Wu, Hoi Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 92-104). / Abstracts in English and Chinese. / Thesis/ Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgments --- p.vi / Table of Content --- p.vii / List of Abbreviations --- p.x / List of Figures --- p.xiv / List of Tables --- p.xv / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Introduction of diabetes mellitus --- p.1 / Chapter 1.1.1 --- Definition and prevalence --- p.1 / Chapter 1.1.2 --- Diagnosis and classification --- p.2 / Chapter 1.1.3 --- Symptoms and complications --- p.4 / Chapter 1.1.4 --- Cause and risk factors --- p.5 / Chapter 1.1.5 --- Prevention and treatment --- p.9 / Chapter 1.2 --- The role of adipose tissue in pathophysiology of T2DM --- p.10 / Chapter 1.2.1 --- Randle's glucose-fatty acid hypothesis --- p.11 / Chapter 1.2.2 --- Ectopic fat storage hypothesis --- p.12 / Chapter 1.2.3 --- Adipose tissue as an endocrine organ --- p.13 / Chapter 1.2.4 --- Low-grade inflammation --- p.15 / Chapter 1.2.5 --- Endoplasmic reticulum (ER) stress --- p.17 / Chapter 1.3 --- Use of berberine in the treatment of T2DM --- p.18 / Chapter 1.3.1 --- Efficacy of berberine in treating diabetes --- p.18 / Chapter 1.3.2 --- Berberine on glucose and lipid metabolism of animals --- p.19 / Chapter 1.3.3 --- Inhibition of adipogenesis --- p.20 / Chapter 1.3.4 --- Activation of AMP-Activated Protein Kinase (AMPK) --- p.20 / Chapter 1.3.5 --- Mitochondrial inhibition --- p.21 / Chapter 1.4 --- Introduction of proteomics --- p.21 / Chapter 1.4.1 --- Why proteomics? --- p.22 / Chapter 1.4.2 --- Gel-based proteomics: Two-Dimensional Gel Electrophoresis --- p.23 / Chapter 1.4.3 --- Gel-free proteomics --- p.25 / Chapter 1.4.4 --- Mass spectrometry --- p.26 / Chapter 1.4.5 --- Proteomics as tool for diabetes research --- p.27 / Chapter 1.5 --- Objectives and significance --- p.32 / Chapter 2. --- Materials and Methods --- p.34 / Chapter 2.1 --- Drug preparation --- p.34 / Chapter 2.2 --- Animal experiment --- p.34 / Chapter 2.3 --- Comparison of proteome of visceral white adipose tissue: obese db/db micevs lean m+/db mice and BBR-treated vs control db/db mice --- p.36 / Chapter 2.3.1 --- Protein sample preparation from adipose tissue --- p.36 / Chapter 2.3.2 --- Protein quantitation --- p.37 / Chapter 2.3.3 --- 2D Gel electrophoresis --- p.37 / Chapter 2.3.4 --- Image analysis --- p.39 / Chapter 2.3.5 --- In-gel digestion and MALDI-ToF MS --- p.39 / Chapter 2.4 --- Cell culture experiment --- p.40 / Chapter 2.5 --- Oil Red O staining --- p.42 / Chapter 2.6 --- Glycerol determination --- p.42 / Chapter 2.7 --- Comparison of proteomes of BBR-treated and control 3T3-L1 adipocytes..… --- p.43 / Chapter 2.7.1 --- Protein sample preparation from 3T3-L1 cells --- p.43 / Chapter 2.7.2 --- Protein quantitation --- p.43 / Chapter 2.7.3 --- 2D Gel electrophoresis --- p.44 / Chapter 2.7.4 --- Image analysis --- p.44 / Chapter 2.7.5 --- In-gel digestion and MALDI-ToF MS --- p.44 / Chapter 2.8 --- Western Immunoblotting --- p.44 / Chapter 2.8.1 --- Protein sample preparation of BBR-treated and control 3T3-L1 --- p.44 / Chapter 2.8.2 --- SDS-PAGE --- p.44 / Chapter 2.8.3 --- Protein blotting --- p.45 / Chapter 2.8.4 --- Membrane blocking and antibody incubations --- p.45 / Chapter 2.8.5 --- Detection of Proteins --- p.46 / Chapter 2.9 --- Statistical analysis --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Comparison of total protein profiles of visceral adipose tissue of obese db/db and lean m+/db mice --- p.47 / Chapter 3.2 --- Effect of berberine on glucose metabolism of obese db/db mice --- p.53 / Chapter 3.3 --- Comparison of the protein profiles of visceral adipose tissue of BBR-treated and control db/db mice --- p.55 / Chapter 3.4 --- Effect of berberine treatment on 3T3-L1 adipocytes --- p.61 / Chapter 3.4.1 --- Berberine treatment inhibited intracellular triglyceride accumulation in both mature and pre-mature 3T3-L1 adipocytes --- p.61 / Chapter 3.4.2 --- Berberine treatment enhanced lipolysis in mature 3T3-L1 adipocytes but inhibited lipolysis in pre-mature 3T3-L1 adipocytes --- p.65 / Chapter 3.4.3 --- Color change in culture media after berberine treatment --- p.65 / Chapter 3.4.4. --- Comparison of protein profiles between berberine-treated and control 3T3-L1 adipocytes --- p.67 / Chapter 3.4.5 --- Western blotting --- p.73 / Chapter 4. --- Discussion --- p.75 / Chapter 4.1 --- Comparison of total protein profiles of visceral adipose tissue of obese db/db and lean m+/db mice --- p.75 / Chapter 4.2 --- "Berberine lowers body weight, reduces fasting blood glucose level and improves glucose-lowering ability of db/db mice" --- p.78 / Chapter 4.3 --- Comparison of the protein profiles of visceral adipose tissue of BBR-treated and control db/db mice --- p.79 / Chapter 4.4 --- Berberine inhibited lipid accumulation in mature and pre-mature 3T3-L1 adipocytes --- p.84 / Chapter 4.5 --- Berberine enhanced lipolysis in mature 3T3-L1 adipocytes but inhibited lipolysis in pre-mature 3T3-L1 adipocytes --- p.84 / Chapter 4.6 --- Comparison of the protein profiles of BBR-treated and control 3T3-L1 adipocytes --- p.85 / Chapter 4.7 --- Western blotting --- p.88 / Chapter 4.8 --- General discussion --- p.89 / Chapter 5. --- References --- p.92

A phytochemical survey to detect alkaloids was performed on extracts of 339 discrete plants parts from a total of 77 species from five genera of Elaeocarpaceae, including 30 species from Queensland, 38 from PNG, and nine from China. An alkaloid detecting reagent, bismuth (III) tetraiodide (Dragendorff's reagent) was used in a preliminary test for alkaloids, with positive ESIMS used to confirm the presence of alkaloids. A total of 35 extracts of various plant parts produced positive results with Dragendorff's reagent. Positive ESIMS detected alkaloids in only 13 of these extracts. Bismuth (III) tetraiodide was demonstrated to produce false positive results with the new non-alkaloidal poly-oxygenated compounds 112 and 113, which were purified from the extract of Sloanea tieghemii. Two new alkaloid producing species, Elaeocarpus habbeniensis, and E. fuscoides were detected from the survey. These species were chemically investigated for the first time. Two other previously investigated species, E. grandis and Peripentadenia mearsii, were also studied. A total of 16 alkaloids, 11 of which are new, were purified from the extracts of these four species. The novel pyrrolidine alkaloids habbenine (114) and peripentonine (123), were isolated from the leaves of E. habbeniensis and Peripentadenia mearsii, respectively. Both of these compounds were purified as inseparable mixtures of diastereomers. The new pyrrolidine alkaloid mearsamine 1 (124), and the novel amino alkaloid mearsamine 2 (125), were also purified from the leaves of P. mearsii. The known pyrrolidine alkaloid peripentadenine (81), was purified from the bark of P. mearsii. Peripentonine (123) was reduced to peripentadenine (81) upon reaction with Pd/C. Four aromatic indolizidine alkaloids were isolated from the extract of the leaves of E. fuscoides. One new compound, elaeocarpenine (122), was isolated from this New Guinean plant. Three known Elaeocarpus alkaloids, isoelaeocarpicine (62), elaeocarpine (60) and isoelaeocarpine (61) were also purified from E. fuscoides. Elaeocarpenine (122) was demonstrated to produce the epimeric compounds elaeocarpine and isoelaeocarpine via reaction with ammonia. The chemical investigation of the Queensland plant E. grandis by two separate purification procedures was performed. An SCX/C18 isolation protocol was used to purify the new indolizidine alkaloids grandisine C (127), D (126), and E (128), in conjunction with the known tetracyclic indolizidine isoelaeocarpiline (63). The second purification of E. grandis was achieved with the use of ammonia in an acid/base partitioning protocol. Grandisine F (129) and G (130), and compounds 131a and b were purified by this procedure, as were 63, 126 and 127. Grandisine F and G were proposed to be ammonia adducts of grandisine D (126). Compound 131a and b were isolated as a mixture of diastereomers. The reduction of grandisine D (126) with Pd/C yielded a mixture of isoelaeocarpine (61) and elaeocarpine (60), whereas the reduction of isoelaeocarpiline (63) produced isoelaeocarpine (61). All of the alkaloids isolated from the Elaeocarpaceae, except grandisine E (128) and 131a and b, were evaluated for binding affinity against the human ? opioid receptor. Every compound except mearsamine 2 (125) possessed a binding affinity of less than 100 ?M. The most active compounds were grandisine F (129), D (126), C (127), elaeocarpenine (122), isoelaeocarpine (61), isoelaeocarpiline (63) and peripentadenine (81). The IC50 values for these compounds were 1.55, 1.65, 14.6, 2.74, 13.6, 9.86 and 11.4 ?M, respectively. The SAR of the active compounds was compared. These observations indicated that the indolizidine alkaloids were more active than the pyrrolidine alkaloids, and a phenol or ketone at position C-12 of the indolizidine alkaloids produced better binding affinity. All of these alkaloids, except 129, were proposed to interact with two of the three binding domains of the ? opioid receptor. Grandisine F (129) was proposed to have a different mode of action than the other alkaloids in the series. Synthetic modifications to isoelaeocarpine (61) and peripentadenine (81) were investigated in an attempt to incorporate an extra aromatic group into these molecules. An extra aromatic group was proposed to provide increased binding affinity to the ? opioid receptor by interaction with the third binding domain of the receptor. Two different aromatic amines were successfully attached to peripentadenine (81) by a reductive amination reaction using NaBH(OAc)3 and a titanium catalyst. The reductive amination of the ketone in isoelaecarpine (61) with various amines and NaBH(OAc)3 or NaBH4 proved unsuccessful.

Part I: Pyrrolidine derivatives were attached a methoxy group on a-C position of pyrrolidine-ring via anodic oxidation. Followed with alkylation and series of transformation under Lewis acids to obtain tropane alkaloids. Part II: Application of intramolecular radical cyclization toward the synthesis of aporphine alkaloids. Tributyltinhydride and AIBN were used to generate aryl radicals. Trapping of aryl radicals with unsaturated alkenes led to products.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Soňa Knápková Supervisor: PharmDr. Daniela Hulcová, Ph.D. Title of diploma thesis: Amaryllidaceae alkaloids as model structures for the development of new potential drugs The family Amaryllidaceae includes a large number of genus of flowering plants as Hippeastrum, Narcissus or Zephyrantes. All of them contain specific Amaryllidaceae alkaloids, which are characteristic of this family due to their chemical structures. The discovery and study of these alkaloids has attracted the attention of many scientists due to the diverse biological activity of these compounds, such as cytotoxic, anticholinesterases, antibacterial and antiviral effects. Plants of the genus Hippeastrum have been used in traditional medicine to treat tumors and inflammatory disorders. This use can be explained by the alkaloids, which it contains. It is mainly lycorine, haemanthamine and pancristatine. These compounds have an antitumor effect. The species Hippeastrum cv. Ferrari is further rich in the alkaloid vittatine. In some research, the simple semisynthetic derivatives of haemanthamine displayed promising inhibitory activities against cholinesterases. For this reason, vittatine was chosen as next lead-structure, for preparation of...

The studies and preliminary screening results reported in this paper developed from an initial interest in what appeared to be mildly toxic qualities of the bulbs of the common daffodil, Narcissus pseudonarcissus, Amaryllidaceae. The literature consulted does not shed much light on the mechanisms of the toxic agent or agents, and there appear to be some inconsistencies in respect to the alkaloids extracted. Part of this may be the result of differences in method of extraction, or perhaps lack of agreement on the identity of the actual plant bulbs used as a source of alkaloids. In regard to the chemical isolation of the alkaloids subsequently studied, the closest attention to the details described in several publications did not produce the results described.

This item was digitized as part of the Million Books Project led by Carnegie Mellon University and supported by grants from the National Science Foundation (NSF). Cornell University coordinated the participation of land-grant and agricultural libraries in providing historical agricultural information for the digitization project; the University of Arizona Libraries, the College of Agriculture and Life Sciences, and the Office of Arid Lands Studies collaborated in the selection and provision of material for the digitization project.

The work in this thesis describes investigations into the development of a tandem radicallDiels-Alder reaction strategy for the construction of indole alkaloids. The synthesis of a key precursor, 2-[(4-methylphenyl)sulfonyl]indole, was investigated and involved two main· strategies: functionalisation of the indole ring using lithiation procedures and assembling the indole as a key step (chapter 2). A method based on the former strategy was deemed to be the most practical and delivered the desired product in moderate yields (35 - 45%). The viability of the first step of the proposed tandem radicallDiels-Alder strategy was investigated by examining the intramolecular addition of vinyl and aryl radicals to a sulfone substituted indole (chapter 3). These studies showed that the addition of sp2 centred radicals to substituted indoles was feasible (20 - 40%). A study using an acetylenic precursor also provided useful information about the relative rates of desulfonylation and radical addition to acetyleneS; de-sulfonylation is a competing process. An alternative tandem palladiumlDiels-Alder reaction was briefly examined, but was thwarted by capricious palladium catalysed cross coupling reactions. Further studies were carried out to extend the scope of the novel ipso substitution reaction (chapter 4). It was found that alkyl radical cyclisations proceed efficiently to give a new ~ethod for the synthesis of fused[1,2-a]indoles (30 - 84%). The feasibility of a related ipso substitution using phenylthio and phenylsulfinyl substituted indoles was also examined. It was found that these reactions are also successful, but proceed in reduced yields compared with the sulfones (24 - 51 %). These results could be attributed to the more favoured reaction of the electron rich carbon centred radical with the more electrophilic 1t bond. The thesis is concluded by a brief discussion of the mechanism of these cyclisations with the available experimental evidence supporting an addition! elimination pathway.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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