Effect of wine on the intestinal transport of 1-methyl-4-phenylpyridinium

The first report on the interactions of polyphenol sources with MPP⁺ uptake by Caco-2 cells tested the effect of intact red or white wine in direct contact with the cell monolayers⁽Reference Monteiro, Calhau and Martel³⁰⁾. As known, wine is produced by the fermentation of crushed grapes from the species Vitis vinifera, and the growing concentration of ethanol during the procedure facilitates the extraction of compounds from the seeds, skins and stems of the fruit⁽Reference German and Walzem⁴⁾. This results in a complex solution where more than 500 different kinds of molecules coexist. Technological variations in the winemaking process allow the production of red wine and white wine from the same grapes. For the latter, grape solids are removed earlier and fermentation goes on only in the presence of the juice. This has great impact on wine composition, mainly on the amount and type of polyphenols present in the final product. Red wine is composed of several different kinds of polyphenols: phenolic acids, stilbenes and flavonoids including flavonols, flavanols and anthocyanins⁽Reference German and Walzem⁴⁾. Since grape seeds and skins are the parts of the fruit with the highest amount of polyphenols, red wine has about six times more polyphenols than white wine (about 1200 and 200 mg/l, respectively)⁽Reference German and Walzem⁴⁾. So, while most polyphenols are usually present in both wines but in lower amounts in white wine (for example, resveratrol, catechins and procyanidins), some others are only found in red wine (for example, some phenolic acids, flavonols and anthocyanins)⁽Reference German and Walzem⁴⁾.

Although the direct treatment of Caco-2 cells with the beverages may be debatable, it is recognised that some wine components may reach the intestinal epithelium intact. On the other hand, treatment with the whole beverages allows the detection of interactions between components of the complex matrix that is wine.

The study of Monteiro et al. ⁽Reference Monteiro, Calhau and Martel³⁰⁾ revealed that red wine induced a concentration-dependent increase in ³H-MPP⁺ uptake into Caco-2 cells. The effect of red wine was partially abolished by the concomitant incubation of the cells with the OCT inhibitor decynium 22⁽Reference Martel, Grundemann and Calhau³⁶⁾. This suggests that there is an involvement of OCT in the effect of red wine upon ³H-MPP⁺ transport, but also that other routes of ³H-MPP⁺ entry into Caco-2 cells may also be affected by red wine. In contrast, white wine caused only a slight decrease in transport ability.

As the two wines tested had approximately the same amount of ethanol (12 %, v/v), it was concluded that the differences between their effects were most likely due to their non-alcoholic components. The fact that red wine has about six times more total polyphenols than white wine, and is about four times richer in high-molecular-weight polyphenols⁽Reference Monteiro, Calhau and Martel³⁰⁾, corroborated this assumption. Incubation of Caco-2 cells with alcohol-free wines or ethanol showed that the effect of alcohol-free red wine was significantly lower than that of red wine, and alcohol-free white wine showed a higher inhibitory potency on ³H-MPP⁺ uptake than white wine. Ethanol, on the other hand, inhibited ³H-MPP⁺ uptake in a concentration-dependent manner. So, the authors concluded that ethanol would exert some other kind of interaction with the remaining wine components, namely a facilitation of compound bioavailability or solubility that could facilitate their effect.

Effect of tea on the intestinal transport of 1-methyl-4-phenylpyridinium

Tea is a beverage prepared by the infusion of leaves of the plant Camellia sinensis. It is largely consumed worldwide, being increasingly related to beneficial health effects⁽Reference Trevisanato and Kim³⁷⁾. Drug interactions have been described after tea intake involving modulation of phase I and II biotransformation enzymes⁽Reference Harris, Jang and Tsunoda¹⁵⁾, and the transport of molecules across cell membranes, namely through P-glycoprotein⁽Reference Jodoin, Demeule and Beliveau³⁸⁾.

Green tea and black tea are produced differently: for the first, leaves are dried only; however, to obtain the second, tea leaves are dried and then allowed to go through a chemical fermentation or oxidation process⁽Reference Trevisanato and Kim³⁷⁾. This reflects on the organoleptic properties of the two beverages and evidently on their chemical composition. Green tea has usually a higher amount of polyphenols (mainly the catechins epigallocatechin-3-gallate (EGCG), gallocatechin, epigallocatechin digallates, epicatechin digallates, 3-O-methyl epicatechin, epigallocatechin (EGC), catechin gallate and gallocatechin gallate) than black tea. Because of oxidation, black tea has a lower catechin level, since these are converted to theaflavins and thearubigins⁽Reference Yang, Maliakal and Meng¹⁴⁾. Tea may also contain flavonoids from other groups, such as myricetin, quercetin and kaempferol and xanthines, especially theophylline and caffeine. Xanthines are more abundant in black tea, whereas green tea is especially rich in EGCG (30 % of dry weight, compared with only 9 % in black tea)⁽Reference Yang, Maliakal and Meng¹⁴⁾.

When testing the effects of different concentrations of green and black tea on ³H-MPP⁺ transport by Caco-2 cells it was found that green tea (0·25 and 0·5 ml/ml) was able to increase by several fold the uptake of ³H-MPP⁺, in a concentration-dependent manner⁽Reference Monteiro, Calhau and Martel²⁹⁾. Moreover, black tea (0·5 ml/ml) also increased ³H-MPP⁺ uptake. However, its effect was significantly lower than that obtained with the same concentration of green tea. In the presence of the OCT inhibitors decynium 22 or corticosterone⁽Reference Martel, Grundemann and Calhau³⁶⁾, the stimulatory effect of tea upon ³H-MPP⁺ transport into Caco-2 monolayers was attenuated, suggesting that uptake of ³H-MPP⁺ may be mediated by both OCT and non-OCT routes.

Effect of isolated polyphenols on the intestinal transport of 1-methyl-4-phenylpyridinium

Grape seed-extracted procyanidins

Procyanidins are a class of flavonoid compounds belonging to the flavan-3-ol subgroup that can be found in several food sources such as tea, apples and cocoa, being especially abundant in red wine. They are polymeric compounds formed of catechin and epicatechin monomers⁽Reference de Pascual-Teresa, Santos-Buelga and Rivas-Gonzalo³⁹⁾. Faria et al. ⁽Reference Faria, Mateus and de Freitas²⁸⁾ studied the effect of a procyanidin extract isolated from grape seeds and of different size fractions of that extract (Table 2). During winemaking, these (and other) compounds are extracted from grape seeds and become soluble in wine⁽Reference Soleas, Diamandis and Goldberg¹¹⁾. The authors began to test the effect of a 60 min incubation period with procyanidins (6, 60 and 600 μg/ml) on ³H-MPP⁺ uptake by Caco-2 monolayers. In the lower concentration, fractions II and III inhibited ³H-MPP⁺ transport. These correspond to the low-molecular-weight flavan-3-ols. In the middle concentration, all fractions inhibited ³H-MPP⁺ uptake except fraction V; however, at the higher concentration, all fractions significantly increased ³H-MPP⁺ uptake, and the effect increased with increasing structural complexity of the fractions. Higher-molecular-weight procyanidins exist in higher concentration in red wine and in much lower amounts in white wine. Therefore, the effects reported by Faria et al. ⁽Reference Faria, Mateus and de Freitas²⁸⁾ are well correlated with those described by Monteiro et al. ⁽Reference Monteiro, Calhau and Martel³⁰⁾ showing opposing effects of red and white wine on ³H-MPP⁺ uptake and strengthen the procyanidin involvement in the effect of wine.

Table 2 Average molecular weights of procyanidins in grape seed fractions, determined by liquid secondary ion MS (adapted from Faria et al. ⁽Reference Faria, Mateus and de Freitas²⁸⁾)

Experiments using the higher concentration of procyanidins, but different incubation periods (3 and 20 min), revealed that the effect of these compounds was also dependent on the time of contact with Caco-2 cells, stimulation of ³H-MPP⁺ uptake being more prominent for the longer incubation time (60 min). Since procyanidins, as good reducing and, therefore, antioxidant agents, are readily oxidisable due to their o-dihydroxyl groups⁽Reference Jovanovic, Steinken and Simic⁴⁰⁾, it was speculated that for the higher incubation time, oxidation of these compounds could change their interaction with or the state of the transporters involved in ³H-MPP⁺ uptake. This hypothesis was confirmed by examining the effect of cell exposure to the oxidised procyanidins. Indeed, oxidised procyanidins tested for 3 min increased ³H-MPP⁺ uptake to a level that was similar to the one found with non-oxidised procyanidins tested for 60 min. Furthermore, incubation for 60 min with oxidised procyanidins resulted in a significantly more pronounced stimulation of ³H-MPP⁺ entry into the cells. To confirm the cellular redox state involvement, both the effects of oxidant and reducing agents on the transport were tested⁽Reference D'Souza, Buckley and Buckley⁴¹⁾. The results obtained with these compounds confirmed that intra- and extracellular oxidation status interferes with the transport. Three possible mechanisms which relate the redox state and OCT regulation have already been previously suggested: (a) changes in transporter affinity for the substrate; (b) changes in interaction of the transporter with the cytoskeleton, which will influence the number of membrane-located transporters; (c) regulation at the gene expression level⁽Reference Ciarimboli and Schlatter⁴²⁾. It is possible that the oxidation level of cysteine residues would alter the occurrence of phosphorylation and dephosphorylation reactions. This attractive hypothesis may indeed constitute the mechanism underlying the effects of procyanidins, because intestinal OC transport has been shown to be regulated by phosphorylation and dephosphorylation mechanisms⁽Reference Martel, Keating and Calhau^43–Reference Calhau, Martel and Soares-da-Silva⁴⁷⁾, and polyphenols are known regulators of intracellular kinases and phosphatases⁽Reference Middleton, Kandaswami and Theoharides⁵⁾.

Anthocyanins and derivative pigments

Anthocyanins are present in flowers, fruits and other vegetables, being an important group of plant pigments⁽Reference Manach, Scalbert and Morand^2, Reference Bravo³⁾. They exist in high amounts in blueberries (Vaccinium myrtillus), having been related to their health-promoting properties⁽Reference Santos-Buelga and Scalbert⁴⁸⁾.

Although growing scientific evidence of anthocyanin bioavailability is being gathered⁽Reference Manach, Williamson and Morand²⁵⁾, few studies have been devoted to the investigation of their possible interference with the absorption of other substrates at the intestinal level. Anthocyanins are glycosides, with the sugar moiety most commonly associated with the 3-position on the C-ring or the 5, 7-position on the A-ring (Fig. 2(a)). Glucose, galactose, arabinose, rhamnose and xylose are the most usually found sugars and can be bound as mono-, di- or tri-saccharide forms. Although there are about seventeen aglycone forms, or anthocyanidins, the most abundant and ubiquitously distributed are delphinidin, petunidin, peonidin, pelargonidin and malvidin. Apart from these more abundant compounds, some anthocyanin derivatives, such as anthocyanin pyruvic acid adducts and vinylpyranoanthocyanin-catechins (portisins), have been identified in low amounts in wine (Fig. 2(b) and (c)). These are more stable than their precursor anthocyanins and display unusual colours (orange and blue), being under investigation for their possible application as natural food dyes. Faria et al. ⁽Reference Faria, Oliveira and Neves⁴⁹⁾ isolated anthocyanins from blueberries and characterised the obtained extract (extract I). This was composed mainly of the anthocyanins delphinidin, cyanidin, malvidin, petunidin and peonidin glucosides. This extract was used to synthesise anthocyanin pyruvic acid adducts (extract II) by reaction of the extract with pyruvic acid and vinylpryranoanthocyanin-catechins (portisins, extract III) after the reaction of anthocyanin pyruvic acid with catechin and acetaldehyde. In a subsequent study⁽Reference Faria, Pestana and Monteiro³¹⁾, the ability of both the original blueberry and the derivate extracts to influence ³H-MPP⁺ apical intestinal uptake was studied, by incubating Caco-2 cells with 100 μg/ml of each extract for 60 min. Of the three tested extracts, only extract II had a significant effect, decreasing ³H-MPP⁺ uptake, an effect which showed concentration dependency. As there are no reports on the ability of OCT to transport anthocyanins or their derivatives, the possibility of an allosteric regulation or interference with the regulation of these transporters should not be excluded. Since molecular weight increases from extract I to III, size was thought not to be relevant to explain why only extract II showed an effect. On the other hand, it was advanced that the presence of a carboxyl group on the D ring of extract II components might play an important role and this was further explored by testing the effect of phenolic acids on the transport (see below).

Fig. 2 General structure of blueberry (Vaccinium myrtillus) anthocyanidin (a), anthocyanidin–pyruvic acid adducts (b) and portisins (c) present in extracts I, II and III, respectively. R1 and R2, independently of each other, are H, OH or O-methyl. R3 is glucose, galactose or arabinose.

Phenolic acids

The effect of four phenolic acids on the intestinal transport of MPP⁺ was tested by Faria et al. ⁽Reference Faria, Pestana and Monteiro³¹⁾. p-Coumaric, caffeic, ferulic, gallic and tannic acids (Fig. 3) were tested at 250 μg/ml for 60 min. Whereas gallic acid had no effect on the transport, the other phenolic acids induced a decrease in cellular ³H-MPP⁺ uptake. The authors suggested that the presence of a vinylphenolic group, with a possible high electronic conjugation, was likely to play a role. They also advanced the hypothesis that the effects observed resulted from ionic interactions between ³H-MPP⁺ and the dissociated acid moieties. As opposed to the other phenolic acids, the structurally more complex compound, tannic acid, increased MPP⁺ uptake by Caco-2 cells. Because this effect more closely resembles the one obtained with more complex flavonoids, such as procyanidins, than the ones obtained in the presence of other phenolic acids and monomeric compounds, molecular size was suggested to contribute to the effects.

Fig. 3 Structures of some phenolic acids. OMe, O-methyl.

Effect of isolated polyphenols on the intestinal transport of thiamin

There is no evidence that phenolic compounds affect thiamin transport into Caco-2 cells⁽Reference Lemos, Calhau and Martel⁵⁷⁾. However, non-alcoholic beverages such as green and black tea, as well as alcohol-free beer and alcohol-free red and white wines, were able to inhibit thiamin transport into Caco-2 cells, when tested acutely. In these same cells, the effect of some alcoholic beverages was tested (red and white wines and lager and stout beer), and the results were very curious. Only lager and stout beers inhibited transport; red and white wines had no effect. In agreement with these results, the inhibitory effect of alcohol-free wines seems to be abolished when ethanol (in the same concentration as that found in intact wines) was added. So, the biological activity of phenolic compounds, as modulators of thiamin uptake, depends on the presence or absence of ethanol. Accordingly with this conclusion, a recent study showed a crucial contribution of ethanol to phenolic bioavailability and/or bioactivity⁽Reference Faria, Pestana and Azevedo⁵⁸⁾.

An important point relates to the effect of xanthohumol. Although xanthohumol had no effect upon thiamin uptake into Caco-2 cells, lager and stout beers (which are both rich in this compound) inhibited its uptake. Additionally, stout beer, a chalcone-enriched beer, was more potent than lager beer. So, it is possible that the phenolic compounds xanthohumol and isoxanthohumol can explain, at least in part, the inhibitory effect found with the beers, as it happens with placental thiamin uptake⁽Reference Keating, Lemos and Azevedo⁵⁹⁾. If this is true, then (as it happens with red wine) the original matrix is important for the biological activity of these compounds.

Part of the results obtained with phenolic compounds in Caco-2 cells⁽Reference Lemos, Calhau and Martel⁵⁷⁾ was also confirmed in the rat. In these experiments, rats consumed red wine for 21 d and, at the end of this period, jejunal thiamin transport was evaluated in Ussing chambers, which allowed measurement of the mucosal-to-serosal apparent permeability to [³H]thiamin⁽Reference Lemos, Azevedo and Martel⁶⁰⁾. The results showed that a chronic consumption of red wine had no effect on thiamin absorption. However, the acute exposure of the rat jejunum to red wine was able to inhibit thiamin absorption, a result apparently contradictory to those observed with Caco-2 cells⁽Reference Lemos, Calhau and Martel⁵⁷⁾. However, two important reasons might explain the discrepancies found: (i) different species were studied (rat v. a human cell line; Caco-2 cells); (ii) different parameters were analysed (intestinal absorption in the rat v. uptake in Caco-2 cells).

Effect of isolated polyphenols on the placental transport of thiamin

As already stated, thiamin is crucial during pregnancy for the normal growth and development of the fetus. As needs for this vitamin increase during pregnancy, the modulation of thiamin transport through trophoblast epithelia constitutes a key point. Thus, any influence on this transport has consequences for fetal development. Indeed, the association between alcoholic abuse and thiamin deficiency is well known⁽Reference Hoyumpa^61, Reference Hoyumpa, Breen and Schenker⁶²⁾, and thus it is possible that deficiency of this vitamin during pregnancy contributes, at least in part, to the developmental abnormalities observed in the fetal alcohol syndrome.

Considering this hot point, Keating et al. ⁽Reference Keating, Lemos and Azevedo⁵⁹⁾ studied the short- and long-term effects of several phenolic compounds on the apical uptake of [³H]thiamin by BeWo cells. These cells are a human choriocarcinoma cell line, commonly used, and well characterised, as a trophoblast cell model. In the short term, no effect of xanthohumol, isoxanthohumol, catechin, epicatechin, resveratrol, quercetin, myricetin, EGCG, rutin and chrysin was found. In the long term (48 h), treatment with xanthohumol or isoxanthohumol significantly decreased thiamin uptake by these cells, but other compounds had no effect. Moreover, the inhibitory effect of xanthohumol and isoxanthohumol was not related to changes in mRNA levels for the thiamin transporters ThTr1 and ThTr2, as evaluated by RT-PCR. Also, these compounds had no effect on human SERT mRNA levels. Human SERT was suggested to be involved in the uptake of thiamin by these cells⁽Reference Keating, Lemos and Azevedo⁵⁹⁾. So, these effects are most probably not related to changes at the transcriptional level.

Altogether, it was found that chalcones, a class of phenolics found especially in beer, chronically inhibit the transport of thiamin. However, isoflavones, present in soya and in several functional foods such as several beverages and yogurts, were not tested. Considering that human SERT seems to be involved in thiamin uptake by BeWo cells, and that soya affects SERT activity⁽Reference Keating, Lemos and Azevedo⁵⁹⁾, it would be interesting to determine the effect of isoflavones upon thiamin placental transport. Indeed, Ito et al. ⁽Reference Ito, Haito and Furumoto⁶³⁾ described an effect of beverages such as St John's wort (which is rich in phenolic compounds) on SERT activity that explains, at least in part, its psychopharmacological effects.

In relation to the results described by Keating et al. ⁽Reference Keating, Lemos and Azevedo⁵⁹⁾ with chalcones, the mechanisms involved in this inhibitory action remain unknown. However, as is known, SERT is inhibited by phosphorylation pathways⁽Reference Kramer, Poblete and Azmitia⁶⁴⁾, and several phenolic compounds interact with the activity of kinases and phosphatases⁽Reference Said, Ortiz and Kumar^56, Reference Kumar, Yanagawa and Ortiz⁶⁵⁾. Thus, it is possible that some phenolic compounds could interfere with transport activities through an indirect effect upon, for instance, phosphorylation and dephosphorylation mechanisms.

Effect of polyphenol-rich drinks and isolated polyphenols on the intestinal transport of folic acid

Because man cannot synthesise FA, this vitamin must be obtained from exogenous sources through intestinal absorption. Therefore, the intestine plays a central role in controlling and regulating FA body homeostasis, and any impairment in FA intestinal absorption may induce a whole-body deficiency state.

Effect upon [³H]folic acid apical uptake by Caco-2 cells

The effect of polyphenolic compounds and some polyphenol-rich drinks (red and white wine, beer, tea and orange juice) upon the intestinal uptake of FA was also investigated by analysing their effect upon ³H-FA or [³H]methotrexate (³H-MTX; an anti-folate) uptake by Caco-2 cells⁽Reference Lemos, Peters and Jansen⁷⁸⁾.

Interestingly enough, all the tested beers (lager, stout and alcohol-free beer), green tea, black tea and orange juice (0·25 and 0·5 ml/ml) significantly inhibited ³H-FA and ³H-MTX apical uptake by Caco-2 cells. Moreover, both red and white wine (0·25 and 0·5 ml/ml) also significantly inhibited the apical uptake of ³H-FA by Caco-2 cells, red wine being more potent than white wine, and the same degree of inhibition was obtained with the alcohol-free wines. On the other hand, ethanol in the same concentration as that present in the red and white wine did also reduce ³H-FA apical uptake, but much less potently⁽Reference Lemos, Peters and Jansen⁷⁸⁾.

Because (1) ethanol had a much more discrete effect than the alcoholic drinks (wines and beers) upon ³H-FA uptake, and (2) alcohol-free drinks (red and white wine and beer) had almost the same effect as wine and beer, other components of these beverages must play a role in their inhibitory effect upon ³H-FA uptake. So, polyphenolic compounds present in all these drinks appear to have an inhibitory effect upon the intestinal absorption of this vitamin. This was confirmed by analysing the effect of some polyphenolic compounds known to be present in wines, beers and/or teas, in these same cells⁽Reference Lemos, Peters and Jansen⁷⁸⁾. When tested acutely, myricetin, EGCG and isoxanthohumol concentration-dependently inhibited ³H-FA uptake (50 % inhibitory concentration (IC₅₀) values of 13, 8 and 36 μm, respectively). Myricetin and EGCG also had a concentration-dependent inhibitory effect upon ³H-MTX uptake (IC₅₀ values of 11 and 10 μm, respectively) (isoxanthohumol was not tested). Other polyphenolic compounds (xanthohumol, resveratrol, quercetin and kaempferol) were found to moderately (20–50 %) inhibit the uptake of ³H-FA and/or ³H-MTX, but only when tested in a high (100 μm) concentration. Moreover, a long-term (2 d) exposure of the cells to isoxanthohumol resulted in inhibition of ³H-FA uptake; the other polyphenols were devoid of effect. Importantly, none of these compounds had a cytotoxic effect at concentrations that modulated ³H-FA and ³H-MTX uptake.

So, these results showed that (1) some polyphenolic compounds were able to significantly inhibit ³H-FA and ³H-MTX uptake by Caco-2 cells, and that (2) these phenolic compounds, when tested for a long period, lose, at least in part, their ability to reduce the apical uptake of ³H-FA in Caco-2 cells. The decrease in the inhibitory effect of polyphenols by long-term exposure to these compounds might result from an increased expression of FA transporter(s), resulting from FA depletion caused by acute exposure to phenolic compounds. This increased expression of FA transporters could compensate for, in long-term treatments, the effect of phenolic compounds.

Interestingly enough, all the compounds that reduced the uptake of ³H-FA and ³H-MTX by Caco-2 cells (the stilbene resveratrol and the flavonols quercetin, myricetin, kaempferol) are very abundant in wines, where they are present in concentrations ranging from 1 μm to more than 300 μm⁽Reference German and Walzem⁴⁾. So, it is possible that the phenolic compounds present in wines are, at least in part, responsible for the inhibitory effect of these beverages upon the intestinal uptake of ³H-FA. This hypothesis is supported by the fact that red wine, that has a much higher content of phenolic compounds than white wine (Table 3), also showed a more potent inhibitory effect than white wine.

Table 3 Total phenolic content of studied beverages (from Lemos et al. ⁽Reference Lemos, Peters and Jansen⁷⁸⁾) (Mean values (n 4) with their standard errors)

On the other hand, because beer constitutes the main dietary source of xanthohumol and other prenylflavonoids such as isoxanthohumol⁽Reference Stevens and Page⁷⁹⁾, and both xanthohumol and isoxanthohumol inhibited ³H-FA uptake, these compounds may be responsible for the inhibitory effect of beers on the intestinal uptake of this vitamin.

As to the inhibitory effect of green and black teas upon both ³H-FA and ³H-MTX by Caco-2 cells, the effect of both teas is possibly due to different polyphenols present in these beverages (see above). Thus, the effect of green tea might be explained by its high content of EGCG⁽Reference Mukhtar and Ahmad¹³⁾, which was one of the most potent inhibitors of the uptake of both ³H-FA and ³H-MTX. Other phenolic compounds such as myricetin, quercetin and kaempferol can also be found in teas⁽Reference Yang, Maliakal and Meng¹⁴⁾ and may also contribute to inhibition of ³H-FA and ³H-MTX uptake. In agreement with the results from Lemos et al. ⁽Reference Lemos, Peters and Jansen⁷⁸⁾, green and black tea extracts, as well as two catechins contained in green tea (EGCG and epicatechin gallate (ECG)), were also found to inhibit FA uptake by Caco-2 cells⁽Reference Alemdaroglu, Wolffram and Boissel⁸⁰⁾.

The difference in the effect of red wine in relation to ³H-FA uptake in Caco-2 cells (inhibition)⁽Reference Lemos, Peters and Jansen⁷⁸⁾ and rat jejunum (no effect)⁽Reference Lemos, Azevedo and Martel⁶⁰⁾ can be explained by, at least, two important differences. First, the duration of the treatment with red wine is different in the two studies (a 48 h in vitro exposure of Caco-2 cells to red wine v. a 21 d in vivo ingestion of red wine). Second, the concentration of red wine in direct contact with the cells or tissue is also different (being higher in the experiments with Caco-2 cells). Finally, it should be noted that the process of digestion alters significantly the composition of red wine, and that this digestion was not assumed in the study by Lemos et al. ⁽Reference Lemos, Peters and Jansen⁷⁸⁾.

As to the nature of the transport mechanism modulated by polyphenols, the observation that ³H-FA and ³H-MTX uptakes in Caco-2 cells were similarly modulated by most of the beverages and phenolic compounds suggests that these compounds share the same transport system in these cells. It is known that MTX and natural reduced folates are substrates of the reduced folate carrier (RFC)⁽Reference Jansen and Jackman^81, Reference Matherly and Goldman⁸²⁾. However, it is not clear whether RFC is the major transporter involved in the intestinal transport of FA. Although many studies suggest a role for RFC in the transport of folates in intestinal cells⁽Reference Balamurugan and Said^83–Reference Wang, Zhao and Russell⁸⁷⁾, recent evidence suggests the involvement of a proton-coupled FA transporter (PCFT). Interestingly enough, PCFT was recently identified as the solute carrier family 46, member 1 (SLC46A1)⁽Reference Qiu, Jansen and Sakaris⁸⁸⁾. So, the effect of polyphenols on FA uptake by Caco-2 may result from an inhibitory effect of these compounds upon RFC and/or PCFT.

In conclusion, the results obtained by Lemos et al. ⁽Reference Lemos, Peters and Jansen⁷⁸⁾ suggest that the effect of several polyphenolic-rich beverages (red and white wine, beer, black and green tea and orange juice), significantly decreasing FA and MTX uptake, can be justified, at least in part, by the effect of their phenolic compounds. So, dietary habits, especially those related to the consumption of polyphenol-containing beverages or phenolic compounds, can modulate the intestinal uptake of both ³H-FA and ³H-MTX. Importantly, they may reduce the therapeutic efficacy of MTX in patients taking it by the oral route. Finally, these results suggest that, in human alcoholism, FA deficiency can result, at least partially, from a decrease in its intestinal absorption.

Effect of isolated polyphenols on the placental uptake of folic acid

Folate is also critically important for normal fetal development, as demonstrated by the well-established association between maternal FA deficiency and pregnancy complications such as pre-eclampsia⁽Reference Lucock^67, Reference Scholl and Johnson⁸⁹⁾ and a higher incidence of fetal neural tube defects⁽Reference Lucock⁶⁷⁾. In line with this, periconceptional supplementation with FA is now widely accepted as a strategy for reducing the risk of neural tube defects⁽Reference Wald^90, Reference Worthington-Roberts, Cohen, Cherry and Merkatz⁹¹⁾.

Using the BeWo choriocarcinoma cell line, Keating et al. ⁽Reference Keating, Lemos and Goncalves⁹²⁾ characterised the effect of several distinct polyphenolic compounds, present in alcoholic and non-alcoholic drinks, upon the placental uptake of FA. Both the short-term (26 min) and long-term (48 h) effect of several compounds (catechin, chrysin, epicatechin, EGCG, isoxanthohumol, myricetin, quercetin, resveratrol, rutin and xanthohumol) upon the uptake of ³H-FA by BeWo cells was determined⁽Reference Keating, Lemos and Goncalves⁹²⁾.

Interestingly enough, ³H-FA apical uptake by BeWo cells was found to be modulated by several dietary bioactive compounds. In the short term, epicatechin and isoxanthohumol inhibited ³H-FA uptake. Interestingly, the maximum effect was quantitatively similar for the two compounds (about 30 % inhibition). Isoxanthohumol seemed to act as a competitive inhibitor, whereas epicatechin caused an increase in both K _m and V _max. The authors hypothesised that epicatechin binds to an allosteric site of the transporter and induces an alteration in the conformation of the active site, thus reducing the affinity for the substrate (increasing K _m) and that, simultaneously, this binding increases the transporter's capacity (increasing V _max) for high concentrations of the substrate.

In the long term, ³H-FA apical uptake by BeWo cells was significantly increased by exposure to xanthohumol, quercetin and isoxanthohumol. At physiological pH, ³H-FA apical uptake by BeWo cells seems to involve both RFC and folate receptor (FR) α⁽Reference Keating, Lemos and Azevedo⁹³⁾. However, the increase in ³H-FA uptake caused by a long-term exposure of BeWo cells to the polyphenols was not accompanied by a change in RFC or FRα mRNA levels. So, the effect of the polyphenols does not seem to result from a modulation of the expression levels of these transporters. Instead, it may otherwise be a result of a direct interaction of the polyphenols with the transporter(s), with a consequent change in the activity of the latter⁽Reference Keating, Lemos and Goncalves⁹²⁾.

The cytotoxic effect as an explanation of the observed effect of polyphenolic compounds upon ³H-FA uptake was excluded. Moreover, in order to assess the specificity of the effect of polyphenolic compounds, the effect of these compounds upon the uptake of [¹⁴C]alanine was also assessed. Interestingly, none of these compounds had any significant effect upon uptake of this compound, except isoxanthohumol, which concentration-dependently and completely reduced uptake of this compound.

In summary, these results suggest a detrimental effect of short-term exposure to epicatechin and isoxanthohumol on placental FA absorption and, on the other hand, a beneficial effect of a long-term exposure to xanthohumol, isoxanthohumol and quercetin on FA absorption at the placental level⁽Reference Keating, Lemos and Goncalves⁹²⁾. Finally, these results also show that, because short- and long-term treatments with these dietary bioactive compounds did not produce parallel results, care should be taken when speculating about chronic effects from acute effects and vice versa⁽Reference Keating, Lemos and Goncalves⁹²⁾.

Effect of isolated polyphenols on the intestinal transport of glucose

Recent studies have suggested that ordinary portions of certain beverages rich in dietary phenols may result in an altered pattern of intestinal glucose uptake. However, this suggestion was based on the in vivo effect of beverages or plant extracts upon glycaemia or glucose tolerance⁽Reference Andrade-Cetto and Wiedenfeld^94–Reference Matsumoto, Ishigaki and Ishigaki⁹⁸⁾, rather than on the direct effect of polyphenolic compounds upon the intestinal absorption of glucose. This latter subject was, however, also investigated in recent years, as shown next.

According to the ‘classical model of sugar absorption’, glucose is actively taken up into the enterocytes from the intestinal lumen by the high-affinity, Na⁺-dependent and phloridzin-sensitive Na⁺/glucose co-transporter 1 (SGLT1) located in the brush border and is then passively released from the enterocytes into the circulation via the Na⁺-independent GLUT2 present in the basolateral membrane (for reviews, see Drozdowski & Thompson⁽Reference Drozdowski and Thompson⁹⁹⁾, Wright et al. ⁽Reference Wright, Martín and Turk¹⁰⁰⁾ and Kellett & Brot-Laroche⁽Reference Kellett and Brot-Laroche¹⁰¹⁾).

Several investigators suggested that some polyphenols decrease SGLT1-mediated glucose uptake. This conclusion was based on experiments using intestinal cells, brush-border membrane vesicles, or SGLT1-expressing Xenopus laevis oocytes⁽Reference Aoshima, Okita and Hossain^102–Reference Wolffram, Block and Ader¹⁰⁹⁾.

Green tea flavonoids were found to inhibit the transport activity of SGLT1. While several flavonoids in green tea were active, this inhibitory activity was most pronounced for (+)-catechin and catechins having galloyl residues such as EGC and EGCG⁽Reference Hossain, Kato and Aoshima^104, Reference Kobayashi, Suzuki and Satsu^106, Reference Shimizu, Kobayashi and Suzuki¹¹⁰⁾. According to Hossain et al. ⁽Reference Hossain, Kato and Aoshima¹⁰⁴⁾, inhibition of SGLT1 by (+)-catechin, ECG and EGCG was independent of glucose concentration, suggesting a non-competitive inhibition mechanism. However, Kobayashi et al. ⁽Reference Kobayashi, Suzuki and Satsu¹⁰⁶⁾ proposed a competitive inhibition mechanism for ECG in relation to SGLT1, although ECG itself is not transported via SGLT1. Because (+)-catechin inhibited glucose uptake weakly, it was concluded that probably it does not suppress glucose uptake in the small intestine under physiological conditions⁽Reference Hossain, Kato and Aoshima^104, Reference Kobayashi, Suzuki and Satsu¹⁰⁶⁾. Tea catechin derivatives have also been reported to inhibit intestinal α-amylase or sucrase, which may be the main mechanism for the suppression of plasma glucose increase after a meal⁽Reference Matsui, Tanaka and Tamura¹¹¹⁾. Nevertheless, a crude extract of tea was shown to inhibit the intestinal absorption of glucose and Na⁺ in rats, although the relative contribution of the individual components involved was not determined⁽Reference Kreydiyyeh, Baydoun and Churukian¹¹²⁾. Additionally, an instant tea preparation administered with a bolus of glucose was postulated to have similar effects in healthy human subjects⁽Reference Bryans, Judd and Ellis¹¹³⁾. Thus, these catechin derivatives present in teas, wine or cocoa act possibly not only as antioxidants but also as inhibitors of glucose uptake in the small intestine, which may be helpful to diabetic patients.

Besides catechins, other polyphenolic compounds were also found to affect the intestinal absorption of glucose. Quercetin-3-O-glucoside inhibited SGLT1, apparently in a competitive manner⁽Reference Cermak, Landgraf and Wolffram^103, Reference Wolffram, Block and Ader^109, Reference Ader, Block and Pietzsch¹¹⁴⁾. Quercetin-4-O-glucoside also inhibited SGLT1, but quercetin-3-O-galactoside, quercetin-3-O-glucorhamnoside (rutin) and the aglycone quercetin were devoid of effect⁽Reference Cermak, Landgraf and Wolffram^103, Reference Ader, Block and Pietzsch¹¹⁴⁾. Glycosides of some other flavonoid classes, such as naringenin-7-O-glucoside, genistein-7-O-glucoside and cyanidin-3,5-O-diglucoside, were ineffective as well⁽Reference Cermak, Landgraf and Wolffram¹⁰³⁾. According to some authors, quercetin glucosides such as quercetin-4-glucoside (the major dietary form of quercetin) are absorbed within the intestine by the active glucose transporter SGLT1, thus being an SGLT1 substrate⁽Reference Walgren, Lin and Kinne^108, Reference Gee, DuPont and Rhodes^115, Reference Hollman, de Vries and van Leeuwen¹¹⁶⁾. However, other studies have concluded that neither quercetin nor any of its glycosylated derivatives are transported by SGLT1⁽Reference Kottra and Daniel¹¹⁷⁾.

Emerging evidence indicates that besides SGLT1, there is also an involvement of the facilitated glucose transporter GLUT2 in the intestinal absorption of glucose⁽Reference Kellett and Brot-Laroche^101, Reference Keating, Goncalves and Lemos^118, Reference Kellett and Helliwell¹¹⁹⁾. It is interesting to note that recent animal studies have suggested that luminal-facing GLUT2 is responsible for a large proportion of glucose uptake from the lumen of the small intestine. Being a major pathway of sugar absorption, GLUT2 is therefore an attractive target of potential agents⁽Reference Kellett and Brot-Laroche^101, Reference Keating, Goncalves and Lemos^118, Reference Kellett and Helliwell¹¹⁹⁾.

Interestingly enough, some polyphenols were also found to interact with this transporter. Quercetin, quercetin-3-O-glucoside, ECG, fisetin, myricetin and gossypin decreased GLUT2-mediated Na⁺-independent diffusive uptake of glucose⁽Reference Cermak, Landgraf and Wolffram^103, Reference Kwon, Eck and Chen^120, Reference Chen, Hsu and Huang¹²¹⁾. In relation to quercetin, its effect upon glucose uptake seems to be GLUT2-specific, because it does not interact at all with either SGLT1 or GLUT5⁽Reference Kwon, Eck and Chen^120, Reference Song, Kwon and Chen¹²²⁾, although it does not appear to be a GLUT2 substrate⁽Reference Kwon, Eck and Chen¹²⁰⁾. In agreement with this observation, Song et al. ⁽Reference Song, Kwon and Chen¹²²⁾ also reported that quercetin was a potent non-competitive inhibitor of GLUT2 expressed in Xenopus oocytes (K _i of 23 μm). In contrast, quercetin-3-O-glucoside and ECG seem to be competitive inhibitors of GLUT2-mediated glucose transport⁽Reference Chen, Hsu and Huang¹²¹⁾. Importantly, when diabetic rats were administered glucose together with quercetin, hyperglycaemia was significantly decreased compared with administration of glucose alone⁽Reference Song, Kwon and Chen¹²²⁾. Because the flavonoid quercetin, a food component, might act as a potent luminal inhibitor of sugar absorption independent of its own transport, flavonols show promise as new pharmacological agents in the obesity and diabetes epidemic⁽Reference Kwon, Eck and Chen¹²⁰⁾. Finally, exposure of Caco-2 cells for 48 h to some anthocyanins results in an increase in their own transport and in GLUT2 expression⁽Reference Faria, Pestana and Azevedo⁵⁸⁾, pointing to the possibility that these compounds are transported via GLUT2.

Some polyphenolic compounds have also been shown to be transported by other mechanisms, at the intestinal level. Indeed, quercetin-4-glucoside was found to be removed from Caco-2 cells by the apically expressed multidrug resistance-associated protein (MRP2), this process being able to decrease the net intestinal absorption of this compound⁽Reference Walgren, Karnaky and Lindenmayer^107, Reference Walgren, Lin and Kinne¹⁰⁸⁾.

In a recent study, the effect of different classes of dietary polyphenols upon the intestinal uptake of glucose was investigated using Caco-2 cells⁽Reference Johnston, Sharp and Clifford¹⁰⁵⁾. Glucose uptake into cells under Na⁺-dependent conditions was inhibited by non-glycosylated polyphenols ((+)-catechin, ( − )-epicatechin, EGCG, EGC and ECG) whereas aglycones (quercetin, apigenin and myricetin), glycosides and phenolic acids (caffeic, ferulic and chlorogenic acids) were without effect. Under Na⁺-free conditions, aglycones (quercetin, apigenin and myricetin) and non-glycosylated polyphenols (EGCG, EGC and ECG) inhibited glucose uptake whereas glycosides and phenolic acids were ineffective. These data suggest that aglycones inhibit GLUT2-mediated uptake and that the non-glycosylated dietary polyphenols EGCG, ECG and EGC are effective against both GLUT2 and SGLT1.

The lack of effect of dietary glycosides (such as naringin, rutin and arbutin) upon both Na⁺-independent (i.e. GLUT-mediated) and Na⁺-dependent (i.e. SGLT1-mediated) uptake of glucose by Caco-2 cells⁽Reference Johnston, Sharp and Clifford¹⁰⁵⁾ is in agreement with previous results⁽Reference Ader, Block and Pietzsch¹¹⁴⁾. However, the glycoside arbutin is transported by SGLT1 in hamster tissue⁽Reference Alvarado and Crane¹²³⁾ and in Xenopus oocytes⁽Reference Lostao, Hirayama and Loo¹²⁴⁾, and its consumption has been associated with ‘arbutin diabetes’⁽Reference Michel¹²⁵⁾. The lack of effect of the phenolic acids on glucose uptake under either Na⁺-dependent or Na⁺-free conditions⁽Reference Johnston, Sharp and Clifford¹⁰⁵⁾ is in contrast with the study by Welsch et al. ⁽Reference Welsch, Lachance and Wasserman¹²⁶⁾ showing that caffeic, ferulic and chlorogenic acids caused an inhibition of Na⁺-dependent glucose uptake by rat brush-border membrane vesicles. The discrepancy in these observations may be related to the differences in the ratio of test substance to substrate⁽Reference Johnston, Sharp and Clifford¹⁰⁵⁾. Still according to these authors, the in vivo anti-hyperglycaemic effect of caffeic acid or chlorogenic acid extracts⁽Reference Andrade-Cetto and Wiedenfeld^94, Reference Hsu, Chen and Cheng⁹⁵⁾ is most probably the result of a direct action on peripheral tissues rather than the result of a blockade of glucose uptake across the intestinal brush-border membrane⁽Reference Johnston, Sharp and Clifford¹⁰⁵⁾. Finally, the results of Johnston et al. ⁽Reference Johnston, Sharp and Clifford¹⁰⁵⁾ concerning the effect of the non-glycosylated polyphenols are in perfect agreement with previous studies, either in vivo or in vitro (see above).

Still according to Johnston et al. ⁽Reference Johnston, Sharp and Clifford¹⁰⁵⁾, the effects of EGCG, ECG and EGC are likely to be the result of steric hindrance caused by incorporation into the membrane with subsequent disruption of the surrounding lipid bilayer, as shown previously by Hossain et al. ⁽Reference Hossain, Kato and Aoshima¹⁰⁴⁾ using transfected Xenopus oocytes as an expression vector.

In conclusion, recent in vitro evidence suggests that there is the potential for a variety of classes of dietary polyphenols to affect intestinal glucose transport in vivo mediated by both SGLT1 and GLUT2 simultaneously. Furthermore, these data suggest that foods and unsweetened beverages rich in these dietary polyphenols might provide a convenient dietary mechanism for regulating the rate of intestinal sugar absorption, an important factor in the management of diabetes, and in the long term might offer some protection against development of the metabolic syndrome or type 2 diabetes.

Effect of isolated polyphenols on the placental transport of glucose

Glucose is essential for the developing fetus, serving as the primary source of energy for metabolism and growth of the feto–placental unit. Because the fetus cannot synthesise it in the amounts required for its optimal development, it must obtain glucose from the maternal circulation. So, the supply of glucose from maternal blood to fetal circulation represents a major determinant of fetal growth and development⁽Reference Battaglia and Meschia^127, Reference Harding and Johnston¹²⁸⁾. Glucose supply to the fetus is mediated by members of the GLUT family of transporters⁽Reference Bissonnette, Black and Wickham^129–Reference Johnson and Smith¹³¹⁾, GLUT1 being the predominant glucose transporter expressed at the placental level⁽Reference Barros, Yudilevich and Jarvis^132–Reference Takata, Kasahara and Kasahara¹³⁴⁾.

The effect of several dietary polyphenols upon the placental transport of glucose was recently studied⁽Reference Araújo, Gonçalves and Azevedo^135–Reference Araújo, Gonçalves and Martel¹³⁷⁾. By using [³H]deoxy-d-glucose (³H-DG), a glucose analogue which is efficiently transported by GLUT family members, several polyphenolic compounds were found to affect the apical uptake of ³H-DG into BeWo cells. When tested in the short term (26 min), resveratrol, EGCG and xanthohumol reduced ³H-DG uptake. Moreover, chrysin and quercetin decreased ³H-DG uptake in a concentration-dependent manner, causing a maximal reduction in ³H-DG uptake to 43 and 79 % of control, respectively. On the other hand, rutin, catechin and epicatechin increased ³H-DG uptake. It was also found that both quercetin and xanthohumol seemed to act as non-competitive inhibitors of ³H-DG apical uptake, whereas EGCG decreased both the K _m and V _max values. The effect of some polyphenolic compounds in association was also tested. Interestingly enough, catechin and epicatechin together decreased the apical uptake of ³H-DG, whereas epicatechin and xanthohumol counterbalanced each one's isolated effect. When tested in a long-term exposure (48 h), rutin and myricetin increased the apical uptake of ³H-DG both isolated and in combination⁽Reference Araújo, Gonçalves and Azevedo^135–Reference Araújo, Gonçalves and Martel¹³⁷⁾.