The isoflavones in kudzu roots, especially the C-glycosylated isoflavone puerarin, have been linked to many health benefits. Puerarin contains a carbon-carbon glycosidic bond that can withstand hydrolysis. The C-glycosylation reaction in the biosynthesis of puerarin has not been thoroughly investigated, with conflicting reports suggesting that it could take place on daidzein, isoliquiritigenin, or 2,7,4ʹ-trihydroxyisoflavanone. Kudzu species were identified for use in comparative transcriptomics. A non-puerarin producing kudzu was identified as Pueraria phaseoloides and a puerarin producing kudzu was identified as Pueraria montana lobata. Through the use of the plant secondary product glycosyltransferase (PSPG) motif, glycosyltransferases (UGTs) were identified from the transcriptomes. The UGTs that had higher digital expression in P. m. lobata were examined further using additional tools to home in on the UGT that could be responsible for puerarin biosynthesis. One of the UGTs identified, UGT71T5, had previously been characterized from kudzu as a C-glycosyltransferase involved in puerarin biosynthesis through in vitro enzyme activity (with daidzein) and a gain of function approach in soybean hairy roots. Previous studies have not supported the end-product of a pathway such as daidzein as the target for C-glycosylation, and no genetic analysis of UGT function had been conducted in kudzu. The activity of recombinant UGT71T5 with daidzein was confirmed in the present work. Following the development of a kudzu hairy root system, UGT71T5 expression was then knocked down by RNA interference (RNAi). When compared to control hairy roots there was a large reduction in puerarin content in the UGT71T5-RNAi roots, confirming the role of this enzyme in puerarin biosynthesis. Isotopic labeling of kudzu plants revealed that labeled daidzein could be directly incorporated into puerarin; however, the percent incorporation of daidzein was substantially lower than that of L-phenylalanine, a compound at the start of the pathway to isoflavone synthesis. The knockdown of 2-hydroxisoflavanone synthase (2-HIS) in kudzu hairy roots blocked formation of puerarin and daidzin (7-O-glycosyldaidzein), and was accompanied by accumulation of C-glycosylated isoliquiritigenin and C-glycosylated liquiritigenin. These compounds were found in low amounts in control hairy roots, but were virtually absent in UGT71T5 knockdown hairy roots. The knockdown of 2-hydroxyisoflavanone dehydratase (2-HID) in kudzu hairy roots resulted in a slight reduction in puerarin but no change to daidzin levels, suggesting that C-glycosylation might stabilize the substrate for 2-HID which can otherwise spontaneously dehydrate. Taken together these results reveal that UGT71T5 is likely the major C-glycosyltransferase involved in puerarin biosynthesis in kudzu. They also provide evidence for an alternative pathway to puerarin biosynthesis through the C-glycosylation of isoliquiritigenin or its immediate precursor. In one pathway, UGT71T5 acts as an operationally soluble enzyme that can directly C-glycosylate daidzein, and in the other pathway UGT71T5 acts as part of a metabolic channel for conversion of a C-glycosylated earlier precursor to puerarin. Other UGT enzymes identified in this work did not show C-glycosyltransferase activity; however, three enzymes showed activity in vitro that could be useful for introducing novel regiospecificity in biochemical synthesis of flavonoid glycosides.

In response to increasing interest in greenhouse production and difficulties imposed by adverse environmental conditions in the southeastern United States, two studies were conducted with green and red-leaf lettuce cultivars. The first study analyzed effects on yield and quality of green and red-leaf lettuce subjected to increasing nutrient solution electroconductivity (EC) across three growing seasons. Results indicated that the interaction between season and cultivar had the greatest effect on growth, flavonoids and phenolics, and leaf mineral content. The second study analyzed effects on yield and quality of increasing solution potassium (K) in red Lollo lettuce at two temperatures. The results suggested that temperature is a stronger regulatory factor than increasing K in the determination of lettuce yield and quality. However, increasing K concentrations to 234.6 mg·L-1 results in higher concentrations of leaf mineral concentrations without compromising lettuce yield and quality.

No description available.

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<p> This thesis describes the isolation and structural determination of flavonoids from the heart wood of Prunus avium as well as synthesis of polyhydroxy pyrrolidine derivatives and aniline core structures as potential antibacterial agents. Nitrogen-synthons containing saturated heterocyclic systems and aniline core structures are important synthons in organic chemistry because of their presence in many biologically-active natural products. Mycobacterial viability is dependent upon the ability of the organism to produce an intact cell wall. Therefore, compounds that interfere with the biosynthesis of the cell wall complex glycons have the potential to become new drugs for the treatment of mycobacterial infections. The oligosaccharide galactan is one of the major structural components of the outer wall of the micro-organism. Galactofuranose is essential for cell growth and survival and therefore, its biosynthesis constitutes a new drug target. The biosynthetic process involves several enzymes having Uridine-diphosphogalacto furanose (UDP-Galf) as the substrate; uridine 5^1 -diphosphogalacto pyranose mutase which catalyzes the interconversion of UDP galacatopyranose to UDP-galactofuranose as well as Galf-transferase. We are seeking and designing molecules that could be mechanistic probes and/or inhibitors of efflux pumps to potentially combat multidrug resistance.</p> <p> The isolation and structure-determination of six naturally occurring Flavan-type Natural products was performed. Such derivatives are known to reverse multiple-drug-resistance (MDR) in persistent microbial infections. The synthesis of pyrrolidine-based antibacterial agents was attempted using two different approaches from tartaric acid. These derivatives were designed as potential transition-state mimics of a carbohydrate processing enzyme specific to TB. A synthetic approach to the aromatic core structure of the antibacterial agent Platensimycin was also investigated. The synergistic use of cytotoxic agents in conjunction with efflux-pump modulators is an emerging area of research in the MDR field; our efforts to make available materials for high-throughput screening in this area will be described.</p> / Thesis / Master of Science (MSc)

Islet amyloid polypeptide (IAPP) is a 37-residue amyloidogenic hormone implicated in the progression of Type II Diabetes (T2D). T2D affects an estimated 422 million people yearly and is a co-morbidity with numerous diseases. IAPP forms toxic oligomers and amyloid fibrils that reduce pancreatic β-cell mass and exacerbate the T2D disease state. Toxic oligomer formation is attributed, in part, to the formation of inter-peptide β-strands comprised of residues 23-27 (FGAIL). Flavonoids, a class of polyphenolic natural products, have been found experimentally to inhibit IAPP aggregate formation. Many of these known IAPP aggregation attenuating small flavonoids differ structurally only slightly; the influence of functional group placement on inhibiting the aggregation of the IAPP(20-29) has yet to be explored. To probe the role of small-molecule structural features that impede IAPP aggregation, molecular dynamics (MD) simulations were performed on a model fragment of IAPP(20-29) in the presence of morin, quercetin, dihydroquercetin, epicatechin, and myricetin. Contacts between Phe23 residues are critical to oligomer formation, and small-molecule contacts with Phe23 are a key predictor of β-strand reduction. Structural properties influencing the ability of compounds to disrupt Phe23-Phe23 contacts include carbonyl and hydroxyl group placement. These structural features influence aromaticity and hydrophobicity, principally affecting ability to disrupt IAPP(20-29) oligomer formation. This work provides key information on design considerations for T2D therapeutics. / Master of Science in Life Sciences / Type II Diabetes (T2D) affects an estimated 422 million people worldwide, with the World Health Organization (WHO) reporting that approximately 1.5 million deaths were directly caused by T2D in 2019. The progression of T2D has been attributed to a protein, called islet amyloid polypeptide (IAPP, or amylin) that is co-secreted with insulin after individuals eat or consumes calories. IAPP has been discovered to form toxic aggregates or clumps of protein material that worsen the disease state and cause a loss of mass of pancreatic cells. There is a large market for therapeutics of T2D and more small molecule drugs are needed to slow progression and severity of T2D. Flavonoids, a class of natural molecules, have been found to inhibit the processes by which IAPP promotes T2D disease progression by stopping the aggregation of IAPP. The structures of these flavonoid compounds differ slightly but show difference in ability to slow IAPP aggregation. By understanding how those differences confer more or less protection against T2D and inhibit IAPP aggregation, we can design more potent and specific drugs to target IAPP. To probe the role of molecular structure in preventing IAPP aggregation, molecular dynamics (MD) simulations — a powerful computational technique — were performed on a model fragment of IAPP in the presence of molecules morin, quercetin, dihydroquercetin, epicatechin, and myricetin. MD simulations provide extremely detailed information about potential drug interactions with a given target, serving as an important tool in the development of new drugs. This work has identified key features and predictors of effective IAPP drugs, providing a framework for the further development of therapeutics against T2D and similar diseases.

Natural products are teeming with potential therapeutic agents. One group of compounds, polyphenols, from plants, exhibit anti-cancer, anti-inflammatory and anti-oxidant activities. Four polyphenolic compounds and their enantiomers were investigated in this thesis; the stilbene, 3-methoxypterostilbene, a structural analog of resveratrol, and the chiral prenylflavonoids from hops (Humulus lupulus L.); 8-prenylnaringenin, 6-prenylnaringenin and isoxanthohumol. A high performance liquid chromatography method for 3-methoxypterostilbene and enantiospecific liquid chromatography-mass spectrometry assays for the three prenylflavonoids were developed and validated. The methods allowed for quantification of these four polyphenols in biological samples and plant-based materials. Content analysis studies of 3-methoxypterostilbene and the three prenylflavonoids in traditional Chinese medicinal plants and hops-containing nutraceuticals were carried out, respectively. The pharmacokinetics of these four compounds were delineated through intravenous and oral administration in rats. 3-Methoxypterostilbene demonstrated greater bioavailability in rats than reported values for resveratrol. Enantiomeric differences in disposition parameters were observed for the three prenylflavonoids along with differences between compounds despite only small structural differences. The in vitro pharmacodynamics of these four compounds were elucidated including anti-oxidant, anti-inflammatory, anti-diabetic and cytochrome P450 modulation activities. All four compounds demonstrated a range of bioactivities related to chronic diseases and potential drug-botanical interactions. Further studies of polyphenols, especially clinical studies, are needed along with enantiospecific study when applicable to continue delineating the importance of bioactivity, pharmacokinetics and safety. Natural products are further developed into nutraceuticals and sold over-the-counter for both human and veterinary use but are not currently required to demonstrate efficacy prior to marketing. In the final section of this thesis, Phycox®, a multi-component veterinary nutraceutical for joint health was investigated for pharmacological activity in an in vitro model of canine osteoarthritis along with select constituents. A pilot single-dose pharmacokinetic study in dogs was also undertaken. Two liquid chromatography tandem mass spectrometry methods were developed and validated to detect constituents in serum. In vitro study results indicated that Phycox® was able to reduce inflammatory mediators similar to the NSAID, carprofen, and acute pharmacokinetic results revealed that detectable concentrations of glucosamine were evident in serum. It is suggested that further clinical studies of Phycox® are warranted to optimize its usage. / May 2016

Po Lai See. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 142-152). / Abstracts in English and Chinese. / TITLE PAGE --- p.p.1 / ACKNOWLEGDEMENTS --- p.p.2 / ABSTRACT --- p.p.3 / 摘要 --- p.p.6 / TABLE OF CONTENTS --- p.p.9 / LIST OF FIGURES AND TABLES --- p.p.16 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter 1.1 --- Estrogen and Estrogen Receptors and its Action --- p.p.18 / Chapter 1.1.1 --- Estrogen --- p.p.19 / Chapter 1.1.2 --- Estrogen Receptors --- p.p.19 / Chapter 1.1.3 --- Structural Differences between ERa and ERp --- p.p.21 / Chapter 1.1.4 --- Functional Differences --- p.p.22 / Chapter 1.1.5 --- Effects of Selective Estrogen Receptor Modulators --- p.p.22 / Chapter 1.1.6 --- Estrogen works --- p.p.23 / Chapter 1.1.7 --- Estrogen Receptors and Breast Cancer --- p.p.24 / Chapter 1.2 --- Flavonoids： Properties and Biological Activities --- p.p.25 / Chapter 1.2.1 --- Chemical Structure and Classification of flavonoids --- p.p.25 / Chapter 1.2.2 --- Biological Properties and Action Mechanism of Flavonoids… --- p.p.27 / Chapter 1.2.3 --- Flavonoids and breast cancer prevention --- p.p.27 / Chapter 1.3 --- Aims and Scopes of Investigation --- p.p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.p.30 / Chapter 2.1.1 --- Flavonoids --- p.p.30 / Chapter 2.1.2 --- Plasmids --- p.p.30 / Chapter 2.2 --- Mammalian cell culture --- p.p.31 / Chapter 2.2.1 --- Maintenance of cells --- p.p.31 / Chapter 2.2.2 --- Preparation of cell stock --- p.p.32 / Chapter 2.2.3 --- Cell recovery from liquid nitrogen stock --- p.p.32 / Chapter 2.3 --- Identification of estrogenic activity in flavonoids --- p.p.33 / Chapter 2.3.1 --- Steady Glo Luciferase Assay --- p.p.33 / Chapter 2.3.2 --- The Biorad Protein Assay kit (a modified Bradford method). --- p.p.33 / Chapter 2.4 --- Viability Assay --- p.p.34 / Chapter 2.5 --- ERE Luciferase reporter gene assay --- p.p.35 / Chapter 2.5.1 --- Transient transfect ion of cell using lipofectamine PLUS reagent --- p.p.36 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.p.37 / Chapter 2.6 --- ERα competitive binding ASSAY --- p.p.37 / Chapter 2.7 --- Apoptotic death assay --- p.p.38 / Chapter 2.8 --- Semi-quantitative RT-PCR Assay --- p.p.40 / Chapter 2.8.1 --- "Isolation of RNA using TRIzol® Reagent (Life Technology,USA) " --- p.p.40 / Chapter 2.8.2 --- Quantitation of RNA --- p.p.41 / Chapter 2.8.3 --- First strand cDNA synthesis --- p.p.41 / Chapter 2.8.4 --- PCR reactions --- p.p.43 / Chapter 2.9 --- Flow Cytometry Analysis --- p.p.43 / Chapter 2.10 --- Total triglyceride and cholesterol measurement --- p.p.44 / Chapter 2.10.1 --- Determination of the total cholesterol --- p.p.45 / Chapter 2.10.2 --- Determination of the total triglyceride --- p.p.46 / Chapter 2.11 --- Manipulation of DNA and RNA --- p.p.46 / Chapter 2.11.1 --- Transformation of DH5α --- p.p.46 / Chapter 2.11.2 --- Mini preparation of plasmid DNA --- p.p.47 / Chapter 2.11.3 --- Preparation of plasmid DNA using QIAGEN-tip 100 midi-prep kit --- p.p.48 / Chapter 2.11.4 --- Preparation of plasmid DNA using QIAGEN-tip 10000 Giga-prep kit --- p.p.49 / Chapter 2.11.5 --- Ethanol preparation of DNA and RNA --- p.p.50 / Chapter 2.11.6 --- Agarose gel electrophoresis of DNA --- p.p.51 / Chapter 2.12 --- Statistical methods --- p.p.52 / Chapter CHAPTER 3 --- Estrogenic and antiproliferative activities on MCF-7 breast cancer cells by flavonoids / Chapter 3.1 --- Introduction --- p.p.53 / Chapter 3.2 --- Results --- p.p.56 / Screening of phytoestrogens for estrogenic activities on MELN cells --- p.p.56 / Cell proliferation activity of phytoestrogens on MCF-7 and MDA-MA231 cells --- p.p.59 / Estrogenic and antiestrogenic activity of phytoestrogens on ERα or erβ transfected hepg2 cells --- p.p.64 / Chapter 3.3 --- Discussion --- p.p.73 / Chapter Chapter 4 --- interaction of baicalein with estrogen receptors / Chapter 4.1 --- Introduction --- p.p.76 / Chapter 4.2 --- Results --- p.p.78 / Estrogen receptor competition assay --- p.p.78 / ERE-Luciferase gene reporter assay --- p.p.82 / Chapter 4.3 --- Discussion --- p.p.88 / Chapter Chapter 5 --- baicalein and genistein display differential actions on er transactivation / Chapter 5.1 --- Introduction --- p.p.90 / Chapter 5.2 --- Results --- p.p.92 / Estrogenic and antiestrogenic activities of genistein and baicalein on ER transactivation --- p.p.92 / Chapter 5.3 --- Discussion --- p.p.105 / Chapter CHAPTER 6 --- APOPTOTIC EFFECTS OF BAICALEIN ON MCF-7 AND MDA-MB-231 CELL LINES / Chapter 6.1 --- Introduction --- p.p.107 / Chapter 6.2 --- Results --- p.p.111 / ER POSITIVE MCF-7 AND ER NEGATIVE MDA-MB-231 cell death assay --- p.p.111 / "Bcl-2, Bax and PS2 mRNA expression " --- p.p.116 / Arrest at sub G1 phase of MCF-7 by baicalein --- p.p.124 / Chapter 6.3 --- Discussion --- p.p.127 / Chapter CHAPTER 7 --- BAICALEIN CAN REDUCE INTRACELLULAR cholesterol and triglceride / Chapter 5.1 --- Introduction --- p.p.129 / Chapter 5.2 --- Results --- p.p.130 / Baicalein has beneficial effect on lipid metabolism --- p.p.130 / Chapter 5.3 --- Discussion --- p.p.139 / Chapter chapter 8 --- Summary --- p.p.140 / BIBLIOGRAPHY --- p.p.142 / APPENDIX 1 ABBREVIATIONS --- p.p.153 / APPENDIX 2 PRIMER LISTS --- p.p.156 / APPENDIX 3 REAGENTS AND BUFFERS --- p.p.157

Xie, Xinhui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 163-193). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ces dernières années, des progrès remarquables ont été accomplis afin d’élucider la biosynthèse des flavonoïdes. Cependant les dernières étapes menant à la formation des proanthocyanidines ou tannins condensés issus de la vigne, restent à ce jour inconnues. Dans le but de déterminer si une ou plusieurs enzyme(s) spécifique(s) sont impliquées dans cette voie de biosynthèse, nous avons développé une approche de protéomique chimique, impliquant des matrices d’affinité constituées de substrats de type flavanols greffés sur un support solide. La validation de ces outils à l’aide de LDOX, une enzyme issue de Vitis vinifera a pu être menée à bien dans le cadre de ces travaux de thèse. / Remarkable progress toward the complete elucidation of the biosynthesis of flavonoids has been accomplished during the last decade, but the final step leading to proanthocyanidins still remain to be elucidated, in particular, the exact nature of starter and extension units as well as the enzymatic or non enzymatic condensation process. In order to answer whether some specific enzymes are involved in the biosynthesis of grapevine proanthocyanidins, we have developped a chemical proteomics approach, with an affinity chromatography-based tool in which a flavanol type substrate is loaded on an appropriate solid support. The validation of these tools with the LDOX enzyme from Vitis vinifera was developped and performed in this Ph.D work.