Aconitum and Delphinium genera are important rich sources of toxic C19-diterpenoid alkaloids. The alkaloidal content of A. napellus and D. elatum seeds have been investigated in detail. After maceration, crude alkaloidal extracts were obtained and then purified by repeated column chromatography (over silica and alumina gels) to sample homogeneity yielding five known C19-diterpenoid alkaloids from A. napellus, aconitine, neoline, 14-O-acetyltalatisamine, 14-O-benzoylaconine, and taurenine, and two others from D. elatum, delpheline (also including its X-ray single crystal analysis) and methyllycaconitine (MLA). These examples showed that mass spectrometry hyphenated with HPLC or other chromatography can be used as a tool for rapid alkaloid content screening of different extracts. NMR spectroscopic (using a variety of techniques and nuclei) data are presented in support of the first report of iminodelsemine A/B as an imino-alkaloid artefact from D. elatum. A detailed chromatographic study across different pH ranges, and over different solid supports, of aconitine and its main degradation product, 14-O-benzoyl-8-O-methylaconine, together with its semi-synthesis and that of its deuterated analogue are reported within studies to minimize artefact formation during the storage or extraction of A. napellus norditerpenoid alkaloids. Likewise, from D. elatum seeds, as a model source of Delphinium alkaloids, we compared the alkaloid yield using different extraction techniques and conventional chromatographic separations. The structures were confirmed by NMR spectroscopy and mass spectrometry. An NMR spectroscopic approach for the pKa determination of some C19-diterpenoid alkaloids has been developed. A modified calculation method for fatty acid composition quantification has also been developed using 1H-NMR spectroscopic methods.

Shum Ka Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 174-182). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Table of content --- p.viii / List of figures --- p.xvi / List of tables --- p.xxii / Abbreviations --- p.xxv / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Importance of authentication of Traditional Chinese Medicines --- p.1 / Chapter 1.1.1 --- Confusing nomenclatures --- p.1 / Chapter 1.1.2 --- Similar morphologies of different medicinal materials --- p.2 / Chapter 1.1.3 --- Toxicities of medicinal materials --- p.2 / Chapter 1.1.4 --- Conservation of natural products --- p.2 / Chapter 1.2 --- TCM listed in the Pharmacopoeia of People's Republic of China --- p.3 / Chapter 1.3 --- Overview of mis-use and intoxication of TCM --- p.4 / Chapter 1.4 --- Ordinances regulating Chinese medicines as natural products --- p.7 / Chapter 1.4.1 --- Laws governing Chinese medicine --- p.7 / Chapter 1.4.2 --- Laws governing endangered species --- p.8 / Chapter 1.5 --- Current technologies in the authentication of Traditional Chinese Medicines and their limitations --- p.9 / Chapter 1.6 --- Historical applications of Radix Aconiti --- p.12 / Chapter 1.7 --- Modern applications of Radix Aconiti --- p.16 / Chapter 1.8 --- Research on Radix Aconiti and its chemical components --- p.17 / Chapter 1.8.1 --- Chemistry --- p.17 / Chapter 1.8.2 --- Pharmacology --- p.19 / Chapter 1.8.3 --- Molecular interaction --- p.22 / Chapter 1.9 --- Brief review on the systematics and phylogeny of Aconitum --- p.23 / Chapter 1.10 --- Historical applications of Radix Aucklandiae and related materials --- p.25 / Chapter 1.11 --- Modern applications of Radix Aucklandiae and related material --- p.27 / Chapter 1.12 --- Research on Aucklandiae and related material and their chemical components --- p.28 / Chapter 1.12.1 --- Chemistry --- p.28 / Chapter 1.12.2 --- Pharmacology --- p.29 / Chapter 1.13 --- Brief review on the systematics and phylogeny of Aucklandia and related medicinal species --- p.31 / Chapter 1.14 --- Authentication by DNA sequencing --- p.33 / Chapter 1.14.1 --- Introduction --- p.33 / Chapter 1.14.2 --- Criteria of sequence markers --- p.36 / Chapter 1.14.3 --- Model used to process polymorphism in DNA sequences --- p.37 / Chapter 1.15 --- Screening for novel markers --- p.38 / Chapter 1.15.1 --- Reason for screening novel markers --- p.38 / Chapter 1.15.2 --- Basic principle --- p.39 / Chapter 1.16 --- Introduction to gas chromatography- mass spectrometry --- p.40 / Chapter 1.16.1 --- Basic principles and components of GC-MS --- p.41 / Chapter 1.16.2 --- Advantages and limitations of GC-MS --- p.42 / Chapter 1.16.3 --- Usage of GC-MS on natural product analysis --- p.43 / Chapter 1.16.4 --- Chemometric analysis --- p.44 / Chapter 1.17 --- Objectives --- p.46 / Chapter Chapter 2. --- Materials and Methods --- p.47 / Chapter 2.1 --- Plant samples --- p.47 / Chapter 2.1.1 --- Samples of Aconitum --- p.47 / Chapter 2.1.2 --- Samples of Aucklandia and related species --- p.51 / Chapter 2.2 --- DNA extraction method --- p.58 / Chapter 2.2.1 --- Reagents --- p.58 / Chapter 2.2.2 --- Methods --- p.59 / Chapter 2.3 --- Chemical extraction methods --- p.61 / Chapter 2.4 --- Chemical standard extraction and purification method --- p.62 / Chapter 2.5 --- DNA sequencing --- p.63 / Chapter 2.5.1 --- Reagents --- p.63 / Chapter 2.5.2 --- Methods --- p.65 / Chapter 2.6 --- Genomic subtraction --- p.70 / Chapter 2.7 --- Search for species-specific markers from the subtraction library --- p.74 / Chapter 2.8 --- Gas chromatography- mass spectrometry --- p.74 / Chapter 2.9 --- GC-MS chemometric analysis --- p.75 / Chapter Chapter 3. --- Authentication of Aconitum by DNA Sequencing --- p.76 / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Methods --- p.77 / Chapter 3.3 --- Results - 5S spacer --- p.77 / Chapter 3.3.1 --- Sequence information --- p.77 / Chapter 3.3.2 --- Sequence similarity --- p.78 / Chapter 3.3.3 --- Phylogram study --- p.81 / Chapter 3.4 --- Results -psbA-trnH --- p.85 / Chapter 3.4.1 --- Sequence information --- p.85 / Chapter 3.4.2 --- Sequence similarity --- p.85 / Chapter 3.4.3 --- Phylogram study --- p.87 / Chapter 3.5 --- Discussion --- p.91 / Chapter 3.5.1 --- Overview of nuclear ribosomal 5S spacer --- p.91 / Chapter 3.5.2 --- Extensive polymorphism of 5S spacer --- p.91 / Chapter 3.5.3 --- Distribution of samples in the phylograms constructed by 5S spacer --- p.93 / Chapter 3.5.4 --- Utility of 5S spacer for authentication --- p.94 / Chapter 3.5.5 --- Overview of psbA-trnH spacer --- p.94 / Chapter 3.5.6 --- Distribution of samples in the phylograms constructed by psbA-trnH spacer --- p.95 / Chapter 3.5.7 --- A distinctive region of inversion --- p.96 / Chapter 3.5.8 --- Utility of psbA-trnH for authentication --- p.97 / Chapter Chapter 4. --- Screening for Novel Markers for Authentication of Aconitum --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Methods --- p.99 / Chapter 4.3 --- Results - subtracted clones --- p.99 / Chapter 4.4 --- Results - SSH6 --- p.104 / Chapter 4.4.1 --- Sequence information --- p.104 / Chapter 4.4.2 --- Sequence similarity --- p.105 / Chapter 4.5 --- Results-SSH15 --- p.107 / Chapter 4.5.1 --- Sequence information --- p.107 / Chapter 4.5.2 --- Sequence similarity --- p.107 / Chapter 4.5.3 --- Phylogram study --- p.109 / Chapter 4.6 --- Results-SSH45 --- p.113 / Chapter 4.6.1 --- Sequence information --- p.113 / Chapter 4.6.2 --- Sequence similarity --- p.113 / Chapter 4.6.3 --- Phylogram study --- p.115 / Chapter 4.7 --- Discussion --- p.119 / Chapter 4.7.1 --- Utility of subtraction in screening markers --- p.119 / Chapter 4.7.2 --- SSH6 --- p.121 / Chapter 4.7.3 --- SSH15 --- p.122 / Chapter 4.7.4 --- SSH45 --- p.123 / Chapter 4.7.5 --- Hybridization in Aconitum --- p.124 / Chapter 4.7.6 --- Inferring species identities of samples from the market --- p.126 / Chapter 4.8 --- Conclusion --- p.128 / Chapter Chapter 5. --- Assessment of Aucklandia lappa and Related Species by GC-MS --- p.129 / Chapter 5.1 --- Introduction --- p.129 / Chapter 5.2 --- Methods --- p.130 / Chapter 5.3 --- Results --- p.130 / Chapter 5.3.1 --- Extraction of essential oil --- p.130 / Chapter 5.3.2 --- GC-MS analysis --- p.131 / Chapter 5.3.3 --- Peak alignment and hierarchical cluster analysis --- p.133 / Chapter 5.3.4 --- Purification of chemical markers from Aucklandia lappa --- p.148 / Chapter 5.3.5 --- Standardization of the purified chemical markers --- p.148 / Chapter 5.3.6 --- Quantitative analysis of chemical markers --- p.152 / Chapter 5.4 --- Discussion --- p.154 / Chapter 5.4.1 --- Analysis of chemical composition --- p.154 / Chapter 5.4.2 --- A comparison on chemometric methods --- p.154 / Chapter 5.4.3 --- Similarity of chemical profiles --- p.156 / Chapter 5.4.4 --- Dendrogram analysis --- p.157 / Chapter 5.4.5 --- Utility of GC-MS in authentication of A. lappa and related species --- p.159 / Chapter 5.4.6 --- Limitations --- p.159 / Chapter 5.4.7 --- Comparison with molecular data --- p.161 / Chapter 5.4.8 --- Contents of dehydrocostuslactone and costunolide --- p.163 / Chapter 5.4.9 --- Locality study --- p.164 / Chapter 5.5 --- Conclusion --- p.165 / Chapter Chapter 6. --- General Discussion --- p.167 / Chapter 6.1 --- DNA sequencing --- p.168 / Chapter 6.2 --- Genomic subtraction --- p.169 / Chapter 6.3 --- Future work on molecular authentication --- p.170 / Chapter 6.4 --- Future work on authentication of Aconitum --- p.170 / Chapter 6.5 --- Gas chromatography- mass spectrometry --- p.171 / Chapter 6.6 --- Future work on authentication by GC-MS --- p.172 / Chapter 6.7 --- Future work on authentication of Aucklandia lappa and related species … --- p.173 / References --- p.174 / Appendix A. Sequence Alignment of 5S Spacer from Aconitum Species --- p.183 / Appendix B. Sequence Alignment of psbA- trnH Spacer from Aconitum Species --- p.188 / Appendix C. Sequences of Subtracted Clones from Aconitum --- p.191 / Appendix D. Sequence Alignment of SSH6 from Aconitum Species --- p.194 / Appendix E. Sequence Alignment of SSH15 from Aconitum Species --- p.195 / Appendix F. Sequence Alignment of SSH45 from Aconitum Species --- p.200 / Appendix G. Gas Chromatograms of Essential Oil Extracts of Aucklandia lappa and Related Species --- p.202