Molecular authentication of three Chinese herbs: baiying, baihuasheshecao and chuanlianzi.

Li Ming.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.Includes bibliographical references (leaves 146-161).Abstracts in English and Chinese.ACKNOWLEDGEMENTS --- p.IABSTRACT --- p.IIITABLE OF CONTENTS --- p.VIILIST OF FIGURES AND TABLES --- p.XIIILIST OF ABBREVIATIONS --- p.XXChapter CHAPTER ONE --- LITERATURE REVIEW --- p.1Chapter 1.1 --- Authentication of Chinese medicines --- p.1Chapter 1.1.1 --- The need for authentication of Chinese medicines --- p.1Chapter 1.1.2 --- Traditional methods for authentication --- p.2Chapter 1.1.3 --- Molecular methods for authentication --- p.4Chapter 1.1.3.1 --- DNA fingerprinting --- p.5Chapter 1.1.3.2 --- DNA sequencing --- p.6Chapter 1.1.3.2.1 --- Choosing a suitable region for DNA sequencing --- p.7Chapter 1.1.3.2.2 --- Chloroplast irnL-trnF region --- p.9Chapter 1.1.3.2.3 --- Complete sequence of ITS rDNA region --- p.10Chapter 1.1.3.2.4 --- 5S rDNA intergenic spacer --- p.11Chapter 1.1.3.2.5 --- Calculation of similarities among sequences --- p.12Chapter 1.1.3.2.6 --- Construction methods of phylograms --- p.12Chapter 1.2 --- The need for molecular authentication of three medicinal herbs --- p.14Chapter 1.2.1 --- The herb Baiying --- p.14Chapter 1.2.1.1 --- The poisoning case reported in Hong Kong --- p.14Chapter 1.2.1.2 --- The identity of genuine Baiying --- p.15Chapter 1.2.1.3 --- Morphological characters of the herb Baiying --- p.15Chapter 1.2.1.4 --- Medicinal values of Baiying --- p.17Chapter 1.2.1.5 --- Xungufeng as the adulterant of Baiying --- p.17Chapter 1.2.1.5.1 --- The toxic chemicals aristolochic acids --- p.18Chapter 1.2.1.6 --- The need for molecular authentication of Baiying --- p.19Chapter 1.2.2 --- The herb Baihuasheshecao --- p.19Chapter 1.2.2.1 --- The identity of Baihuasheshecao --- p.19Chapter 1.2.2.2 --- Morphological characters of the herb Baihuasheshecao --- p.20Chapter 1.2.2.3 --- Medicinal uses --- p.23Chapter 1.2.2.4 --- Chemical profile --- p.24Chapter 1.2.2.5 --- Adulterants of Baihuasheshecao --- p.24Chapter 1.2.2.6 --- Chemical studies of H. diffusa and H. corymbosa --- p.25Chapter 1.2.2.7 --- Existing methods for authentication --- p.26Chapter 1.2.2.8 --- The need for molecular authentication of Baihuasheshecao --- p.28Chapter 1.2.3 --- The herb Chuanlianzi --- p.28Chapter 1.2.3.1 --- The identity of Chuanlianzi --- p.28Chapter 1.2.3.2 --- Medicinal values --- p.29Chapter 1.2.3.3. --- The bioactive chemical --- p.31Chapter 1.2.3.4 --- Kulianzi as the substitute of Chuanlianzi --- p.31Chapter 1.2.3.5 --- Poisoning cases reported due to ingestion of Kulianzi --- p.32Chapter 1.2.3.6 --- Comparative studies of Chuanlianzi and Kulianzi --- p.32Chapter 1.2.3.7 --- The need for molecular authentication of Chuanlianzi --- p.33Chapter CHAPTER TWO --- OBJECTIVE --- p.35Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.36Chapter 3.1 --- Plant and herb samples --- p.36Chapter 3.2 --- Total DNA extraction --- p.48Chapter 3.2.1 --- Cetyltriethylammonium bromide extraction --- p.48Chapter 3.2.2 --- Commercial kit extraction --- p.49Chapter 3.3 --- DNA amplification --- p.50Chapter 3.4 --- DNA fingerprinting --- p.51Chapter 3.4.1 --- DNA concentration determination --- p.51Chapter 3.4.2 --- ISSR fingerprinting --- p.52Chapter 3.5 --- Agarose gel electrophoresis --- p.53Chapter 3.6 --- Purification of PCR product --- p.53Chapter 3.7 --- Cloning of PCR product --- p.54Chapter 3.7.1 --- Ligation --- p.54Chapter 3.7.2 --- Transformation --- p.55Chapter 3.7.3 --- Cell cultivation --- p.55Chapter 3.7.4 --- Plasmid extraction --- p.55Chapter 3.7.5 --- Insert confirmation --- p.56Chapter 3.8 --- Determination of DNA concentration --- p.56Chapter 3.9 --- DNA sequencing --- p.57Chapter 3.9.1 --- Cycle sequencing --- p.57Chapter 3.9.2 --- Purification of cycle sequencing products --- p.57Chapter 3.9.3 --- DNA analysis --- p.58Chapter 3.10 --- Sequence analysis --- p.58Chapter CHAPTER FOUR --- MOLECULAR AUTHENTICATION OF BAIYING --- p.59Chapter 4.1 --- Results --- p.59Chapter 4.1.1 --- Sequence alignment --- p.59Chapter 4.1.2 --- Percentage similarity analysis --- p.68Chapter 4.1.3 --- Phylogram study --- p.71Chapter 4.2 --- Discussion --- p.79Chapter 4.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Baiying and Xungufeng --- p.79Chapter 4.2.2 --- Molecular authentication of Baiying --- p.80Chapter 4.3 --- Conclusion --- p.81Chapter CHAPTER FIVE --- MOLECULAR AUTHENTICATION OF BAIHUASHESHECAO --- p.82Chapter 5.1 --- Results --- p.82Chapter 5.1.1 --- ITS region used for DNA sequencing --- p.82Chapter 5.1.2 --- Sequence alignment --- p.82Chapter 5.1.3 --- Percentage similarity analysis --- p.88Chapter 5.1.4 --- Phylogram study --- p.90Chapter 5.2 --- Discussion --- p.98Chapter 5.2.1 --- Evaluation of complete sequence of ITS region in differentiation of Hedyotis species --- p.98Chapter 5.2.2 --- Molecular authentication of retailed Baihuasheshecao --- p.99Chapter 5.2.3 --- Analysis of conflicting data between this study and published results --- p.99Chapter 5.2.3.1 --- Comparison of ITS-1 region --- p.101Chapter 5.2.3.2 --- Comparison of ITS-2 region --- p.104Chapter 5.2.3.3 --- Proposed reasons for the conflicting data --- p.108Chapter 5.3 --- Conclusion --- p.109Chapter CHAPTER SIX --- MOLECULAR AUTHENTICATION OF CHUANLIANZI --- p.110Chapter 6.1 --- Results --- p.110Chapter 6.1.1 --- DNA sequencing --- p.110Chapter 6.1.1.1 --- Complete sequence of ITS region used for DNA sequencing --- p.110Chapter 6.1.1.1.1 --- Sequence alignment --- p.111Chapter 6.1.1.1.2 --- Percentage similarity analysis --- p.113Chapter 6.1.1.2 --- 5S rDNA intergenic spacer used for DNA sequencing --- p.113Chapter 6.1.1.2.1 --- Sequencing alignment --- p.114Chapter 6.1.1.2.2 --- Percentage similarity analysis --- p.122Chapter 6.1.1.2.3 --- Phylogram study --- p.128Chapter 6.1.2 --- ISSR fingerprinting --- p.136Chapter 6.2 --- Discussion --- p.138Chapter 6.2.1 --- DNA sequencing results --- p.138Chapter 6.2.2. --- ISSR fingerprinting results --- p.139Chapter 6.2.3 --- Investigation of the identity of retailed Chuanlianzi --- p.140Chapter 6.2.4 --- Taxonomic interpretation for Melia species --- p.141Chapter 6.2.5 --- Kulianzi involved in this study --- p.141Chapter 6.3 --- Conclusion --- p.141Chapter CHAPTER SEVEN --- CONCLUSION --- p.143BILBIOGRAPHY --- p.146APPDENDIX - MATERIAL PREPARATION --- p.16