingerprint are unknown yet (Xie et al., 2006). And the latter („intensive‟ quality control) means chromatographic fingerprint coupled with chemometrics to enhance the high capability for processing the array of the parameters. While the chromatographic fingerprint can also serve as „quality data bank‟ in which the intact suit of ingredients has been reserved for sustainable in-depth study. Fingerprinting has been introduced and accepted by the

WHO as a strategy for identification and quality evaluation of herbal medicinal products (WHO,

1991). Both the U.S. Food and Drug Administration (FDA) (FDA, 2004) and the European

Medicines Agency (EMEA, 2001) clearly state that the appropriate fingerprint chromatograms should be used to assess the consistency of botanical products. In 2004, the State Food and Drug

Administration (SFDA) of China officially required all injections made from herbal medicine to be standardized by chromatographic fingerprints (SFDA, 2004).

50 Chromatographic techniques used for fingerprint analysis of herbal medicines include

TLC, GC, HPLC, and hyphenation procedures. Among them TLC is a convenient method for determining the quality and possible adulteration of herbal products due to the advantages of its simplicity, versatility, high velocity, specific sensitivity and simple sample preparation. Even nowadays, TLC is still frequently used for the analysis of herbal medicines since various pharmacopoeias such as American Herbal Pharmacopoeia (AHP) (Upton, Santa Cruz, US,

2002), Chinese drug monographs and analysis (Wagner, K¨otzting/Bayer, Wald, Germany,

1997), Pharmacopoeia of the People‟s Republic of China (Pharmacopoeia Commission, Beijing,

1997), etc. still use TLC to provide first characteristic fingerprints of many herbs. It is worth noting that the technique of TLC is also being updated in progress and the different techniques like rotation planar chromatography (RPC), overpressured-layer chromatography (OPLC), and electroplanar chromatography (EPC) are reported (Nyiredy, 2003). Over the past decades, GC has been a popular and useful analytical tool for the analysis of volatile compounds in herbal medicines with the advantage of high sensitivity of detection for almost all the volatile chemical compounds. Especially, with the use of hyphenated GC–MS instrument, reliable information on the identity of the compounds is available as well. However, the most serious disadvantage of

GC is that it is not convenient for analysis of the samples containing polar and non-volatile compounds. For this, it is necessary to use tedious sample work-up which may include derivatization. Therefore, liquid chromatography becomes another necessary tool to be applied for the comprehensive analysis of the herbal medicines. HPLC is a versatile method for analysis of almost all compounds in the herbal medicines when coupled with UV detector, ELSD, or hyphenated techniques, such as HPLC–MS, HPLC-NMR. Some new techniques, including

51 micellar electrokinetic capillary chromatography (MECC) (Albert et al., 1997), high-speed counter-current chromatography (HSCCC), low-pressure size-exclusion chromatography (SEC)

(Pervin et al., 1995), reversed-phase ion-pairing HPLC (RP-IP-HPLC) (Karamanos et al., 1997;

Loganathan et al., 1990), and strong anion-exchange HPLC (SAX-HPLC) (Rice et al., 1989) have been developed for good separation for some specific extracts of some herbal medicines.

In order to evaluate the similarity and difference between a number of chromatographic fingerprints obtained from the same herbal medicine or from different species, several chemometric approaches such as Hierachical Cluster Analysis, Principle Component Analysis,

Pattern Recognition and K-Nearest Neighbour Analysis were employed to deal with the chromatographic fingerprint, and a great number of such papers have been published (Debeljak et al., 2005; Gan et al., 2006; Gong et al., 2003, 2004, 2006; Li et al., 2004; Mok et al., 2006;

Nederkassel et al., 2006; Xu et al., 2006). It is worth noticing that the principle behind these methods is statistical basis. Therefore, a reliable sample size is critical and should be calculated properly before experiments to ensure the validity of the results.

3.2 Background and objectives

Flavonoids have been reported as characteristic components of Scutellaria genus. More than 600 flavonoids have been reported from about 60 Scutellaria species, of which over 60 were isolated from S. baicalensis (see section 1-2). Previous studies for quality control of S. baicalensis were focused on the qualitative and quantitative determination of a few major flavonoids existed, including baicalein, baicalin, wogonin and wogonoside. Different chromatographic techniques, such as HPLC-UV (Katsura et al., 1982; Ma et al., 2003; Miyaichi et al., 1986; Stojakowska et al., 1998; Takino et al., 1987; Tomimori et al., 1985; Zhang et al.,

52 1998), HPLC–ESI tandem mass spectrometry (Guo et al., 2005; Zhang et al., 2007; Wu et al.,

2004a), CE (Myoung et al., 1999; Wang et al., 2005; Yu et al., 2007), MECC (Liu et al., 1994) have been applied to evaluate the quality of raw material as well as the preparation claiming to contain S. baicalensis.

To date, only a few papers have touched the field related to the assessment of the quality of American skullcap and the discrimination between skullcap and its adulterant, germander.

Gafner et al. (Gafner et al., 2003) reported methods, including HPTLC, and digital photomicroscopy to differentiate S. lateriflora and its potential adulterants T. canadense and T. chamaedrys. In the HPTLC analysis, three flavonoids isolated from S. lateriflora, e.g. baicalein, baicalin, and ikonnikoside, two phenylpropanoids from T. canadense, e.g. verbascoside and teucrioside, were used as markers. Gao‟s group (Gao et al., 2008) developed a HPLC-UV method to compare the flavonoid biomarker content (baicalein, baicalin, and wogonin) of eleven commercial tinctures derived from S. lateriflora aerial parts (n=7) and S. baicalensis (n=4).

Results showed that the concentrations of these flavonoids varied widely. Makino (Makino et al., 2008) analyzed the amounts of four flavonoids, including baicalein, baicalin, wogonin, and wogon-7-O-glucuronide, in the roots, stems, and leaves of S. baicalensis and S. lateriflora, and in the commercial products prepared from S. lateriflora. Experimental results indicated that these flavonoids were detected in the roots of S. baicalensis, however, not in its aerial parts. The contents of these flavonoids in the aerial parts of S. lateriflora were higher than that in the roots.

Recently, Zhang et al established an HPLC method to analyze twelve phenolic compounds, including 10 flavonoids and two phenylethanoid glycosides, in commercial American skullcap products from different sources (Zhang et al., 2009). The total analyzed ingredients varied

53 widely for different samples and the relative proportions of ingredients in each sample were significantly different. They suggested that the chemical variability of the commercial American skullcap products might come from the plant raw materials that had variable chemical compositions. In another study, an HPLC-DAD/ESI-MS method was used to distinguish between S. lateriflora and its adulterants using the phenylpropanoid verbascoside and teucrioside as markers (Lin et al., 2009). Although the above mentioned methods had made progress, these methods are limited in their ability to ensure identity of skullcap plant material being used in the product.

Hence the main objective of our study was to develop a HPLC-PDA fingerprinting method that can be applied to authenticate S. lateriflora, to distinguish it from its potential adulterants, and to compare the chemical profiles of different species in this genus. In the present study three classes of compounds, including eleven flavonoids isolated from Scutellaria species, two phenylpropanoids verbascoside and teucrioside and one diterpene teucrin A isolated from T. canadense, were used as markers to provide comprehensive analysis for the above mentioned purposes. Since the UV patterns are different and specific for each type of compounds investigated, analysis of UV spectra of the known markers and relevant peaks in samples will be quite valuable for identification of different species.

3.3. Experimental sections

3.3.1. Reagents and chemicals

All solvents, including acetonitrile, water and acetic acid, were of HPLC grade and provided by Fisher Scientific (Fair Lawn, JN, USA) and Sigma (St. Louis, MO, USA), respectively. The eleven standard flavonoids used in this study; namely baicalein (17), baicalin

54 (18), oroxylin A (5), wogonin (6), chrysin (4), quercetin (24), rutin (25), daidzein (30), naringenin (27), hesperetin (28), and hesperidin (29), were isolated from Scutellaria species by authors (see Chapter 2 for details). The phenylpropanoids, including verbascoside (I) and teucrioside (II), one diterpene, teucrin A (III), were isolated at the National Center for Natural

Products Research (NCNPR). Their structures (see Figure 3-1) were elucidated based on the 1D and 2D NMR spectra, HRMS data as well as comparison with reported data. The purity of these standards was confirmed by chromatographic methods (TLC and HPLC).

3.3.2. Standard solutions

An individual stock solution of the standard compounds was prepared at a concentration of1 mg/mL in methanol. 10 μL of each standard flavonoid solution was mixed and the mixture was further diluted to a concentration of 50 μg/mL which was then used for HPLC analysis. 3

μL of each standard phenylpropanoid solution was mixed with 30 μL of teucrin A standard solution to get the working solution for HPLC analysis. All these solutions were stored in refrigerator.

3.3.3. Sample preparation

Ten species (total fourteen samples), including seven scutellaria related species and three

Teucrium species, were investigated in this study. The data associated with each species, such as source and code number are listed in Table 3-1.

General extraction procedure - The dried powder of each plant material (about 1.000 g) was extracted with 20 mL MeOH under ultrasonication for 30 min followed by centrifugation for

5 min at 1550 rpm. The supernatant was transferred to a 100 mL flask. The extraction was repeated two more times and the respective supernatants were combined and evaporated to

55 dryness. 2 mL of HPLC grade methanol were added to the dried extract and transferred to 5 mL volumetric flask. The flask was rinsed with about 1.5 mL methanol twice and the final volume was adjusted to 5 mL with methanol.

Prior to injection, this solution was mixed thoroughly and filtered through 0.45 μm membrane. The first 0.5 mL was discarded and the remaining volume collected for HPLC analysis. Each sample solution was injected in triplicate.

Three dietary supplements claiming to contain S. lateriflora were purchased through commercial sources (see Table 3-2). The dosage form of these three samples is tincture. 2.0 mL of sample solution was filtered through 0.45 μm membrane. The first 0.5 mL was discarded and the remaining volume was collected then diluted to proper concentration for HPLC analysis.

3.3.4 Instrumentation and HPLC conditions

HPLC-PDA analysis was performed on a Waters 2695 Alliance Separations Module

(Waters, Milford, MA) connected with a Waters 2996 Photodiode Array Detector and separation was investigated using two different HPLC columns, a Luna column RP-18 and a Gemini RP-18 column (250 x 4.6 mm i.d.; 5 µm particle size for each of column) from Phenomenex (Torrance,

CA, USA). The column was equipped with a 2 cm LC-18 guard column (Phenomenex Inc.).

The temperature of the column was maintained at 25 °C. The mobile phase consisted of acetonitrile (A) and water (B), all containing 0.2% acetic acid, which were applied in the following gradient: 0-5 min, 15%-20% A; 5-30 min, 20%-25% A; 30-60 min, 25%-55% A; 60-

65 min, 55% A; 65-70 min, 55%-100% A, then hold 5 min. Each run was followed by a 5 minutes washing procedure with 100% acetontrile. The flow rate was adjusted to 1 mL/min, and the injection volume was 10 µL. Re-equilibration was done with 15% A for 12 min. Total run

56 time was 75 min.

OH R8 OH R O 7 HO O

R6 R O R3 5 OH O

R5=R6=R7=OH, R8=H baicalein (17) R3=OH quercetin (24) R5=R6=OH, R7=O-GlcUA, R8=H baicalin (18) R3=O-rutinosyl rutin (25) R5=R7=OH, R6=R8=H chrysin (4) R5=R7=OH, R8=OCH3, R6=H wogonin (6) R5=R7=OH, R6=OCH3, R8=H oroxylin A (5)

R3' R4' R O HO O 7

OH O O OH R7=OH, R3'=H, R4'=OH naringenin (27) daidzein (30) R7=OH, R3'=OH, R4'=OCH3 hesperetin (28) R7=O-rutinosyl, R3'=OH, R4'=OCH3 hesperidin (29)

OH HO OH HO HO O OH O O O O OH HO O OH CH HO 3 O O H H O O O O CH OH OH O 3 O

O HO HO OH OH verbascoide (I) teucrin A (III) teucrioside (II)

Figure 3-1. Chemical structures of fourteen standard compounds used in this study

57 Table 3-1. Species Analyzed in Present Study species code number source S. amoena 534:1/28/2003; SCAML Da Li, Yun Nan, China S. baicalensis 522:1/28/2003; SCABL (leaf) S. baicalensis 503:1/27/2003; SCABL (root) Nei Meng Gu, Wu Chuan Xian, China S. barbata 533:1/28/2003; SCABL Si Chuan, China S. barbata 535:1/28/2005 SCABL Shen Yang, China S. integrifolia SAR01204 unknown S. lateriflora #198 w/H-tet unknown S. lateriflora 1180:6/4/2004; SCLAL Starwest Botanicals , Inc. USA S. lateriflora 7828:11/11/2009; SCLAL Bazaar of India S. lateriflora 3257: 04/17/2007: SCLAL FRONTIER NATURAL PRODUCTS CO.OP (www.frontiercoop.com) S. likiangensis SCLIL Yun Nan, China S. rehderiana 524:1/28/2003; SCREL Liao Ning, China T. chamaedry 4849;11/16/2008; TECHL Monsento T. canadense 6544;06/03/2009; TECAL Missouri Botanical Garden 2893;02/22/2008; TECAL American Mercentile Corperation T. fruticans 8073;12/09/2009; TEFRL Missouri Botanical Garden

Table 3-2. Products Claiming to Contain S. lateriflora product code dosage form Scullcap 7825:11/11/2009; SCLAL tincture (59-65% alcohol by volume) Scullcap 7827:11/11/2009; SCLAL alcohol-free extract (1:1) Scullcap 7828:11/11/2009; SCLAL alcohol-free extract (1:1)

3.3.5. Method development

The qualified standards described in Chapter 2, and authenticated species S. lateriflora

(1180:6-4-2004; SCLAL) were used for analytical method development in this study.

58 Initial fingerprinting method development began by extracting authenticated S. lateriflora with methanol. The extract was then analyzed on two different HPLC columns, a Luna column

RP-18 and a Gemini RP-18 column, with a gradient solvent system (shown in Table 3-3). Three

UV wavelengths, including 250, 280 and 350 nm, were chosen for analysis. Regardless of the gradient solvent system, a level baseline could be achieved only using a Luna column. Then, an optimized mobile phase gradient for the Luna column was determined for the fingerprint analysis. Initially, a mobile phase consisting of acetonitrile (A) and water (B), all containing

0.1% acetic acid, was used to investigate the separation of the extract of S. lateriflora. The mobile phase was pumped at 1 mL/min under gradient manner (see Table 3-3). The HPLC chromatogram of the plant extract is shown in Figure 3-2. In order to achieve good resolution for a series of peaks to elute in less than 20 min in the HPLC chromatogram attempts were made to modify gradient system (shown in Table 3-4) and the separation of the peaks in less than 20 min was achieved (shown in Figure 3-3). Subsequently, the continued method development involved in applying further modified mobile phase (shown in Table 3-5) to get better separation for the peaks occurring between 45-60 min. Even though separation was achieved under these conditions, the peak shapes were not satisfactory due to peak tailing (see Figure 3-4). Therefore, the concentration of acetic acid in binary mobile phase was adjusted from 0.1% to 0.2% and peak shapes were optimized (shown in Figure 3-5). Once the fingerprint method had been developed through the use of authenticated plant material, the mixture of standard solutions and other species had to be tested to ensure its usefulness. All species of Scutellaria and Teucrium tested are listed in the experimental section (Table 3-1). This method was applied successfully to the mixture of eleven standard flavonoids (shown in Figure 3-6 and Table 3-7) and most of

59 Scutellaria related samples investigated. Meanwhile, using this method good separation was achieved for the mixture of three standards isolated from T. canadense (see Figure 3-7) and

Teucrium samples. The proper sample concentration was obtained by testing a series of concentrations for each sample.

The HPLC fingerprint method in the present study was validated by analysis of chromatograms in the intra-and inter-day precision tests. Five peaks were selected as characteristic peaks in the chromatogram of the S. lateriflora (see Figure 3-8) as they had relatively large peak areas and good resolution. Peak 4th was used as the reference peak. The relative peak retention time (RPRT) and relative peak area (RPA) of each characteristic peak related to the reference peak were calculated for expression of the HPLC fingerprint pattern. The detection wavelength was 250 nm. Four individual samples of S. lateriflora on three consecutive days under optimized HPLC conditions were analyzed.

60 Table 3-3. HPLC Gradient Condition I for Fingerprint Analysis time (min) flow (mL/min) %ACN w/ 0.1% HOAc %water w/ 0.1% HOAc 0.01 1.00 20.0 80.0 5.00 1.00 20.0 80.0 30.00 1.00 30.0 70.0 55.00 1.00 55.0 45.0 65.00 1.00 55.0 45.0 70.00 1.00 100.0 0.0 75.00 1.00 100.0 0.0

0.20 AU

0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 Minutes

Figure 3-2. HPLC chromatogram of S. lateriflora under the gradient elution system shown in Table 3-3.

Table 3-4. HPLC Gradient Condition II for Fingerprint Analysis time (min) flow (mL/min) %ACN w/ 0.1% HOAc %water w/ 0.1% HOAc 0.01 1.00 15.0 85.0 5.00 1.00 20.0 80.0 30.00 1.00 25.0 75.0 55.00 1.00 50.0 50.0 65.00 1.00 55.0 45.0 70.00 1.00 100.0 0.0 75.00 1.00 100.0 0.0

61 0.20

AU 0.10

0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 Minutes

Figure 3-3. HPLC chromatogram of S. lateriflora under the gradient elution system shown in Table 3-4.

Table 3-5. HPLC Gradient Condition III for Fingerprint Analysis time (min) flow (mL/min) %ACN w/ 0.1% HOAc %water w/ 0.1% HOAc 0.01 1.00 15.0 85.0 5.00 1.00 20.0 80.0 30.00 1.00 25.0 75.0 60.00 1.00 55.0 45.0 65.00 1.00 55.0 45.0 70.00 1.00 100.0 0.0 75.00 1.00 100.0 0.0

0.20

AU 0.10

0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 Minutes

Figure 3-4. HPLC chromatogram of S. lateriflora under the gradient elution system shown in Table 3-5.

62 Table 3-6. HPLC Gradient Condition IV for Fingerprint Analysis ime (min) flow (mL/min) %ACN w/ 0.2% HOAc %water w/ 0.2% HOAc 0.01 1.00 15.0 85.0 5.00 1.00 20.0 80.0 30.00 1.00 25.0 75.0 60.00 1.00 55.0 45.0 65.00 1.00 55.0 45.0 70.00 1.00 100.0 0.0 75.00 1.00 100.0 0.0

0.20 AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

Figure 3-5. HPLC chromatogram of S. lateriflora under the gradient elution system shown in Table 3-6.

0.20 2′

1 4 AU 0.10 ′ 3′ ′ 5′

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

Figure 3-8. HPLC chromatographic fingerprinting profile of S. lateriflora (250 nm, for precision study)

63 250 nm

0.20 11.062 Extracted 18.152 Extracted 26.799 Extracted 206.3 201.6 0.08 214.5 0.15 0.06 25 29 0.06 276.9 18

AU 0.10 254.5

0.10 354.2 0.04 0.04

AU AU AU 282.8 316.0

0.05 0.02 0.02 329.1 407.9 0.00 0.00 0.00 0.00 300.00 400.00 300.00 400.00 300.00 400.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 nm nm nm

Minutes 30.777 Extracted 37.441 Extracted 43.967 Extracted 247.4 208.6 215.6 0.10 0.15 30 24 0.20 27 288.7

0.10 254.5 371.8

AU AU AU 300.6 280 nm 0.05 0.10 4 0.05 28 391.1 435.6 27 0.00 0.00 0.00 30 17 5 300.00 400.00 300.00 400.00 300.00 400.00 nm nm nm 0.10 45.950 Extracted 47.091 Extracted 55.738 Extracted

AU 6 215.6 218.0 25 29 18 0.15 24 0.30 28 17 0.10 6

0.20 0.10 274.5 274.5

AU AU AU 286.4 322.0 0.05 0.00 0.10 0.05 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 440.5 0.00 0.00 0.00 Minutes 300.00 400.00 300.00 400.00 300.00 400.00 nm nm nm

56.453 Extracted 57.444 Extracted 350 nm 0.20 213.3 215.6 0.15 0.20 267.4 4 271.0 5

0.10 AU AU 316.0 0.10 313.7 0.10 0.05

0.00 0.00 AU 0.05 300.00 400.00 300.00 400.00 nm nm 0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

Figure 3-6. HPLC chromatogram of a mixture of eleven standard flavonoids under optimized gradient system.

Table 3-7. Retention Times and UV Absorptions of Standard flavonoids Obtained Under Optimized HPLC Conditions compound retention time (min) UV λmax (nm) rutin (25) 11.06 254, 354 hesperidin (29) 18.15 283 baicalin (18) 26.80 214, 277, 316 daidzein (30) 30.78 247, 301 (sh) quercetin (24) 37.44 209, 254, 372 naringenin (27) 43.97 216, 289 hesperitin (28) 45.95 286 baicalein (17) 47.09 216, 276, 322 wogonin (6) 55.74 218, 275 chrysin (4) 56.45 213, 267, 314 oroxylin A (5) 57.44 216, 271, 316

64 10.924 Extracted I 1.00 0.20 II 250 nm 0.80 I

0.60 329.1 AU 0.10 AU 0.40 III 0.20 0.00 0.00 300.00 400.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 nm Minutes 12.053 Extracted 280 nm 0.60 II 0.40

0.40 329.1

AU AU 0.20 0.20

0.00 300.00 400.00 0.00 nm 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes 43.678 Extracted 219.1 350 nm 0.40 II

AU 0.20 I

0.00 300.00 400.00 nm

Figure 3-7. HPLC chromatogram of a mixture of three standard compounds isolated from T. candense under optimized gradient system.

3.4. Results and discussion

The optimized chromatographic conditions were obtained after evaluation of different

HPLC columns and running different mobile phases (described in experimental section). The complex nature of the extract necessitated a relatively long analytical run to improve resolution.

Use of a PDA detector was extremely valuable for peak identification during the initial method development. Multiple injections showed low standard error, indicating that the results were highly reproducible. Precisions of RPRT and RPA of all characteristic peaks were within the acceptable relative standard deviation (RSD) (n=4) 3.0%, indicating that the conditions used in the qualitative chromatographic fingerprint analysis are satisfactory. The chemical fingerprint method developed in this study was applied to verify S. lateriflora species present in ground plant material and to distinguish between S. lateriflora and its potential adulterants, germander.

Also, it was applied to compare the chemical fingerprint profiles of different species in this genus

65 The comparison of the fingerprint profiles of S. lateriflora with its potential adulterants is shown in Figure 3-9. Meanwhile, the mixture of three markers isolated from T. candense is included. The comparison of the chromatograms of two sets of standard mixtures at 250, 280 and 350 nm (shown in Figure 3-6 and 3-7) found that selecting 250 nm as the detection wavelength resulted in an acceptable response and enabled the detection of all 14 standards used in this study. In Figure 3-9, the chromatogram of authenticated S. lateriflora species exhibits very different fingerprint profile compared to that of Teucrium species. Since the UV spectra of three standard markers isolated from T. canadense are different from that of flavonoids, the combined analysis of the chromatograms and the UV spectra of a series of peaks occurring in these two species allowed us to differentiate S. lateriflora from its potential adulterants

66 0.20 S. lateriflora

0.10 AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

T. chamaedy (6 times dilution)

0.50 AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.60 T. candense (2893, 4 times dilution)

0.40 AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.20 T. candense (6544)

AU 0.10

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.60 T. fruticanes (8073, 3 times dilution)

0.40 AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes I

0.20 II std. mix-3 AU 0.10 III

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

Figure 3-9. Comparison of HPLC profiles of S. lateriflora with its potential adulterants detected at wavelength 250 nm.

67 Some species such as S. amoena, S. likiangensis, and S. rehderiana have been used as substitutes as “Huang Qin” in China (Song et al., 1981). In the present study we found the fingerprint profiles of these species are similar to that of S. baicalensis (shown in Figure 3-10) which provided scientific support for this conventional use. As to the chromatograms of S. lateriflora and S. baicalensis, their fingerprint profiles are not very similar. In addition, the

HPLC profile of S. barbata is different from that of S. baicalensis. In China, S. barbata named

“banzhilian” was used for different indication as S. baicalensis. From the chemical point of view, the present results provide scientific evidence for their different uses.

Three samples of S. lateriflora obtained from different sources were investigated in this study and Figure 3-11 details the fingerprint comparison of them with an authenticated sample.

The chromatogram of sample from India showed similar HPLC profile to that of authenticated sample, but the fingerprints of the other two samples varied widely. Two samples of S. barabata obtained from different sources were compared and the chromatograms of them are similar

(shown in Figure 3-12). For the different parts of the plant investigated, for example, S. baicalensis, the contents of flavonoids in the leaves were different from those in the root (see

Figure 3-13) which is consistent with the results reported ((Makino et al., 2008)). This method was also applied to analyze three commercial products claiming to contain S. lateriflora and the comparison of their HPLC profiles are shown in Figure 3-14. The results indicated that the contents of flavonoids in the product of skullcap 7825 are similar to that of skullcap raw materials, but different from the product of skullcap 7827 and 7828.

68 4 28 27 17 std. mix-11 0.10 30

AU 29 18 6 5 25 24

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes 0.60 S. amoena (4 times dilution)

0.40 AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.20 S.baicalensis (503, 5 times dilution)

AU 0.10

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes S.likiangensis (1.5 times dilution) 0.50

AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes 0.60 S. rehderiana (2 times dilution)

0.40 AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.40 S. lateriflora (1180)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes 0.40 S. integriflora (1.8 times dilution)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes S. barbata (533)

0.20 AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes Figure 3-10. Comparison of HPLC profiles of Scutellaria related species detected at wavelength 280 nm.

69 0.40 S. lateriflora (1180)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes S. lateriflora (7828) 0.20

AU 0.10

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes 0.40 S. lateriflora (3257, 2 times dilution)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes 0.40 S. lateriflora (#198, 4 times dilution)

0.20 AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes Figure 3-11. Comparison of HPLC profiles of S. lateriflora obtained from different sources detected at wavelength 280 nm.

std. mix-11

0.10 AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.20 S. barbata (533:1-28-2003) AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.40 S. barbata (535:1-28-2005)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes Figure 3-12. Comparison of HPLC profiles of S. barabata from different sources detected at wavelength 280 nm.

70 4 28 5 27 17 std. mix-11 0.10 30 6

AU 29 18 25 24

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.40 S.baicalensis (503, 4 times dilution)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes S. baicalensis (522, 2 times dilution)

0.50 AU

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes Figure 3-13. Comparison of HPLC profiles of different part of S. baicalensis detected at wavelength 280 nm. 4 28 27 17 5 std. mix-11 30 6 25 29 18 24

0.40 S. lateriflora (1180)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

0.40 skullcap (7825)

AU 0.20

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

1.00 skullcap (7827, 5 times dilution)

AU 0.50

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

1.00 skullcap (7828, 5 times dilution)

AU 0.50

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Minutes

Figure 3-14. Comparison of HPLC profiles of commercial products claiming to contain S. lateriflora detected at wavelength 280 nm.

71 3.5. Conclusion

The HPLC-PDA fingerprinting analysis method described in this study is the first detailed report of qualitative analytical method capable of using two sets of standards. The mixture of eleven standard flavonoids allows not only analyzing the presence or absence of desired markers in different Scutellaria species but also helping in comparison of the HPLC profiles of all Scutellaria species. Meanwhile two sets of standards used in this study allow us to differentiate S. lateriflora from its potential adulterants. The developed method in the present study can be applied for the authenticity of S. lateriflora plant material and distinguishing it from germander; also it will assist in the quality and safety assessment of commercial products claiming to contain S. lateriflora.

72 Chapter 4

Identification of Phenolic Compounds in Scutellaria lateriflora Using

High-Performance Liquid Chromatography-Electrospray Tandem Mass Spectrometry-

Ultraviolet Photodiode Array

4.1. Introduction

The plant kingdom represents an extraordinary reservoir of novel molecules, of which only a small percentage has received phytochemical investigation (Wolfender et al., 1996). It is estimated that about 2% of all carbon photosynthesized by plants is converted into flavonoids or closely related compounds (Markham, 1982). Flavonoids constitute a highly prominent class of polyphenolic compounds and embrace a diversity of chemical structures. The structure of flavonoids is composed of three rings with a basic C15 phenyl-benzopyrone skeleton (Figure 4-

1). The substitution pattern of C ring, regarding the site of the B-ring as well as the keto, hydroxyl, methoxyl, and glycosyl groups, defines the different flavonoid groups, including flavones, flavonols, flavanones, flavanols, and isoflavones, etc. (Figure 4-2). In plant, flavonoids are often present as O- or C-glycosides. The O-glycosides have sugar substituents bonded to a hydroxyl group of the aglycone, whereas the C-glycosides have sugar substituents bonded to a carbon of the flavonoid aglycone, generally at position 6-C and 8-C. The flavonoids act in plants as antioxidants, antimicrobials, photoreceptors, visual attractors, and feeding repellants (Pietta,

2000). Many studies have suggested that flavonoids exhibit a broad array of pharmacological activities, which include anti-inflammatory, antiallergic, antiviral, antioxidant, cancer preventive activities and other benefits to human health which was summarized in chapter 1.

73 3' 2' 4' 8 B 7 O 2 5' A C 6' 6 3 5 4

Figure 4-1. Basic ring structure of flavonoids with labelling convention

O O O

OH O O O flavone flavonol flavanone

O O

OH O O flavanol isoflavone

Figure 4-2. Basic structures of the major flavonoid classes

From the view point of phytochemical identification of flavonoids, difficulties mainly arise because many of them are isomers, especially given that the biological activities of flavonoids correlate with specific structural features. Therefore, 2D NMR spectroscopy is commonly used for the unambiguous assignment of the structures of flavonoids, even in the case of simple known compounds, such as wogonin, oroxylin A, and gankwanin. The introduction of

LC-MS in the analysis of crude plant extract has represented an important step for the on-line identification of unknown compounds or for dereplication of known constituents. Mass

74 spectrometer is a universal detector. It can achieve very high sensitivity and provide information on the molecular weight as well as chemical structure (Wolfender et al., 1994).

To date, about seventeen flavonoids, including flavone, flavanone, and their glycosides, five diterpenoids and three minor dihydropyranocoumarins, have been reported from the aerial parts of American skullcap (Gafner et al., 2003; Li et al., 2009; Maurizio et al., 1998; Zhang et al., 2009). The flavonoid components far outnumber the diterpenoids found in the aerial parts of this plant. From the HPLC chromatogram of this plant (shown in chapter 3), it can be concluded that some flavonoids have not been identified yet. Therefore, detailed identification of the indigenous flavonoid components is necessary for better understanding of its traditional uses and authentication of skullcap as well as its products. Quantitative determination of its major flavonoids has been reported recently (Gao et al., 2008; Lin et al., 2009). However, no efforts have been attributed to develop LC-MS/MS methods for the identification of all the main phenolic compounds.

For the purpose of phytochemical screening of flavonoids in S. lateriflora, a method of analysis was developed based on liquid chromatography with photodiode array and tandem mass spectrometric detection (LC-DAD/ESI-MSn) to identify the flavonoids of S. lateriflora.

In this study, thirteen flavonoids and one stilbene derivative were identified in S. lateriflora, seven of which are described for the first time in S. lateriflora.

4.2. Experimental sections

4.2.1. Reagents and chemicals

All solvents, including acetonitrile, water and acetic acid, were of HPLC grade and provided by Fisher Scientific (Fair Lawn, JN, USA) and Sigma (St. Louis, MO, USA),

75 respectively.

Twelve standard flavonoids used in this study (shown in Figure 4-3), namely baicalein

(17), baicalin (18), chrysin (4), oroxylin A (5), wogonin (6), gankwanin (22), quercetin (24), rutin (25), naringenin (27), hesperetin (28), hesperidin (29), and daidzein (30), were isolated from Scutellaria species by authors (see Chapter 2 for details). Their structures were elucidated based on the 1D and 2D NMR spectra, HRMS data as well as comparison with reported data.

The purity of these standards was confirmed by chromatographic methods (TLC and HPLC).

OH R4' OH R8 R7 O HO O R 6 R3 R5 O OH O

R5=R6=R7=OH, R8=R4'=H baicalein (17) R3=OH quercetin (24) R5=R6=OH, R7=O-GlcUA, R8=R4'=H baicalin (18) R3=O-rutinosyl rutin (25) R5=R7=OH, R6=R8=R4'=H chrysin (4) R5=R7=OH, R8=OCH3, R6=R4'=H wogonin (6) R3' R5=R7=OH, R6=OCH3, R8=R4'=H oroxylin A (5) R4' R5=R4'=OH, R7=OCH3, R6=R8=H genkwanin (22) R7 O HO O OH O

O OH R7=OH, R3'=H, R4'=OH naringenin (27) R7=OH, R3'=OH, R4'=OCH3 hesperetin (28) daidzein (30) R7=O-rutinosyl, R3'=OH, R4'=OCH3 hesperidin (29)

Figure 4-3. Chemical structures of twelve standard flavonoids investigated

4.2.2. Standard solutions

Standard flavonoid solutions were prepared at a concentration of 1 mg/mL in methanol.

Working solutions were obtained by twenty folds dilutions of concentrations from 1mg/mL to 50

μg/mL. All these solutions were stored in refrigerator.

76 4.2.3. Sample preparation

The aerial parts of S. lateriflora investigated were purchased from Starwest botanicals

(http://www.starwest-botanicals.com) and authenticated by Dr. Vaishali Joshi at the National

Center for Natural Products Research, University of Mississippi, where a voucher specimen

(voucher no. 2120) has been deposited.

The dried powder of the aerial parts (1.0520 g) of S. lateriflora was extracted with 20 mL

MeOH under ultrasonication for 30 min followed by centrifugation for 5 min at 1550 rpm. The supernatant was transferred to 100 mL flask. This process was repeated twice and respective supernatants were combined and evaporated to dryness. The residue was dissolved in 2 mL of

HPLC grade methanol and transferred to 5 mL volumetric flask. The flask was rinsed with about

1.5 mL methanol twice and the final volume was adjusted to 5 mL with methanol.

Prior to injection, this solution was mixed thoroughly and 2 mL of it was filtered through

0.45 μm membrane. The first 0.5 mL was discarded and the remaining volume was collected into the HPLC sample vial for analysis.

4.2.4. LC/ESI-MSn conditions

Mass spectrometry experiment was performed on a LCQ ion trap instrument (Finnigan

MAT. Thermo Scientific, Waltham, MA, USA) with an electrospray source. Ultrahigh pure helium (He) was used as the collision gas and high purity nitrogen (N2) as the nebulizing gas.

The operating parameters were optimized for baicalin in the negative ion mode . The optimized

MS conditions were as follows: electrospray voltage of the ion source at 3.5 kV with the shealth gas at 45 arbitrary units (AU), capillary voltage at 28 V, auxiliary gas at 15 AU, capillary temperature at 300 C, and tube lens offset voltage at -10 V. For full scan MS analysis, the

77 spectra were recorded over the range of m/z 100–1000. A syringe pump was used for the direct injection of pure standard dissolved in MeOH (about 50 μg /mL). Following each first-order MS scan, the most intense ion (usually pseudomolecular ion during compound elution) was selected and fragmented up to the MS4 stage by CID, each successive most-abundant fragment ion being selected again as precursor ion for the next step. The collision energy for CID was between 30-

45%, and the isolation width of precursor ions was 1.0 U. The data were analyzed using the

XCalibur software.

The HPLC system employed in the LCQ ion trap instrument consists of a model TSP

P4000 quaternary pump coupled with a mode TSP AS3000 autosampler and a model TSP

UV6000 diode-array detector. The chromatographic analysis was conducted at ambient temperature and the chromatographic separations were carried out on the same column using the same elution program as developed in chapter 3.

4.3. Results and discussion

In the present study, we have examined the mass fragmentation behavior of twelve standard flavonoids present in Scutellaria species in the negative ion mode and obtained fragmentation patterns for each compounds. Thereafter we have characterized the phenolic compounds from the methanol extract of S. lateriflora. For the identification of the different compounds from the extract, sample was injected in full scan mode in order to choose the compounds of interest. Then, the MS-MS scan mode was selected based on the predominant ions obtained in the mass spectrum in full scan mode. The structure of each compound was identified by the elucidation of its fragmentation pathway as well as by the comparison of its fragmentation information to that of standard flavonoid obtained and reported data.

78 For the convenience of discussion the CID-MS-MS spectra, the fragments recorded for the two bonds cleavages of C ring have been annotated according to the nomenclature proposed by Ma et al (Ma et al., 1997). Scheme 4-1, adapted from Ma et al. shows the various retrocyclization fragments observed in this study. The i,jA- and i,jB- represent product ions containing intact A and B rings, the superscript i and j indicate the C-ring bonds that have been broken.

1,3A- R3' R3' OH 1,2A--CO 1,2A- OH B B R7O O 1 R7O O 1 R5' R5' A C 2 A C 2 4 3 4 3 0,2A- OR3 OH O OH O

1,3 - 1,3A- 0,4 - 0,4 - B B -H2O B OH R 8 B HO 0O 1 A C 2 4 R6 3 OH O

1,4A- Scheme 4-1. Nomenclature adopted for the different retrocyclization cleavage fragments of deprotonated flavones, flavonols and flavanones observed in this study (adapted from Ma)

4.3.1 Fragmentation pathways of twelve standard flavonoids

In this study the ESI-MSn fragmentation behaviors as well as their corresponding UV characteristics of twelve flavonoids (their structures shown in Figure. 4-3), including flavones

(baicalein, baicalin, chrysin, wogonin, oroxylin A, gankwanin), flavonols (quercetin and rutin), flavanones (hesperetin, hesperidin and naringenin), and isoflavone (daidzein), have been investigated in detail and summarized in Table 4-1. Some common features, for instance the

79 - losses of small neutral molecules and/or radicals from [M-H] ions, such as CO (28 Da), CO2 (44

. Da), H2O (18 Da), and/or CH3 (15 Da), can be observed. It has been demonstrated that the

- losses of CO and CO2 from the [M-H] ion are due to the contraction of C ring (Fabre et al.,

2001) and other characteristic neutral losses are very useful for determination of the presence of specific functional groups. Negative ion mode was chosen for ESI-MSn analysis in this study, since background noise and interfering peaks derived from mobile phase were found to be much less than in the positive mode.

Fragmentation pathways of hydroxylated flavones

The deprotonated molecular ion of baicalein with tri-hydroxyl groups on A ring, [M−H]− at m/z 269, was obtained after full-scan ESI-MS. In the ESI-MS2 experiment, the product ion at

− m/z 251 as base peak was observed, which is resulted from the loss of H2O from [M−H] at m/z

269. Same product ion could not be observed for chrysin in this study as well as for luteolin reported (Fabre et al., 2001), in which two hydroxy groups are located on C-5 and C-7 position, respectively. Therefore, the ion involved in the loss of H2O can be observed only when the flavone has vicinal hydroxy groups. The ion at m/z 241 is due to the loss of CO, and the m/z 223 ion is due to the successive loss of H2O and CO (see Scheme. 4-2). Baicalin is an O-glucuronide of baicalein. Upon CID, the glycosidic bond was easily cleaved to generate an ion at m/z 269 attributed to the neutral loss of a glucuronic acid (∆m = 176 Da). In the MS3 experiment, ion at m/z 269 produced the same fragments as baicalein described above.

80 Table 4-1 LC-UV/MSn Characteristics of Twelve Standard Flavonoids compound UV [M-H]- MSn ions m/z (relative abundance) λmax m/z (nm) baicalein 216, 269 MS full scan: 269 (100); (17) 274, MS2 [269]: 251 (100), 241 (30), 223 (32), 197 (20), 169 (7); 322 MS3 [251]: 223(100), 213 (7) baicalin 216, 445 MS full scan: 891 (100), 445 (83),269 (62); (18) 275, MS2 [445]: 269 (100), 175 (16) 316 wogonin 216, 283 MS full scan: 283 (100); (6) 274 MS2 [283]: 268; MS3 [268]: 240 (56), 239 (100), 224 (14), 223 (34), 212 (22), 211 (34), 163 (44); MS4 [239]: 211 (100) oroxylin A 215, 283 MS full scan: 283 (100); (5) 271, MS2 [283]: 268; 317 MS3 [268]: 240 (36), 239 (100), 224 (12), 223 (20), 212 (38), 211 (25), 163 (7); MS4 [239]: 211 (100) gankwanin 215, 283 MS full scan: 283 (100); (22) 266, MS2 [283]: 268; 336 MS3 [268]: 240 (9), 239 (3) hesperetin 286 301 MS full scan: 301 (100); (28) MS2 [301]: 286 (100), 283 (48), 257 (59), 242 (99), 233 (4), 215 (9), 199 (12), 125 (9); MS3 [286]:258 (97), 242 (100), 201 (36), 174 (22), 164 (16) hesperidin 283 609 MS full scan: 609 (100); (29) MS2 [609]: 301 (100) quercetin 205, 301 MS full scan: 301 (31); 603 (100), 905 (31); (24) 255, MS2 [301]: 273 (13), 257 (11), 193 (5), 179 (100), 151 (45); 370 MS3 [273]:245 (17), 229 (100), 163 (16); MS3 [257]:239 (79), 229 (100), 211 (67); MS3 [179]: 169 (30), 151 (100) rutin (25) 206, 609 MS full scan: 609 (100); 1219 (69); 255, MS2 [609]: 301 (100), 300 (91), 271 (13); 355 MS3 [301]:273 (15), 271 (97), 257 (12), 255 (57), 179 (100) narigenin 216, 271 MS full scan: 271 (100); (27) 287 MS2 [271]: 177 (17), 151 (100); MS3 [177]:133 (12), 109 (100); MS3 [151]: 107 (100); chrysin 212, 253 MS full scan: 253 (100); (4) 267, MS2 [253]: 225 (2), 209 (20), 181 (2), 151 (4) 311(sh)

81 daidzein 247, 253 MS full scan: 253 (100); 507 (54); (30) 302 MS2 [253]: 225 (16), 209 (7); (sh)

Fragmentation pathways of methoxylated flavone isomers

Wogonin (8-OCH3), oroxylin A (6-OCH3), and genkwanin (7-OCH3) are three methoxylated flavone isomers with the same MW of 284. They exhibited a significant [M-H-

−• CH3] radical anion at m/z 268 as base peak and no other fragmentation was observed in their

2 − MS spectra. The loss of a CH3 radical (15 Da) from the [M-H] ion explained the presence of a methoxy group. The MS3 product ion spectra of these three compounds, obtained under the

3 −• same conditions, are shown in Figure 4-4. In MS experiment of the [M-H-CH3] ion of genkwanin, no any other fragmentation, except low signal intensity ions at m/z 240 (9, -CO) and

82 239 (3, -COH•), were observed. This suggested that this radical anion is probably rearranged to a stable 7-keto-diconjugated-3,4-enol derivative avoiding any further fragmentation for this 7- methoxy-4′-hydroxyflavone aglycone (Fabre et al., 2001). But six ions were observed in MS3 spectra for both wogonin and oroxylin A. The product ion at m/z 240 was produced by the loss of CO from 4-position, and ion at m/z 224 was produced by loss of CO2 from 1-O and 4-CO as discussed in the literature (Fabre et al., 2001). The notable ions are those at m/z 239 and 223, and the base peak at m/z 239. It was proposed that these ions are due to the respective losses of

• • COH and CO2H (Wu et al., 2004b) and is as shown in Scheme 4-3. The mechanism for the loss of COH• may be loss from 4-CO and one hydrogen atom from the molecule, thus the molecule is

• easy to form steady conjugated system. A similar fragmentation for the loss of CO2H from the loss of 1-O and 4-CO and one hydrogen atom has been observed. The ion at m/z 212 was produced by the loss of two CO, the position is not known. Although the fragments of wogonin were almost identical to those of oroxylin A in MS3 experiment, interesting differences between these two isomers could be observed from MS3 spectra when we compared these two spectra carefully (show in Table 4 -2). The relative abundance of ion 0,2A- at m/z 163 is higher than that of the ion at m/z 212 for wogonin. On the contrary, the relative abundance of ion 0,2A- at m/z 163 is lower than that of the ion at m/z 212 for oroxylin A. This allowed us to distinguish these two isomers. Based on the different fragmentation behaviors of their MS3 spectra these three isomers can be differentiated.

(a) (b)

83 wogonin.268 #7 RT: 0.15 AV: 1 NL: 5.66E3 oroxylin A_268.3 #11 RT: 0.19 AV: 1 NL: 3.50E4 T: - c Full ms3 [email protected] [email protected] [ 100.00-400.00] T: - c Full ms3 [email protected] [email protected] [ 100.00-400.00] 239.25 239.31 100 100

90 90

80 240.18 80 70 70

60 163.16 60 50 50 212.10 240.27 40 40 223.10 212.19 268.22

30 30 Relative Abundance Relative Abundance 211.31 268.19 198.02 223.25 20 267.33 20 195.30 184.02 163.21 196.16 10 165.96 10 267.26 250.01 149.90 269.18 102.87 137.59 150.06 268.91 110.14 139.35 168.13 250.20 0 0 100 120 140 160 180 200 220 240 260 280 300 100 120 140 160 180 200 220 240 260 280 300 m/z m/z

(c) gankawain268 #8 RT: 0.18 AV: 1 NL: 4.18E4 T: - c Full ms3 [email protected] [email protected] [ 70.00-400.00] 268.26 100

90

80

70

60

50

40

30 Relative Abundance

20 269.29 10 240.23 151.12 171.15 212.18 271.18 0 100 150 200 250 300 350 400 m/z Figure 4-4. ESI-MS3 spectra of three methoxylated flavone isomers: (a) wogonin; (b) oroxylin A; (c) gankwanin

84

85 Table 4-2. ESI-MSn Product Ions of Three Methoxylated flavonoids scan mode ion(s) produced wogonin oroxylin A gankwanin MS full scan [M-H]- 283 283 283 .- [M-H-CH3] 268 268 268 2 .- MS [283] [M-H-CH3-CO] 240 (56) 240 (36) 240 (9) .- [M-H-CH3-COH] 239 (100) 239 (100) 239 (3) .- [M-H-CH3-CO2] 224 (14) 224 (12) .- [M-H-CH3-CO2H] 223 (34) 223 (20) 3 .- MS [268] [M-H-CH3-2CO] 212 (22) 212 (38) 0,2A- 163 (44) 163 (7)

Fragmentation pathways of flavanones

The fragmentation of three flavanones, narigenin, hespertin, and hesperidin, were investigated.

The MS2 spectra of narigenin yielded the 1,3A- ion at m/z 151 as the base peak. Also the ion at m/z 177 with low relative intensity was observed, which was resulted from the loss of B

3 ring system. The ion at m/z 107 in the MS experiment was produced by the loss of CO2 from the ion at m/z 151 (see Scheme 4-4).

The MS2 fragmentation pathway of hesperetin is much more complex than that of narigenin (shown in Scheme 4-5). There are two main fragmentation pathways for hespertin.

- The first pathway begins with the loss of CH3 from the anion [M-H] to form the radical anion

−• 3 [M-H-CH3] , followed by the losses of CO and CO2 in the MS experiment to form the ions at m/z 258 and 242, respectively. The second main pathway related to almost all other fragmental

1,4 - ion at m/z 257, 233, and 125, corresponding to the neutral loss of CO2, C3O2, and A ion, from the [M-H]- ion.

86

87

88 Fragmentation pathways of flavonols

The fragments proposed for quercetin consecutive to CO and CO2 losses (b and d) are similar to the corresponding luteolin fragments reported by Fabre (Fabre et al., 2001). Further losses of CO2 and CO from b and d, respectively, give rise to the resonance-stabilized ion c (see

Scheme 4 -6). The most interesting fragment concerns the base peak at m/z 179. This 1,2A- diagnostic ion undergoes further loss of CO giving rise to a 1,2A- - CO ion at m/z 151. It is worth mentioning that this specific pathway, involving the 1,2 bonds, observed in this study, was consistent with the result observed for fisetin by other group (Fabre et al., 2001). In the MS2 experiment of rutin, the ion at m/z 301 as base peak was observed suggesting the loss of rutinose residue.

Fragmentation pathways of isoflavonoidh

In this study the consecutive loss of CO from pseudomolecular ion of daidzein can be observed in negative mode (see Scheme 4-7). Also it is worth noticing that the fragmentation pathway of isoflavonoid daidzein is analogous to that of flavone chrysin in negative mode. But the ratio of relative abundance of product ions at m/z 225 and 209 for these two types of compounds with the hydroxy group at different ring is different (shown in table 4-3).

Table 4-3. Relative Intensity of Ions in ESI-MS/MS of Chrysin and Daidzein

compound m/z=225 [M-H-CO] (%) m/z=209 [M-H-CO2] (%)

chrysin 2 20

daidzein 16 7

89

90 4.3.2 Characterization of phenolic compounds from S. lateriflora using HPLC-DAD/ESI-MSn

The LC-MS chromatogram (TIC) of the methanolic extract of skullcap in negative ion mode is shown in Figure 4-5. The molecular mass of each component at its retention time (tR) in full scan mode was recorded. Fourteen phenolic compounds were identified by the elucidation of their fragmentation pathways and by comparison of their MS fragmentation patterns as well as

UV spectra (see Figure 4-6 and Table 4-4) with standard flavonoids or reported data.

(a)

RT: 0.00 - 74.98 30.27 74.32 NL: 1.09E6 39.18 48.42 1000000 36.08 58.90 Total Scan PDA 2.63 900000 41.85 55.78 Scutellaria 11.07 Ext Conc 800000 3.58 71.90 CID=20 12.83 700000 53.22 71.55 600000 27.95 5.42 17.60 uAU 59.48 69.55 500000 20.80 400000

300000 61.02 65.60 200000

100000

0 0 10 20 30 40 50 60 70 Time (min) (b) RT: 0.00 - 74.99 35,36,37 35.85 NL: 100 2.66E7 Base Peak 90 M S 80 Scutellaria Ext Conc 70 CID=20

60 34 30.50 50 19,38,39 39.34

40 42.51 Relative Abundance Relative 30 33,18 44.301748.91 20 17.96 27.30 13.92 6,4,5 10 20.14 2.30 12.96 32 58.98 62.65 69.68 0 0 10 20 30 40 50 60 70 Time (min) Figure 4-5. HPLC-ESI-MSn analysis of the extract of skullcap. (a)HPLC-UV chromatogram monitored at 280 nm; (b) LC-negative ion ESI-MS total ion current (TIC) chromatogram.

91

92 Table 4-4. LC-UV/MSn Identification of Phenolic Compounds in S. lateriflora

- n Peak tR UV .λmax [M-H] ESI-MS (MS ) m/z identification no. (min) (nm) m/z (relative abundance) 32 24.50 251, 301 417 ESI-MS: 417, 255; 5-(β-D-glucosyloxy)- MS2 [417]: 255 (7), 237 3-hydroxy-trans- (100), 211 (18) stilbene-2-carboxylic acidb 33 27.72 275, 350 445 ESI-MS: 445, 269; norwogonin-7-O- (sh) MS2 [445]: 269 (100), 251 glucuronide b (83), 241 (15), 223 (30), 197 (18), 171 (9), 151 (5), 137 (5) 18 28.44 268, 315 445 ESI-MS: 445, 269; baicalin a MS2 [445]: 269 (100), 251 (72), 241 (37), 223 (37), 197 (22), 171 (9), 145 (5) 34 30.36 242, 282, 447 ESI-MS: 447, 271; 5,6,7-trihydroxy 348 (sh) MS2 [447]: 271 (100); flavanone-7-O- MS3 [271]: 253 (49), 243 glucuronide b (100), 227 (24), 199 (13), 167 (dihydrobaicalin) (8) 35 36.22 232, 282 445 ESI-MS: 445, 269; galangin-7-O- MS2 [445]: 269 (76), 241 glucuronide b (65), 225 (100), 197 (45), 183 (7), 181 (9), 171 (8) 36 36.62 237, 282 461 ESI-MS: 461, 285; dihydrooroxylin A-7- MS2 [461]: 285 (100), 270 O-glucuronide b (22); MS3 [285]: 270 (31), 269 (100), 243 (6), 166 (11) 37 37.59 268, 308 459 ESI-MS: 459, 283; oroxylin A-7-O- MS2 [459]: 283 (100), 268 glucuronide b (51), 175 (5), 113 (11) MS3 [283]: 268 (100), 239 (8)

93 Table 4-4. LC-UV/MSn Identification of Phenolic Compounds in S. lateriflora (Cont.)

- n Peak tR UV .λmax [M-H] ESI-MS (MS ) m/z identification no. (min) (nm) m/z (relative abundance) 19 39.67 236, 268 459 ESI-MS: 459, 283; wogonin-7-O- MS2 [459]: 283 (100), 268 (70), glucouronide b 175 (4); MS3 [283]: 268 (100), 137 (4) 38 39.80 237, 280, 489 ESI-MS: 489, 313; 5,7-dihydroxy-6,8- 329 MS2 [489]: 313 (100), 298 (83), dimethoxy flavone- 283 (26); 7-O-glucuronide b MS3 [313]: 298 (100), 283 (3), 269 (5), 254 (5), 214 (3), 193 (3), 167 (3) 39 40.34 239, 291 461 ESI-MS: 461, 285; dihydrowogonin-7- MS2 [461]: 285 (100), 270 (32); O-glucuronide b MS3 [285]: 270 (100), 243 (6), 223 (5), 166 (8) 17 47.50 270, 320 269 ESI-MS: 269, 251; baicalein a MS2 [269]: 251 (100), 241 (33), 223 (30), 197 (18), 169 (7) 6 56.22 280 283 ESI-MS: 283.18, 268.36; wogonin a MS2 [283]: 268 (100); MS3 [268]: 240 (53), 239 (100), 224 (14), 223 (36), 212 (20), 211 (34), 163 (44) 5 58.06 272, 316 283 ESI-MS: 283.18, 268.36; MS2 [283]: 268 (100); oroxylin A a MS3 [268]: 240 (32), 239 (100), 224 (14), 223 (20), 212 (39), 211 (27), 163 (8) 4 56.62 271, 253 ESI-MS: 253, 209; chrysin a 320(sh) MS2 [253]: 225 (4), 209 (26), 181 (2), 151 (6) a Identified by comparing the retention time, UV spectra and MSn data with standard flavonoids. b Identified by UV and MSn data in comparison with literature data.

94 Compound 32 exhibited a UV spectrum with characteristic bands at 251 nm and 301 nm, which are not consistent with the typical UV spectral pattern of flavonoids, suggesting a nonflavonoid type of compounds. The characteristics of the UV spectrum of this compound are identical with those of trans-stilbene (Beale et al., 1953; Suzuki et al., 1960). The ESI-MS in full scan mode gave an ion [M-H]- at m/z 417 and an ion [M-H-164]- at m/z 255 produced by the loss of a glucose residue. In the MS2 experiment, the ion at m/z 237 represented the base peak

- resulted from the loss of H2O of [M-H-164] , suggesting vicinal hydroxy groups or a hydroxy and one free carboxylic group present in the molecule. The ion at m/z 211 was attributed to the neutral loss of CO2, supporting the presence of carbony group. The proposed fragmentation pathway of 32 was shown in scheme 4-8. Based on these results compound 32 was tentatively identified as 5-(β-D-glucosyloxy)-3-hydroxy-trans-stilbene-2-carboxylic acid, which has been isolated from Scutellaria scandens (Miyaichi et al., 1988a).

O C O

OH O COOH -H2O m/z=237 O OH m/z=255 -CO2 O m/z=211

Scheme 4-8. Proposed fragmentation pathways of compound 32 agylcone

Compound 33, 18 and 35 are isomers with the same ion [M-H]- at m/z 445 in full scan mode. The product ion [M-H-176]- at m/z 269 was observed as base peak in MS2 experiment for

95 these three isomers, indicating the neutral loss of a glucuronic acid reside from [M-H]- ion.

Since the glucoside bond of O-glucuronide was easily cleaved to generate its aglycone, this neutral loss is very useful for the identification of O-glucuronides. In the MS2 experiment of compound 33 and 18, ion at m/z 269 of both compounds produced the identically diagnostic neutral losses of H2O (18 Da), CO (28 Da), and the losses of both H2O and CO (46 Da), resulting in the ion at m/z 251, 241 and 223, respectively, whose fragmentation mechanism was consistent with the standard baicalein. Since the tR value as well as the UV spectrum of compound 18 were identical to those of authentic baicalin, it was identified as baicalin. Compound 33 exhibited the same UV behavior as norwogonin 7-O-glucuronide (Oi et al., 1988), indicating 33 is norwogonin-7-O-glucuronide. In the MS2 experiment of compound 35, no product ion attributed to the neutral loss of H2O from its precursor ion at m/z 269, indicating absence of vicinal hydroxy groups in A ring, whereas product ions at m/z 241 and 225, which resulted from the neutral losses of CO and CO2 respectively, were predominant, suggesting 35 is galangin-7-O- glucuronide. The MS2 fragmentation behaviors as well as UV spectrum of this compound were identical to those of reported data (Han et al., 2007).

The peak 34 showed [M-H]- ion at m/z 447 and aglycone [M-H-176]- signal at m/z 271 in full scan mode, which are indicative of the presence of a glucuronic acid residue. Further MS2 spectra of m/z 271 yielded the ions at m/z 253, 243, 227, and 167. The ion at m/z 253 resulted from the loss of H2O, revealing the presence of two OH groups in ortho position. The ions at m/z 243 and 227 resulted from the neutral losses of CO and CO2 from the corresponding [M-H-

176]- ion. The ion at m/z 167 was attributed to the 1,3A-, suggesting the occurrence of three OH groups in A ring. The molecular mass of this compound was 2 Da greater than that of baicalin.

96 Also band I, associated with the absorption ascribed to the B-ring cinnamoy system, was weak in its UV spectrum, while band II, associated with absorption involving the A-ring benzoy system, showed bathochromic shift with respect to the value of baicalin, indicating a flavanone-type compound. Based on the evidence that all 7-O-glucuronides showed essentially identical UV spectra with their corresponding aglycones (Liu et al., 2009), 7-OH glycosylation was proposed for 34. On the basis of comparing the UV spectrum of 34 with reported data (Liu et al., 2009), this compound was characterized as 5,6,7-trihydroxy-flavanone-7-O-glucuronide.

Peak 37 and 19 are isomers with the molecular weight of 460 Da. They showed the same product ion [M-H-176]- at m/z 283, which indicated the loss of a glucuronic acid moiety as

−• discussed above. For the aglycone part of these two compounds a significant [M-H-CH3] radical anion at m/z 268 presented as base and sole peak in their MS2 spectra, suggesting the presence of a methoxy group for both compounds. To further investigate the structures of these two isomers, tandem mass experiments were employed on the two [M-H-176-15]- ions at m/z

269. Although the products ions of m/z 269 for these two compounds were identical, the relative intensities of ion at m/z 212 and m/z 163 were different. Comparison with the results obtained for authentic wogonin and oroxylin A suggested that peak 37 and 19 are oroxylin A-7-O- glucuronide and wogonin-7-O-gluconide, respectively.

Compounds 36 and 39 both gave an [M-H]- ion at m/z 461 and their agylcone ion at m/z

285 (-176 Da), which suggested the presence of a glucuronic acid moiety. The UV spectra of these two compounds only exhibited band II, indicating a flavanone skeleton. The MS2 spectra of the ion at m/z 285 yielded an ion at m/z 270, which resulted from the loss of a CH3 radical (15

Da) from the ion [M-H-176]- at m/z 285. In addition, the ion 1,3A- at m/z 166, which are the

97 characteristic of flavanone, suggested that these two compounds are either dihydrowogonin or dihydrooroxylin A. Compound 36 was identified as dihydrooroxylin A-7-O-glucopyranoside since its UV spectrum was consist with the literature (Miyaichi et al., 1995) and 39 as dihydrowogonin-7-O-glucopyranoside.

Compounds 38 exhibited an [M-H]- ion at m/z 489. CID of this compound produced ion

[M-H-176]- at m/z 313, which was resulted from the neutral loss of a glucuronic acid residue.

Fragments of m/z 298 and 283, due to the sequential losses of CH3 radicals, indicated the presence of two methoxy groups. In the MS3 experiment of m/z 313, the product ion at m/z 298 was base peak. The product ion at m/z 269 was produced by the loss of COH•, and ion at m/z

0,2 - 254 was produced by loss of CO2 from m/z 298. The ion A at m/z 193 along with above mentioned two ions at m/z 269 and 254 were indicative for the second methoxy group on A ring.

The UV spectrum of 38 was same as 5,7-dihydroxy-6,8-dimethoxy flavone-7-O-glucuronide reported in the literature (Liu et al., 2009). Thus, compound 38 was identified as 5,7-dihydroxy-

6,8-dimethoxy flavone-7-O-glucuronide.

- The peak at tR=12.9 min in TIC showed an [M-H] ion at m/z 623 as well as product ion

[M-H-162]- at m/z 461 and [M-H-162-146]- at m/z 315 resulting from the sequential losses of glucosyl (162 Da) and rhamnosyl (146 Da) residues, and revealing the presence of one glucose and one rhamnose moieties. The molecular weight of the agylcone of this compound is 316.

This compound was tentatively characterizated as isorhamnetin-7-O-rhamnosylglucoside by Guo et al (Han et al, 2007). The same compound was also observed by Yao‟s group (Liu et al., 2009) from S. baicalensis, and they doubted the result from Guo et al since the UV spectrum for isorhamnetin and its 7-O-glycosides reported were characterized by two bands at 253 and 370

98 nm, which are quite different from that of the compound studied with absorptions at 290 and 330 nm. No conclusion was made based on the data obtained by Yao‟s group. In the present study same UV spectral behavior and MS fragmentation patterns were also observed. We endeavored to elucidate its structure based on the data obtained, but further MS/MS experiments are needed to get sufficient information.

Compound 17, 6, 5, and 4 were positively characterized as baicalein, wogonin, oroxylin

A, and chrysin, respectively, by the comparison of their UV spectrum and MSn fragmentation patterns with corresponding standards.

4.4. Conclusion

As has been demonstrated, the MS-MS spectra of the deprotonated molecules [M-H]- for four types of flavonoids yielded different fragmentation profile. The diagnostic fragment ions resulted from the cleavage of two bonds at positions 1/3, 0/2 and 0/4 of the C-ring are very helpful for the structural determination of the A- and B-ring substitution patterns. Furthermore, loss of internal sugar moiety linked to aglycone in the flavonoid glycoside together with those fragment ions mentioned above are also useful for the determination of the properties of the linkage between the sugar and aglycone (O- or C-glycosides), and the position of sugar

. substitution in the aglycone. In addition, losses of H2O and CH3, etc. are valuable in the determination of the presence of some functional groups. Based on the differences in the relative abundances of some specific ions isomeric methoxylated flavonoids can be discriminated. These fragmentation patterns have been used as guidelines for the determination of the compounds in crude plant extracts.

LC/ESI-MSn is a very promising complementary tool for the characterization of

99 flavonoids. The LC/MS-MS method developed in the NCNPR was successfully employed to separate and identify the major flavonoids from S. lateriflora, which provides knowledge of both its chemical composition and its potential use as a source of natural antioxidants. In this study, we have demonstrated how the combination of UV spectra and ESI-MSn spectra allows a fast characterization of thirteen flavonoids and one stilbene derivative without time-consuming isolation of compounds even if reference compounds are not available or on hand. It is worthy noted that the choice of appropriate conditions for collision induced fragmentation is very important to assure acquisition of enough fragmentation information for structural characterization of these types of compounds. Also special care should be taken during the interpretation of MS data since there are many isomers co-existed in the skullcap extract.

100 Chapter 5

Evaluation of Bioactivities of Methanol Extracts of S. baicalensis L. and S. lateriflora

Georgi and Flavonoids Isolated from Them

5.1. Introduction

American skullcap has been reputed as a nerve tonic, sedative, and anticonvulsant among

Native Americans and Europeans (Millspaugh, 1974). It has been traditionally used to treat epilepsy and other neurological disorders such as nervous tension states, hysteria, chorea, anxiety, and insomnia. Despite its extensive uses, there are limited data on its bioactive constituents, the pharmacological properties and its safety issues. Results from in vivo animal behavior trials suggested that the aqueous extract of plant material of American skullcap has anxiolytic property, and that the flavonoids, and possibly the amino acids contents, may be responsible for this activity (Awad et al., 2003). Two flavone-glycosides, scutellarein and ikonnikoside I isolated from American skullcap, have been found to bind to one of the serotonin receptors, the 5-HT7 receptor, in brain (Gafner et al., 2003). The 5-HT7 receptor is thought to play a role in the pathogenesis of various ailments such as sleep disorders, depression, and migraine (Gafner et al., 2003). Significant anxiolytic effects have been demonstrated in a double blind, placebo-controlled study of healthy volunteers and results indicated that commercial

American skullcap products have clinical benefits as a relaxing nervine with no overt evidence of toxicity or side effects (Wolfson et al, 2003).

Chinese skullcap has attracted attention since 1980‟s, and a number of studies related to the evaluation of the bioactivities of aqueous extract of this species as well as its major

101 flavonoids, including baicalein, baicalin, and wogonin, have been reported. The results showed that both aqueous extract and the flavonoids isolated from it demonstrated diverse bioactivities, as summarized in chapter 1.

The aim of the present study is to evaluate the bioactivities of crude extracts of the two above mentioned skullcap species as well as thirteen individual flavonoids isolated from them to provide scientific support for the traditional usages of these plants. In the present study, the methanol extracts of S. baicalensis George (SBE), and S. lateriflora L (SLE) together with thirteen flavonoids, including baicalein (17), baicalin (18), chrysin (4), oroxylin A (5), wogonin

(6), genkwanin (22), viscidulin I (23), quercetin (24), rutin (25), naringenin (27), hesperetin (28), hesperidin (29) and daidzein (30), isolated from these two species, were tested in a panel of bioassays to determine their anticancer, antioxidant, anti-inflammatory, estrogenic activity and cytotoxic activities. In addition, their activities on peroxisome proliferator-activated receptor- alpha and -gamma (PARP and PPARγ) were also evaluated.

5.2. Materials and methods

5.2.1. Materials

The aerial parts of S. lateriflora were purchased from Starwest botanicals

(http://www.starwest-botanicals.com) and authenticated by Dr. Vaishali Joshi at NCNPR,

University of Mississippi, where a voucher specimen (voucher no. 2120) has been deposited.

S. baicalensis ethanol extract was purchased from Chinese Herbs Direct and authenticated by comparison of its TLC profiles with that of the authentic sample provided by

Dr. Vaishali Joshi. A voucher specimen (Voucher No. 1181) has been deposited in the repository of NCNPR.

102 Both samples were extracted with MeOH and the MeOH extracts were dried in vacuo for bioassay.

Thirteen standard flavonoids used in this study were isolated from S. baicalensis and S. lateriflora. Their structures were elucidated based on the 1D and 2D NMR spectra, HRMS data as well as comparison with reported data. The purity of these standards was confirmed by chromatographic methods (TLC and HPLC).

103 R3' R2' R4' R8 R7 O R5' R6' R6 R5 O

R5=R6=R7=OH, R8=R4'=H baicalein (17) R5=R6=OH, R7=O-GlcUA, R8=R4'=H baicalin (18) R5=R7=OH, R6=OCH3, R8=R4'=H oroxylin A (5) R5=R7=OH, R8=OCH3, R6=R4'=H wogonin (6) R5=R4'=OH, R7=OCH3, R6=R8=H, genkwanin (22) R5=R7=OH, R6=R8=R4'=H chrysin (4)

R3' R2' R4' HO O HO O

R6' R3 O OH O OH

R3=R2'=R6'=OH, R3'=R4'=H viscidulin I (23) daidzein (30) R3=R3'=R4'=OH, R2'=R6'=H quercetin (24) R3=O-rutinosyl, R3'=R4'=OH, R2'=R6'=H, rutin (25)

R3' R4'

R7 O

R3 OH O

R7=R4'=OH, R3=R3'=H naringenin (27) R7=R3'=OH, R4'=OCH3, R3=H hesperetin (28) R3=H, R7=O-rutinosyl, R3'=OH, R4'=OCH3 hesperidin (29)

Figure 5-1. Structures of flavonoids tested

104 5.2.2. Methods

5.2.2.1. Assays for in vitro cytotoxicity

The samples were tested for their in vitro cytotoxicity against a panel of normal cell lines

(Vero: monkey kidney fibroblasts and LLC-PK11: pig kidney epithelial cells) and solid tumor cell lines (HepG2: human hepatocellular carcinoma, SK-MEL: human malignant melanoma, KB: human epidermal carcinoma (oral), BT-549: human breast carcinoma, and SK-OV-3: human ovary carcinoma) (Mustasa et al., 2004) All cell lines were obtained from American Type

Culture Collection (ATCC) and grown in RPM1-1640 medium supplemented with 10% bovine calf serum (BCS) and amikacin (60 mg/L). HepG2 cells were grown in DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS). Cells (25,000 cells/well) were seeded in the well of a 96-well plate and incubated for 24 h. Samples were added and followed by incubation for 48 h. The number of viable cells was determined using the supravital dye Neutral Red.

Briefly, the cells were washed with saline and incubated for 3 h with a solution of neutral red.

The cells were washed again to remove extracellular dye. A solution of acidified ethanol was added to liberate the incorporated dye from viable cells and the absorbance was read at 450nm.

5.2.2.2. Assay for antioxidant activity (inhibition of intracellular ROS generation)

Antioxidant activity was determined by the DCFH-DA method. Myelomonocytic HL-60 cells (1 × 106 cells/mL, ATCC) were suspended in RPMI-1640 medium (Roswell Park Memorial

o Institute) supplemented with 10% FBS and antibiotics at 37 C in 5% CO2: 95% air. The cell suspension (125 µL) was added to a 96-well plate. After treatment with test compounds for 30 min, cells were stimulated with 100 ng/mL phorbol 12-myristate-13-acetate (PMA, Sigma) for

30 min. 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes, 5 µg/mL) was

105 added and cells were further incubated for 15 min. Levels of DCF were measured on a

SpectrMax plate reader with excitation wavelength at 485 nm and emission at 530 nm.

Antioxidant activity of test samples is determined in terms of % decrease in DCF production compared to the vehicle control. The cytotoxicity to HL-60 cells was also determined after incubating the cells (2 x 104 cells/well in 225 µL) with test samples for 48 h by XTT {2,3-bis (2- methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} method

(Zhao et al, 2008). Briefly, 25 µL of XTT-PMS (phenazine methosulfate) solution (1 mg/mL

XTT solution supplemented by 25 µM of PMS) was added to each well. After incubating for 4 h at 37oC, absorbance was measured on a plate reader (EL312e; Bio-Tek instruments, Winooski,

VT) at a dual wavelength of 450 - 630 nm.

5.2.2.3. Assay for anti-inflammatory activity (inhibition of NF-kB)

The SW1353 human chondrosarcoma cells were cultured in 1:1 mixture of dulbecco's modified eagle medium/nutrient mixture F-12 (DMEM/F12) supplemented with 10% FBS, 100

U/mL penicillin G sodium, and 100 µg/mL streptomycin at 37 C in an atmosphere of 5% CO2 and 95% humidity. NF-kB luciferase plasmid construct was provided by Dr. David Pasco,

NCNPR, the University of Mississippi.

Cells (1.2 x 107) were washed once in an antibiotic and FBS-free DMEM/F12, and then resuspended in 500 µL of antibiotic-free DMEM/F12 containing 2.5% FBS. NF-kB luciferase plasmid construct was added to the cell suspension at a concentration of 50 μg/mL and incubated for 5 min at room temperature. The cells were then electroporated at 160 V and one 70-ms pulse using BTX disposable cuvettes model 640 (4-mm gap) in a BTX Electro Square Porator T 820

(BTX I, San Diego, CA). After electroporation, 1.25 x 105 cells per well were plated in 200 µL

106 of DMEM/F12 containing 10% FBS and antibiotics. After 24 h, cells were treated with different concentrations of test compounds for 30 min and then induced with PMA (70 ng/mL) for 8 h.

Cells were lyzed by adding 40 µL of a 1:1 mixture of lucLite reagent and PBS containing 1 mM calcium and magnesium (Packard Instrument Company, Meriden, CT). Light output was detected on SpectraMax. Relative luciferase units (RLU) were 12-15 folds higher in PMA induced cells versus uninduced controls (Ma, et al, 2007).

5.2.2.4. Assay for estrogenic activity

Yeast cells (Saccharomyces cerevisiae) expressing the human estrogen receptor alpha, were cultured in 25 mL of growth medium for 24 h at 30 ˚C in a shaking incubator (Tabanca et al., 2004). For the assay, seeded assay medium was prepared by adding 1.5 mL of this culture and 250 L of chlorophenol red β-D-galactopyranoside (CPRG, 10 mg/mL) to 25 mL of fresh growth medium (Routledge et al., 1996). Sample was dissolvd in DMSO (10 mM for pure compounds and 10 mg/mL for extracts). Six serial dilutions were made on a 96-well plate and

10 L of diluted sample was transferred to the assay plate followed by addition of 190 L of seeded assay medium to each well. Test concentrations ranged from 7.8 µg/mL to 250 µg/mL for extract and 7.8 to 250 for pure compounds in duplicates. 17 β -Estradiol (E2) was used as positive control and a dose-response curve with concentrations ranging from 3.1 nM to 100 nM was run in parallel to samples in every assay. The highest concentration of DMSO in the vehile control was 0.25 %. The plates were shaken for 5 min and incubated for 72 h at 30 ºC. At the end of incubation the plates were shaken, and the absorbance was measured at 540 nm.

Absorption at 630 nm was subtracted to correct for turbidity. % Maximal E2 response was calculated as described earlier (Zhao et al, 2008).

107 5.2.2.5. Assay for the activation of PPARα and PPARγ in CHO cells.

Chinese hamster ovary (CHO) cells were cultured in Ham‟s F12 medium containing 5% newborn calf serum (NCS), 50 U/mL penicillin G and 50 µg/mL streptomycin sulfate at 37 oC in

an atmosphere of 90% humidity and 5% CO2.

The assay for activation of PPARα and PPARγ was performed as described earlier

(Jaradat et al., 2001) with some modifications.

The plasmids were kindly provided by Dr. Dennis Feller, Department of Pharmacology,

School of Pharmacy, the University of Mississippi. CHO cells were transfected for PPARα with

25 µg of PPARα plasmid and for PPARγ with 25 µg of PPRE-aP2-tk-luc reporter and 25 µg of the expression construct pcMV-PPARγ. Electroporation was performed (Electro square porator model T820) at 155 V for 70 msec and one pulse. Transfected cells were seeded into 96-well tissue culture plate at a density of 5 x 104 cell/well and incubated for 24 h. Tested compounds at different concentrations (0.1-30 µM) were added and after incubation with compounds for 24 h, cells were lysed and the luciferase activity was determined by using luciferase assay system

(Promega). Plates were read on Spectra Max (Molecular Devices Corporation).

Fold induction in the activity of PPARα and PPARγ was calculated in comparison to the vehicle control (0.3% DMSO) and presented as mean ± SME (n=3). Wy14643 and ciglitazone were used as positive control for PPARα and PPARγ, respectively.

5.3. Results and discussion

5.3.1. In vitro cytotoxicity

The cytotoxicities of SBE and SLE together with thirteen flavonoids were evaluated against a panel of normal cell lines and solid tumor cell lines. No cytotoxicity was observed for

108 most of the samples up to a highest concentration of 50 μM for purified compounds and 50

μg/mL for the extracts in any of the cell lines tested (data shown in Table 5-1). Based on these results a conclusion might be made that the toxicity of American skullcap is very low and the reports of hepatotoxic reactions of American skullcap is likely due to its adulterant germanders.

5.3.2. Antioxidant activity

Potent antioxidant activity was observed for the SEB and SLE with IC50 values of 2.5

μg/mL and 1.5 μg/mL, respectively. These results are consistent with published data (Cole et al.,

2008; Wojcikowshki et al., 2007). Compounds 17, 18, 6, 24 ,25, and 29 showed strong inhibition of ROS generation with IC50 values of 0.52, 0.32, 0.7, 0.7, 0.13, and 0.9 μM, respectively (see Table 5-2) in comparison to the IC50 value of 0.01 μM for the positive control, vitamin C. In order to determine the specificity of the antioxidant effect, their cytotoxicity to

HL-60 cells was also tested. The purpose of this experiment is to rule out the possibility that antioxidant activity was not observed due to their toxicity. No cytotoxicity was observed for all samples at the concentration of 12.5 μM or 62.5 μM (as indicated in Table 5-2).

109 Table 5-1. Cytotoxicities (IC50) of Skullcap Extracts and Flavonoids Isolated from Them sample VERO PK-11 HepG2 SK-MEL KB BT-549 SK-OV-3 SBE NA* NA* NA* NA* NA* NA* NA* SLE NA* NA* NA* NA* NA* NA* NA* baicalein (17) >50µM NA NA 35 µM NA NA NA baicalin (18) NA NA NA NA NA NA NA wogonin (6) NA NA NA NA NA NA NA oroxylin A (5) NA NA NA NA NA NA NA genkwanin (22) NA NA NA NA NA NA NA chrysin (4) NA NA NA NA NA NA NA viscidulin I (23) NA NA NA NA NA NA NA quercetin (24) NA NA NA 35 µM NA 50 µM NA rutin (25) NA NA NA NA NA NA NA daidzein (30) NA NA NA NA NA NA NA naringenin (27) NA NA NA NA NA NA NA hesperetin (28) NA NA NA NA NA NA NA hersperidin (29) NA NA NA NA NA NA NA doxorubicin >5 0.5 0.5 0.9 0.9 1 0.6 NA*: no cytotoxicity observed at 50µg/mL. NA: no cytotoxicity observed at 50µM.

110 Table 5-2. Antioxidant Activities of Skullcap Extracts and Flavonoids Isolated from Them

sample antioxidant activity IC50 cytotoxicity

(μM) (HL-60 cells) (μM)

SBE 2.5# NC#

SLE 1.5# NC#

baicalein (17) 0.52 NC*

baicalin (18) 0.32 NC*

oroxylin A (5) NA NC

wogonin (6) 0.7 NC*

genkwanin (22) NA NC

chrysin (4) NA NC

viscidulin I (23) NA NC

quercetin (24) 0.7 NC*

rutin (25) 0.13 NC*

daidzein (30) NA NC

naringenin (27) NA >62.5

hesperetin (28) 2.3 NC

hesperidin (29) 0.9 NC*

vitamin C 0.01

NA: no activity up to 62.5 μM; NC: no cytotoxicity up to 62.5 μM; NC*: no cytotoxicity up to 12.5 μM. # : IC50 in μg/mL.

5.3.3. Anti-inflammatory activity

The anti-inflammatory activity was determined in terms of inhibition of NF-κB mediated

111 transcription in SW1353 cells induced by PMA and the results were shown in Table 5-3. A moderate activity was observed for compounds 6, 4, 24, and 25 with IC50 values of 39, 33, 40, and 39 μM, respectively, in comparison with the positive control, parthenolide (IC50 of 4.1 μM).

In this experiment, a luciferase construct with binding sites for Sp-1 was used as a control because this transcription factor is unresponsive to inflammatory mediators such as PMA.

Hence, the measurement of Sp-1-mediated luciferase expression is useful for detecting agents that nonspecifically inhibit luciferase expression because of cytotoxicity, inhibition of luciferase enzyme activity or light output (Ma et al., 2007). None of the samples inhibited Sp-1 dependent luciferase expression, indicating that their effect on NF-κB was specific.

112 Table 5-3. Anti-inflammatory Activity of Skullcap Extracts and Flavonoids Isolated from Them: Effect on NF- κB Mediated Transcription

sample IC50 (μM)

NF- κB SP-1

SBE NA# NA

SLE >50# NA

baicalein (17) NA NA

baicalin (18) NA NA

oroxylin A (5) >50 NA

wogonin (6) 39 NA

genkwanin (22) NA NA

chrysin (4) 33 NA

viscidulin I (23) NA NA

quercetin (24) 40 NA

rutin (25) 39 NA

daidzein (30) NA NA

naringenin (27) NA NA

hesperetin (28) NA NA

hesperidin (29) NA NA

parthenolide 4.1 6.2

NA: no activity up to 50 μM. # : IC50 in μg/mL.

5.3.4. Estrogenic activity

Phytoestrogens comprise a large group of plant-derived compounds, which include

113 isoflavonoids, lignans, stilbenes and flavonoids. They have attracted scientific interest due to their possible medical applications in the prevention of certain cancers and treatment of osteoporosis and cardiovascular diseases (Munro et al., 2003; Ross et al., 2002; Stopper et al.,

2005). The estrogenic effects vary according to the phytoestrogenic substance studied, the experimental system, and species. The mode of action of phytoestrogens seems to be complex, and their efficacy and safety need to be evaluated individually (Fitzpatrick et al., 2003;

Moutsatsou et al., 2007; Ren et al., 2001; Sirtori et al., 2005).

In the present study, yeast estrogen screen (YES) assay was used to compare the estrogenic activity of 17 β -Estradiol on estrogen receptor (ER)-α with two crude extracts SBE and SLE and thirteen flavonoids isolated from SBE and SLE. Estrogenic response of E2 at 100 nM was defined as 100% and % maximal 17 β -Estradiol response of sample tested was calculated at the highest effective dose in comparison to 100 nM 17 β -Estradiol. The extracts

SBE and SLE did not show any significant effect at the highest dose of 250 µg/mL. Out of the tested compounds naringenin and daidzein showed strong estrogenic effects with 98% maximal

17 β -Estradiol response at 250 µM (naringenin) and 40% maximal 17 β -Estradiol response at

62.5 µM (daidzein) (Table 5-4). For most of the compounds highest effective dose was 250 µM except for diadzein. The effect of daidzein was maximum at 125 and 62.5 µM. Toxic effect at the highest dose of 250 µM could be responsible for a low estrogenic effect of daidzein at this concentration. Previously, the estrogenic potency of naringenin was controversially discussed.

Strong estrogenic activity of naringenin was observed by Zand et al. (Rosenberg Zand et al.,

2000). Whereas, Ruh et al. reported that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells (Rhu et al.,

114 1995). Daidzein, which is one of the two major isoflavonic phytoestrogens in soy bean, has been shown to have dual function, i. e. the estrogenic and anti-estrogenic properties in the presence of high or low 17 β -Estradiol levels (Ni et al., 2010). Different results for the same compound might be due to the differences in the test system used. The major conclusion that could be drawn from the present study is that subtle changes in the structure of compounds, for instance naringenin and hesperetin, can bring about a large change in their estrogenic activity.

Table 5-4. Estrogenic Response of Scutellaria Extracts and Flavonoids Isolated from Them sample % max E2 response % max E2 response % max E2 response at 250 µM at 125 µM at 62.5 µM SBE NA# SLE NA# baicalein (17) NA baicalin (18) NA oroxylin A (6) 30% 18% wogonin (5) 13% 10% genkwanin (22) 16% 9% chrysin (4) 9% viscidulin I 23) NA quercetin (24) 11% rutin (25) NA daidzein (30) 28% 39% 40% naringenin (27) 98% 90% 66% hesperetin (28) 10%

NA#: no activity up to 250 µg/mL. NA: no activity up to 250 µM. E2: 17β-estradiol. % max E2 response: % maximal 17 β-estradiol response was calculated at the indicated doses in comparison to 100 nM E2 as 100%.

5.3.5. Activation of PPARα and PPARγ in CHO cells

115 PPARs are ligand-activated transcription factors that belong to the subfamily of nuclear receptors (Schoonjans et al., 1997). They are pivotal regulators of body lipid homeostasis and are proposed as possible therapeutic targets for metabolic disorders. They show broad, but isoform-specific, tissue expression patterns. Currently, PPARs subfamily has been divided into

PPARα, PPARβ (also called PPARδ and NUC1) and PPARγ. Three PPAR isoforms differ in their tissue distribution and ligand specificity (Forman et al., 1996). PPARα is predominantly expressed in tissues exhibiting high catabolic rate of fatty acids (heart, liver, and kidney), whereas PPARβ expression is ubiquitous, and its physiological role is unclear. PPARγ is expressed predominantly in adipose tissue, the adrenal gland, spleen, large colon and the immune system (Chawla et al., 1994; Kliewer et al., 1994; Tontonoz et al., 1994). Therefore, PPARα is a major regulator of fatty acid metabolism and catabolism within the body (Spiegelman, 1998).

PPARγ plays an important role in regulating adipocyte differentiation and glucose homeostasis

(Berger et al., 2005). Agonists of PPARα and PPARγ have been developed as antihyperlipidemic and antidiabetic agents.

In the present study two skullcap extracts (SBE and SLE) together with thirteen flavonoids isolated from these extracts were first tested for their activation effects on PPARα and

PPARγ in CHO cells. Induction of PPARs activity by each compound was tested at concentrations ranging from 0.1 µM to 30 µM and fold induction was calculated against vehicle control. Wy 14643 and ciglitazone were used as positive controls to evaluate the ability of tested samples to activate PPARα and PPARγ, respectively. The results are summaried in Table 5-5 and shown in Figure 5-2 and 5-3. In this study a fold induction of 2 or more was considered as significant induction in comparison to vehicle control.

116 SLE significantly stimulated the PPARα activities in a dose-dependent manner. At the dose of 10 µg/mL, the extract increased PPARα activity up to 2.6 folds over the vehicle control.

It is more potent than the activity of SBE, which showed 1.6 fold increase in activity over the vehicle control (see figure 5-2). At the dose of 30 µg/mL, the fold of PPARα induction of SLE is up to 4.9 compared to 2.4 of SBE. Of the tested flavonoids, compounds baicalein (17), baicalin (18), wogonin (6), genkwanin (22), chrysin (4), quercetin (24), daidzein (30), naringenin

(27), and hesperetin (28) exhibited significant activation of PPARα at 10 µM (2.8, 2.4, 2.5, 2.0,

3.5, 2.1, 2.2, 2.2, and 2.9, respectively) (shown in Table 5-5). Most of them induced the PPARα activity in a dose-dependent manner. The activity of these compounds was comparable to the activity of the positive control Wy 14643 (1.8 fold) (Table 5-5 and Figure 5-2). However, there was a cytotoxic effect in CHO cells with 30 µM of chrysin and hesperetin and a decrease in the activation of PPARα was observed (Figure 5-2). Although another group (Fang et al., 2008) reported that quercetin inhibited the activation of all three isoforms of PPARs (PPARγ IC50 =

56.3 μM; PPARα IC50 = 59.6 μM; PPARβ IC50 = 76.9 μM) in Cos-7 cells. Our studies showed activiations at 10 μM. Oroxylin A (5), viscidulin I (23), rutin (25), and hesperidin (29), had no effect on activation of PPARα, and the data are shown in Table 5-5.

Only baicalein (17), wogonin (6), and chrysin (4) showed significant induction of PPARγ activity at a concentration of 10 µM (fold induction of 2.3, 2.0, and 3.4, respectively). However, oroxylin A (5), quercetin (24), and daidzein (30) demonstrated an increase in the PPARγ activity at a concentration of 30 µM (2.6, 6.9, and 2.7 folds, respectively), which were comparable to positive control ciglitazone (3.0 fold induction). Consistent with our results, chrysin was previously reported to increase the PPARγ activity in a dose-dependent manner, with the EC50 of

117 approximately 10 µM (Liang et al., 2001). It is worth noticing that compounds baicalein (17), wogonin (6), and chrysin (4) demonstrated dual PPARα and PPARγ activity at the concentration of 10 µM (see Figure 5-4), whereas quercetin (24) and daidzein (30) showed the same effects at a concentration of 30 µM (Figure 5-5). To date, there is no report on the effects of skullcap extracts on PPARs activation. The present study, for the first time, sheds some light on the fact that the skullcap extracts have the potential to act as PPARα agonists. In our study, some of flavonoids, including baicalein (17), baicalin (18), wogonin (6), chrysin (4), etc. were also found to significantly induce PPARα activity. Hence it may be concluded that the induction for

PPARα activation produced by extracts may be due to the presence of these flavonoids, any of these, or a combination of them having direct or indirect effects on PPARα. The mechanism of action needs further detailed studies using individual compounds. Toxicity studies (data shown before) have already proven that no toxicity was observed at a dose far above the dose used in this investigation. Therefore, compounds, such as baicalein (17), baicalin (18), wogonin (6), chrysin (4), quercetin (24), daidzein (30), etc. with significant PPARα activity might serve as new leads with less adverse effects for treatment of PPARα-related diseases, such as obesity and dyslipidemia. A growing body of evidence suggests that combined treatments with PPARα and

PPARγ agonists may potentially improve insulin resistance and alleviate atherogenic dyslipidemia. A considerable amount of effort has thus been made in the development of dual

PPARα/γ agonists for the treatment of diabetes as well as the associated dyslipidemia.

Muraglitazar (Devasthale et al., 2005), tesaglitazar (McIntyre et al., 2003), KRP-297 (MK- 767)

(Calkin et al., 2003), and TAK-559 (Sakamoto, J. et al., 2004) are examples of late stage dual

PPARα/γ clinical candidates which have demonstrated efficacy in glucose normalization and

118 correction of lipid abnormalities in diabetic patients. However, the development of all of these advanced clinical candidates has been discontinued for multiple reasons, mainly due to considerable side effects (Barlocco, 2005; Colca, A.C. 2007; Conlon, D. 2006; Najman, D.M.

2006; Nissen, S.E. 2006; Parra et al., 2006). Our discovery of the compounds with dual regulator function for PPARα/γ discovered in this study, such as baicalein (17), wogonin (6), and chrysin (4) etc. might lead to PPAR agonists with improved risk-benefit profile for the treatment of diabetic patients with the additional risk factor of dyslipidemia. Future studies will be carried out to determine structure–activity relationships of potential PPARα and PPARγ agonists screened in this study using molecular modeling. The elucidation of structure–activity relationships would help in identifying the key structural features of the ligand molecules for activation of each subtype of the PPAR, which in turn, will assist us in design and discovery of more safe and effective agonists.

5.4. Conclusion

In the present study the methanol extracts of American skullcap and Chinese skullcap along with thirteen flavonoids isolated from them were evaluated their activities on a panel of bioassays. Neither extracts of SBE and SLE nor pure compounds tested showed cytotoxicity against a panel of mammalian normal and solid tumor cell lines at the high tested concentrations, suggesting that American skullcap has very low toxicity and the previous reports of hepatotoxic reactions of American skullcap might not be due to skullcap itself but could be due to its adulterant germander. Significant antioxidant activity was observed for both skullcap extracts as well as compounds, such as baicalein (17), baicalin (18), wogonin (6), quercetin (24), rutin (25), and hesperidin (29). The antioxidant activity of American skullcap was firstly observed in the

119 present study and was found to be stronger than Chinese skullcap. Naringenin (27) and daidzein

(30) showed marked estrogenic effects in the YES assay. Only a moderate anti-inflammatoy activity was observed with wogonin (6) and chrysin (4) as indicated by the effect on NF-κB mediated transcription. In this study, the most significant finding was the identification of

PPARs activation of skullcap extracts as well as their components, such as baicalein (17), baicalin (18), wogonin (6), and chrysin (4), etc. Results of our study provide useful information on possible new application of skullcap, especially American skullcap, for the treatment of diabetic-related diseases. In addition, the activation of PPARs by pure flavonoids suggests their potential to become leads as PPARα and PPARγ agonists.

120 Table 5-5. Fold Induction of Tested Samples on PPARs at Different Concentrations PPARα PPARγ 30 µM 10 µM 1 µM 0.1 µM 30 µM 10 µM 1 µM 0.1 µM SBE* 2.4±0.9 1.2±0.15 1.3±0.55 1.9±0.30 1.7±0.45 1.2±0.25 SLE* 4.9±0.85 2.6±0.7 1.3±0.1 1.4±0.55 1.4±0.20 0.9±0.25 baicalein 4.2±0.98 2.8±0.73 1.3±0.22 1.1±0.24 2.4±0.62 2.3±0.49 1.2±0.18 0.9±0.11 (17) baicalin 3.1±0.47 2.4±0.64 0.8±0.16 0.9±0.29 1.6±0.55 1.3±0.25 1.0±0.40 1.1±0.40 (18) oroxylin A 1.3±0.30 1.2±0.10 1.2±0.60 0.9±0.20 2.6±0.05 1.3±0.05 1.1±0.25 1.0±0.25 (5) wogonin 4.1±0.76 2.5±0.47 1.4±0.36 1.1±0.29 2.9±0.70 2.0±0.40 1.4±0.33 1.2±0.16 (6) genkwanin 2.6±0.62 2.0±0.82 2.1±0.87 1.2±0.44 1.7±0.15 1.8±0.20 1.2±0.15 1.0±0.15 (22) chrysin (4) 3.1±0.05 3.5±0.00 1.9±0.10 1.4±0.20 2.0±0.05 3.4±0.05 1.4±0.50 1.1±0.15 viscidulin 1.1±0.10 0.8±0.10 0.8±0.00 1.1±0.05 1.1±0.05 1.2±0.00 1.1±0.05 0.9±0.20 I (23) quercetin 2.8±0.45 2.1±0.45 1.0±0.15 1.1±0.20 6.7±0.15 1.2±0.60 1.0±0.55 0.9±0.10 (24) rutin (25) 0.7±0.05 1.1±0.05 0.7±0.00 0.6±0.00 1.1±0.05 1.0±0.05 0.9±0.10 1.0±0.00 daidzein 5.3±0.65 2.2±0.40 1.2±0.4 1.4±0.55 2.7±0.10 1.8±0.25 1.8±0.55 1.0±0.10 (30) naringenin 4.9±1.70 2.2±0.55 1.2±0.15 1.2±0.70 1.5±0.40 1.3±0.33 1.0±0.18 0.9±0.20 (27) hesperetin 2.1±0.20 2.9±0.55 1.8±0.95 1.2±0.25 1.7±0.29 1.1±0.24 0.9±0.13 0.7±0.13 (28) hesperidin 1.0±0.10 0.9±0.20 0.7±0.10 0.7±0.00 1.1±0.30 1.3±0.10 1.0±0.40 0.9±0.15 (29) Wy 14643 1.9±0.05 1.8±0.05 1.2±0.00 0.9±0.10 ciglitazone 3.0±0.05 3.1±0.25 1.6±0.20 1.6±0.05

*The concentration unit of the extract is µg/mL.

121 7 30 µM 10 µM 6

5

4

3

2

1 Fold Induction of PPARα Induction Fold of 0

SBE* SLE* rutin (25) chrysin (4) Wy 14643 baicalin (18) wogonin (6) baicalein (17) oroxylin A (5) quercetin (24) daidzein (30) genkwanin (22)viscidulin I (23) naringeninhesperetin (27)hesperidin (28) (29)

Figure 5-2. Effect of tested samples on PPARα activation.

30 µM 8 10 µM 7

6

5

4

3

2

FoldInduction of PPARγ 1

0

SBE* SLE* rutin (25) chrysin (4) ciglitazone baicalin (18) wogonin (6) daidzein (30) baicalein (17)oroxylin A (5) quercetin (24) genkwanin (22)viscidulin I (23) naringeninhesperetin (27)hesperidin (28) (29)

Figure 5-3. Effect of tested samples on PPARγ activation.

122 4.5 PPARα activation 4 PPARγ activation

3.5

3

2.5

2

1.5

1

0.5

0 Fold Induction of atDual 10 PPARs µM

SBE* SLE* Wy 14643 chrysin (4) wogonin (6) baicalein (17) ciglitasone

Figure 5-4. Dual effect of tested samples on PPARs activation at 10 µM.

9 PPARα activation 8 PPARγ activation 7

6

5

4

3

2

1

0 Fold Induction µM 30 of Dual at PPARs

SBE* SLE*

Wy 14643 chrysin (4) wogonin (6) daidzein (30) ciglitasone baicalein (17) quercetin (24)

Figure 5-5. Dual effect of tested samples on PPARs activation at 30 µM.

123

Second Project

Phytochemical Investigation of Pfaffia paniculata (Martius) Kuntze

124 Chapter 6

Introduction of Pfaffia paniculata (Martius) Kuntze

Around ninety species of Pfaffia (Amaranthaceae) are known in Central and South

American. In Brazil, twenty-seven species have been described to date, among which Pfaffia paniculata (Martius) Kuntze, commonly nameed suma, is the most employed species in commercial preparations in Brazil as “Brazilian ginseng” and has been commonly used for three centuries for the same indications as American and Asian ginseng (Taniguchi et al., 1997;

Vasconcelos, 1982). It is a large, rambling, shrubby ground vine with an intricate, deep, and extensive root system. It is indigenous to the Amazon basin and other tropical parts of

(southern) Brazil, Ecuador, Panama, Paraguay, Peru, and Venezuela. Although harvested wild, suma has been domesticated and farmed for quite some time. For instance, it has been cultured successfully in Sichuan, Zejiang, and Guangxi province in China (Ling et al., 2006). Since its first botanical recording in 1826, it has been referred to by several botanical names, including

Pfaffia paniculata, Hebanthe paniculata, Gomphrena paniculata, Gomphrena eriantha, Iresine erianthos, Iresine paniculata, Iresine tenuis, Pfaffia eriantha, Xeraea paniculata

(http://www.rain-tree.com/plants.htm).

Traditionally suma has been used as a food that provided physical stamina and endurance. The indigenous peoples of the Amazon region have used suma root for generations for a wide variety of health purposes, including as a general tonic, as an energy rejuvenating, and sexual tonic, and as a general cure-all for many types of illnesses, such as diabetes, ulcers, cancer etc (De Oliveira, 1986; Taniguchi et al., 1997). The benefits of suma were such that the

125 indigenous people of the jungle call it “para todo,” a Spanish word that means “for all things.”

In herbal medicine throughout the world today, suma is considered as a tonic and an adaptogen.

The herbal definition of an adaptogen is a plant that increases the body's resistance to adverse influences by a wide range of physical, chemical, and biochemical factors and has a normalizing or restorative effect on the body as a whole (Panossian et al., 1999). Therefore, suma was named as Brazilian ginseng with many applications same as regular ginseng.

The root of this plant is such a rich source of β-ecdysterone that it is the subject of a

Japanese patent for the extraction methods employed to obtain it from suma root.

Approximately 2.5 g of β-ecdysterone can be extracted from 400 g of powdered suma root

(0.63%) (Wakunaga, 1984). Also it has very high saponin content (up to 11%) (De Oliveira et al., 1980). The specific saponins found in the roots of suma include a group of novel nortriterpenoid saponins, named Pfaffic acid (hexacyclic nortriterpene) and their glycosides, pfaffosides A-F. Besides these, a mixture of stigmasterol and sitosterol, their glycosides, and allantoin have been reported (Nakai et al., 1984; Nishimoto et al., 1984; Takemoto et al., 1983).

Nutritionally, suma root contains 19 different amino acids, a large number of electrolytes, trace minerals, iron, magnesium, zinc, vitamins A, B1, B2, E, K, and pantothenic acid. Its high germanium content probably accounts for its properties as an oxygenator at the cellular level.

Besides, its high iron content may account for its traditional use for anemia (Zucchi et al., 2005).

Pharmacological studies showed that suma has anticancer, anti-inflammatory, and sexual stimulatory activities.

Anti-inflammatory activity

The alcoholic extract of P. paniculata dried roots was studied for analgesic

126 antiinflammatory activity in the rat paw oedema test, writhing test, hot plate test and increased vascular permeability test. P. paniculata inhibited the carrageenin-induced rat paw oedema and increased vascular permeability and showed analgesic activity on inflammatory pain but not on noninflammatory pain. Moreover the extract was devoid of local irritant action (Mazzanti et al.,

1994).

Anticancer activity

The powdered root of P. paniculata orally administered to mice at a daily dose of 200 mg/Kg, for 10 days reduced the ascetic volume of Ehrlich tumor cells in mouse peritoneal cavity, as well as the ascetic fluid volume. The result indicates that this effect is related to the anti- inflammatory activity (Matsuzaki et al., 2003). Later, the same research group studied the effects of the ethanolic extract, butanolic residue, and aqueous residue of ethanolic extract of P. paniculata on animal survival and tumor growth in mice bearing Ehrlich tumor. The results demonstrated antineoplastic effects on survival time of tumor bearing-mice following the treatment with the butanolic residue of ethanolic extract of P. paniculata (Matsuzaki et al.,

2006). Also, a human breast tumor cell line, the MCF-7 Cells treated with butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. These results showed that this butanolic residue indeed contains cytotoxic substances (Nagamine et al., 2009). The active components of this plant, at least in relation to survival parameter, are likely to be present in the butanolic fraction. Since the main constituents of butanolic residue obtained from the ethanolic extract are saponins, indicating that saponins may be responsible for the antineoplastic effects. In fact, pfaffic acid and its glycosides termed pfaffosides A-F, isolated from P. paniculata, have shown in vitro inhibitory effects on the

127 growth of B-16 melanoma (Nakai et al., 1984) and the pfaffosides as well as pfaffic acid derivatives in suma were patented as antitumor compounds in several Japanese patents in the mid-1980s (Takemoto et al., 1984). These results support the hypothesis proposed above. In another study, antiangiogenic activity was observed in the mouse cornea after the animal was treated with 1000 mg/Kg of the methanol extract of P. paniculata. The anticancer activity mediated by the P. paniculata may result from the intense stimulation of macrophages, natural killer cells and cytotoxic T lymphocytes (Watanabe et al., 2000). Increased macrophage activity was also observed by Pinello‟s group, suggesting that increased macrophage activity may be one of the effects contributing to inhibition of the Ehrlich ascetic tumor growth in mice (Pinello et al., 2006). On the other hand, chemopreventive effects of P. paniculata have been demonstrated in vivo. Watanabe et al. reported an inhibition of enlargement of thymic lymphoma in mice receiving 750 mg/Kg/week of a P. paniculata preparation for 24 week (Watanabe et al., 2000).

This is consistent with the study of Silva et al. that demonstrated chemopreventive effects using a mouse hepatocarcinogensis model, when these animals received 2.0 % of the powdered roots of this plant (v./v.) mixed in their chow (Silva et al., 2005).

Toxicity study was performed in which doses of 400 and 200 mg/Kg of powdered suma root were administrated orally for 10 days to mice. The chosen doses are higher than that recommended for humans, which varied from 100 to 300 mg per day. The evaluated parameters were the body weight, the presence of serum Alanine transaminase (ALT) indicative of necrosis in the liver, and the histopathological examination. None of these parameters showed alterations in mice treated with P. paniculata (Matsuzaki et al., 2003). In addition, oral intake of powdered

P. paniculata root for 30 days induced no adverse reactions in mice, suggesting that P.

128 panicualta can be consumed safely for long periods (Oshima et al., 2003). In another study, the ethanolic extract of P. paniculata administrated by intraperitoneal injection (i.p.) to mice did not produce behavioral effects up to 5 g/Kg in mice. Sedation, motor incoordination, loss of reflexes, hypothermia and mortality were observed only with the dose of 10.0 g/Kg (Mazzanti et al., 1994).

Sexual stimulatory activities

P. paniculata extract, when administrated orally to male rats with the dose of 0.25, 0.5, and 1.0 mL/Kg, improved the copulatory performance of sexually sluggish/impotent rats, but had no effects on the copulatory behavior of sexually potent rats. The highest dose of 1.0 mL/Kg increased the percentage of rats achieving ejaculation and significantly reduced mount, intromission and ejaculation latencies, post-ejaculatory interval and intercopulatory interval without affecting locomotor activity. The selective effect might act mainly by increasing central noradrenergic and dopaminergic tone, and possibly oxytocinergic transmission (Arletti et al.,

1999; Oshima et al., 2003). These results provide experimental support to the folk reputation of

P. paniculata as sexual stimulant.

Gingseng is one of the most widely used herbal medicines in the world for its physical, mental and sexual effects. It is one of the best-selling herbs in the Unite States (Yuan et al.,

2004). The general population might not be aware that different kinds of ginseng are prepared from plants belonging to different species even different family, for example, Chinese or Korean ginseng from Panax ginseng, American ginseng from Panax quinquefolium, Brazilian ginseng from Pfaffia paniculata, and Siberian ginseng from Eleutherococcus senticosus, etc. (Janetzky et al., 1997; Walker et al., 1994). More than fifty preparations, which were contained or produced

129 from plant P. paniculata are available on USA market (Lafont et al., 2003), but no analytical methods currently available to identify P. paniculata raw materials, and its products. Therefore, the primary objective of this project was to investigate the chemical components which will be used as marker compounds for the quality control of suma raw material as well as the products claiming to contain this species.

130 Chapter 7

Phytochemical investigation of Pfaffia paniculata (Martius) Kuntze

7.1 Introduction

As mentioned in Chapter 6, only very limited data associated with the chemical constituents of P. paniculata were reported even though it has been widely used as “Brazilian ginseng” for three centuries. Previously, three types of secondary metabolites have been reported from it, including phytosterols, pfaffic acid and their glycosides, named pfaffosides A-F

(saponins) and allantoin (Nakai et al., 1984; Nishimoto et al., 1984; Takemoto et al., 1983;

Wakunaga, 1984).

Herein, the detailed phytochemical investigation of P. paniculata was carried out. Two new nortriterpenoids pfaffine A and B (40-41), one new compound pfaffine C (42a and b), along with eleven known compounds were isolated from the roots of this plant (see Figure 7-1).

The structures of the new compounds were elucidated on the basis of extensive spectroscopic

(1D, 2D-NMR, HR-ESI-MS) and chemical degradation studies. Those known compounds were identified as ecdysone (43) (Chan et al., 2005), 20-hydroxyecdysone (44) (Wang, T. et al., 2004), pterosterone (45) (Coll et al., 1994), rapisterone (46) (Baltaev et al., 1987), pfaffic acid (47)

(Takemoto et al., 1983), pfameric acid (48) (Shiobara et al., 1993), mesembryanthemoidigenic acid (49) (Ikuta et al., 1986), calenduloside E 6′-methyl ester (50) (Melek et al., 1996), oleanolic acid 28-O-β-D-glucopyranoside (51) (Gupta et al., 2003), (+)-angelicoidenol-2-O-β-D- glucopyranoside (52) (Inoshiri et al.,1988), Vinillic acid (53) (Conrad et al., 2001), and iresinoside A (54) (Shiobara et al., 1992) by comparison of spectroscopic data with those

131 reported in the literature. All the fourteen compounds except ecdysone are reported for the first time from this plant.

132 29

20 H 18 22 25 11 26 13 17 H COOH 9 COOGlc 28 1 H OH 10 5 H 27 HO 6 H HO H 24 23 3 ,16 -dihydroxy-30-noroleana-12(29)-dien-28-oic acid 3 -hydroxy-30-norolean-12,20(29)-dien-28-oic acid (40, new compound) -28-O- -D-glucoside (41, new compound)

HO COOH

H COOH H H H OH HO HO H H pfaffic acid (47) pfameric acid (48)

OH OH H COOH HO OH H MeO O O H COOH H H O H HO H

mesembryanthemoidigenic acid (49) calenduloside E 6'-methyl ester (50)

H COOGlc CH2OH OH H OHO O HO H HO OH oleanolic acid 28-O- -D-glucopyranoside (51) (+)-angelicoidenol-2-O- -D-glucopyranoside (52)

Figure 7-1. Structures of compounds isolated from Pfaffia paniculata

133

134 7.2 Experimental sections

7.2.1. General experimental procedures

Optical rotations were measured in MeOH using a Rudolph Research Auto Pol IV polarmeter with a sodium lamp (589 nm) and a 0.5 dm microcell. High-resolution mass spectra

(HRESIMS) were performed on Bruker MicroTOF. NMR spectra were recorded in pyridine-d5 on either a Varian 600 or Brucker Avance 400 NMR spectrometers. All chemical shifts (δ) are given in ppm with reference to solvent and coupling constant (Ј) is given in Hz. IR spectra were obtained on a Perkin Elmer 100 FT-IR spectrometer. Column chromatography was carried out on silica gel (40 µm for flash chromatography, J. T. Baker) and C18-reversed phase silica (230-

400 mesh, Sigma-Aldrich). The fractions were monitored by TLC on silica gel plates (0.25 mm,

Merck) as well as on RP-18 F254 plates (0.25 mm, Merck). Spots were visualized by heating at

105 °C for 1-2 min after spraying with anisaldehyde/H2SO4 reagent. HPLC was performed on an octadecyl silane (ODS) column [Phenomenex prodigy 5μ ODS (3) 100A] and elutes were monitored by a UV detector at 240 nm. L-cysteine methyl ester hydrochloride, dioxane, D- and

L-glucose were obtained from Sigma-Aldrich (USA).

7.2.2. Plant material

The roots of Pfaffia paniculata Kuntze was purchased from American Mercantile

Corporation ([email protected]) and authenticated by Dr. V. Joshi at the

National Center for Natural Products Research, University of Mississippi, where a voucher specimen (No. 3096) has been deposited.

7.2.3. Extraction and isolation

The powder of the roots (2.4 Kg) of P. paniculata was extracted by stirring in MeOH (3 x

135 2L) at room temperature three hrs each time and the MeOH extracts were combined. The MeOH was evaporated under vacuum to get a brown viscous residue (437 g). The concentrate was suspended in water and then partitioned with hexanes, chloroform and n-BuOH successively (see

Chart 7-1). The n-BuOH soluble fraction (72.1 g) was subjected to VLC on silica gel (600 g) eluting with CHCl3 (2 L), CHCl3-MeOH-H2O [930:80:10 (1 L), 830:180:10 (1 L), 680:300:50

(2.5 L)] and MeOH (1.5 L) successively to give six fractions (A-F). Fraction B [CHCl3-MeOH-

H2O 830:180:10 (1 L), 2.5438 g] was subjected to reversed-phase C-18 flash chromatography

(100g, column: 4 x 15 cm) eluting with a gradient of MeOH-H2O (40:60 to 100:0 with elution volume of 1603 mL at the flow rate of 4 mL/min, 18 mL for fraction size) to afford twelve fractions. Subfraction 7 (between 780 to 950 mL, 0.2312 g) was separated by silica gel column eluted with a gradient of CHCl3:EtOAc:MeOH or CHCl3:MeOH several times to get vinillic acid

(53, 10.2 mg). Subfraction 8-9 (between 958 to 1240 mL, 0.4154 g) was subjected to normal- phase silica gel flash chromatography (Biotage Flash 12+M, 90g, column: 1.2 x 15 cm), eluted with CHCl3-EtOAc-MeOH-H2O (15:8:4:1 with elution volume of 1000 mL at a flow rate of 5 mL/min and fraction volume of 18 mL for each) to afford pfameric acid (48, between 530 to 756 mL, 4.0 mg). Subfraction 10 (between 1242 to 1502 mL, 0.4268 g) was separated by silica gel flash chromatography (Biotage Flash 40+M, 100g, column: 4 x 15 cm], 96 fractions were collected through elution with a gradient of CHCl3-EtOAc-MeOH 1698 mL (1:1:2 to 1:1:8) at the flow rate of 6 mL/min. Fractions 12-25 (between 72 to 150 mL, 287.2 mg) were subjected to normal-phase followed by reversed-phase silica gel VLC several times to afford pfaffic acid (47,

6.3 mg) and emsembryanthemoidigenic acid (49, 9.3 mg). Fractions 26-33 (between 156 to 198 mL, 25.6 mg) were further purified by reversed-phase C-18 flash chromatography (Biotage Flash

136 12+M, 9g, column: 1.2 x 15 cm) eluting with MeOH-H2O mixtures of increasing polarity (40:60 to 60:40 with elution volume of 2000 mL at a flow rate of 3 mL/min and fraction volume of 15 mL for each) afford pfaffine A (40, between 1638 to 1716 mL, 4.5 mg). Fraction C [CHCl3-

MeOH-H2O 680:300:50 (0.5 L), 0.4916 g] was subjected to silica gel VLC [500g, column: 4 x

15 cm) eluting with CHCl3-MeOH-H2O (7:2:1, 3 L with fraction volume of 15 mL for each) to afford four subfractions. Subfraction I (between 615 to 750 mL, 125.5 mg) was chromatographied on RP-C18 VLC (10g, column: 3 x 13 cm) eluting with MeOH-H2O [(80:20

(200 mL), 84:16 (100 mL)] and MeOH (50mL)] to afford calenduloside E 6′-methyl ester (50, between 235 to 250 mL, 2.1 mg). Subfraction II (between 915 to 1110 mL, 103.7 mg) was subjected to RP-C18 VLC [10g, 3 x 13 cm] eluting with a gradient of MeOH-H2O [74:26 (200 mL), 75:25 (100 mL), 78:22 (200 mL), and MeOH (50 mL)], 86 fractions were collected.

Fractions 56-66 (between 285 to 355 mL, 13.7mg) were purified by preparative HPLC with a gradient of MeOH-H2O as eluting solvent (60:40 to 90:10) at the flow rate of 2 min/mL to obtain pfaffine B (41, 4.0mg). Fraction 81-86 (between 435 to 450 mL, 10.2 mg) was purified by crystallization from MeOH to give oleanolic acid 28-O-β-D-glucopyranoside (51, 4.8 mg).

Subfraction III (between 1305 to 1525 mL, 99.2mg) was subjected to NP- as well as RP-C18 silica gel flash chromatography to give rapisterone (7, 2.0 mg). Subfraction IV (between 1635 to

1740 mL, 103.1 mg) afforded ptersterone (6, 13 mg) and (+)-angelicoidenol-2-O-β-D- glucopyranoside (52, 2.9 mg) after repeated NP- and RP-C18 flash chromatographies. Fraction

D [CHCl3-MeOH-H2O 680:300:50 (between 500 to 1000 mL), 0.7686 g] was chromatographied on RP-C18 VLC (140g, column: 6 x 30 cm) eluting with a gradient of MeOH-H2O [5:95 (200 mL), 15:85 (200 mL), 20:80 (200 mL), 30:70 (mL), 40:60 (200 mL), 40:40 (200 mL), 20:80 (200

137 mL), and MeOH (200 mL)] to afford ecdysone [43, MeOH-H2O 15:85 (200 mL), 100.3 mg] and

20-hydroxyecdysone [45, MeOH-H2O 30:70 (200 mL), 286.2 mg], respectively. Fraction F

[CHCl3-MeOH-H2O 680:300:50 (between 1000 to 2000 mL), 0.9636 g] was chromatographied on RP-C18 VLC (140g, column: 6 x 30 cm) eluting with a gradient of MeOH-H2O [10:90 (200 mL), 20:80 (200 mL), 30:70 (mL), 40:60 (200 mL), 40:40 (200 mL), 20:80 (200 mL), and

MeOH (200 mL)]. Four fractions were obtained. Subfraction VI was applied to Sephadex LH-

20 eluting with MeOH to obtain two yellow pigments iresinoside A (54, 6.2 mg) and pfaffine C

(42, 5.4 mg).

138 Pfaffia paniculata 2.4 Kg MeOH, 8 L

MeOH ext. residue ANJ-35 437 g solvent partition

hexane portion CHCl3 portion n-butanol portion ANJ-3-36-1 ANJ-3-36-2 ANJ-3-36-3 72.1 g 3.8186 g 7.5411 g silica gel VLC CHCl3 CHCl3:MeOH:H2O

ANJ-3-39-1-6 ANJ-3-39-7-8 ANJ-3-39-11-14 2.5432 g ANJ-3-39-9 ANJ-3-39-10 ANJ-3-39-15

RP 18-silica gel NP-VLC RP-C18 VLC CHCl :MeOH:H O 3 2 MeOH:H2O

ANJ-3-46-7 ANJ-3-46-8 & 9 ANJ-3-46-10 ANJ-3-85-7 0.1233 g 0.4145 g 0.4628 g ANJ-3-85-1-4 ANJ-4-13-3 ANJ-4-13-4

sil-gel sil-gel, CHCL3:MeOH DCM"MeOH or RP-C18, MeOH:H2O sil-gel, CHCl3:MeOH CHCl3:MeOH RP-C18 VLC sil-gel, CHCl3:MeOH RP-C18, MeOH:H2O MeOH:H O RP-C18, MeOH:H2O 2 JL-6-95-1 compound 48 compound 41, 45, 46, 47, 50,51, 52 JL-6-88-4 compound 40, 47, 49 silica gel repeatedly sephadex LH-20, MeOH CHCL3:EtOAC:MeOH or sil-gel, CHCL3:MeOH CHCl3:MeOH

compound 53 compound 42, 54 JL-6-77-3 ANJ-3-67-1-3 ANJ-3-67-4 ANJ-3-67-7-11

RP-C18 VLC MeOH:H2O sil-gel, CHCL3:MeOH sil-gel, CHCL3:MeOH HPLC, MeOH:H2O compound 43, 44 JL-6-63-6 JL-6-93-1 JL-6-94-4

Chart 7-1. Scheme for isolation of chemical constituents from Pfaffia paniculata

139 25 Pfaffine A (40): white powder; [α] D + 11.3 (c 0.1, MeOH); IR νmax = 3357 (OH), 2937,

1650 (C=O), 1443, 1031 cm-1; 1H- (600 Hz) and 13C- NMR (150 Hz) data, see Table 7-1; HR-

ESI-MS: m/z = 455.31863 [M-H]- (calcd. for 455.31614).

25 Pfaffine B (41): white powder; [α] D + 8.7 (c 0.46, MeOH); IR νmax = 3356 (OH), 2928,

1731 cm-1 (C=O), 1380, 1027; 1H- (600 Hz) and 13C- NMR (150 Hz) data, see Table 7-2; HR-

ESI-MS: m/z =625.37055 [M + Na]+ (calcd. for 625.37109).

25 Pfaffine C (42): yellow powder; [α] D - 67.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε)

275 (0.72), 380 (1.21) nm; IR νmax = 3284 (OH), 2923, 1672 (C=O), 1602 (aromatic –C=C–);

1531, 1514, 1259, 1163, 1060, 1019, 831 cm-1; 1H- (600 Hz) and 13C- NMR (150 Hz) data, see

Table 7-3 and 7-4, respectively; HR-ESI-MS: m/z = 469.3183 [M-H]- (calcd. for 469.3151).

A solution (3.0 mg) of compound 41 in 1N HCl (1.5 mL) was stirred at 80 C for 4 h.

After cooling, the solution was neutralized with NH4OH and partitioned between EtOAc (2 mL) and H2O (2 mL). The aqueous layer after drying was dissolved in pyridine (0.3 mL) and 0.1 M

L-cysteine methyl ester hydrochloride in pyridine (1.0 mL) was added. The mixture was heated at 60 C for 1h and an equal volume of Ac2O was added and heated one more hour. The acetylated thiazolidine derivatives were analyzed by GC using a capillary column DB-5 ms (30 m x 0.25 mm x 0.25 µm). Temperatures of injector and detector were 250 C for both. A temperature gradient system was used starting at 100 C for 1 min and increasing to 250 C at a rate of 10 C/min. D-glucose was identified by comparison of its retention time (tR 22.21 min) with that of authentic sample after being treated in the same manner.

7.3. Results and discussion

Structural elucidation of compounds

140 Compound 40 was obtained as a white powder, [α]D +11.3 (c 0.1, MeOH). Its molecular formula C29H44O4 was deduced from the negative-ion high-resolution electrospray ionization mass spectrum (HR-ESI-TOF-MS), showing a pseudomolecular ion [M-H]- at m/z 455.3186

(calcd. for 455.3161), together with 13C NMR spectrum (29 carbon signals). Its IR spectrum exhibited absorption bands due to free hydroxy (3357 cm-1), carbonyl groups (1650 cm-1). The

1 H NMR spectrum of 40 displayed five singlet resonances for tertiary methyl groups at δH 1.36,

1.27, 1.06, 1.06, and 0.92, two singlets at δH 4.81 and 4.77 assigned to an exomethylene group, two oxymethine protons at δH 3.48 and 4.66, and an olefinic proton at δH 5.52 (brd) (shown in

Table 7-1 and Figure 7-3). The 13C NMR spectrum showed 29 carbon signals including five methyls, ten methylenes, six (two of which are oxygenated) methines, and eight quaternary carbons (shown in Figure 7-4 and 7-5). The two signals at δ 122.3 and 144.4 ascribable to C-12 and C-13 suggested a ∆12 oleanene skeleton (Mahato et al., 1994). On the basis of the 1H-1H

COSY (shown in Figure 7-6), HMQC (shown in Figure 7-7), DEPT ( shown in Figure 7-5) spectra, and molecular formula C29H44O4, compound 40 was suggested to be noroleanic acid type triterpene. The 20(29)-exomethylene group was ascertained by long-range correlations between

H-19α, β (δH = 2.75, 2.29) /C-20 (δC = 150.1), H-19α, β /C-29 (δC = 106.6), H-21α (δH = 2.60) /C-

20,29, H-29a, b (δH = 4.81, 4.77)/C-19 (δC = 42.3), H-29a, b /C-21 in the HMBC spectrum of 40

(Figure 7-8). The positions of the two hydroxy groups were established on the basis of the

HMBC and nuclear overhauser effect spectroscopy (NOESY) experiments. The HMBC spectrum of 40 (shown in Figure 7-8) showed correlations between the oxymethine proton at δH

= 3.48 and the carbons C-4 (δC = 39.5), C-23 (δC = 28.9), and C-24 (δC = 16.7), conforming the location of one hydroxy group at C-3. The 3β-OH equatorial orientation was established using

141 H-3 α coupling constant (Jtrans = 10.8 Hz) and the correlation observed in the NOESY spectrum

(shown in Figure 7-9), between the two axial protons H-3α (δH = 3.48) and H-5 (δH = 0.89).

Furthermore, the HMBC spectrum exhibited correlations between the second oxymethine proton at δH = 4.66 and the carbons C-17 (δC = 50.8), C-22 (δC = 33.9), and C-28 (δC = 183.0). These data suggested the second hydroxy group to be located at C-16. The NOESY spectrum displayed important correlations between the proton H-16 (δH = 4.66) and the methyl protons CH3-27 (δH =

1.36), H-19 α (δH = 2.75), and H-21 α (δH = 2.60), suggesting α-axial orientation of the proton H-

16. Therefore, the structure of compound 40 was established as 3β, 16β-dihydroxy-30-norolean-

12, 20(29)-dien-28-oic acid, for which the name pfaffine A was adopted.

142 Table 7-1. NMR Data of Compound 40 in Pyridine-d5 No. 1H [δ (ppm), mult., 13C COSY HMBC J (Hz)] [δ (ppm)] 1α 1.02 (1H, m) 39.0 H-2 1β 1.57 (1H, m) H-2 2 1.82 (2H, m) 28.2 H-1α, β, 3 3 3.48 (1H, dd, 10.8, 3.6) 78.1 H-2 C-4, 23, 24 4 39.5 5 0.89 (1H, brd, 10.8) 55.9 H-6α, 6β C-3, 4, 6, 7, 9, 10, 23, 24, 25 6α 1.60 (1H, m) 18.9 H-5, 6β 6β 1.39 (1H, m) H-5, 6α C-5 7α 1.57 (1H) 33.5 C-5, 8 7β 1.40 (1H) C-5 8 39.8 9 1.65 (1H, dd, 10.2, 7.2) 47.4 H-11 C-5, 8, 10, 11, 14, 25, 26 10 37.4 11 1.90 (1H, m) 23.9 H-9, 12 C-9 12 5.52 (1H, brd) 122.3 H-11 C-9, 11, 14, 18 13 144.4 14 44.6 15α 1.81 (1H, m) 38.9 H-16, 15β C-14, 16, 17, 27 15β 2.44 (1H, t, 12.0) H-16, 15α C-14, 16, 17, 27 16 4.66 (1H, dd, 11.4, 4.2) 65.5 15α, β C-17, 22, 28 17 50.8 18 3.53 (1H, dd,13.8, 4.8) 50.3 H-19α, β C-12, 13, 14, 16, 17, 19, 28 19α 2.75 (1H, t, 13.8) 42.3 H-18, 19β C-17, 20, 29 19β 2.29 (1H, dd 13.8, 4.8) H-18, 19α C-17, 20, 21, 29 20 150.1 21α 2.60 (1H, brt, 13.8) 30.7 H-21β, 22α, β C-20, 22, 29 21β 2.28 (1H, brd, 16.2) H-21α, 22α, β 22α 3.08 (1H, d 12.0) 33.9 H-21α, β, 22β C-17, 20, 21 22β 1.81 (1H, m) H-21α, β, 22α C-17, 20 23 1.27 (3H, s) 28.9 C-3, 4, 5 24 1.06 (3H, s) 16.7 C-3, 4, 5 25 0.92 (3H, s) 15.7 C-1, 5, 9, 10 26 1.06 (3H, s) 17.8 C-7, 9, 14 27 1.36 (3H, s) 27.0 C-8, 14, 15 28 183.0 29a 4.81 (1H, s) 106.6 C-19, 20, 21 29b 4.77 (1H, s) C-19, 20, 21

143 H 29 H

H H

20 H H 18 25 12 28 16 COOH 1 9 OH H 3 5 HO H H

Figure 7-2. Key HMBC and NOE correlations for compound 40. Green line indicates HMBC and red line shows NOE correlations

1 Figure 7-3. H-NMR spectrum of compound 40 in pyridine-d5

144

13 Figure 7-4. C-NMR spectrum of compound 40 in pyridine-d5

Figure 7-5. DEPT spectrum of compound 40 in pyridine-d5

145

1 1 Figure 7-6. H- H COSY spectrum of compound 40 in pyridine-d5

Figure 7-7. HSQC spectrum of compound 40 in pyridine-d5

146

Figure 7-8. HMBC spectrum of compound 40 in pyridine-d5

Figure 7-9. ROESY spectrum of compound 40 in pyridine-d5

147 Compound 41 was obtained as a white powder, [α]D + 8.7 (c 0.46, MeOH). The positive

HR-ESI-MS of compound 41 gave an [M + Na]+ ion at m/z 625.37055 consistent with the

+ pseudomolecular formula C35H54O8Na (calcd. for [M+Na] 625.37109). Compound 41 displayed 35 carbon resonances in its 13C NMR spectrum (see Figure 7-11), of which 29 were assigned to a noroleanic acid type triterpene moiety and 6 to a monosaccharide portion. For the

2 aglycon portion, along with five methyl resonances (δC = 28.9, 26.1, 17.6, 16.6, 15.7), four sp - hybridized carbon signals (δC = 123.4, 143.5, 107.4, 148.6), and one signal for hydroxymethine group (δC = 78.7) could be observed (shown in Figure 7-12). These data, when correlated with

1 data from H NMR spectrum (shown in Figure 7-10) [five methyl singlets at δH = 1.26, 1.24,

1.13, 1.05, 0.93, one olefinic proton at δH = 5.48, and one oxymethine at δH = 3.46], confirmed that the aglycon of 41 possessed a noroleanic acid type triterpene skeleton. A detailed analysis of the NMR spectroscopic data (1H, 13C, HSQC, and H-H COSY) of the aglycon moiety of 41 in comparison with those of 40 showed that the difference of 41 from 40 is only for the absence of the secondary alcoholic function at C-16. The 1H NMR spectrum for the sugar portion of compound 41 showed one anomeric proton signal at δH = 6.31 ( 1H, d, J=8.4 Hz). The chemical shifts of all the individual protons and carbons of the sugar unit were assigned on the basis of

COSY (see Figure 7-13) and HSQC (see Figure 7-14) spectral analysis. The sugar unit,

3 identified as β-glucopyranosyl which was deduced from its JH-1,H-2 coupling constant of 8.4

(Woldemichael et al., 2001), was placed at C-28 of the aglycon on the basis of the HMBC correlation (shown in Figure 7-15) between the anomeric proton at δH = 6.31 and the C-28 carbon resonance at δC = 175.8. The configuration of the glucose unit was established as D after acid hydrolysis of 41 followed by derivatization and GC analysis (Hara et al., 1987) (see

148 Experimental section). Thus the structure of compound 41 was deduced as 3β-hydroxy-30- norolean-12,20(29)-dien-28-oic acid-28-O-β-D-glucoside based on the above evidence and was given the trivial name pfaffine B.

Table 7-2. NMR Data of Compound 41 in Pridine-d5 No. 1H [δ (ppm), 13C HMBC COSY NOSEY mult., J (Hz)] [δ (ppm)] 1α 1.00 (1H, dt) 39.1 H-1β, 2 H-3 1β 1.56 (1H, m) H-1α 2α 1.84 (1H, m) 28.3 H-1, 3 H-3 2β 1.84 (1H, m) 3 3.46 (1H, brd, 78.2 H-23, 24 H-2 H-1α, 2α, 5, 23 7.2) 4 39.5 H-23, 24 5 0.87 (1H, d, 55.9 H-23, 24, 25 H-6β H-1α, 3, 23, 9, 6α, 7α 11.4) 6α 1.55(1H, m) 18.9 H-5 H-6β H-5, 23 6β 1.37 (1H, m) H-5, 6α H-26, 24, 25 7α 1.50 (1H, brd 33.2 H-26 H-7β H-5, 27, 9 14.4) 7β 1.37 (1H, m) H-7α 8 40.0 H-6α, 7β, 9, 26, 27 9 1.66 (1H, t, 8.7) 47.8 H-25, 26 H-11 H-1α, 5, 7α, 11, 27 10 37.4 H-5, 9, 25 11 1.92 (2H, m) 23.9 H-9 H-9, 12 12 5.48 (1H, brd) 123.4 H-11, 18 H-11 H-11, 18, 19β 13 143.5 H-11, 18, 27 14 42.2 H-15β, 26, 27 15α 1.22 (1H) 28.2 H-27 H-15β 15β 2.38 (1H, dt) H-15α, 16α H-26, 16β 16α 2.20 (1H, dt) 23.7 H-18, 22β H-15β, 16α H-27 16β 2.09 (1H, m) H-16α H-15β 17 47.4 H-18, 22β, 19β 18 3.16 (1H, dd, 48.2 H-19α, β H-19β, 22β 13.8, 4.8) 19α 2.63 (1H, t, 41.8 H-18, 29α, β H-18, 19β H-27 13.8) 19β 2.23 (1H, m) H-18, 19α H-12, 29α 20 148.6 H-19α, 19β, 21β

149 21α 2.23 (1H, m) 30.2 H-19β, 22β, 29α, H-21β, 22β β 21β 2.07 (1H, m) H-21α 22α 2.03 (1H, m) 37.7 H-21α, 16β H-22β 22β 1.71 (1H, dt) H-21α, 22α H-18 23 1.26 (3H, s) 28.9 H-3, 5, 24 H-5, 24, 6α 24 1.05 (3H, s) 16.6 H-3, 5, 23, H-25, 23 25 0.93 (3H, s) 15.7 H-5, 9 H-24, 26 26 1.13 (3H, s) 17.6 H-9 H-24, 6β, 15β 27 1.24 (3H, s) 26.1 H-15β H-9, 7α, 16α, 19α 28 175.8 H-18, 22β, 1′ 29a 4.78 (1H, s) 107.4 H-19α, 21α H-19β 29b 4.71 (1H, s) H-21β, α 1′ 6.31 (1H, d, 95.9 H-2′ H-2′ H-5′, 3′ 8.4) 2′ 4.21 (1H, t, 8.4) 74.2 H-1′, 3′ H-4′ 3′ 4.29 (1H, t, 9.0) 78.9 H-2′ H-2′, 4′ H-1′, 5′ 4′ 4.35 (1H, t, 9.0) 71.2 H-3′, 5′ H-2′ 5′ 4.03 (1H, brd, 79.4 H-4′, 6′ α, H-1′, 3′, 6′ α, β 9.0) β 6′α 4.40 (1H, dd, 62.4 H-5′, 6′ β H-5′ 11.4, 3.6) 6′β 4.47 (1H, brd, H-5′, 6′ β H-5′ 10.2) 3-OH 5.75 C-2, 3, 4

150

1 Figure 7-10. H-NMR spectrum of compound 41 in pyridine-d5

13 Figure 7-11. C-NMR spectrum of compound 41 in pyridine-d5

151

Figure 7-12. DEPT spectrum of compound 41 in pyridine-d5

1 1 Figure 7-13. H- H COSY spectrum of compound 41 in pyridine-d5

152

Figure 7-14. HSQC spectrum of compound 41 in pyridine-d5

Figure 7-15. HMBC spectrum of compound 41 in pyridine-d5

153

Figure 7-16. ROESY spectrum of compound 41 in pyridine-d5

Compound 42 was obtained as a yellow powder. Its molecular formula was determined

- to be C29H30O12 by the negative-ion HRMS, showing a pseudomolecular ion [M-H] at m/z

469.3183 (calcd. for 469.3151). The IR spectrum exhibited absorption bands due to free hydroxy (3284 cm-1), carbonyl groups (1672 cm-1), and aromatic ring system (1602 cm-1). The characteristic UV absorption at λ 275 and 380 nm suggested the existence of a α，β-unsaturated

α-pyrone ring (Scott, 1964). The 1H and 13C NMR spectra (see Figure 7-18 and 7-19) exhibited two sets of signals in a ratio of ca 3:2, indicating that compound 42 is a mixture of two components. Further purification using preparative RP-HPLC resulted in obtaining two compounds. On the re-examination of each elute after removal of the solvent, two peaks were

154 again detected. These results suggested that compound 42 is consisted of two interchangeable isomers (shown in Figure 7-17). Two sets of AB olefinic signals at 6.58 (1H, d, J=16.2 Hz)/7.29

(1H, d, J=16.2 Hz) (major) and 5.92 (1H, d, J=12.3 Hz)/6.68 (1H, d, J=12.3 Hz) (minor) were assigned to trans and cis double bonds, indicating that compound 42 was a mixture of trans and cis isomers. Two p-substituted phenyl groups were suggested for each component by four sets of

1 A2B2 quartets in H NMR spectrum, e.g. 7.34 (1H, d, J=9.0 Hz)/6.64 (1H, d, J=9.0 Hz) and 7.25

(1H, d, J=9.0 Hz)/6.64 (1H, d, J=9.0 Hz) for the major component, and 7.25 (1H, d, J=8.4

Hz)/6.68 (1H, d, J=8.4 Hz) and 7.22 (1H, d, J=8.4 Hz)/6.63 (1H, d, J=8.4 Hz) for the minor one.

A CH3OOCCH2CH- unit for each component was deduced from the correlation of H-9 at δH =

4.80/4.77 ppm (major/minor) and H2-10 at δH = 3.25/3.20 ppm (major/minor) in COSY spectrum

(see Figure 7-20), and the HMBC (see Figure 7-22) correlations of C-11 at δC = 175.14/175.05 ppm (major/minor) with H-10 and methyl protons at δH = 3.60/3.59 ppm (major/minor). A glucose moiety was readily identified by the 1H and 13C NMR data listed in Table 7-3 and 7-4.

The β-configuration of this glucose was determined by the large coupling constant (J=7.8 Hz) of the anomeric proton and the chemical shift of the anomeric carbon at δC = 100.98/101.26 ppm

(major/minor). There are five pairs of carbon at δC = 165.83/165.72 (C-2), 109.85/110.83 (C-3),

166.92/166.25 (C-4), 98.82/100.12 (C-5), 160.97/160.44 (C-6) ppm (major/minor) and one pair of proton at δH = 6.47/6.58 (H-5) (major/minor) remained in the carbon and proton NMR respectively. These signals and the HMBC correlations between H-5 and C-3,4,6 suggested a tri-substituted pyran-2-one unit. The HMBC correlations (shown in Figure 7-23) of H-9/C-2,3,5 and H-7/C-5,6 confirmed the pyran-2-one ring and established the connections of C-9 to C-3 and

C-7 to C-6. The HMBC correlations of H-9/C-1′′,2′′,6′′ and H-2′′,6′′/C-9 indicated C-1′′ of one

155 of the phenyls connected to C-9. The connection of another phenyl group to C-8 was established by the HMBC correlations of H-8/C1′,2′,6′ and H-2′,6′/C-8. The glucose moiety was determined to connect to C-5 by the HMBC correlation of the anomeric proton and C-5. The 1H and 13C

NMR data of compound 42a are similar to those of compound 54 which was firstly reported from P. iresinoides (Shiobara et al, 1992), except that of the carboxylic group in 54 was esterified by a methyl group in 42a (see Table 7-3 and 7-4). Thus, the structure of compound 42 was determined as depicted. The absolute configuration of C-9 needs to be determined.

OH OH HO HO OH OH HO O O HO Z-E isomerization HO E Z O OH aqueous MeOH O OH H H O O

O O COOCH3 COOCH3 OH

Figure 7-17. Z-E isomerization of compound 42 in aqueous MeOH

156

1 Figure 7-18. H-NMR spectrum of compound 42 in MeOH-d4

13 Figure 7-19. C-NMR spectrum of compound 42 in MeOH-d4

157

1 1 Figure 7-20. H- H COSY spectrum of compound 42 in MeOH-d4

Figure 7-21. HSQC spectrum of compound 42 in MeOH-d4

158

Figure 7-22. HMBC spectrum of compound 42 in MeOH-d4

OH

HO H OH H HO 4' H O 2' HO H H 5 H 7 O 6'' OH 1' 8 6 6' 4'' H O H H 1 1'' 2 9 2'' 10 O H H COOCH3 11 Figure 7-23. Key HMBC correlations of compound 42a

159 Table 7-3. 1H-NMR Data of Compound 42a and 42b [δ (ppm), mult., J (Hz)] No. compound 42a compound 42b compound 54 4 6.47 (1H, s) 6.58 (1H, s) 6.51 (1H, s) 7 6.59 (1H, d, 16.2) 5.93 (1H, d, 12.3) 6.66 (1H, d, 16.2) 8 7.31 (1H, d, 16.2) 6.76 (1H, d, 12.3) 7.29 (1H, d, 16.2) 9 4.80 (1H, m) 4.77 (1H, m) 4.84 (1H, m) 10 3.25 (2H, m) 3.20 (2H, m) 3.00 (2H, brd, 7.8) 11-Me 3.60 (3H, s) 3.59 (1H, s) 2′, 6′ 7.34 (1H, d, 9.0 for each) 7.25 (1H, d, 8.4 for each) 7.41 (1H, d, 9.0 for each) 3′, 5′ 6.69 (1H, d, 9.0 for each) 6.68 (1H, d, 8.4 for each) 6.78 (1H, d, 9.0 for each) 2′′, 6′′ 7.25 (1H, d, 9.0 for each) 7.22 (1H, d, 8.4 for each) 7.28 (1H, d, 9.0 for each) 3′′, 5′′ 6.64 (1H, d, 9.0 for each) 6.63 (1H, d, 8.4 for each) 6.64 (1H, d, 9.0 for each) Glc-1 5.11 (1H, d, 7.8) 4.80 (1H, d, 7.8) 5.01 (1H, d, 7.8) Glc-2 3.54 (1H, m) 3.48 (1H, m) 3.63 (1H, t, 8.4) Glc-3 3.45 (1H, m) 3.45 (1H, m) 3.51 (1H, t, 9.0) Glc-4 3.35 (1H, m) 3.35 (1H, m) 3.40 (1H, t, 9.0) Glc-5 3.51 (1H, m) 3.51 (1H, m) 3.54 (1H, m) Glc-6 3.69 (1H, dd, 12.0, 6.3) 3.69 (1H, dd, 12.0, 6.3) 3.69 (1H, dd, 12.3, 6.3) 3.92 (1H, dd, 12.0, 2.1) 3.92 (1H, dd, 12.0, 2.1) 3.93 (1H, dd, 12.3, 2.1)

160 Table 7-4. 13C-NMR Data of Compound 42a and 42b [δ (ppm)] No. compound 42a compound 42b compound 54 2 165.83 165.72 166.6 3 109.85 110.83 110.6 4 98.28 100.12 99.4 5 166.92 166.25 166.6 6 160.97 160.44 159.9 7 115.46 118.36 117.5 8 137.3 139.6 138.1 9 37.04 37.01 39.0 10 37.28 37.14 41.9 11 175.14 175.05 181.1 11-Me 52.15 52.13 1′ 126.1* 126.2* 128.6 2′, 6′ 130.48 132.10 130.4 3′, 5′ 118.18 117.66 116.9 4′ 164.4* 162.8* 160.4 1′′ 133.91 133.68 135.7 2′′, 6′′ 129.97 130.00 130.3 3′′, 5′′ 115.84 115.85 115.8 4′′ 157.05 157.10 156.6 Glc-1 100.98 101.26 101.6 2 74.71 74.64 74.8 3 78.20 78.22 78.2 4 71.22 70.69 71.4 5 78.62 78.30 78.7 6 62.48 61.87 62.7

*: Signals exchangeable

161 The behavior of compound 42 on HPLC can be explained by a trans-cis isomerization to afford a equilibrium of mixture in aqueous MeOH, by which a similar reaction was reported to occur for yangonin , that is, 6-(p-methoxystyryl)-4-methoxy-2-pyrone (Smith et al, 1984). In the present study, compound 54 was purified as a single E-isomer using preparative normal-phase

TLC and organic solvents. Since compound 42 and several related compounds were not resolved on normal-phase TLC, the fraction containing compound 42 and others was then subjected to HPLC separation using a Phenomenex prodigy 5μ ODS column and gradient elution of methanol-water. The peak at tR=7.2 min was collected and when examined by HPLC this peak gave two peaks. Attempts to obtain the individual compounds were unsuccessful because each compound apparently re-equilibrated to give the mixture after separation was carried out again. In order to confirm the proposed isomerization behavior of compound 42 NMR techniques were applied. Compound 54 was prepared in MeOH-d4 for NMR analysis. After one day standing in daylight the compound was stable and the NMR spectrum kept the same (shown in Figure 7-24). When one drop of deuterium oxide was added into the solution, two sets of signals were detected after 20 min. The signals at δH = 6.76 and 5.93 ppm (J=12.3) corresponding to a cis-double bond became more and more intense with the time increased.

After 20 hrs two sets of signals were at the ratio of ca. 1:1. Therefore, it can be concluded that trans-cis isomerization occurred in aqueous solution but in organic solvents such as chloroform and methanol virtually no change occurred.

162 MeOH, 1d

MeOH+D2O, 20min

MeOH+D2O, 2h

MeOH+D2O, 20h

Figure 7-24. Z-E isomerization test of compound 54 in aqueous MeOH

163 7.4. Conclusion

In the present study, four types of compounds, including seven triterpenoids, four steroids, one monoterpenoid, two pigments and vanillic acid, have been isolated from the roots of P. paniculata. The compounds reported herein will be served as markers for quantification of raw material as well as preparations claiming to contain P. paniculata. Also, efforts have been made to investigate the isomerization of pigments.

164 Chapter 8

Concluding Remarks

American skullcap has been used more than three decades for its reputed tonic, tranquilizing, and antispasmodic remedies. Currently it is one of the most popular botanicals with many products on the USA market. The hepatotoxic reactions have been reported after skullcap-containing preparations were ingested. Meanwhile, it was reported to be adulterated with germander, e.g. Teucrium chamaedry, and T. canadense, which is a group of plants with hepatotoxicity. Due to the lack of stringent regulations from FDA there is a paucity of scientific evidence to evaluate the safety, quality, and efficacy of these products. Therefore, safety, efficacy, and quality control of the products claiming to contain this species are of major concern. Among them control quality of skullcap products presents a formidable analytical challenge in view of lack of information about the authenticity of raw materials, complexity of the constituents in the plant materials, and unavailability of qualified reference standards. In this dissertation, investigation of the chemical constituents, development of analytical method for quality control, and evaluation of biological activities of the raw materials as well as the compounds isolated from it have been carried out.

8.1. Contributions of this dissertation

Chromatographic separation of the MeOH extract of the aerial parts of American skullcap resulted in the isolation of sixteen compounds, including three minor coumarins of which two are new compounds, six flavonoids, four triterpenes, two steroids, and one fatty acid. The structures of all the compounds were elucidated via spectroscopic (NMR, UV, and FT-IR) and spectrometric (HRESI-MS) methods. The absolute configuration of two new coumarins was

165 determined by comparison of the experimental and theoretical calculated CD spectra. As a part of our efforts in phytochemical study HPLC-DAD/ESI-MSn method has been developed to characterize thirteen flavonoids and one stilbene derivative without time-consuming isolation of compounds. The ESI-MSn fragmentation pathways of four types of flavonoid standards have been examined and analyzed. These fragmentation patterns provide very useful guides for the characterization of different types of flavonoids. Based on the results we obtained it can be concluded that flavonoids are the major constituents in the American skullcap, some of which are also reported from other species in this genus, such as S. baicalensis. In order to compare and obtain more marker compounds, the chemical components of S. baicalensis, which is the most often used herb medicine in this genus, were also studied and seventeen flavonoids have been isolated.

Using eleven major flavonoids isolated from S. baicalensis as well as S. lateriflora as chemical markers a simple HPLC-PDA fingerprinting method has been established. In present study, two phenylpropanoids, verbascoside and teucrioside, and one diterpene, teucrin A isolated from T. canadense were also used as markers to provide comparative analysis. The results indicated that this method can be applied to authenticate S. lateriflora, to distinguish it from its potential adulterants, to compare the chemical profiles of different species in this genus, and to assess the quality of the products claiming to contain S. lateriflora. Since the UV patterns are different and specific for each type of compounds investigated, analysis of UV spectra of the known markers and relevant peaks in samples is quite valuable for the identification of each peak.

Another major contribution of this dissertation is to put the two skullcap extracts and

166 thirteen flavonoids isolated from them into one study to evaluate their bioactivities on a panel of bioassay. Neither extracts of SBE and SLE nor pure compounds tested showed cytotoxicity against a panel of mammalian normal and solid tumor cell lines at the highest test concentrations, suggesting that the reports of hepatotoxic reactions on American skullcap might not due to skullcap itself but its adulterant germander. Significant antioxidant activity was observed for both skullcap extracts as well as their components, such as baicalein, baicalin, wogonin, quercetin, rutin, and hesperidin. The antioxidant activity of American skullcap was first observed in present study and it is stronger than Chinese skullcap. Naringenin and daidzein showed strong estrogenic effects in the YES assay. Moderate anti-inflammatoy activity was observed with wogonin and chrysin on NF-κB mediated transcription test. The most significant finding was the identification of PPARs activation of skullcap extracts as well as their characteristic components, including baicalein, baicalin, wogonin, and chrysin, etc. Results of our study provide useful information on possible new application of skullcap, especially

American skullcap, for the treatment of diabetic-related diseases. In addition, the capability of pure flavonoids on PPARs suggests their potential to become leads of PPARα and PPARγ agonists.

Pfaffia paniculata is another species studied in this dissertation. It is the most employed species in commercial preparations in Brazil as “Brazilian ginseng” and has been commonly used for three centuries as the same indications as American and Asian ginseng. However, all these so-called ginsengs belong to different species even different family. More than fifty preparations which contain or are produced from P. paniculata are available on USA market, but no analytical method existed to identify P. paniculata raw materials, and its products. As a final

167 remark we investigated the chemical components of P. paniculata and obtained four types of compounds, including seven triterpenoids, four steroids, one monoterpenoid, two yellow compounds and vanillic acid, among which two triterpenoids pfaffine A and B, and one yellow compound pfaffine C are new. All fourteen compounds except ecdysone are reported for the first time from this plant. The compounds reported herein will be served as markers for quantification of raw material as well as preparations claiming to contain P. paniculata. From the NMR-based stability study of pigment iresinoside A, it can be concluded that trans-cis isomerization of this type of compound occurred in aqueous solution but in organic solvents such as chloroform and methanol virtually no change occurred.

8.2. Future work

The characteristic components isolated from both of skullcap species, including baicalein, baicalin, wogonin, and chrysin, etc. showed significant PPARs activation effect. The mechanism of action will be studied using isolated individual compounds. Meanwhile, structure–activity relationships of potential PPARα and PPARγ agonists screened in this study will be carried out using molecular modeling. The evaluation of structure–activity relationships would help in identifying the key structural features of the ligand molecules for activation of each subtype of the PPAR, which in turn, will assist us in design and discovery of more safe and effective agonists.

NMR-based metabolomics analysis has been applied to a wide array of fields, such as on the identification of disease biomarkers and investigation of microbial fermentation processes and biotransformations, etc. Studying plant metabolomics is particularly challenging due to the occurrence of substantially more and complex compounds compared with animals and

168 microorganisms and still in its infancy. Some pioneer applications to plant metabolomics analysis have been presented recently, mostly concentrating on the impact of environmental factors on plants. We will try to use NMR-based metabolomics analysis to differentiate

American skullcap from its potential adulterant and to assess the quality of its products. This method may complement the HPLC-PDA fingerprint method discussed in this dissertation from different points of view.

In order to evaluate and control the quality of the products produced from P. paniculata quantitative analysis method will be set up. Primary analysis result indicated that the content of ecdysones in P. paniculata is overwhelming. HPLC-PDA or HPLC-MS method will be applied to quantitatively analyze ecdysones using ecdysone and 20-hydroxyecdysone obtained in this dissertation as markers.

8.3. Related publications

The publications listed below are direct consequence of the evolution of this dissertation during my Ph. D. program.

[1] Li, J.; Wang, Y.H.; Smillie, T.; Khan, I.A. Identification of Phenolic Compounds in

Scutellaria lateriflora Using High-Performance Liquid Chromatography-Electrospray Tandem

Mass Spectrometry-Ultraviolet Photodiode Array. in preparation

[2] Li, J.; Wang, Y.H.; Khan, I.A. Chemical Fingerprint Analysis of Scutellaria Species,

Germander and Dietary Supplements Claiming to Contain Skullcap Using HPLC-PDA. in preparation.

[3] Li, J.; Jadhav, A.N.; Khan, I.A. Triterpenoids from Brazilian Ginseng, Pfaffia paniculata.

Planta Medica 2010, 76, 635-639.

169 [4] Li, J.; Ding, Y.Q.; Li, X.C.; Ferreira, D.; Khan, S.; Smillie, T.; Khan, I.A. Scuteflorins A and

B, Dihydropyranocoumarins from Scutellaria lateriflora J. Nat. Prod. 2009, 72(6), 983-987.

[5] Li, J.; Jadhav, A.N.; Khan, I.A. New Terpenoids from Pfaffia paniculata Kuntze. Planta

Medica 2009, 75(4), 424-425.

[6] Li, J.; Khan, I.A. Chemical Investigation of Scutellaria lateriflora L. Planta Medica 2008,

74(3), 355.

[7] Li, J.; Khan, I.A. The Advanced Bioactivity Studies of Scutellaria baicalensis Georgi and Its

Phenolic Compounds Acta Horticulturae 2006, 720, 157-169.

170

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193 VITA

Born in Jining, a small town, in Neimeng Gu province, China, I grew up witnessing the popularity of Traditional Chinese Medicine (TCM) as primary health care in my country. Out of curiosity about the magical effects of TCM, I went to Beijing University of Chinese Medicine, one of the top medical universities, especially in traditional herbal medicine, in 1982. I enrolled in a special five-year pharmacy program, which is a combination of conventional pharmacy and traditional herb medicine. After graduation in 1987, I worked in the QA lab in a TCM company, responsible for evaluating the quality of raw materials of TCM based on taxonomic examination

(macroscopic and/or microscopic) and chemical (thin-layer chromatography) analysis. Soon after, I found my interest was in research. After passing the required exams for the application of graduate program with highly competitive scores, I was matriculated as a master degree graduate student in the Institute of Materia Medica, Chinese Academy of Medical Sciences &

Peking Union Medical College, the No. 1 institute in pharmaceutical research in China in 1989.

During that time my knowledge on phytochemistry and analytical chemistry was extensively increased and broadened. My thesis was on the phytochemical and analytical studies on the pharmaceutical plants in genus Salvia. After many trials, eleven phenolic compounds, which are characterized as high polarity and instability, were isolated from the species Salvia bowleyana.

HPTLC quantitative method was then developed to determine the quantity of seven depsides in

52 plant samples in the genus Salvia. The analytical part of my thesis won the first prize of the

First National Excellent Pharmaceutical Analysis Paper Evaluation sponsored by Chinese

Pharmaceutical Society in 1992.

I was appointed as a research associate by the Institute of Medicinal Biotechnology,

194 Chinese Academy of Medical Sciences after I finished my master program in 1992 and promoted to assistant professor and associate professor late. My work focused on bioassay-guided isolation and identification of bioactive compounds from plants. After collaboration with other professors in several projects, as a PI, one of my projects entitled “Chemical and Biological

Studies on the Protein Kinase C Inhibitor from Spatholubus suberctus” was granted by National

Scientific Foundation of China (NSF). In 1998 I was promoted to vice chairman of Department of Biochemistry. The experience of being vice chairman enhanced my ability of management and organization of research work.

In 1999 I was employed as an imported drug reviewer by the Center for Drug Evaluation,

State Drug Administration (SFDA in China) and promoted to a leader of imported drug team and vice director of the department of imported drug later. My job was to review CMC parts

(chemistry, manufacturing and quality control) of imported drug applications. During the four years of reviewing imported drugs from leading pharmaceutical companies worldwide I learned a plethora of knowledge involved in new drug development. In 2000 I was honored as an excellent reviewer in the Center and was awarded to visit Bayer Healthcare Pharmaceuticals in

Germany as a visiting scholar in 2001. There, I was trained in the New Drug Development and

Research Center, Bayer for half year learning the strategy and state-of-the-art technology in research and development of new drugs

In 2003 I enrolled in the Ph.D. program in the Department of Pharmacognosy, School of

Pharmacy, the University of Mississippi. During this special journey not only was my knowledge in the field of natural product chemistry, biochemistry as well as ecology broadened but I also benefited from the wonderful international experience.

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