Over 30 years ago, it was shown that consumption of tea is in accordance with low non-haem iron bioavailability [90
]. Consequently, flavonoids, or polyphenols, from the tea were recognized as the main cause for low non-haem iron absorption. Today, flavonoids, among them primarily quercetin, are considered as one of the main dietary inhibitors of iron absorption in the duodenum. Even though the exact mechanism of how flavonoids inhibit non-haem iron absorption is still not fully elucidated, it is strongly believed that its power to chelate iron is mainly responsible for this action [92
]. In contrast, it was shown that quercetin may operate as a substrate for Dcytb by increasing its reduction potential and providing more Fe2+
for cellular uptake by DMT1 [94
In diseases connected with an imbalance in iron homeostasis, organ-specific iron accumulation is present. In order to bring iron levels back into balance, chelato therapeutics are applied. Potent chelato therapeutics should be able to go through iron-over loaded tissues, complex iron by forming stable and redox-inactive iron and transfer it to Tf in the circulation. Known chelato therapeutic drugs fulfil more or less listed requirements [95
]. However, it has been proven in vitro that quercetin is able to decrease intracellular iron and to transfer it to Tf. These significant findings suggest that quercetin could be a valuable representative of chelato therapeutics for iron-redistribution therapy. Yet, this fact still needs to be proven with in vivo studies [96
]. On the other hand, it is clear that flavonoids should be avoided in IDA, especially during oral consumption of iron either as a natural constituent of the diet or as a food supplement.
Furthermore, flavonoids were shown to be potent in regulation of systemic iron metabolism. Namely, Bayele et al. [97
] reported that quercetin increased expression of hepcidin, a main iron regulatory hormone, which might involve the Nrf2 pathway. Other authors showed in cells that quercetin is able to activate Nrf2 pathway by supporting its nuclear translocation and transcriptional activity [98
]. In view of the fact that levels of FPN and H and L ferritin are also known to be transcriptionally up regulated by Nrf2 pathway quercetin could affect iron homeostasis and help cells defending against oxidative stress. Moreover, Vanhees et al. [99
] showed that prenatal exposure to quercetin caused hepcidin induction in adult mice.
4.4.1. Flavonoids as Iron Chelators
Flavonoids are known for their numerous health benefits which are mostly attributed to their ability to scavenge highly reactive free radical species. However, flavonoids’ antioxidative potential is, at least partially, associated with their ability to chelate iron. By chelating iron, flavonoids reduce the accessibility of iron to oxygen and consequently diminish oxygen high toxicity, e.g., by inhibiting the production of HO•
in Fenton reaction [99
The exact mechanism by which certain flavonoids reduce bioavailability of non-haem iron is not fully understood, but it is proposed that flavonoids are able to chelate non-haem iron [92
Like most other flavonoids, it was proven that quercetin possesses a high ability to chelate iron [103
]. The preferred site for iron chelation by flavonoids, such as quercetin, is its 3-hydroxyl and 4-carbonyl group. Specifically, for complexes containing one iron and one quercetin, the binding strength has an order 3–4 > 4–5 > 3′–4′. Moreover, the 3–4 chelation site is also preferred for complexes which are formed between one iron and two or three quercetin molecules (see Figure 5
]. In addition, it is estimated that quercetin, like most other flavonoids, forms a complex with Fe3+
with a greater stability than Fe2+
. Even though when quercetin initially forms a complex with Fe2+
will autooxidise to Fe3+
Regarding inhibition of iron absorption by quercetin, it was clearly demonstrated in vivo that chelation of iron by the 3-hydroxyl group of quercetin is an important determinate of iron uptake in duodenum [93
]. The authors confirmed that the decrease in duodenal iron transfer is due to chelation of iron by quercetin which increases apical uptake of iron, but prevents basolateral transport. Further information that supports this hypothesis is the fact that the quercetin–Fe complex is considerably stable in gastrointestinal conditions. Namely, it was shown in vitro, by mimicking conditions that occur in the stomach, that the recovery of quercetin–Fe complex is up to 45%, which supports the importance of chelation of iron by quercetin in the human body [106
]. However, the precise place of iron chelation by quercetin is still uncertain. It is still unknown whether chelation occurs in the duodenal lumen or the cytosol of duodenal enterocytes. One explanation could be that iron is chelated by quercetin in the duodenal lumen by forming the apical–membrane–permeable quercetin–Fe complex that cannot cross the basolateral membrane of enterocyte. Despite its great size, there are in vitro reports that support transport of the quercetin–Fe complex across the cell membrane in both directions [96
]. Furthermore, there is evidence that the quercetin–Fe complex is transported by GLUTs transporters [107
], which could also be the case in vivo. Furthermore, even though it was shown that quercetin can be transported via GLUTs 1, 3 and 4 transporters, quercetin is lipophilic enough so it can easily cross lipid bilayers without interaction with transporters [80
]. A second possibility is that that quercetin could influx into the cell and then form a complex with free iron. Additionally, it was shown that quercetin may operate as a substrate for Dcytb by increasing its reduction potential and providing more Fe2+
for cellular uptake by DMT1 [94
]. Knowing this, quercetin could firstly chemically reduce non-haem iron and thus increase apical uptake followed by formation of the quercetin–Fe complex inside the cell. Therefore, both luminal and cytosolic iron chelation, or their combination, can provoke iron accumulation within duodenal mucosa in vivo. However, in both cases the quercetin–Fe complex could remain in the enterocyte due to the inhibition of FPN function or simply because the quercetin–Fe complex would be too bulky to be transported by FPN. This explanation can be applied to all polyphenols that have a noticeable capacity to chelate iron, particularly those which are present in the diet and thus can directly affect iron absorption.
This phenomenon was previously shown for other polyphenols, particularly for (–)-epigallocatechin-3-gallate, but in in vitro conditions using Caco-2 cells as a model system [101
]. Kim et al. [101
] reported their finding as unexpected as it was common to think that polyphenols inhibit iron absorption by preventing mainly apical uptake of non-haem iron.
Furthermore, the quercetin-Fe complex inside the cell could be a negative signal for the iron regulatory protein/iron responsive element (IRE/IRP) system and thus destabilize FPN mRNA. Thus, by chelating iron, quercetin could lower free iron levels inside the cell and thus trigger the post-transcriptional IRE/IRP control system, such that when iron levels in tissue are reduced, the expression of FPN is decreased [110
]. Furthermore, the possibility that quercetin or its metabolites have direct inhibitory effects on the function of FPN should not be discounted. In addition, bearing in mind that many proteins that have a pivotal role in iron homeostasis beside FPN, such as ferritin, DMT1, TfR1 and Hif-2α, are also regulated by the IRE/IRP system the role of flavonoids which are potent to chelate iron becomes even more important.
Together all these mechanisms could account for the increased mucosal iron retention observed in our studies [93
]. Furthermore, if a quercetin–Fe complex is formed inside the cell, it could be proposed that quercetin could affect absorption of haem iron too. Namely, quercetin could also prevent the export of free iron for the haem source, after haem degradation by HO-1 which occurs in the cytosol after its absorption.
Potentially listed modes of action could be ascribed to all dietary polyphenols that have demonstrable capacity to chelate iron and this information could be useful in the design of iron chelators based on the structure of these common dietary polyphenols.
In marked contrast, work using Caco-2 cells showed that certain flavonoids promote iron bioavailability (i.e., epicatechin, kaempferol [111
]). However, these are still in vitro results that needs to be confirmed in vivo.
4.4.2. Flavonoids as Regulators of Systemic Iron Metabolism
It was shown that flavonoids could play important role in regulation of systemic iron metabolism. Namely, a couple of studies have shown that flavonoids have a great effect on hepcidin levels in vivo. However, results are contradictory.
Bayele et al. [97
] reported that intraperitoneal quercetin increased hepcidin expression which might involve the Nrf2 pathway, which also correlated with changes in serum iron levels and Tf saturation, as well as with reduction in FPN mRNA. Vanhees et al. [99
] showed that prenatal exposure to quercetin caused hepcidin induction in adult mice and the authors hypothesized that after birth, when pups were no longer exposed to quercetin, improved bioavailability of dietary iron sensed as body iron overload. Lesjak et al. [102
] showed that quercetin increased hepcidin mRNA levels in both liver and spleen. Increased levels of hepcidin were followed by decreased FPN levels.
However, previous results of others indicate contradictory results on how different dietary polyphenols affect hepcidin levels. Mu et al. [113
] reported that the polyphenol myricetin inhibits hepcidin expression induction in vivo by the BMP/SMAD signalling pathway. Quercetin and myricetin are very similar in structure, with myricetin having an extra hydroxyl group. The differences in effect of these two similar polyphenols on iron absorption indicate the complexity of responses to polyphenols. Furthermore, Zhen et al. [114
] and Patchen et al. [115
] showed that genistein, a main polyphenol from soya, and ipriflavone, synthetic analogue derived from abundant dietary polyphenol daidzein, respectively, both strongly promote hepcidin expression in vivo. Recent studies by Grillo et al. [116
] and Zhang et al. [117
] indicate that other natural products apart from flavonoids could also have a major role in iron metabolism in vivo and may have potential in therapy of iron metabolism disorders. Namely, Grillo et al. [116
] showed that hinokitiol, a natural product of terpenoid structure found in wood that strongly complex iron, could restore iron transport in vivo very effectively. Zhang et al. [117
] elucidated that prenylated flavonoid glycoside icariin, which can be found in some Chinese herbal medicinal plants, are able to induce hepcidin expression in mice and change serum and tissue iron concentrations by activating Stat3 and Smad1/5/8 signaling pathways.
Interestingly, it was shown that quercetin directly regulates FPN expression [93
]. Namely, in Caco-2 cells exposed to quercetin there was a significant dose-dependent decrease in FPN protein and mRNA and this was associated with a significant decrease in iron transport across Caco-2 cell monolayers. This occurrence was associated with interactions between miRNA and 3′UTR of FPN mRNA. This data suggests a possible great role for miR-17 and potentially other microRNAs in mediating diet-gene interactions that can influence nutrient bioavailability [93