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The possibility of acetone shock loadings to phenol acclimated systems resulting in sequential substrate utilization and increased effluent phenol concentrations was evaluated. Phenol acclimated batch and continuous-flow systems, developed with seed from a municipal wastewater treatment plant, were shock loaded with acetone, bacto-peptone, and domestic primary effluent. Phenol and acetone utilization rates were then monitored using direct injection gas-liquid chromatography. The results of the investigation indicated that, under the described experimental conditions, qualitative shock loading of phenol acclimated/utilizing cultures had no significant effect on effluent phenol concentrations. Variations of system pH, however, were found to have extreme effects. / Master of Science

Using a model system of glycosylated and aglycon standards consisting of rutin and quercetin respectively, and a series of pre-packed solid phase extraction cartridges, including C18 Extra-Clean, DSC-18, DPA-6S, Oasis HLB and Amberlite XAD-2. The experimental findings also showed that use of a commercial hesperinidase preparation, resulted in adequate hydrolysis of the glycosylated standard rutin. Based on these findings, the phenolic compounds and flavonoids from maple sap and syrup were separated using the Amberlite XAD-2 column, where the glycosylated fractions eluted with 60% aqueous methanol solution and the aglycon fractions eluted with a methanol:acetonitrile mixture (1:1, v/v). The recovered glycosylated fractions were subjected to enzymatic hydrolysis using the hesperinidase preparation and the liberated phenolic compounds and flavonoids, as well as the sugar components were analyzed by high-performance liquid chromatography (HPLC).

Comparative high-performance liquid chromatography (HPLC) and gas-liquid chromatography (GC) analyses of selected phenolic and flavonoid standards were developed using a wide range of detectors, including ultraviolet diode-array (UV-DAD) and electrochemical (EC) detectors for HPLC and flame ionization detector (FID) and mass spectrometry (MS) for GC. The results demonstrated that the limits of detection obtained with HPLC-EC analysis were 10 to 500-times higher for phenolic acid standards and 2 to 50-times higher for flavonoid standards than those obtained with the HPLC-UV analysis. HPLC-EC was more sensitive than GC/FID for all investigated standards, especially for vanillin and syringaldehyde. The results indicated that GC/FID/MS analysis of phenolic and flavonoid standards was more efficient than that of HPLC, providing a fast analysis with better resolution and baseline separation of all standards with minimum co-elution. The only co-elution encountered in GC/FID was with coniferol and p-coumaric acids. For HPLC analysis, (-)-epicatechin, caffeic and homovanillic acids were co-eluted at 28.04 min and sinapic and ferulic acids at 34.57 min. Phenolic compounds and flavonoids were extracted from maple sap and maple syrup with ethyl acetate and the recovered compounds were subjected to HPLC and GC analyses. Tentative identification of phenolic compounds and flavonoids in maple sap and maple syrup indicated the presence of protocatechuic acid, hydroxycinnamic acid derivatives, (+)-catechin, (-)-epicatechin, vanillin, coniferol, syringaldehyde, flavanols and dihydroflavonols related compounds. In addition, the identification by GC/MS of protocatechuic acid, vanillin, syringaldehyde, coniferol and p-coumaric acid was made by comparing mass spectrum characteristics of individual peak from total ion chromatogram (TIC) to that of standard compounds. The seasonal variation of selected phenolic compounds and flavonoids present in maple sap and maple syrup was also invest

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Leung, Yu Tim. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-128). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.iv / Table of Contents --- p.v / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xiii / Chapter 1. --- Introduction / Chapter 1.1 --- Flower herbal teas --- p.1 / Chapter 1.2 --- R. rugosa --- p.3 / Chapter 1.2.1 --- The phytochemistry of R. rugosa --- p.3 / Chapter 1.3 --- Secondary metabolites --- p.4 / Chapter 1.4 --- Classification of secondary metabolites --- p.6 / Chapter 1.5 --- Phenolic compounds --- p.6 / Chapter 1.5.1 --- Phenylpropanoid compounds --- p.6 / Chapter 1.5.2 --- Lignins --- p.7 / Chapter 1.5.3 --- Coumarins --- p.7 / Chapter 1.5.4 --- Stilbenes --- p.8 / Chapter 1.5.5 --- Tannins --- p.8 / Chapter 1.5.6 --- Flavonoids --- p.9 / Chapter 1.6 --- Oxidative Stress --- p.13 / Chapter 1.6.1 --- Diseases related to ROS --- p.13 / Chapter 1.6.2 --- Significant chemical or biochemical conversion of ROS --- p.14 / Chapter 1.6.3 --- Sources of ROS --- p.15 / Chapter 1.7 --- Natural dietary antioxidants --- p.15 / Chapter 1.7.1 --- Vitamin C --- p.15 / Chapter 1.7.2 --- Vitamin E --- p.16 / Chapter 1.7.3 --- Carotenoids --- p.16 / Chapter 1.7.4 --- Phenolic compounds --- p.16 / Chapter 1.8 --- Cancinogenesis --- p.17 / Chapter 1.9 --- Cell cycle --- p.18 / Chapter 1.9.1 --- Cell cycle of eukaryotic cells --- p.18 / Chapter 1.9.2 --- Checkpoints of cell cycle --- p.18 / Chapter 1.10 --- Cancer cell lines --- p.19 / Chapter 1.11 --- The growth phases of cancer cell lines --- p.20 / Chapter 1.12 --- Antiproliferative effects of phenolic compounds --- p.21 / Chapter 1.13 --- Genotoxicity of phenolic compounds --- p.22 / Chapter 1.14 --- Objectives --- p.23 / Chapter 2. --- Methods and Materials / Chapter 2.1 --- Extraction of active substances --- p.40 / Chapter 2.2 --- Determination of antioxidant activities TEAC assay --- p.40 / Chapter 2.3 --- Determination of hydroxy 1 radical scavenging activity by the deoxyribose assay --- p.41 / Chapter 2.4 --- Determination of phenolic contents by Folin´ؤCiocalteu assay --- p.43 / Chapter 2.5 --- Determination of total flavonoid by aluminum chloride colorimetric method --- p.43 / Chapter 2.6 --- Determination of oxidative DNA damage by comet assay --- p.44 / Chapter 2.7 --- Cell lines propagation --- p.49 / Chapter 2.8 --- Determination of antiproliferative activities by MTT assay (colorimetric) --- p.50 / Chapter 2.9 --- Determination of antiproliferative activities by BrdU labeling assay --- p.52 / Chapter 2.10 --- Cell cycle analysis by flow cytometry --- p.55 / Chapter 2.11 --- Determination of genotoxicity by SOS chromotest --- p.57 / Chapter 3. --- Results / Chapter 3.1 --- Dermination of antioxidant activities by TEAC assay --- p.59 / Chapter 3.1.1 --- Trolox Standard Reference --- p.59 / Chapter 3.1.2 --- TEAC of the seven flower extracts --- p.59 / Chapter 3.2 --- Hydroxyl radical scavenging activity by deoxyribose assay --- p.60 / Chapter 3.3 --- Determination of phenolic contents by Folin´ؤCiocalteu assay --- p.60 / Chapter 3.4 --- Determination of total flavonoids by colorimetirc aluminium chloride assay --- p.61 / Chapter 3.5 --- "The Inter-correlation between the antioxidant activities, total phenolic and flavonoid contents of flower extraction powders" --- p.61 / Chapter 3.6 --- Determination of oxidative DNA damage by comet assay --- p.62 / Chapter 3.7 --- Determination of antiproliferative activities by MTT assay --- p.63 / Chapter 3.7.1 --- Antiporoliferative activities on HepG2 --- p.63 / Chapter 3.7.2 --- Antiproliferative activities on MCF7 --- p.63 / Chapter 3.7.3 --- IC50 of R. rugosa extract on both HepG2 and MCF7 --- p.64 / Chapter 3.8 --- "The Inter-correlation between antioxidant activities, total phenolic contents, flavonoid contents, and the antiproliferative activities of flower extraction Powders" --- p.64 / Chapter 3.9 --- Determination of DNA synthesis by BrdU labeling analysis --- p.65 / Chapter 3.10 --- Cell cycle analysis by flow cytometry --- p.65 / Chapter 3.11 --- Determination of genotoxicity by SOS chromotest --- p.66 / Chapter 4. --- Discussions / Chapter 4.1 --- Extraction method --- p.90 / Chapter 4.2 --- Comparison of TEAC of the dry flowers with other foods --- p.90 / Chapter 4.3 --- Correlation between ABTS+ and hydroxyl scavenging ability of flower extraction powder --- p.91 / Chapter 4.4 --- Comparison of phenolic contents of the fry flowers with other foods --- p.92 / Chapter 4.5 --- Correlation between total phenolic contents and flavonoid contents of flower Eextraction powders --- p.92 / Chapter 4.6 --- "Correlation between total phenolic, flavonoid content and antioxidant activities of flower extraction powders" --- p.93 / Chapter 4.7 --- Factors affecting the antioxidant power besides total phenolic contents --- p.94 / Chapter 4.8 --- Synergistic effect of phenolic compounds --- p.94 / Chapter 4.9 --- Toxicity of drinking flower herbal tea --- p.95 / Chapter 4.10 --- Recommended dose of flower herbal teas --- p.96 / Chapter 4.11 --- Antiproliferative activities of flower extracts by MTT assay --- p.97 / Chapter 4.12 --- Antiproliferation activities of flower extraction Powders by Brdu labeling assay --- p.98 / Chapter 4.13 --- Protective effects of flower extraction powder on oxidative DNA damage determined by comet assay --- p.99 / Chapter 4.14 --- Cell cycle analysis --- p.100 / Chapter 4.15 --- Further Studies --- p.101 / Chapter 5. --- Conclusion --- p.102 / Chapter 6. --- References --- p.103

<p><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a>ABSTRACT</a></p> <p>Phenols are ubiquitous in our surroundings including biological molecules such as L-Dopa metabolites, food components, such as whiskey and liquid smoke, etc. This dissertation describes a new method for detecting phenols, by reaction with Gibbs reagent to form indophenols, followed by mass spectrometric detection. Unlike the standard Gibbs reaction which uses a colorimetric approach, the use of mass spectrometry allows for simultaneous detection of differently substituted phenols. The procedure is demonstrated to work for a large variety of phenols without <i>para</i>‐substitution. With <i>para</i>‐substituted phenols, Gibbs products are still often observed, but the specific product depends on the substituent. For <i>para</i> groups with high electronegativity, such as methoxy or halogens, the reaction proceeds by displacement of the substituent. For groups with lower electronegativity, such as amino or alkyl groups, Gibbs products are observed that retain the substituent, indicating that the reaction occurs at the <i>ortho</i> or <i>meta</i> position. In mixtures of phenols, the relative intensities of the Gibbs products are proportional to the relative concentrations, and concentrations as low as 1 μmol/L can be detected. The method is applied to the qualitative analysis of commercial liquid smoke, and it is found that hickory and mesquite flavors have significantly different phenolic composition.</p> <p>In the course of this study, we used this technique to quantify major phenol derivatives in commercial products such as liquid smoke (catechol, guaiacol and syringol) and whiskey (<i>o</i>-cresol, guaiacol and syringol) as the phenol derivatives are a significant part of the aroma of foodstuffs and alcoholic beverages. For instance, phenolic compounds are partly responsible for the taste, aroma and the smokiness in Liquid Smokes and Scotch whiskies. </p> <p>In the analysis of Liquid Smokes, we have carried out an analysis of phenols in commercial liquid smoke by using the reaction with Gibbs reagent followed by analysis using electrospray ionization mass spectrometry (ESI-MS). This analysis technique allows us to avoid any separation and/or solvent extraction steps before MS analysis. With this analysis, we are able to determine and compare the phenolic compositions of hickory, mesquite, pecan and apple wood flavors of liquid smoke. </p> <p>In the analysis of phenols in whiskey, we describe the detection of the Gibbs products from the phenols in four different commercial Scotch whiskies by using simple ESI-MS. In addition, by addition of an internal standard, 5,6,7,8-tetrahydro-1-napthol (THN), concentrations of the major phenols in the whiskies are readily obtained. With this analysis we are able to determine and compare the composition of phenols in them and their contribution in the taste, smokey, and aroma to the whiskies.</p> <p>Another important class of phenols are found in biological samples, such as L-Dopa and its metabolites, which are neurotransmitters and play important roles in living systems. In this work, we describe the detection of Gibbs products formed from these neurotransmitters after reaction with Gibbs reagent and analysis by using simple ESI‐MS. This technique would be an alternative method for the detection and simultaneous quantification of these neurotransmitters. </p> <p>Finally, in the course of this work, we found that the positive Gibbs tests are obtained for a wide range of <i>para</i>-substituted phenols, and that, in most cases, substitution occurs by displacement of the <i>para</i>-substituent. In addition, there is generally an additional unique second-phenol-addition product, which conveniently can be used from an analytical perspective to distinguish <i>para</i>-substituted phenols from the unsubstituted versions. In addition to using the methodology for phenol analysis, we are examining the mechanism of indophenol formation, particularly with the <i>para</i>-substituted phenols. </p> <p>The importance of peptides to the scientific world is enormous and, therefore, their structures, properties, and reactivity are exceptionally well-characterized by mass spectrometry and electrospray ionization. In the dipeptide work, we have used mass spectrometry to examine the dissociation of dipeptides of phenylalanine (Phe), containing sulfonated tag as a charge carrier (Phe*), proline (Pro) to investigate their gas phase dissociation. The presence of sulfonated tag (SO₃^-) on the Phe amino acid serves as the charge carrier such that the dipeptide backbone has a canonical structure and is not protonated. Phe-Pro dipeptide and their derivatives were synthesized and analyzed by LCQ-Deca mass spectroscopy to get the fragmentation mechanism. To confirm that fragmentation path, we also synthesized dikitopeparazines and oxazolines from all combinations of the dipeptides. All these analyses were confirmed by isotopic labeling experiments and determination and optimization of structures were carried out using theoretical calculation. We have found that the fragmentation of Phe*Pro and ProPhe* dipeptides form sequence specific b₂ ions. In addition, not only is the ‘mobile proton’ involved in the dissociation process, but also is the ‘backbone hydrogen’ is involved in forming b₂ ions. </p> <p> </p>