Dietary flavonoids: Bioavailability and biosynthesis

Flavonoids are one of the largest groups of natural plant products and are found in most, if not all, fruits and vegetables. As dietary components, flavonoids have widespread biological properties and have been associated with several health benefits, including reduced risk of cancer and cardiovascular disease. The flavonol, quercetin, one of the most ubiquitous dietary flavonoids, is found principally as glycoside conjugates in plants and quercetin-3-glucosylrhamnoside (rutin) is one of the more common forms. To establish the role of rutin as a protective agent in vivo, it is critical to understand the chemical nature of the absorbed forms and their in vivo concentrations in the circulatory and excretory system. The bioavailability of rutin is not very well understood as reflected by the varying peak plasma concentrations (Cmax) and the time taken to reach the peak plasma concentration (Tmax) reported in the literature. This may, in part, be due to the analytical techniques used which include acid or glucuronidase/sulphatase treatments to release the parent aglycone prior to quantitative analysis by low resolution, isocratic reverse-phase HPLC. In addition, there has been some debate on the types of rutin catabolites produced as a result of colonic breakdown. There is therefore a need for further more detailed information on the absoiption, metabolism and bioavailability of rutin. The underlying objective in Chapter 3 was to investigate the metabolism and absoiption of rutin in vivo after ingestion of tomato juice supplemented with 164 ?moles (l00mg) of rutin. Rutin was found to be extensively metabolised and made bioavailable to humans reflected in the identification of many phase II metabolites and catabolites. To investigate the role of colonic microflora in the breakdown of rutin, an in vitro faecal fermentation study was carried out as discussed in Chapter 4. Accumulation of quercetin in the fermentation vessel as early as 4 h after incubation indicated that deglycosylation was the initial step in the colonic breakdown of rutin. The addition of glucose to the fermentation media enhanced the deglycosylation process by almost 20 h. The study in Chapter 5 aimed at achieving this objective. An attempt was made to direct the synthesis of genistein in arabidopis and in tomato by introducing isoflavone synthase (IFS), which is the key enzyme for the entry into the isoflavonoid biosynthetic pathway. The full length IFS cDNA from soybean roots was isolated and cloned into binary vectors, pGlasglow and pCHF3 habouring the CaMV 35S promoter and into pER8 vector carrying an estrogen inducible G10-90 promoter. Using agrobacterium mediated transformation, the IFS gene was introduced in arabidopsis and tomato. Due to the nature of the pER8 construct and its G10-90 promoter, a 3-10 fold higher gene expression level was observed in arabidopsis transgenic plants transformed by pER8 binary vector than those transformed with pGlasglow. High levels of gene silencing was observed when using the CaMV 35S in arabidopsis and total gene silencing was observed when the same promoter was used in tomatoes. HPLC-PDA-MS2 analysis of leaf extracts in the highly and moderately expressed IFS arabidopsis lines failed to detect the presence of genistein. Western blot analysis implied that IFS proteins were synthesized and accumulated in the leaves of arabidopsis and were present 'freely' in the cytoplasm rather than being membrane bound as they would in their natural environment. It was, therefore, hypothesised that the 'free' IFS was not able to access the substrate, naringenin which may be compartmentalized in a pre-existing multienzyme complex, and as a consequence, IFS was unable to synthesize genistein. (Abstract shortened by ProQuest.)