Theses and Dissertations from UMD
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Item Purple Pepper Plants, An Anthocyanin Powerhouse: Extraction, Separation and Characterization(2014) Taylor, Cassandra Lynn; Mignerey, Alie C; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Dissertation: PURPLE PEPPER PLANTS, AN ANTHOCYANIN POWERHOUSE: EXTRACTION, SEPARATION AND CHARACTERIZATION Cassandra Lynn Taylor Doctor of Philosophy, 2014 Dissertation directed by: Professor Alice C. Mignerey Department of Chemistry and Biochemistry Most plants have multiple anthocyanins present that produce their color. In contrast, the foliage of the purple pepper plant (Capsicum annuum L.) contains high concentrations of a single anthocyanin delphinidin-3-p-coumaroylrutinoside-5-glucoside (Dp-3-p-coumrut-5-glc) in the foliage, making it very unique. This provides an excellent platform to extract the single anthocyanin at high concentrations. A food-grade extraction method was developed using 1% hydrochloric acid and 200 proof ethanol (1% HCl/EtOH) in order to remove the intact anthocyanin. A separation method using High Performance Liquid Chromatography (HPLC) was developed to identify Dp-3-p-coumrut-5-glc. The retention time was compared with the Blue Ribbon Iris, a known source of Dp-3-p-coumrut-5-glc. The HPLC results confirmed the presence of Dp-3-p-coumrut-5-glc in the pepper extract, but the chromatograms also demonstrated the presence of additional highly colored compounds. The extract was injected onto the HPLC and the major anthocyanin peak (Dp-3-p-coumrut-5-glc) was collected over the course of multiple injections. The collected fractions were dried down and re-solubilized in 1% HCl/methanol for analysis by mass spectrometry. A HPLC coupled to a photodiode array detector and an electrospray ionization tandem mass spectrometer (LC-PDA-ESI-MS/MS) was utilized to characterize Dp-3-p-coumrut-5-glc. The precursor compound was confirmed at m/z 919 with product ions at m/z 757, 465 and 303 by comparing against plant extracts of freeze-dried purple pepper foliage, Chinese eggplant and Chinese celery. The extract's structure was elucidated by Nuclear Magnetic Resonance (NMR) by analyzing both proton (1H) and carbon (13C) spectra. The 1H and 13C data matched very well with previous NMR data of Dp-3-p-coumrut-5-glc elucidated in eggplant peels. The major difference was that the trans isomer of Dp-3-p-coumrut-5-glc greatly dominated over the cis in the purple pepper extract.Item Trapping Labile Adducts Formed Between an ortho-Quinone Methide and DNA(2012) McCrane, Michael Patrick; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exogenously generated electrophiles are capable of alkylating DNA. If not repaired, the resulting DNA adducts can lead to mutations and either cancer or cell death. Electrophilic ortho-quinone methides (o-QM) are reactive intermediates that alkylate DNA and are generated during xenobiotic metabolism of a variety of compounds including environmental toxins and therapeutic agents. Identifying the full alkylation profile of o-QM towards DNA would allow for the genotoxicity of o-QM precursors to be better understood. From model studies based on nucleosides, o-QMs react most readily, but reversibly with the strong nucleophiles 2'-deoxycytidine (dC) N3, 2'-deoxyguanosine (dG) N7, and 2'-deoxyadenosine (dA) N1 and less efficiently, but irreversibly with the weak nucleophiles dG N1, dG N2, and dA N6. The reverse reactions complicate analysis of their products in DNA, which requires enzymatic digestion and chromatographic separation. Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) can transform the reversible o-QM-DNA adducts into irreversible derivatives capable of surviving such analysis. To facilitate this analysis, a series of oxidized o-QM-dN adducts were synthesized as analytical standards. Initial oxidative trapping studies with an unsubstituted o-QM and dC demonstrated the necessity of an alkyl substituent para to the phenolic oxygen to block over-oxidation. A novel o-QM included a methyl group para to the phenolic oxygen that successfully blocked the over-oxidation allowing for generation of a stable MeQM-dC N3 oxidized product. Further oxidative trapping studies with MeQM and dG resulted in the formation of three stable MeQM-dG oxidized products (guanine N7, dG N1, and dG N2). Initial studies with duplex DNA optimized the enzymatic digestion and confirmed that the assay conditions were compatible with oxidative trapping. The low yielding MeQM alkylation of duplex DNA needs to be scaled up prior to the oxidative trapping studies. Alternative studies quantified the release of MeQM from DNA with the use of 2-mercaptoethanol as a nucleophilic trap. These studies revealed single stranded DNA as a superior carrier of MeQM than duplex DNA and, therefore, a better target DNA for the oxidative trapping studies due to increased yield of MeQM adducts. With the increased MeQM-DNA yield, the intrinsic selectivity and reactivity of MeQM towards DNA can be determined.Item Promotion of Value-Added Uses of Soybeans(2011) Slavin, Margaret; Yu, Liangli; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Soybeans contain a wealth of health-promoting phytochemicals, the amounts of which are known to vary considerably across genotypes and growing conditions. The present work is dedicated to better understand these variations and to improve the methods for investigating soy phytochemicals. To address the first of these concerns, low-linolenic soybeans and colored seed coat soybeans were investigated for their health beneficial components, as well as their chemical and biological properties. A study of low-linolenic soybeans showed their similar antioxidant activity, total phenolics, and isoflavones to normal fatty acid soybeans. The potential for a different trend in tocopherols and lutein was observed between low-linolenic and regular fatty acid soybeans, but the limited study size prevented a definitive conclusion. This work was the basis for further studies on low-linolenic soybeans not included in this report. Subsequently, a study of eighteen soybeans of brown, green, yellow and black seed coat colors concluded that black seed coat soybeans had significantly higher scavenging activity against hydroxyl, peroxyl, and ABTS+ radicals. Black seed coat soybeans also contained higher total phenolic contents and isoflavones than the other colored soybeans, and were the only color to contain the anthocyanin cyanidin-3-glucoside. However, when soybean extracts were tested for their ability to prevent colon cancer cell proliferation, seed coat color and chemical composition were not necessarily predictive of an extracts' bioactivity. Lastly, a new analytical method was developed and validated for quantification of phytosterols, tocopherols and carotenoids, three separate classes of soy lipophilic phytochemicals, in a single extraction and HPLC run. A ternary, isocratic solvent system of acetonitrile, methanol and water (48:22.5:29.5, v/v/v) was used to achieve separation on a phenyl column. Evaporative light scattering detection (ELSD) was used to quantify beta-sitosterol, stigmasterol, campesterol, alpha-, delta- and gamma- tocopherols, while lutein was quantified with visible light absorption at 450 nm. This method offers a more efficient alternative to separate, individual methods for quantifying lutein, tocopherols and sterols in soybeans.Item A Novel Pre-fluorescent Nitroxide Probe for the Highly Sensitive Determination of Peroxyl and Other Radical Oxidants(2009) Jia, Min; Blough, Neil V; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Peroxyl and other radical oxidants react with stable cyclic nitroxides, such as the piperidinyl and pyrrolidinyl nitroxides to form initially the one electron oxidation product, the oxoammonium cation. For most of the nitroxides studied thus far, the oxoammonium cation can in part be regenerated to the nitroxide through reduction by solution constituents. The reaction mechanisms, however, remain a matter of debate. Further, the highly-sensitive, quantitative determination of peroxyl and other radical oxidants has yet to be achieved, posing a major hurdle to a further understanding of the impact of peroxyl radicals in many biological and environmental processes. A unique, amino-pyrrolidinyl nitroxide, 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) is shown to undergo an irreversible reaction with peroxyl radicals and other radical oxidants to generate a diamagnetic product. When a fluorophore, fluorescamine is covalently linked through the amino group on the nitroxide, the resulting compound (3-apf, or I) has very low fluorescence quantum yield. Upon reaction with peroxyl and other radical oxidants, the quantum yield of the product increases dramatically (~100 fold), and thus 3-ap or 3-apf can be used as a highly sensitive and versatile probe to determine oxidant production optically, either by monitoring the changes in fluorescence intensity using a spectrofluorometer, by HPLC analysis with fluorescence detection, or by a combination of both approaches. By changing the [O2]/[nitroxide] ratio, it is shown that peroxyl radicals can be detected and quantified preferentially in the presence of other radical oxidants, such as *NO2 and CO3*-. When decreasing the [O2]/[nitroxide] ratio, the oxidation product decreases, with a concomitant increase of the alkoxylamine product resulting from reaction of 3-ap (3-apf) with carbon centered radicals. Preliminary studies suggest that the reactions of 3-ap and 3-apf with peroxyl radical produce different final products. High resolution mass spectrometry and NMR studies indicate that 3-ap is oxidized to form a cyclic peroxide structure, while 3-apf is oxidized to form a cyclic -NH-O- structure, with this difference resulting possibly from the presence of the fluorescamine moiety in 3-apf. Detection of photochemically produced peroxyl radicals is achieved by employing 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) alone, followed by derivatization with fluorescamine, while detection of thermally-generated peroxyl radicals employs 3-apf. Preliminary applications include the detection of peroxyl radicals generated thermally in soybean phosphatidylcholine liposomes by 3-apf and produced photochemically in tap water by 3-ap.Item Total Synthesis of (3R,3'R,6'R)-Lutein, (3R,3'R)-Zeaxanthin and Their Stereoisomers(2008) Chang, An-Ni; Khachik, Frederick; DeShong, Philip; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)(3R,3'R,6'R)-Lutein (1) and (3R,3'R)-zeaxanthin (5) are dietary carotenoids that are found in most fruits and vegetables. Numerous studies have shown that 1 and 5 play an important role in the prevention of age-related macular degeneration (AMD) that is the leading cause of blindness. To date, the metabolic pathways of 1 and 5 in ocular tissues of an animal model in relation to AMD have not been explored. This is primarily because of the lack of a viable method for the synthesis of 1 and 5 that can be labeled with a stable isotope. Among the eight possible stereoisomers of lutein, only 1 has been previously prepared by total synthesis in 14 steps in an overall yield of 0.5%. The total synthesis of lutein, zeaxanthin, and their stereoisomers from (rac)-alpha-ionone has been accomplished by a C15+C10+C15 coupling strategy. Therefore, (3R,3'R,6'R)-lutein (1, 8%), (3R,3'S,6'S)-lutein (2, 7%), (3R,3'S,6'R)-lutein (3, 6%), and (3R,3'R,6'S)-lutein (4, 7%) were each prepared in a high optical purity in 7 steps. 3-Hydroxy-alpha-ionylideneacetaldehyde served as a common precursor to afford luteins 1 - 4 by a much shorter synthetic sequence and a higher overall yield than that of a published method for 1. The other four stereoisomers of lutein can be similarly prepared. (3R,3'R)-Zeaxanthin (5, 12%) and (3S,3'S)-zeaxanthin (6, 11%) were prepared in 8 steps from (rac)-alpha-ionone via 3-hydroxy-alpha-ionone which was transformed into 3-hydroxy-beta-ionone (3R-42, 22%) and its enantiomer (3S-42, 21%), respectively. The key intermediates, 3R-42 and 3S-42 were converted into the corresponding C15-Wittig salts 3R-16 and 3S-16 that were used in a double Wittig reaction with the C10-dialdehyde 17 to afford 5 (98% ee) and 6 (98% ee). Utilizing Wittig salts 3R-16 and 3S-16, (3R)-beta-cryptoxanthin (135, 8%) and (3S)-beta-cryptoxanthin (136, 9%) were each prepared in optical purity of 98%. The most important feature of the strategies presented here is its application to the total synthesis of isotopically labeled and optically pure lutein, zeaxanthin, and their stereoisomers for metabolic studies. This synthesis also provides access to the C15-precursors of optically active carotenoids with 3-hydroxy-alpha- and 3-hydroxy-beta-end groups that are otherwise difficult to synthesize.Item Chronic Ingestion of (3R,3'R,6'R)-Lutein and (3R,3'R)-Zeaxanthin in Female Rhesus Macaque Primates(2006-05-08) London, Edra; Khachik, Frederick; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries in individuals over the age of 65. High intake of the dietary carotenoids lutein (L) and zeaxanthin (Z) is believed to reduce the risk of AMD. This study investigated the effects of long-term supplementation of primates with high doses of L or Z, and their 1:1 combination, and whether high supplemental doses cause ocular toxicity. Eighteen female rhesus macaques were divided into 4 groups: control (n=3), L-treated (n=5, 9.34 mg/kg L and 0.66 mg/kg Z), Z-treated (n=5, 10 mg/kg Z), and L/Z-treated (n=5, L and Z each at 0.5 mg/kg). At 6 month intervals beginning at baseline, plasma samples were analyzed by HPLC for L, Z, and their metabolites. Carotenoid analysis of tissues, ocular examinations, and toxicity assays were performed. High-dose supplementation of primates with L or Z significantly increased plasma, and ocular and other tissue concentrations of these carotenoids and their metabolites in most cases. Supplementation with a 1:1 dose of L and Z increased plasma concentrations of these carotenoids after 6 months, but baseline and month 12 levels in plasma and ocular tissues were not significantly different. Supplementation of primates with L or Z at high doses does not cause ocular or kidney toxicity.