Proyección de la demanda

Several reviews have been published on the bioavailability of biofl avonoids and its implication for their physiological functioning (D’Archivio et al., 2007; Prasain and Barnes, 2007; Scalbert and Willamson, 2000; Singh et al., 2008; Willamson and Manach, 2005; Yang et al., 2008). The term bioavailability indicates the extent of utilization of orally taken food ingredients or drugs in the body (Stahl et al., 2002), but the values are dealt with differently among researchers in different fi elds. In the nutritional research fi eld, bioavailability has simply been evaluated from the recovered amount of ingested molecules in the urine. In the pharmacokinetic research fi eld, however, the term is defi ned as the rate of pharmacological utilization of a dose of drugs in the body after adminis- tration. Orally administered drugs usually enter the body through several paths, known as ADME (absorption from gastrointestinal tract, distribution into plasma and tissues, phase I and phase II metabolism in tissues, and excretion into urine or feces). To evaluate the effi cacy of utilization of an administered dose of any particular drug, bioavailability is evaluated by a comparison of the area under the plasma concentration curves (AUC) obtained for intravenous and orally administered drugs, respectively (Ichiyanagi et al., 2006a). A drug which is administered intravenously skips gastrointestinal absorption and metabolism processes, but follows the same liver metabolism and tissue distribution processes as an orally administered drug. Thus, it might be more useful to calcu- late the overall utilization profi le in the body after oral ingestion in order to fi nd the bioavailability value. Prasain and Barnes (2007) discussed the defi nition and signifi cance of bioavailability more extensively in their review.

5.2.1 FACTORS AFFECTING BIOAVAILABILITY

As described above, bioavailability means the rate of utilization of an administered dose of a certain target molecule and thus is independent of the quantity of the respective molecule contained in the food. However, this value is useful in order to estimate a reasonable dose required for the expected functioning of either medicines or food factors.

The absorption of food ingredients including fl avonoids usually occurs in the gut tract, and thus the determining factor for absorption is primarily the amount of the target compound reaching the enterocyte in a form suitable for absorption (Scholz and Willamson, 2007). In addition to an inherent instability due to their chemical nature, biotransformation by intestinal microfl ora and degradation is a critical factor affecting the bioavailability of orally ingested fl avonoids (Scalbert and Willamson, 2000; Willamson and Manach, 2005). Flavonoids exist mainly in sugar-conjugated form in dietary plants. They undergo intestinal hydrolysis by microfl oral glucosidase or hydrolase to release the respective aglycone, or in host cells when they are taken orally and then transported across the gut membrane into plasma. They are also subjected to metabolic transformation, such as methylation of the free phenolic group mediated by cathecol O-methyl transferase (COMT) (Day et al., 2000; Hur et al., 2000).

Such biotransformation might reduce the apparent bioavailability of the original parent molecules and, when the metabolites are physiologically active, a large discrepancy might be expected as is shown in biofl avonoids between the apparent bioavailability and the physiological effects observed

by the intake of original food factors. Such an example is isofl avone. It is well known that an isofl a- vone such as daidzein is transformed by intestinal microfl ora into the active metabolite, (S)-equol, in which estrogen-like activity is much higher than in the original parent molecule (Setchell et al., 2002; Setchell et al., 2005).

Moreover, during intestinal uptake, fl avonoids, either intestinally transformed or in intact form, undergo glucuronate or sulfate conjugation reactions in enterocytes, and are also subjected to fur- ther metabolism after absorption into plasma, mainly in the liver. Indeed, the quercetin glycoside level is quite low in blood plasma after oral administration; the metabolites and their secondary modifi ed glucuronate or sulfate conjugates appear as the major component, and the plasma level of the original ingested form is quite low (Corona et al., 2006). Therefore, bioavailability must be evaluated not only in terms of the original parent molecule but also of the metabolites generated. Recently, highly sensitive tandem mass spectrometry (MS) studies have addressed this issue and have been reviewed by many authors (Prasain and Barnes, 2007).

Mennen et al. (2008) have recently challenged our whole understanding of the benefi cial health effects of dietary fl avonoids, by targeting several polyphenols and their metabolites in a spot of urine as the biomarker of polyphenol intake.

The intestinal mucosa, on the other hand, are not a simple barrier against xenobiotics but work for selective absorption, biotransformation, and excretion back to the lumen (Sergent et al., 2008). The presence of ATP-binding cassette (ABC)/multiple drug resistance (MDR) family transloca- tors on the lumen-side membrane of enterocytes is also a factor which negatively modulates the net intestinal uptake of fl avonoids (O’Leary et al., 2003; Zjhang et al., 2004). MDR is a membrane transporter protein which acts to prevent the disadvantageous uptake of xenobiotics and thus con- fers multidrug tolerance in cancer chemotherapy (Benet et al., 1999). Flavonoids as micronutrients are also xenobiotic substances and thus a signifi cant effl ux of tea isofl avones from the basal to the apical side was shown in the Caco-2 monolayer model; the effl ux rate was greater in epicatechin (EC) than in epigallocatechin (EGC) and epigallocatechin gallate (EGCG) (Chan et al. 2007).

Basically, absorption effi cacy depends on the amount or concentration of respective fl avonoids to be absorbed in the gut. When intestinal absorption is not mediated by specifi c membrane trans- porters, the absorption of molecules is generally regulated by the physicochemical properties of a molecule such as its hydrogen bonding character, molecular weight, and log P value (P being the partition coeffi cient between oil and water), known as Lipinski’s rule (Zjhang et al., 2004). Thus, molecules having a large molecular size, high polarity, and large apparent size (hydration) are poorly absorbed so long as any specifi c transporters are not available (Vaidyanathan and Wall, 2001). This rule is also adapted to polyphenols including fl avonoids, although the P value here is usually less than 5. Since aglycones and metabolites are more hydrophobic and have a small molecular size, they are absorbed more easily in diets than the parent fl avonoids with conjugated sugar (Hong et al., 2002). In the same way, the uptake effi ciency of EGCG was lower than that of EC and ECG when compared among tea catechin aglycones (Hong et al., 2003).

As discussed above, sugar conjugation decreases the lipophilicity of molecules, which is a critical requirement for the intestinal absorption of dietary ingredients, but on the other hand, it improves their stability. Moreover, it is known that the type of sugar affects the absorption effi ciency in sev- eral fl avonoids such as quercetin, where glucoside was much more easily absorbed than rutinoside (Erlund et al., 2000). This was because quercetin glucoside is better hydrolyzed by brush-border glu- cosidase in the small intestine in order to release the aglycone, whereas rhamnoside is hydrolyzed by microfl oral enzymes in the colon. Thus, intestinal stability is the factor controlling bioavailability.

Flavonoids in nature are acylated in addition to glycosidation. The extent and type of the acylated group also affect the bioavailability of fl avonoids. Henning and Heber compared the bioavailability of gallated and nongallated fl avan-3-ols in tea and found acylation resulted in decreased absorp- tion. This rule is also adapted to anthocyanins from the purple sweet potato (Suda et al., 2002) and cooked black carrot (Kurilich et al., 2005), where it was shown that the plasma level of diacylated anthocyanin (peonidin-3-caffeoyl sophoroside-5-glucoside) in purple potato was six times lower

than in nonacylated anthocyanin (aglycon) after oral administration. It was, however, noticed that

p- coumaroyl delphinidin from eggplant showed better absorption than nonacylated delphinidin.

Although the mechanism of enhanced absorption is unclear, the type of acylation may alter the absorption effi ciency, probably due to improved stability (Ichiyanagi et al., 2006b).

In addition to the factors modulating ADME mentioned above, the physicochemical properties of molecules, including stability, protein binding, and (coexisting) matrix effect, are also fac- tors which affect bioavailability. For example, polyphenols bind to salivary proteins, particularly proline-rich proteins and basic residues of histatins, and thus are prevented from interacting with gut enzyme proteins such as α amylase, α glucosidase, and protease (Bennick, 2002; Griffi ths, 1986; McDougall and Stewart, 2005). Recent studies have indicated a signifi cant binding of fl avonoids to blood plasma protein. This protein binding usually restricts the tissue uptake of fl avonoids, that is, restricts the molecules from reaching the target site of action. Moreover, the profi le of bound fl avonoids is different from that of free fl avonoids in the plasma, indicating that protein binding alters the functional role of the diet from that which would have been expected from the compo- sition of existing active ingredients (Manach et al., 1995). In relation to this, the effect of milk protein on the bioavailability of tea polyphenols has been extensively studied. Primarily, it was reported that milk lowers the bioavailability of catechin when tea was added with milk (Reddy et al., 2005) but others reported that milk did not alter the plasma level of catechin (Van het Hof et al., 1998; Serafi ni et al., 1996; Richelle et al., 2001). A similar discussion has taken place about the effect of milk on cocoa polyphenols, but recent studies including double blind cross-over studies on healthy human subjects rather concluded that the effect of milk on cocoa polyphenols was marginal and does not give rise to signifi cant physiological effects (Keogh et al., 2007; Roura et al., 2007).

The effect of the addition of milk to black tea was also assessed for its ability to modulate oxidative stress and antioxidant status in adult male volunteers using catechin as a marker. Milk addition may not obviate the ability of black tea to modulate the antioxidant status of subjects and the consumption of black tea with/without milk prevents oxidative damage in vivo. This rule was typically observed when the tea catechin family was studied for their intestinal absorption and back fl ux behavior, mediated by MDR using the Caco-2 cell monolayer system (Reddy et al., 2005).

5.2.2 ANTHOCYANIN BIOAVAILABILITY

Anthocyanin has a unique property which makes it different from other fl avonoids in several respects; for example, the chemical structure is variable dependent on pH. In an acidic pH below 4, the major form is fl avylium cation, but in an alkaline pH above 8 it is in the quinoidal form. Both are rather stable. However, at a neutral pH covering physiological pH, it turns to a colorless open ring structure, chalcone, and eventually decomposes (Rahman, 2008). In particular, aglycones are more instable than their glycosides. Moreover, it is known that anthocyanins are absorbed in their intact form in a different way from other fl avonoids such as quercetine (Erlund et al., 2000). These characteristics of anthocyanin defi ned their bioavailability as quite low. Nevertheless, many human and animal experiments have indicated a high potentiality in the prevention of diseases, particu- larly cardiovascular and neurodegenerated diseases (Konishi and Ichiyanagi, 2008). Ichiyanagi et al. (2006a) closely studied the bioavailability of 15 anthocyanins in bilberry by the pharmacokinetic approach and showed that the values varied among the anthocyanins in bilberry from 0.028% to 0.6%, still quite low compared to other fl avonoids. When the plasma uptake among anthocyanins was compared with the same aglycone but a different type of conjugated sugar in rats, galactosyl anthocyanin showed a higher plasma concentration than glucosyl or arabinosyl anthocyanins after oral administration (Ichiyanagi et al., 2006a; Konishi and Ichiyanagi, 2008; Rahman, 2008). Sugar conjugation stabilizes anthocyanin aglycone but it is not yet clear whether the stability of galactosyl conjugate is superior to other sugar conjugates, and involvement of an unknown transporter prefer- entially mediates galactosyl anthocyanin. Wu and coworkers (2005) studied the effect of conjugated

sugar on intestinal absorption and found that cyanidin-3-O-rutinoside is better absorbed than cyani- din-3-O-glucoside after oral administration of blackcurrant extract. They indicated that the higher plasma level of rutinoside is due to its stability in the small intestine compared to glucoside (Wu et al., 2005).

Since the natural content of anthocyanin in daily diets is quite large as a micronutrient—for exam- ple, one serving of blueberry supplies 10–100 mg of anthocyanins (Grotewold, 2006)—improved bioavailability will be of practical importance to exploit the benefi cial health effects of fl avonoids anthocyanins in daily life.