Quercetin is a flavonoid that is found in all plant foods. As with other flavonoids, its role in higher plants is to function as an antioxidant [1
]. In humans it is postulated that not only is it an antioxidant, but it can also act directly on cells as an anti-inflammatory agent [2
]. It has properties demonstrated in vivo and in vitro consistent with protection against cardiovascular disease [3
]. There is also evidence for anticancer [4
] and antiviral [5
] effects. However, the bioavailability of quercetin is low [6
] and its absorption can be affected by macronutrients [7
]. In plants, quercetin is in the form of glycosides, which are converted to the aglycone by β-glycosidases in the intestine before being absorbed into the enterocytes [8
]. Here they are metabolised to quercetin conjugates. Previously, we studied quercetin aglycone solubilisation in simple bile salt (BS) micelles of composition relevant to the duodenal lumen [10
]. In the present work aspects of quercetin’s solubilisation are extended to mixed micelles, PC liposomes and lysoPC micelles, which are all lipid phases found in the small intestine at different stages of digestion. Since flavonoids are absorbed and conjugated by human epithelial intestinal CaCo-2 cells [11
], we studied quercetin uptake into CaCo-2/TC7 cells in the presence and absence of mixed micelles with the aim of getting information about whether micelle solubilisation influences its bioavailability. To our knowledge, this is the first study of the effect of lipid micelles on quercetin uptake by intestinal cells.
The UV-visible spectrum of quercetin is sensitive to the pH and polarity of its environment [10
]. The structure of quercetin and its spectra as a function of pH and presence of BS are shown in Figures 1 and 3A of our previous publication [10
]. The molecule is planar, consisting of two aromatic rings (A and B) linked by a γ-pyrone ring. We have found that the wavelength of maximum absorption of the long wavelength peak (Peak B) has a bathochromic shift when quercetin moves from an aqueous phase into a more hydrophobic phase (for example bile salt micelles). Thus, we were able to show that the adsorption site on simple bile salt micelles of composition mimicking the hydrophobicity of bile salts in humans was less polar than that on sodium dodecyl sulphate micelles. (This was confirmed by pyrene fluorescence, where the vibronic fine structure of the spectra depends on the polarity of the pyrene environment.) The effect of changing pH is most clearly seen in the short wavelength peak (Peak A). The first ionisation of quercetin (HQ ➔ H+
) gives rise to a peak at 270 nm. Therefore, the ratio of the maximum absorbance of peak A to that at 270 nm gives a measure of the relative amounts of HQ and Q−
. Using this ratio, we can measure the relative affinity of the ionised and neutral forms of quercetin for micelles or other lipid phases and from peak B assess the relative polarity of the binding sites.
In vivo, quercetin’s bioavailability is enhanced if fed with oils [13
]. Its oil/water partition coefficient favours partition into oil (log P = 1.8), but this value is amongst the lowest of those for flavonoids [16
]. This reflects quercetin’s relatively polar nature. It is possible that the role of the oil is to stimulate bile production so as to solubilise the quercetin in lipid micelles. Alternatively, in spite of the rather low partition coefficient, the oil and in particular the oil–aqueous phase interface could provide a quercetin store for subsequent passage into the enterocytes. Indeed, we [10
] and others [17
] have shown that in simple micelles and lipid membranes quercetin is solubilised close to the polar groups of the organised structures. This may also be true of other nutrients and drugs that have an intermediate polarity between strongly hydrophobic molecules (such as vitamin D and cyclosporin) and hydrophilic water soluble molecules (such as vitamin C and aspirin). Therefore, quercetin is not only of interest as a flavonoid, but also as a model compound for other similar compounds of interest. When considering the relative bioavailability of compounds, their positive affinity for lipid phases found in the small intestine is often referred to as solubilisation. However, the interaction may be a binding process, as is the case for quercetin and pyrene. This could be thought of as a solubilisation in the region of the binding sites. Therefore, in this paper we have used the terms solubilisation and binding to mean the same: a positive interaction with the lipid phases.
We have studied the binding of quercetin to lipid phases present in the small intestine and found there is preferential binding of uncharged quercetin (HQ) over ionised quercetin (Q−
) to mixed and lysoPC micelles. The same is true for simple BS micelles [10
]. Only in the case of PC liposomes do we find evidence for Q−
binding. A consensus view is that quercetin binds to the surfaces of organised lipid phases [10
]. Only in the case of planar lipid membranes, where there is solvent present is there evidence for HQ penetrating between acyl chains [26
]. The hydrophobicity of the binding sites decreases in the order BS > mixed micelles > PC = lysoPC and the affinity of quercetin for the lipid phases follows the reverse order. This is understandable because quercetin is a relatively polar molecule and will have an affinity for more polar surfaces. The bile salt mixture we have used mimics the average hydrophobicity of bile salts found in human bile. Quercetin has a low affinity for these BS micelles. However, the solubility data (Table 2
) and our previous paper [10
] indicate monomeric BSs can interact with uncharged quercetin (HQ).
Our approximate estimate for the affinity of unionised quercetin, HQ, for PC liposomes, expressed as a binding constant, is relatively high (45 mM−1). It is calculated from the best fit to the data over the whole range of concentration of PC studied and assumes all the quercetin is bound at saturation. If about half the PC is available for quercetin binding, the saturation of sites at about 0.15 mM PC by 22.5 µM quercetin suggests (75/22.5 = 3.3) PC molecules form each binding site. Half the PC molecules being available assumes uni-lamellar liposomes. The fact that transparent and translucent liposome suspensions give the same results is evidence that this is true. (The size of the transparent liposomes was 150 nm; translucent liposomes were not measured.)
Spectra at pH 7.15 (Figure 4
B) indicate that ionised quercetin, Q−
, adsorbs to the liposomes. This contrasts with mixed micelles, where there is no evidence of Q−
binding. The reason for the difference lies in the negative charge carried by the conjugated bile salts. The disc model [27
] for the mixed micelles consisting of phospholipid, fatty acid and bile salts postulates a lipid bilayer structure with the potentially exposed hydrophobic fatty acid chains coated with bile salts. Quercetin preferentially binds to the surface of the bilayer part of the disc as it has a low affinity for the bile salts and the negative charge on the bile salts precludes Q−
binding. In fact, our attempts using UV-visible spectroscopy to measure a Kb
for HQ binding to simple BS micelles gave inconsistent results because the affinity was so low (Using the fluorescent properties of quercetin which are more sensitive than UV-visible spectra would be a better method to measure Kb
for quercetin—simple BS micelle interactions).
The importance of negative charge influencing quercetin adsorption is shown by OA addition to PC liposomes giving an inhibition of Q− binding. The observation that OA does not significantly affect quercetin adsorption to mixed micelles is presumably due to the negative charge on the bile salts. Simple micelles of lysoPC showing preferential binding of HQ is unexpected, given that lysoPC and PC have the same polar head groups. LysoPC is susceptible to hydrolysis and it may be that some long-chain fatty acid was present in the micelles.
When mixed micelles are diluted, the intermicellar concentration of BSs is reduced. This means the micelle size increases as the lipid lamellae have fewer available BSs to shield the hydrophobic fatty acid chains from the aqueous phase. At low concentrations, as micelles are forming from phospholipid and bile salts, phospholipid lamellar polar surface will predominate and form a substrate for quercetin and pyrene binding. This explains the increased quenching of pyrene by quercetin as they both adsorb to the surface, increasing the chance of their mutual interaction. (Whether this is by fluorescent energy transfer or exciplex formation is yet to be determined.) At higher concentrations there is less lamellar surface for adsorption of quercetin and pyrene. The situation is different for simple bile salts, where the bile salt concentration must be high enough to form a surface for adsorption. This is why quercetin quenching of pyrene fluorescence for simple BS micelles shows the opposite trend to mixed micelles—increased quenching at high micelle concentrations.
Our results shed light on why the bioavailability of quercetin is low. To be bioavailable, a nutrient must first be bioaccessible (that is, solubilised) so it can be absorbed by the enterocytes. As a nutrient passes down the gastrointestinal tract it will be subjected to changing conditions of pH and solubilising agents (micelles and lipids). In this context, and in the light of our results on quercetin’s properties and solubilisation, we discuss here how quercetin’s bioaccessibility changes as it passes from the stomach to the distal small intestine and how the presence of oil can enhance its bioavailability. We suggested in the Introduction that the enhancement of quercetin bioavailability by oil might be due to the oil stimulating bile secretion into the duodenum. Our results indicate that this hypothesis is false. Bile components inhibit quercetin uptake into CaCo-2 cells.
In the stomach
the pH is less than 4. With a pKa1
of 7.08, quercetin will be unionised (HQ) and free HQ can pass through the stomach wall. However, HQ is poorly soluble so it will tend to precipitate. Nevertheless, quercetin absorption from rat stomach has been observed [28
]. Quercetin’s solubility in oil is higher. Therefore, in oil it is protected from precipitation. Lipolysis by gastric lipase is limited [29
] so quercetin in oil can pass through to the duodenum.
In the proximal duodenum
lipid bilayers and micelles (simple and mixed) are present [30
]—all potential solubilising structures. However, the pH is >7 so quercetin will ionise with Q−
≥ HQ. Q−
could potentially reach the enterocytes in the free state or bound to PC, but diffusion through the negatively charged mucosal membranes will be restricted. We have found that mixed micelles, mimicking those in the upper small intestine, inhibit uptake of quercetin into CaCo-2 cells, which are considered to be able to model small intestine enterocytes [31
]. Therefore, it seems even mixed micelles do not provide a pathway for enhancing quercetin absorption. We have shown that monomeric bile salts interact with unionized quercetin. The negative charge of the bile salts will further inhibit the diffusion of quercetin into the enterocytes. The lipolysis of emulsified oil by pancreatic lipases is known to enhance the passage of oil-soluble nutrients to micelles [33
]. Therefore, although the micelle solubilisation of quercetin is low, oil can provide a quercetin reservoir for slow release of HQ, to overcome the limitations we have described.
Moving towards the distal small intestine
, the pH first falls and then becomes alkaline. In the acid region, diffusion of HQ into the enterocytes is possible, but inhibited while there are still significant concentrations of micelles and monomeric bile salts, as described above. Increasing concentrations of lysolipids can potentially solubilise quercetin, but they must compete with the tendency of quercetin to precipitate. The reservoir of oil is diminished by lipolysis so provides a smaller source of protected quercetin. At the terminal ileum, where the pH is >7.4 [34
], the quercetin will be more soluble but will not pass easily across the mucosal membranes.
Studies on quercetin uptake in different regions of the rat small intestine from fats loaded with quercetin have shown that most quercetin is absorbed in the ileum [14
]. As the uptake media were the same for each section of the intestine, this effect was attributed to the smaller thickness of the mucus layer in the ileum. It would be of interest to measure uptake from media with the different concentrations of lipids appropriate for the different intestinal regions.
Of course, there are other factors that should be taken into account to understand quercetin’s bioavailability. For example, the environment of quercetin in the intestines is altered by the presence of foods. As well as fats, proteins and carbohydrates can alter the kinetics and absorption of plant phenols [7
]. Further, we have not considered the possibilities of endocytosis of insoluble quercetin across the mucosal membranes and the effect of cholesterol, which is present in bile. The colon provides another site for absorption, but efficient metabolism of quercetin by the enterobacteria [36
] will limit the amount that can be absorbed.