A study of quality and safety of herbal medicine adulteration and herb drug interactions

A STUDY OF QUALITY AND SAFETY OF HERBAL MEDICINE: ADULTERATION AND HERB-DRUG INTERACTIONS HOU PEI LING (B.Sc., BMU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS First of all, I would like to extend my gratitude to my supervisor Associate Professor Koh Hwee Ling and co-supervisor Associate Professor Eli Chan Wing Yuen for their scientific guidance and continuous encouragement in my graduate project I would like to thank the help from the technical staff in our Department, including Ms Ng Sek Eng, Ms Oh Tang Booy, Mr Tham Mun Chew and Miss Molly Nam Wan Chern, for their kind support in chemicals supply, instruments set-up, etc My sincere thanks also go to all classmates and lab mates, especially Yiran, Zou peng, Tung Kian, Agnes, Jianhong, Johannes, Dingfung, Li Lin, Liu Xin, Xie Feng and Xiaohua for their timely assistance, friendship and discussion I also acknowledge the financial support of a Graduate Research Scholarship provided by the National University of Singapore This dissertation is dedicated to my loving family, without their endless support and encouragement, I would not have accomplished it I LIST OF PUBLICATIONS PUBLICATION Hou Peiling, Zou Peng, Low Min-Yong, Chan Eli and Koh Hwee-Ling Structural identification of a new acetildenafil analogue from pre-mixed bulk powder intended as a dietary supplement Food Additives and Contaminants 2006, 23(9): 870-875 Zou Peng, Hou Peiling, Oh Sharon Sze-Yin, Low Min-Yong and Koh Hwee-Ling Electrospray tandem mass spectrometric investigations of tadalafil and its analogue Rapid Communications in Mass Spectrometry 2006, 20(22): 3488-3490 Zou Peng, Hou Peiling, Low Min-Yong and Koh Hwee-Ling Structural elucidation of a tadalafil analogue found as an adulterant of a herbal product Food Additives and Contaminants 2006, 23(5): 446-451 Zou Peng, Oh Sharon Sze-Yin, Hou Peiling, Low Min-Yong and Koh Hwee-Ling Simultaneous determination of synthetic phosphodiesterase-5 inhibitors found in a dietary supplement and pre-mixed bulk powders for dietary supplements using high-performance liquid chromatography with diode array detection and liquid chromatography-electrospray ionization tandem mass spectrometry Journal of Chromatography A 2006, 1104(1-2): 113-122 Hou Peiling, Chan Eli and Koh Hwee-Ling Rapid profiling and structural characterization of the integral phthalides and related bioactive constituents in Ligusticum chuanxiong Hort by high performance liquid chromatography-photodiode array detection (PDA)-electrospray ionization mass spectrometry Manuscript in preparation Hou Peiling, Chan Eli and Koh Hwee-Ling Herb-drug interactions between Ligusticum chuanxiong Hort and aspirin in Sprague-Dawley rat Manuscript in preparation POSTER PRESENTATION Hou Peiling, Chan Eli and Koh Hwee-Ling A study of Traditional Chinese herb Ligusticum chuanxiong Hort and its active components on rat liver CYP 3A and CYP 2D activities Poster presentation, International Pharmaceutical Federation II (FIP) Conference 2007, April 22-25, 2007 Amsterdam, Holland Hou Peiling, Zou Peng, Zhou Shufeng, Chan Eli and Koh Hwee-Ling An update on drug-herb interactions Poster presentation, The 5th Combined Scientific Meeting incorporating The 4th Graduate Student Society-Faculty of Medicine (GSS-FOM) Scientific Meeting, May 12-14, 2004, Singapore III TABLE OF CONTENTS ACKNOWLEDGEMENTS Ⅰ LIST OF PUBLICATIONS Ⅱ TABLE OF CONTENTS Ⅳ SUMMARY Ⅷ LIST OF TABLES XII LIST OF FIGURES XV LIST OF SCHEMES XVII LIST OF ABBREVIATIONS AND SYMBOLS XVIII Chapter Introduction 1.1 Quality issues 1.1.1 Factors affecting quality 1.1.2 Methods for the quality control of herbal medicines 1.1.2.1 Chemical identification 1.1.2.2 Molecular-based methods 1.2 Safety issues of Herbal Medicines 11 13 1.2.1 Introduction 13 1.2.2 Herb-drug interactions 14 1.2.3 The mechanisms of herb-drug interactions 15 1.2.3.1 Pharmacokinetic interactions 16 1.2.3.2 Pharmacodynamic interactions 1.2.4 Approaches employed in the studies of herb-drug interactiona 20 21 1.2.4.1 Introduction 21 1.2.4.2 In vitro models 22 1.2.4.3 In vivo studies using experimental animal models 25 1.2.4.4 Clinical studies 26 1.3 Herb-drug interaction between L chuanxiong and aspirin 30 1.4 Rationale, Hypotheses and Objectives 34 IV Chapter Adulteration: Structural identification of a new acetildenafil analogue 37 2.1 Introduction 37 2.2 Objective 46 2.3 Experimental 46 2.3.1 Sample and reagents 46 2.3.2 Extraction and isolation 47 2.3.3 Instrumentation 48 2.4 Results and discussion 49 2.4.1 LC-UV 49 2.4.2 MS/MS and high-resolution MS analysis 49 2.4.3 IR analysis 52 2.4.4 Structural identification of compound with NMR 52 2.5 Conclusion 55 Chapter Chemical analysis of Ligusticum chuanxiong Hort by high performance liquid chromatography and high performance liquid chromatography-diode array detection (DAD)electrospray ionization mass spectrometry 56 3.1 Introduction 56 3.2 Objective 59 3.3 Experimental 59 3.3.1 Chemical and reagents 59 3.3.2 Materials and sample preparations 60 3.3.3 Analysis 62 3.3.3.1 HPLC Analysis 62 3.3.3.2 High resolution LC-MS Analysis 63 3.3.3.3 LC-MS/MS Analysis 64 3.3.4 Accuracy and Precision 3.4 Results and discussion 3.4.1 Quantification of tetramethylpyrazine and ferulic acid in L chuanxiong by HPLC and LC-MS/MS methods 3.4.2 Analysis of phthalides and other bioactive components using 65 66 66 69 different kinds of MS methods 3.4.2.1 Evaluation of LC-MS method for profiling the 69 constituents in L chuanxiong V 3.4.2.2 Rapid profiling and structural characterization of 75 non-phthalide components 3.4.2.3 Rapid profiling and structural characterization of 93 phthalides 3.5 Conclusion Chapter Studies of effects of Ligusticum chuanxiong Hort and its active components on rat hepatic CYP3A and CYP2D activities 113 115 4.1 Introduction 115 4.2 Objective 121 4.3 Materials and Methods 121 4.3.1 Chemicals and reagents 121 4.3.2 Preparation of L chuanxiong extract 122 4.3.3 Animal treatments 122 4.3.4 Preparation of rat hepatic microsomes 123 4.3.5 Determination of the microsomal protein concentration 124 4.3.6 Determination of total microsomal P450 content 4.3.7 Determination of the activities of CYP 3A in rat hepatic 125 microsomal samples using midazolam as substrate 126 4.3.8 Determination of the activities of CYP 2D in rat hepatic microsomal samples using bufuralol as substrate 127 4.3.9 Effects of high dose of L chuanxiong extract on CYP 2D in rats in vivo using dextromethorphan as substrate 129 4.3.10 Data Analysis 133 4.4 Results 133 4.4.1 Growth characteristics and hepatic cytochrome P450 in rats 134 4.4.2 Protein concentration and P450 content in rat hepatic microsomes 135 4.4.3 Ex vivo CYP 3A activity in rats using midazolam as substrate 137 4.4.4 Ex vivo CYP 2D activity in rats using bufuralol as substrate 141 4.4.5 In vivo CYP 2D activity in rats using dextromethorphan as substrate 143 4.5 Discussion 149 VI 4.6 Conclusion 152 Chapter Herb-drug interactions between Ligusticum chuanxiong Hort extract and Aspirin 153 5.1 Introduction 153 5.2 Objective 163 5.3 Experimental 164 5.3.1 Chemicals and reagents 164 5.3.2 Prepared solutions 164 5.3.3 Preparation of L chuanxiong extract and vehicle solution 165 5.3.4 Animals 165 5.3.5 Ex vivo inhibition of platelet aggregation experiment 166 5.3.6 Pharmacokinetic study 167 5.3.7 LC-MS/MS Analysis of aspirin and salicylic acid 168 5.3.8 Protein binding 169 5.3.9 Pharmacokinetic data analysis and Statistical analysis 171 5.4 Results 171 5.4.1 Effects of interaction between aspirin and L chuanxiong on antiplatelet activity 171 5.4.2 Pharmacokinetic study 173 5.5 Discussion 183 5.6 Conclusion 186 Chapter Conclusion and Future work 188 Reference List 193 Appendix 222 VII SUMMARY The work presented in this thesis aims to develop methods and apply them for the assessment of quality and safety of herbal medicines Quality is the paramount issue which affects the efficacy and safety of herbal medicines Analytical methods were developed to evaluate the quality of herbal medicines and related products, while ex vivo and in vivo methods to assess potential herb-drug interactions were explored With regards to assessment of quality, an adulterant was detected in the extract of a premixed bulk powder for dietary supplement and structurally elucidated It was determined to be an analogue of acetildenafil (hydroxyacetildenafil) Its structure was identified by nuclear magnetic resonance (NMR), fourier transform infrared (IR), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution mass spectrometry (HRESIMS) This is the first report of hydroxyacetildenafil found as an adulterant in a dietary supplement Its structural information would be helpful for screening phosphodiesterase type enzyme (PDE-5) inhibitors and their analogues to control the quality of dietary supplements and herbal products Ligusticum chuanxiong is a Chinese herb that improves blood circulation and is selected as a model herb for the assessment of quality and safety (herb-drug interactions) High performance liquid chromatography (HPLC), LC-MS/MS and high resolution liquid chromatography-mass spectrometry (LC-MS) methods were VIII successfully developed for the chemical analysis of L chuanxiong and its reported chemical markers (tetramethylpyrazine and ferulic acid) The concentrations of ferulic acid were found to be low in all the extracts whereas tetramethylpyrazine could not be detected by HPLC, and even LC-MS/MS at a low detection limit of 1.3×10-8 g Hence, the results suggest that these two analytes could not be used as marker compounds to assess the quality of L chuanxiong A novel and efficient LC-ESI-MSn method was developed and successfully applied for the quality control of L chuanxiong A total of 70 compounds (mainly different kinds of phthalides) were identified or tentatively characterized based on a combination of retention times, high resolution mass data and their multistage fragmentation behaviors One of the important safety issues is herb-drug interaction To assess the potential herb-drug interaction of L chuanxiong, the effects of the ethanol extract of L chuanxiong and its reported active components tetramethylpyrazine and ferulic acid on the most important drug-metabolizing enzymes, cytochrome P450 (CYP) 3A and 2D activities were determined in Sprague-Dawley rat liver 14-day pre-treatment with L chuanxiong extract (10 g herb·kg-1·d-1) significantly increased CYP2D activity (p