Serial Review: Flavonoids and Isoflavones (Phytoestrogens: Absorption, Metabolism, and Bioactivity)Flavonoids: antioxidants or signalling molecules?☆
Introduction
Flavonoids have been proposed to exert beneficial effects in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. Many of the biological actions of flavonoids have been attributed to their antioxidant properties, either through their reducing capacities per se or through their possible influences on intracellular redox status. The precise mechanisms by which flavonoids exert their beneficial or toxic actions remain unclear. However, recent studies have speculated that their classical hydrogen-donating antioxidant activity [1], [2], [3] is unlikely to be the sole explanation for cellular effects [4], [5], [6]. This premise is based on a number of lines of reasoning. Firstly, flavonoids are extensively metabolised in vivo, resulting in a significant alteration in their redox potentials. It has become clear that the bioactive forms of flavonoids in vivo are not those forms found in plants, for example, the glycosides and, in some instances, aglycone, but instead conjugates and metabolites arising from these on intestinal absorption [reviewed in [7], [8], [9], [10], [11]]. In particular there is now strong evidence for the extensive phase I deglycosylation and phase II metabolism of the resulting aglycones such as quercetin, hesperetin, naringenin, and epicatechin to glucuronides, sulphates and O-methylated forms during transfer across the small intestine [7], [12] and then again in the liver (Fig. 1). Further transformation has been reported in the colon, where the enzymes of the gut microflora degrade flavonoids to simple phenolic acids [13], which may also be absorbed and subsequently further metabolised in the liver (Fig. 1).
The uptake of flavonoids and their in vivo metabolites is dependent on cell type [14]. However, this is most probably due to a greater level of intracellular metabolism and faster rate of export from some cells rather than simply differing levels of passive diffusion into the cell. Generally, flavonoids may undergo three forms of intracellular metabolism: (1) Conjugation with thiols, particularly GSH; (2) oxidative metabolism; and (3) P450-related metabolism. For example, the intracellular metabolism of quercetin in human dermal fibroblasts has been shown to involve the formation of intracellular oxidation products, the generation of 2′-glutathionyl quercetin (Fig. 2), and the demethylation of O-methylated forms of quercetin [15]. In contrast, epicatechin and its O-methylated metabolites entered fibroblasts to a lesser extent and do not undergo measurable cellular metabolism [4]. Cell-generated metabolites, such as 2′-glutathionyl quercetin, are of interest as they may be capable of mediating cellular effects whether beneficial or toxic in cells.
Metabolic modifications of flavonoids will alter their “classical” antioxidant nature, which is defined mainly by the presence of a B-ring catechol group (dihydroxylated B-ring) capable of readily donating hydrogen (electron) to stabilise a radical species [16], [17], [18]. Other structural features important for antioxidant nature include the presence of 2,3 unsaturation in conjugation with a 4-oxo- function in the C-ring and the presence of functional groups capable of binding transition metal ions, such as iron and copper [16] (Fig. 3, quercetin as example). Circulating metabolites of flavonoids, such as glucuronides and O-methylated forms, and intracellular metabolites, for example, flavonoid–GSH adducts, have a reduced ability to donate hydrogen [4] and are less effective scavengers of reactive oxygen and nitrogen species relative to their parent aglycone forms (Spencer et al., unpublished observations). Indeed, studies have indicated that although such conjugates and metabolites may participate directly in plasma antioxidant reactions and by scavenging reactive oxygen and nitrogen species in the circulation, their effectiveness is reduced relative to their parent aglycones [19], [20], [21], [22], [23]. Secondly, concentrations of flavonoids and their metabolite forms accumulated in vivo, for example, in the plasma or in organs such as the brain [24], are lower (high nanomolar, low micromolar) than those recorded for small molecule antioxidant nutrients such as ascorbic acid and α-tocopherol [25]. Consequently, flavonoids are unlikely to express beneficial action in vivo through outcompeting antioxidants such as ascorbate, which are present at higher concentrations (high micromolar). Accumulating evidence suggests that the cellular effects of flavonoids may be mediated by their interactions with specific proteins central to intracellular signalling cascades [26]. In particular, investigation has indicated that they may interact selectively within the mitogen-activated protein kinase (MAP kinase) signalling pathway [27], [28]. Recent experiments have indicated that flavonoids are capable of protecting neurons against oxidative stress more effectively than ascorbate, even when the latter was used at 10-fold higher concentrations [29], which supports a nonantioxidant activity.
Section snippets
Flavonoid interactions with intracellular signalling cascades
Flavonoids might exert modulatory effects in cells independent of classical antioxidant capacity through selective actions at different components of a number of protein kinase and lipid kinase signalling cascades such as phosphoinositide 3-kinase (PI 3-kinase), Akt/PKB, tyrosine kinases, protein kinase C (PKC), and MAP kinases [6], [28], [30], [31], [32], [33], [34]. Inhibitory or stimulatory actions at these pathways are likely to profoundly affect cellular function by altering the
Oxidative stress activation of MAP kinase as a target for flavonoids in the nervous system
The best characterised of the MAP kinase pathways are the mitogenic ERK1/2 and the stress-activated JNK and p38 cascades. The potential modulation of MAP kinase signalling pathways by flavonoids is significant as ERK1/2 and c-jun amino-terminal kinase (JNK) are involved in growth factor-induced mitogenesis, differentiation, apoptosis, and various forms of cellular plasticity [63], [64], [65], [66], [67]. ERK and JNK are generally considered as having opposing actions on neuronal apoptosis [68].
Specific actions of flavonols
Although the flavonol quercetin is one of the most frequently researched flavonoids, with both its beneficial [97], [98] and deleterious effects [15], [34], [99], [100], [101] on different cell types being well described, its mechanism of action remains unclear. Quercetin has been thoroughly investigated for its abilities to express antiproliferative effects [102], [103] and induce cell death predominantly by an apoptotic mechanism in cancer cell lines [100], [104], [105], [106]. For example,
Specific actions of flavanols
Much interest has centred on the beneficial effects of flavanols, such as epicatechin, epicatechin gallate (EGC), and epigallocatechin gallate (EGCG), and there is growing evidence that the cytoprotective nature of these polyphenols is based on their interactions within signalling pathways. For example, epicatechin and one of its major in vivo metabolites, 3′-O-methyl epicatechin, have been shown to elicit strong cytoprotective effects against oxidative stress in fibroblasts and neurons [4], [5]
Conclusions
In summary, the emerging view is that flavonoids are likely to exert beneficial and/or toxic actions on cells not through their potential to act as antioxidants but rather through their modulation of protein and lipid kinase signalling cascades. The basis for this conclusion is that flavonoids and their metabolites are unlikely to act as major antioxidants in vivo in the presence of small molecule antioxidants such as ascorbic acid and α-tocopherol, the concentrations of which are at much
Acknowledgements
The authors acknowledge the support of the Biotechnology and Biological Sciences Research Council for financial support of some of the research discussed in this review.
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This article is part of a series of reviews on “Flavonoids and Isoflavones (Phytoestrogens): Absorption, Metabolism, and Bioactivity”. The full list of papers may be found on the home page of the journal.