by Kwok Chiu Nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 1-3 (4th gp.)). / Abstract also in Chinese. / TABLE OF CONTENTS --- p.i / ABSTRACT --- p.iv / 摘要 --- p.vi / LIST OF FIGURES --- p.vii / LIST OF TABLES --- p.x / ABBREVIATION --- p.xi / Chapter CHAPTER ONE --- RESEARCH BACKGROUND / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- Alkaloids --- p.1 / Chapter 1.1.2 --- Diterpenoid alkaloids --- p.2 / Chapter 1.1.3 --- Aconitine-type alkaloids --- p.2 / Chapter 1.1.4 --- Toxicity --- p.4 / Chapter 1.1.5 --- Safety concerns --- p.4 / Chapter 1.2 --- Summary of the Previous Work --- p.8 / Chapter 1.3 --- Objectives and Outline of the Present Work --- p.13 / Chapter CHAPTER TWO --- INSTRUMENTATION AND EXPERIMENTAL / Chapter 2.1 --- Instrumentation --- p.15 / Chapter 2.1.1 --- High performance liquid chromatography (HPLC) --- p.15 / Chapter 2.1.2 --- Triple-stage quadrupole (TSQ) mass spectrometer --- p.17 / Chapter 2.1.2.1 --- Atmospheric pressure chemical ionization (APCI) --- p.17 / Chapter 2.1.2.2 --- Electrospray ionization (ESI) --- p.20 / Chapter 2.1.2.3 --- Quadrupole system --- p.20 / Chapter 2.1.2.4 --- Ion detection system --- p.22 / Chapter 2.1.2.5 --- Data system --- p.22 / Chapter 2.2 --- Experimental --- p.22 / Chapter 2.2.1 --- Sample and reagents --- p.22 / Chapter 2.2.2 --- Sample preparation --- p.23 / Chapter 2.2.3 --- High performance liquid chromatography conditions --- p.23 / Chapter 2.2.4 --- Mass spectrometry conditions --- p.25 / Chapter 2.2.4.1 --- Atmospheric pressure chemical ionization conditions --- p.25 / Chapter 2.2.4.2 --- Electrospray ionization conditions --- p.25 / Chapter CHAPTER THREE --- SELECTION AND OPTIMIZATION OF HPLC/MS METHOD / Chapter 3.1 --- Introduction --- p.26 / Chapter 3.2 --- Experimental --- p.29 / Chapter 3.3 --- Results and Discussion --- p.29 / Chapter 3.3.1 --- Triethylamine concentration --- p.31 / Chapter 3.3.2 --- Ammonium acetate concentration --- p.34 / Chapter 3.3.3 --- Acetic acid concentration --- p.37 / Chapter 3.3.4 --- HPLC/MS interface --- p.40 / Chapter 3.3.5 --- MS/MS conditions --- p.40 / Chapter 3.4 --- Conclusions --- p.43 / Chapter CHAPTER FOUR --- DETERMINATION OF ACONITINE-TYPE ALKALOIDS IN ACONITE ROOTS / Chapter 4.1 --- Introduction --- p.48 / Chapter 4.2 --- Experimental --- p.48 / Chapter 4.3 --- Results and Discussion --- p.50 / Chapter 4.3.1 --- Selection of internal standard --- p.50 / Chapter 4.3.2 --- Method validation --- p.50 / Chapter 4.3.2.1 --- Precision of measurement --- p.50 / Chapter 4.3.2.2 --- Accuracy of measurement --- p.50 / Chapter 4.3.2.3 --- Limits of detection and quantitation --- p.58 / Chapter 4.3.3 --- Determination of aconitine-type alkaloids in aconite roots --- p.58 / Chapter 4.4 --- Conclusions --- p.60 / Chapter CHAPTER FIVE --- CONCLUSIONS AND FUTURE WORK / Chapter 5.1 --- Conclusions --- p.67 / Chapter 5.2 --- Future Work --- p.68 / ACKNOWLEDGMENT --- p.A1 / APPENDIX --- p.A2 / REFERENCES --- p.R1

Mesmo antes do advento da escrita, o homem já buscava na natureza a cura para suas doenças. Dentre as diferentes classes de produtos naturais usados para esse fim, destaca-se a dos alcalóides, por exibir relevante diversidade estrutural e farmacológica, e que tem como fonte importante o gênero Aspidosperma, do qual foram isolados alcalóides que têm sido usados nas terapias atuais. Nesse contexto, o presente trabalho teve como objetivo investigar os alcalóides obtidos a partir de diferentes partes de duas espécies ainda pouco exploradas: Aspidosperma ramiflorum e Aspidosperma tomentosum quanto à sua composição e atividades antifúngica, antimicrobiana e antitumoral. Foram identificados através de Cromatografia à Gás acoplada à Espectrometria de Massas (CG-EM) e Ressonância Magnetica Nuclear os alcalóides beta-ioimbina, nos pericarpos e arilos de A. ramiflorum, uleína, nas folhas, ramos e sementes de A. tomentosum, além de quebrachamina e razinilama, nos arilos e sementes dessa espécie. Adicionalmente, com base na literatura e nos resultados das análises por CG-EM verificou-se em A. tomentosum a presença de desmetileno-oxa-uleína, nas folhas, de eburnamonin-19-ona nos ramos e de di-hidro-razinilama, de aspidospermidina e também de di-hidro-razinilama e de 1-acetil-17-metóxi-aspidospermidina nas sementes e arilos dessa mesma espécie. Nenhum dos extratos de alcalóides totais ou alcalóides isolados apresentou atividade antibacteriana relevante. Verificou-se atividade antitumoral em extratos de alcalóides dos pericarpos de A. ramiflorum e dos ramos, folhas e arilos de A. tomentosum. Observou-se ainda atividade antifúngica em todos os extratos de alcalóides ensaiados, além de atividade específica contra isolados de Criptococcus neoformans resistentes ao fluconazol para os alcalóides quebrachamina e beta-ioimbina. O extrato de alcalóides totais das folhas de A. tomentosum, além da atividade frente a essas leveduras, também se mostrou ativo frente a duas espécies de Candida (C. krusei e C. parapsilosis). Concluiu-se a partir dos resultados obtidos que as duas espécies estudadas podem ser consideradas fontes promissoras de alcalóides que poderão ser utilizados como modelos para novos fármacos antifúngicos e antitumorais. / Even before the advent of writing, man already searched the nature for the cure of his illnesses. Among the different classes of natural products used for this purpose, monoterpenoid indole alkaloids exhibit large structural and pharmacological diversities and these compounds are commonly found in the Aspidosperma genus. In this context, the present work had as main goals to investigate the alkaloids from different parts of two poorly studied species, Aspidosperma ramiflorum and Aspidosperma tomentosum, regarding their composition and antibacterial, antifungal, and antitumoral activities. Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Nuclear Magnetic Ressonance analysis allowed the identification of the following alkaloids: beta-yohimbine, in the pericarp and arils of A. ramiflorum; uleine, in leaves, branches and seeds of A. tomentosum; quebrachamine and rhazinilam, in the arils and seeds of the same species. Moreover, based on literature data and the results of the GC-MS analysis was characterized in A. tomentosum the presence of oxo-uleine, in the leaves and stems, eburnamonine, in the stems, and rhazinilam, aspidospermidine, dihydrorhazinilam and 1-acetyl-17-methoxy-aspidospermidine like alkaloids in the seeds and arils. Neither crude alkaloid extracts nor isolated alkaloids presented a relevant antibacterial activity. An antitumoral activity could be detected in crude alkaloids extracts from pericarps of A. ramiflorum and from stems, leaves and arils of A. tomentosum. On the other hand, antifungal activity was observed in all crude alkaloid extracts assayed, besides a specific activity of the alkaloids quebrachamine and beta-yohimbine against fluconazol resistant Criptococcus neoformans. Additionally, it was observed that crude alkaloid extract from leaves of A. tomentosum was also active against two Candida species (C. krusei e C. parapsilosis), as well as to the C. neoformans isolate. From these results we can conclude that the two studied species has to be considerated as promising sources for antifungal and antitumor alkaloids to be used as models to design new drugs.

It was found that narciclasine retarded the growth of human cancer cells and plant suspension cells in dose-dependent manner. The inhibitory mechanism of narciclasine was found to be apoptosis for the DNA histogram showed an apoptotic peak in narciclasine-treated A375 cancer cells. The fluorescent signal dUTP fluorescein was found in the narciclasine-treated A735 cancer cell in TUNEL assay. The Annexin-V-FLUOS stained A375 cancer cell at 24-hour treatment with no PI found. These results suggest that narciclasine triggered early apoptosis in A375 cancer cell. Immunoblot analysis of the apoptotic signalling pathway showed that narciclasine induced apoptosis through the intrinsic pathway. Narciclasine induced the cleavage of caspase-9 but not the caspase-8, which was triggered by cytochrome c release from mitochondrial intermembrane space into cytosol. The activated caspase-9 triggered caspase cascade (e.g. cleavage of caspase-3, caspase-6 and caspase-7) which induced the cleavage of PARP. / Narciclasine is an isoquinoline alkaloid extracted from the bulb of Narcissus tazetta. It shows a wide range of biological activities such as antitumour, antiviral and plant growth inhibitory activities. However, little information is available regarding such inhibitory activities. The objective of this study is to elucidate the mechanisms of the cytotoxic effects of narciclasine in different cell models. / On the other hand, narciclasine triggered programmed cell death (PCD) in plant cells as proved by the increased intensity of Evans blue in narciclasine-treated suspension cells. Fluorescent microscopy showed that narciclasine induced PCD in tobacco BY2 cell with the dUTP fluorescein stained in narciclasine-treated cell. The induction of PCD was in dose-dependent and time-dependent manner. / Proteomic studies showed that narciclasine may affect A375 cancer cell and rice meristemic cells in similar manner. Narciclasine may affect the metabolism and defence system of both A375 cancer cell and rice meristemic cells through down-regulating the expression of metabolic enzymes (e.g. triosephosphate isomerase in A375 cancer cell and fructose bisphosphate aldolase in rice root tip) and defensive proteins (e.g. peroxiredoxin in A375 cancer cell and catalase in rice root tip). Narciclasine down-regulated the heat-shock proteins (HSP) which is involved in regulating cellular homeostasis and promoting cell survival. Therefore, narciclasine reduced HSP to lower the cell survival ability and induced the caspase cascade or caspase-like activity in A375 cancer cell and rice respectively. / To summarize, narciclasine induced apoptosis in A375 cancer cell and programmed cell death in tobacco BY2 cell. / Wong, Chi Fai. / "October 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4576. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 230-255). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Chiral alpha-branched amines are common substructures of bioactive synthetic targets such as alkaloids and amino acids. Direct asymmetric amine synthesis by addition to the C=N bond of carbonyl imino derivatives is promising and efficient to introduce the stereogenic center and carbon-carbon bond in one step. Furthermore, disconnection of either C-C bond at the amine stereogenic center would be the most versatile method to achieve this objective; we could make the choice depending on the different synthetic strategies, such as the availability of precursors and the presence of complicating structural features. In our group, we disclosed that manganese carbonyl mediates stereoselective photolytic radical addition of alkyl iodides to chiral imino acceptors, which is a powerful tool to form a new C-C bond and generate a chiral center. Qualitative mechanistic studies confirm the importance of free radicals, imply that this is a nonchain (or short chain length) free-radical process, and reveal that organomanganese compounds are not a viable source of alkyl radical for the addition reactions under the conditions in our lab. In my thesis, we have extended the application of our methodology. At the beginning of my research, our Mn-mediated addition methodology was first applied to accomplish the couplings of iodides and ketone N-acylhydrazones, generating quaternary carbon stereocenters and offering access to a variety of alpha-alkylated alanine analogs. These radical additions complement enolate alkylation methodologies, as they occur under nonbasic conditions and permit introduction of both primary and secondary alkyl groups with relative ease. The versatility with respect to the iodide is a distinguishing feature of the Mn-mediated coupling that foreshadows application to more complex targets. Secondly, a Mn-mediated radical-ionic annulation strategy was validated as a synthetic route to quinine. Intermolecular radical addition to C=N bonds has rarely been applied as a strategic bond construction in natural product synthesis; this synthesis of quinine offers the strongest demonstration yet of the utility of such reactions in application toward complex multifunctional targets.

Author: Jana Öhlschlegelová Title: Alkaloids of family Amaryllidaceae: genus Hippeastrum Diploma thesis Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany 2019, 74 p. Keywords: Hippeastrum, Amaryllidaceae, alkaloids, antiproliferative activity, Alzheimer's disease, cholinesterase inhibitors, galanthamine The aim of this diploma thesis was to unify current findings about alkaloids isolated from selected plants of the Hippeastrum genus in the Amaryllidaceae family. The fytochemical characteristics of the examined species was introduced, and a group of alkaloids which were isolated from these plant species was composed. Also, the biological activity was evaluated. Up to date, at least 13 plant species of the Hippeastrum genus were examined from the fytochemical perspective. Out of these species examined, 56 different alkaloids with defined structure were isolated. The isolated alkaloids are divided into several groups based on their structure. Namely, these are lycorine, homolycorine, crinine, galanthamine, narciclasine, tazettine, haemanthamine and montanine structural types. Also, alkaloids which differed structurally from these basic types were found in several plants studied. In the substances gained, the antiproliferative activity, inhibitory activity...

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Botany Consultant: Ing. Kateřina Macáková, Ph. D. Student: Markéta Šilhová Title of Thesis: Interactions of alkaloids with transition metals II. Copper is an important component of the human body. It is involved in the right functioning of organ systems and is also a part of many important body enzymes. It is necessary to maintain a balanced amount of copper in the body in order to avoid excess or deficiency, which can lead to various diseases. The aim of this diploma thesis was to determine the copper chelating and copper reducing effects of isoquinoline alkaloids berberine chloride, canadine, corydaline, sculerine, sinactine, stylopine, tetrahydropalmatine, allocryptopine, protopine, corycavamine and cryptopine. In experimental measurements the determination of chelation of copper ions with hemytoxyline and the determination of chelation and reduction of copper ions using disodium salt of bathocuproindisulfonic acid was performed. The highest reducing activity was exhibited by scoulerine, whose structure in comparison with other alkaloids contains hydroxyl groups and which has been exhibited in the past to inhibit the growth of tumour cell. The lowest reducing activity was measured for protopine alkaloids and...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Radka Rzepecká Supervisor: Ing. Kateřina Macáková, Ph.D. Title of Thesis: Interactions of alkaloids with transition metals I. Copper is one of the essential trace elements that are necessary for the proper functioning of the organism. Copper is a significant component of many enzymes that affect various metabolic processes in the human body. It is important that the level of copper in the body is regulated because its deficit or excess lead to a variety of pathological conditions. Alkaloids are secondary plant metabolites that are distinguished by numerous biological activities. In this thesis the copper-chelating and copper-reducing activity of eleven isoquinoline alkaloids was measured: boldine, isocorydine, (+)-bulbocapnine, (+)-corydine, glaucine, (-)-sinoacutine, (-)-californidine, (-)-escholtzine, platycerine, (-)-fumaricine, and (+)- parfumine. Alkaloid activity was measured at four (patho) physiological pH values by a verified spectrophotometric method using two indicators: bathocuproinedisulfonic acid disodium salt and hematoxylin. Based on the results, structure-effect relationships were derived. The results show that none of the tested substances was able to chelate copper ions. In...

Pyrrolizidine alkaloids are cytotoxic, carcinogenic, and anti-carcinogenic in vivo and in vitro, and they produce many hazardous effects in humans and animals. Pyrrolizidine alkaloids (PAs) also cross-link with DNA and/or protein. However, whether such cross-linking is important to the toxic action of PAs is not known. In addition, the exact mechanism underlying these DNA cross-links or cytotoxicity is also not clear. In three separate studies, I characterized the nature of PA-induced DNA cross-links and the relationships between PA structures and cross-linking potency. In the first study (Chapter II), I found that cross-linking potency of PA congeners coincided with their abilities to cause cytopathologic effects. Macrocyclic a,p-unsaturated diesters PAs and their pyrrolic metabolites were the most potent inhibitors of colony formation, and inducers of cytopathologic changes and megalocyte formation. The macrocyclic α, β-saturated diester PA and open diesters PAs slightly inhibited colony formation, and slightly changed cell morphology. Retronecine and indicine N-oxide were completely inactive. In the next study (Chapter Ill), I found that pyrrolic macrocyclic metabolites were more potent DNA cross-linkers than their parent compounds as determined by alkaline elution. The pyrroles of the macrocyclic diester PAs were potent DNADNA (inter- and/or intra) cross-linkers in BstEll-digested λ-phage DNA or pBR322 plasmid DNA but dehydroretronecine and indicine N-oxide were not. I also examined which DNA sequences were more susceptible to PA-induced cross-links by using a series of restriction endonucleases to determine sequence specificity. The most favorable cross-linking site for PAs appeared to be 5'd(GG) and 5'-d(GA) although other sites, 5'-d(CC) or 5'-d(CG), might be also preferable cross-linking targets. In the next study (Chapter IV), I characterized the nature of DNA-protein interactions induced by PAs, because I found in previous studies that PA-induced cross-links are largely protein associated. In PA or pyrrolic PA exposed cells, cross-linked proteins with molecular weights 40 - 60 kD were detected. Two-dimensional electrophoretic analysis revealed that these proteins were probably acidic in nature. In an in vitro system utilizing pBR322 or Bst Ell-digested λ-phage DNA. dehydrosenecionine induced DNAprotein cross-links with BSA, indicating that such interactions might be related to amino acid composition of protein. These results confirmed that PA-induced DNA cross-links (DNA-DNA, DNA-protein cross-links) are influenced by three structural features: the C1 ,2 unsaturation of pyrrolizidine ring, α, β-unsaturation, and size of the macrocyclic diester ring. The ability to form cross-links was closely related to the known toxic potencies of these PAs. From this research, I also conclude that DNA crosslinking is the most critical event leading to PA-related diseases and that crosslinking is due to pyrrolic metabolites of PAs, not via a common metabolite as was once thought.

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