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Research aims of the projectFlavonoids have become increasingly popular in terms of health protection. However, those flavonoids that are most frequently encountered as constituents of human diet are not necessarily the most active ones within the human body, either because they exhibit a lower intrinsic activity or because they are poorly absorbed from the intestine, highly metabolized or rapidly eliminated. Knowledge on the bioavailability and on the metabolism of flavonoids is thus essential in identifying those compound that are the most likely ones to have biological activity. The aims of this project are (1) to analyze kinetic parameters and metabolite profiles of structurally related flavonoids in different organs of mice and to relate them to organ-specific flavonoid prooxidative, pro-apoptotic and cytotoxic actions, (2) to detect in transporter-deficient mice transport proteins important for flavonoid uptake/distribution and (3) to mechanistically analyze the results received in the in vivo studies by use of cell culture systems expressing distinct transport proteins and detoxication enzymes. State of the art of current research Quercetin is one of the most abundant flavonoids in human diet and is also considered to be one of the most promising members of this class both for general chemoprevention and for cancer chemotherapy. The pharmacokinetics of this flavonoid have therefore repeatedly been studied in rats as well as in humans. It was reported that rats adapted to quercetin (0.2% quercetin diet) maintained plasma concentrations (quercetin + metabolites) of approximately 100 µM (Manach et al. 1997). In humans, low micromolar plasma concentrations of quercetin/quercetin metabolites are measured after administration of onions (a plant source known to contain quercetin as glucosides), e.g. 7.65 µM after ingestion of 100 mg quercetin equivalent (Graefe et al. 2001). Controversal data are availabe on quercetin blood metabolites: Some authors find no free quercetin in blood samples (Moon et al. 2000) while others report that free quercetin was also detected (Erlund et al. 2000). Much about the mechanisms of gastrointestinal absorption as well as on the excretion via bile and urine of polyphenols remains so far unknown. Most polyphenols are probably too hydrophylic to penetrate the gut wall by passive diffusion, but membrane carriers that could be involved in polyphenol absorption have until now not been identified. Quercetin glucoside absorption occurs in the small intestine, and the efficiency of absorption is higher than for the aglycone itself. The underlying mechanism by which glucosylation facilitates quercetin absorption has been partly elucidated. It was suggested that glucosides could be transported into enterocytes by the sodium-dependent glucose transporter (SGLT1) (Hollman et al. 1999; Walgren et al. 2000). Another pathway involves the lactase phloridzine hydrolase, a glucosidase of the brush border membrane of the small intestine. More detailed studies on the transport of flavonoids have been performed using Caco-2 cells and rat small intestine, where flavonoid glycosides are absorbed using SGLT1 and the monocarboxylate transporter (MCT) as described for epicatechin-3-gallate [Vaidyanathan and Walle 2003). The multidrug resistance (MDR)-associated proteins 2 and 3 have been shown to be an important efflux transporter of flavonoids, mainly for the glucuronide and sulfate conjugates. Up to now, little is known about the distribution of quercetin metabolites in tissues and cells when nutritional doses of polyphenols are consumed. It is at present unclear whether the metabolites still possess pharmacological activity (Spencer et al. 2004). Data obtained from animal studies indicate that some polyphenol metabolites may accumulate in certain target tissues rather than just equilibrate between blood and tissues. The group of P. Proksch is experienced in and has access to all relevant analytical techniques and equipment needed for the chromatographic separation, detection and identification (using hyphenated techniques such as HPLC-DAD and HPLC-MS as well as “classic” spectroscopic techniques such as 1H and 13C NMR) of flavonoids and of other plant derived phenolic constituents [PP 1, 4, 43, 45, 46]. Based on previous and ongoing projects that focus on the isolation and structural identification of phenolic plant constituents an extensive in house library of flavonoids and related compounds is available that will funnelled into this project. These compounds will also be made available to other projects in the Research Training Group. Structural analysis of different flavonoids are performed in a joint German-Chinese research project focussing on “Content of bioactive flavonoids in Chinese vegetables” (DFG, Pr 229/9-1, 9-2). The group of W. Wätjen is experienced in molecular biology and cell culture techniques. They have published on the antioxidative/antiapoptotic as well as pro-oxidative and pro-apoptotic actions of structurally related flavonoids, isoflavones and lignans [WW 1 – 5, 7], in part in cooperation with Prof. A. Bast (Maastricht, NL) and Prof. G. Degen (Dortmund) [2, 5, 7]. Title of thesis - “Mechanistic studies on absorption and metabolism of dietary flavonoids in a mouse model” By the use of different knockout mice deficient in candidate flavonoid transporters in the intestine, the mechanism of flavonoid uptake in vivo will be studied, and the importance of the transporters identified will be verified in primary human IEC. Feeding
experiments in mice are
carried out to
investigate uptake
and distribution of flavonoids, parameters of oxidative stress
and organ-specific flavonoid cytotoxicity and induction of
apoptosis. Transporter-deficient mice are used to analyse requirements of
flavonoid uptake (e.g. mdr1a,b-/-, mdr1,a,b+/+, FVB
mice; cooperation with Prof. E. Petzinger, Giessen). In cell culture
experiments, further mechanistic analysis using pharmacological inhibitors
of transporters, overexpression of transporters, and transporter knockdown
via RNA interference is performed. |
