Chocolate: Modern Science Investigates an Ancient Medicine

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1 Chocolate: Modern Science Investigates an Ancient Medicine Dietary Intake and Bioavailability of Polyphenols 1 Augustin Scalbert* 2 and Gary Williamson *Laboratoire des Maladies Métaboliques et Micronutriments, INRA, Saint-Genès-Champanelle, France and Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. ABSTRACT The main dietary sources of polyphenols are reviewed, and the daily intake is calculated for a given diet containing some common fruits, vegetables and beverages. Phenolic acids account for about one third of the total intake and flavonoids account for the remaining two thirds. The most abundant flavonoids in the diet are flavanols (catechins plus proanthocyanidins), anthocyanins and their oxidation products. The main polyphenol dietary sources are fruit and beverages (fruit juice, wine, tea, coffee, chocolate and beer) and, to a lesser extent vegetables, dry legumes and cereals. The total intake is 1 g/d. Large uncertainties remain due to the lack of comprehensive data on the content of some of the main polyphenol classes in food. Bioavailability studies in humans are discussed. The maximum concentration in plasma rarely exceeds 1 M after the consumption of mg of a single phenolic compound. However, the total plasma phenol concentration is probably higher due to the presence of metabolites formed in the body s tissues or by the colonic microflora. These metabolites are still largely unknown and not accounted for. Both chemical and biochemical factors that affect the absorption and metabolism of polyphenols are reviewed, with particular emphasis on flavonoid glycosides. A better understanding of these factors is essential to explain the large variations in bioavailability observed among polyphenols and among individuals. J. Nutr. 130: 2073S 2085S, KEY WORDS: polyphenol phenolic acid flavonoid flavonoid glycoside dietary intake bioavailability gut absorption metabolism colonic microflora glucosidase antioxidant Polyphenols are receiving increasing interest from consumers and food manufacturers for several reasons. Epidemiological studies have suggested associations between the consumption of polyphenol-rich foods or beverages and the prevention of diseases. Fruit and vegetable consumption prevents cancers (Steinmetz and Potter 1996). It may also prevent stroke (Ness and Powles 1997), whereas wine consumption might prevent coronary heart disease (Criqui and Ringel 1994, Renaud and De Lorgeril 1992). The consumption of tea may protect against cancers (Yang and Wang 1993) and coronary heart diseases (Tijburg et al. 1997), and that of soy may protect against breast cancer and osteoporosis (Adlercreutz and Mazur 1997). A second reason is linked to the fundamental chemical nature of polyphenols. Polyphenols are reducing agents, and together with other dietary reducing agents, such as vitamin C, vitamin E and carotenoids, they protect the body s tissues against oxidative stress. Commonly referred to as antioxidants, they may prevent various diseases associated with oxidative stress, such as cancers, cardiovascular diseases, inflammation 1 Presented at the symposium Chocolate: Modern Science Investigates an Ancient Medicine, held February 19, 2000 during the 2000 Annual Meeting and Science Innovation Exposition of the American Association for the Advancement of Science in Washington, D.C. Published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were John W. Erdman, Jr., University of Illinois-Urbana-Champaign; Jo Wills, Mars, United Kingdom and D Ann Finley, University of California, Davis. 2 To whom reprint requests should be addressed. and others. Last, polyphenols are the most abundant antioxidants in our diets. However, the considerable diversity of their structures makes them different from other antioxidants. Several thousands of natural polyphenols have been identified in plants, many of them in plant foods (Shahidi and Naczk 1995), although only a more limited number are at significant levels in most human diets. The chemical structure of polyphenols will affect their biological properties: bioavailability, antioxidant activity, specific interactions with cell receptors and enzymes and other properties. To understand their impact on human health, it is essential to know the nature of the main polyphenols ingested, their dietary origin, the amounts consumed in different diets, their bioavailability and the factors controlling their bioavailability. These aspects are discussed in this review. Nature of dietary polyphenols A phenolic molecule is often characteristic of a plant species or even of a particular organ or tissue of that plant. It is therefore impossible to know precisely the nature of all of the polyphenols that we ingest. In contrast, it is desirable to know the main classes of the polyphenols consumed, the main foods that contain them and their content in these foods. The main classes of polyphenols are defined according to the nature /00 $ American Society for Nutritional Sciences. 2073S

2 2074S SUPPLEMENT FIGURE 1 Chemical structures of the main classes of polyphenols. of their carbon skeleton: phenolic acids, flavonoids and the less common stilbenes and lignans (Fig. 1). Phenolic acids are abundant in foods. The most frequently encountered are caffeic acid and, to a lesser extent, ferulic acid. Ferulic acid is associated with dietary fiber and is linked through ester bonds to hemicelluloses. One of the main food sources of ferulic acid is wheat bran (5 mg/g) (Kroon et al. 1997). Caffeic acid is also found in the form of esters. The most frequently encountered caffeoyl ester is chlorogenic acid, which is present in many fruits and vegetables and in coffee. One cup of instant coffee (200 ml) contains mg of chlorogenic acid (Clifford 1999). Other phenolic acid derivatives are hydrolyzable tannins. Phenolic acids are esterified to a polyol, usually glucose. The phenolic acids are either gallic acid in gallotannins (mango fruit) or other phenolic acids derived from the oxidation of galloyl residues in ellagitannins (blackberry, raspberry, strawberry, wine and brandy aged in oak barrels) (Fig. 1) (Clifford and Scalbert 2000). Their occurrence is much more limited than that of condensed tannins. Flavonoids are the most abundant polyphenols in our diets. They can be divided into several classes according to the degree of oxidation of the oxygen heterocycle: flavones, flavonols, isoflavones, anthocyanins, flavanols, proanthocyanidins and flavanones. The occurrence of some of these flavonoids is restricted to a few foodstuffs. The main source of isoflavones is soy, which contains 1 mg of genistein and daidzein/g dry bean (Reinli

3 DIETARY POLYPHENOL INTAKE AND BIOAVAILABILITY 2075S and Block 1996). These two isoflavones have received considerable attention due to their estrogenic properties and their suggested role in the prevention of breast cancer and osteoporosis (Adlercreutz and Mazur 1997). Citrus fruits are the main food source of flavanones. The most widely consumed is hesperidin from oranges ( mg/l of juice) (Rousseff et al. 1987). Other flavonoid types are common to various food sources. Quercetin, the main flavonol in our diet, is present in many fruits and vegetables as well as in beverages. It is particularly abundant in onions (0.3 mg/g fresh weight) (Hertog et al. 1992) and tea (10 25 mg/l) (Hertog et al. 1993a), which represent the main sources of flavonols in the Dutch diet (Hertog et al. 1993b). Flavones are less common and were identified in sweet red pepper (luteolin) and celery (apigenin) (Hertog et al. 1992). The main flavanols are catechins. They are very abundant in tea. Young shoots contain mg of catechin, gallocatechin and their galloylated derivatives/g of dry leaves (Hara et al. 1995). An infusion of green tea contains 1 g/l catechins (Lee et al. 1995). In black tea, their content is reduced to about half this value due to their oxidation into more complex polyphenols during fermentation (Ding et al. 1992). Other sources are red wine (270 mg/l) (Frankel et al. 1995) and chocolate (Arts et al. 1999). Proanthocyanidins are polymeric flavanols. They are present in plants as complex mixtures of polymers with an average degree of polymerization between 4 and 11. They are responsible for the astringency of food and are usually present in association with flavanol catechins. Common sources are fruits such as apple, pear and grape, beverages such as red wine and tea, as well as chocolate (Santos-Buelga and Scalbert 2000). Anthocyanins are pigments of red fruits such as cherries, plums, strawberries, raspberries, blackberries, grapes, red currants and black currants. Their contents vary from 0.15 (strawberries) to 4.5 mg/g (cherries) in fresh fruit (Clifford 1996). The average content in red wines is 26 mg/l (Frankel et al. 1995). Stilbenes are not widespread in food plants. Nevertheless, one of them, resveratrol, which was revealed during the screening of medicinal plants, has recently received great attention for its anticarcinogenic properties (Jang et al. 1997) and presence in wine. However, its very low concentration in wine (0.3 2 mg/l in red wines) (Frankel et al. 1995) makes the attribution of protective effects to this molecule unlikely. Lignans have been identified in human plasma and urine (Adlercreutz and Mazur 1997). Their dietary origin is established, but their precursor in food is still unknown. The only foods that contain considerable quantities of lignans are flaxseed and flaxseed oil (Axelson et al. 1982). When fed to humans or animals, they are metabolized by the gut microflora into the mammalian lignans. Lignans are recognized as phytoestrogens due to their estrogen agonist and antagonist properties. Other dietary polyphenols are not well-defined chemical entities and result from the oxidative polymerization of flavonoids and phenolic acids. This may occur during ripening or food processing (grinding, fermentation, storage, cooking and other processes). These ill-defined phenolic compounds are the main polyphenols in black tea and wine, particularly aged wine (Santos-Buelga and Scalbert 2000). Polyphenol content in food and dietary intake The structural diversity of dietary polyphenols is not limited to differences in the structure of the carbon skeleton and in the oxidation state of the heterocycle of flavonoids. It is further complicated by varying patterns of hydroxylation of the phenolic rings, by glycosylation of most flavonoids, by acylation with phenolic acids and by the existence of stereoisomers, among others. The structural diversity of polyphenols makes the estimation of their content in food difficult. Their average content in some food servings is given in Table 1. Values are only indicative because they vary widely according to varieties: by a factor of 1:4 for flavonoids and phenolic acids in apple (Amiot et al. 1992, Hammerstone et al. 2000) and in the same proportions for quercetin in yellow and red onions (Tsushida and Svzuki 1996). The white varieties of onions are devoid of flavonols. Polyphenols are not evenly distributed in plant tissues, and food fractionation during processing may result in a loss or enrichment of some phenolic compounds. In apple, quercetin is found in the peel (1 mg/g fresh weight); the peeled fruit contains no other flavonols (Burda et al. 1990). Similarly, polyphenols in wheat grain are principally contained in the outer layers (aleurone cells, seed coat) and are lost during the refining of flour (Shahidi and Naczk 1995). Conversely, pressing may result in the solubilization of phenolic compounds in juices otherwise present in the unconsumed parts of the fruit. This is the case of phloridzin from apples, which is confined to its skin and especially to the pips (Spanos and Wrolstad 1992). For a number of reasons, including structural diversity, lack of standardized analytical methods and variation of content in a particular foodstuff, it is extremely difficult to estimate the average daily intake of polyphenols. Most authors refer to the data published 25 y ago (Kühnau 1976). A daily intake of 1 g of total phenols was reported, but the methods used to obtain this result were not detailed. In Table 1, we present the content of various classes of polyphenols in some foods and beverages commonly consumed in Western diets. Two different approaches were used to estimate polyphenols: i) specific compounds such as chlorogenic acid in potato or coffee, quercetin in onions or catechins in tea were estimated individually by chromatographic techniques or ii) total phenols were estimated by reduction of the Folin-Ciocalteu reagent (Scalbert 1992). Values obtained by the first method are usually lower than those estimated by the Folin assay (Table 1). One reason is that some polyphenols in a given food source may escape determination by chromatography. These can be unknown compounds, compounds present as traces that were not considered in the characterization of food sources or compounds that are not resolved by chromatography, such as proanthocyanidin polymers and oxidized polyphenols (Santos- Buelga and Scalbert 2000) as in apple, wine, tea or beer. A second reason is that other reducing agents may be present in food. Ascorbic acid also reduces the Folin reagent (1 mg is equivalent to 0.70 mg catechin often used as a standard in this assay) (Singleton and Rossi 1965). For example, the ascorbic acid content of potato, tomato, onion, apple and orange juice (17, 24, 8, 12 and 54 mg/100 g fresh weight, respectively) (Souci et al. 1986) would account for 40 and 46% of the estimated total phenols in potato and tomato but for only 6 and 4% in polyphenol-rich onion and apple. It has been claimed on the basis of the Folin assay, that vegetables (dry legumes included) provide 218 mg of total phenols/d in an average U.S. diet (Vinson et al. 1998). Due to the contribution of ascorbic acid to the Folin values, the actual value should be lower. No similar survey of total phenol content in fruits has been published. Fruits are usually richer in polyphenols than vegetables, with total phenol contents as high as 1 2 g/100 g fresh weight for some fruits, such as plum and persimmon (Macheix et al. 1990). They often contain high amounts of proanthocyanidins (apple, plum, grape and

4 2076S SUPPLEMENT TABLE 1 Polyphenol content of a typical serving of commonly consumed foodstuffs and beverages (mg) Flavonoids Flavanols Total phenols Foodstuff (quantity) Phenolic acids Flavonols Catechin monomers Proanthocyanidins Flavanones Anthocyanins Chromatography Folin assay 3 Reference Vegetables Potato, 200 g Hughes and Swain 1962, Vinson et al Tomato, 100 g Fleuriet and Macheix 1985, Crozier et al. 1997, Vinson et al Lettuce, 100 g Vinson et al Onion, 20 g Winter and Herrmann 1986, Hertog et al. 1992, Vinson et al Fruits Apple, 200 g Hertog et al. 1992, Spanos and Wrolstad 1992, Vinson 1998, Hammerstone et al Cherry, 50 g Macheix et al. 1990, Clifford 1999 Other foods Wheat bran, Kroon et al g Dark chocolate, 20 g Beverages Orange juice, 100 ml Red wine, 125 ml Coffee, 200 ml Black tea, 200 ml Waterhouse et al. 1996, Adamson et al. 1999, Hammerstone et al Rousseff et al. 1987, Henn and Stehle Ricardo da Silva et al. 1991, Ricardo da Silva et al. 1992, Frankel et al Brown et al. 1990, Clifford Brown et al. 1990, Ding et al. 1992, Hertog et al. 1993a 1 Oligomers up to decamers. 2 Oligomers up to trimers. 3 Estimated by the Folin-Ciocalteu colorimetric assay as catechin or gallic acid equivalents. Values are in excess for samples containing ascorbic acid (see text). 4 Folin values for cherry and wheat bran are missing, and chromatographic estimates are used instead. persimmon) and anthocyanins (cherry and other red fruits) not commonly found in vegetables (with the exception of eggplant and dry legumes) (Clifford 1996, Santos-Buelga and Scalbert 2000). The consumption of cereal products contributes to the phenolic acid intake only when whole grains are used for their manufacture. Chocolate is also very rich in polyphenols, and a minor consumption of chocolate may significantly contribute to total polyphenol intake and more particularly to the catechin (Arts et al. 1999) and proanthocyanidin intake. A major source of polyphenols is beverages (red wine, coffee, tea and fruit juices). For those regularly consuming wine, coffee or tea, these beverages will likely be the major source of polyphenols. Orange juice is not as rich in polyphenols. Vitamin C (50 mg/100 ml) (Souci et al. 1986) accounts for 40% of the estimated total phenols, with the remaining fraction corresponding to flavanones. Beer and chocolate drinks also provide proanthocyanidins. A total phenol content in beer of mg/l was measured (Leupold and Drawert 1981), but part of this may be in fact derived from Maillard products (Maillard and Berset 1995). The total polyphenol intake can be calculated from the polyphenol contents in food and food consumption tables. Kühnau (1976) determined a flavonoid intake in the United States of 1 g/d, but no details were given on the methods used to determine this figure. A person who would consume in 1 d the different servings of foodstuffs and beverages shown in Table 1 would effectively ingest 1 g of flavonoids and phenolic acids, regardless of the method of polyphenol estimation used (chromatography or Folin colorimetric assay). Some general trends regarding the main sources of polyphenols and main polyphenols consumed can be deduced from the data presented in Table 1: contribution to polyphenol intake is shared more or less equally by food and beverages. Phenolic acids account for approximately one third of the total phenols, and flavonoids account for two thirds. This propor-

5 DIETARY POLYPHENOL INTAKE AND BIOAVAILABILITY 2077S tion will largely depend on the consumption of coffee (Clifford 1999). Heavy coffee drinkers will likely consume more phenolic acids than flavonoids. The proportion of the different flavonoids will also largely vary according to the foodstuffs consumed. For persons consuming fruits or beverages such as red wine, tea, chocolate or beer, the most abundant flavonoids will be flavanols (catechins plus proanthocyanidins), anthocyanins and their oxidation products (Clifford 1996, Santos- Buelga and Scalbert 2000). It is anticipated that taken together, they will account for more than two thirds of the total polyphenol dietary intake. It appears that polyphenol intake depends to a large extent on dietary habits and preferences. This concerns not only the consumption of polyphenols as a whole or that of the different classes of polyphenols but also that of each individual phenolic compound. This has been clearly shown for quercetin, for which differences in consumption of 3 and 34 mg/d have been reported for the 10th and 90th percentiles of a Dutch cohort (Hertog et al. 1993b). The evaluation of polyphenol dietary intake still lacks precision. Most of the data on polyphenol content in food originate from scattered sources. A more comprehensive and thorough survey of the occurrence in food of the various types of polyphenols must be undertaken using well-standardized methods. So far, this has been done for flavonols, flavones (Hertog et al and 1993b) and isoflavones (Reinli and Block 1996). The intake of flavonols (largely quercetin) and flavones by the Dutch population has been established as 21 and 2 mg/d, respectively (Hertog et al. 1993b). For isoflavones, an average dietary intake of mg/d was determined for the Japanese (Kimira et al. 1998, Wakai et al. 1999). The consumption in Western countries is significantly lower due to the limited consumption of soy products (Kirk et al. 1999). It thus appears that the intake of flavonols, flavones and isoflavones is relatively low compared with that of phenolic acids and other flavonoids, such as proanthocyanidins, anthocyanins and oxidized polyphenols. The consumption of compounds such as quercetin and genistein does not exceed 2 4% of the total polyphenol dietary intake in Western diets. They have been the most largely studied phenolic compounds in human nutrition due to their particular biological activities. However, attention should also be paid to the other phenolic compounds that also contribute to the prevention of oxidative stress and that may have some, still ignored, more specific biological activities. Bioavailability of polyphenols Biological properties of polyphenols depend on their bioavailability. Indirect evidence of their absorption through the gut barrier is the increase in the antioxidant capacity of the plasma after the consumption of polyphenol-rich foods. This has been observed for a wide array of foodstuffs such as tea (Serafini et al. 1996, van het Hof et al. 1997), red wine (Duthie et al. 1998, Fuhrman et al. 1995, Maxwell et al. 1994, Serafini et al. 1998, Whitehead et al. 1995) or black currant and apple juice (Young et al. 1999). More direct evidence on the bioavailability of a few phenolic compounds has been obtained by measuring their concentrations in plasma and urine after the ingestion of either pure compounds or of foodstuffs with known content of the compound of interest. The chemical structure of polyphenols determines their rate and extent of intestinal absorption and the nature of the metabolites circulating in the plasma. The few bioavailability studies in humans show that the quantities of polyphenols found intact in urine vary from one phenolic compound to another (Table 2). They are particularly low for quercetin and rutin, a glycoside of quercetin ( %), but reach higher values for catechins in green tea, isoflavones in soy, flavanones in citrus fruits or anthocyanidins in red wine (3 26%). Interindividual variations have also been observed: 5 57% of the naringin consumed with grapefruit juice is found in urine according to the individual (Fuhr and Kummert 1995). A major part of the polyphenols ingested (75 99%) is not found in urine. This implies they have either not been absorbed through the gut barrier, absorbed and excreted in the bile or metabolized by the colonic microflora or our own tissues. Only very rare measurements of the intestinal absorption of polyphenols in humans are available. Half of the quercetin glucosides contained in onions fed to ileostomized volunteers were absorbed in the small intestine (Hollman et al. 1995). Absorption level of rutin, a rhamnoglucoside of quercetin, was one half to one third that of quercetin glucoside and required deglycosylation by the intestinal microflora before absorption through the colon barrier (Hollman et al. 1997). The same bioavailability studies (Table 2) have also shown that the concentrations of intact flavonoids in human plasma rarely exceed 1 M when the quantities of polyphenols ingested do not exceed those commonly ingested with our diets. These maximum concentrations are most often reached 1 2 h after ingestion (Aziz et al. 1998, Balant et al. 1979, Hollman et al. 1996, Kivits et al. 1997, Lee et al. 1995, Unno et al. 1996), except for polyphenols, which are absorbed only after partial degradation by the colon microflora. With regard to rutin, the maximum concentration of quercetin in the plasma is reached 9 h after ingestion (Hollman et al. 1997). For most flavonoids absorbed in the small intestine, the plasma concentration then rapidly decreases (elimination half-life period of 1 2 h). This fast excretion is facilitated by the conjugation of the aglycone to sulfate and glucuronide groups (see later). The elimination half-life period for quercetin is much higher (24 h) (Hollman et al. 1997). This slow elimination of quercetin is possibly explained by its particularly high affinity for plasma albumin (Dangles et al. 2000, Manach et al. 1995). The maintenance of a high concentration in plasma thus requires a repeated ingestion of the polyphenols over time, as has been observed with volunteers consuming tea every 2 h (van het Hof et al. 1999). However, the half-life of metabolites formed in the colonic microflora is longer due to the long residence time of polyphenols in the colon. More than 2 d are needed for the phytoestrogen metabolites equol (Lu et al. 1995), enterodiol and enterolactone (Nesbitt et al. 1999) to reach the baseline concentrations in plasma and urine after the consumption of soy milk and flaxseed, respectively. Chemical and biochemical factors affecting polyphenol bioavailability Polyphenols exist in foods and beverages in various chemical forms that determine their gut absorption. Chemical structures will also influence the conjugation reactions with methyl, sulfate or glucuronide groups and the nature and amounts of metabolites formed by the gut microflora absorbed at the colon level. Understanding the structural factors that influence absorption and metabolism is essential to determine the polyphenols that are better absorbed and that lead to the formation of known active metabolites. Gut absorption. Flavonoid glycosides. Certain classes of polyphenols, such as flavonols, isoflavones, flavones and anthocyanins, are usually glycosylated. The linked sugar is often

6 2078S SUPPLEMENT TABLE 2 Bioavailability in humans of polyphenols consumed alone or in foods1 Polyphenol Source Quantity of polyphenol ingested Maximum concentration in plasma Excretion in urine Reference mg M % Phenolic acids Caffeic acid Jacobson et al Flavonols Quercetin Onion Hollman et al Quercetin Apple Hollman et al Quercetin-4-O-rhamnoglucoside Pure compound Hollman et al Quercetin-4-O-glucoside Pure compound Hollman et al Quercetin Onion Aziz et al Quercetin Mixed black currant and apple juice, 1000 ml/d for 7 d Young et al Catechins Epigallocatechin gallate Green tea infusion, 1.2 g ND Lee et al Epigallocatechin Epicatechin gallate 33 ND ND Epicatechin Epigallocatechin gallate Green tea infusion, 5g Unno et al Epigallocatechin gallate Green tea infusion, 6g 5.0 Maiani et al Epigallocatechin gallate Green tea extract Nakagawa et al Catechin Red wine, 120 ml Donovan et al Catechin Pure compound Balant et al Isoflavones Genistein Soy milk Xu et al Daidzein Genistein Soy proteins, 60 g/d for 1 mo Cassidy et al Daidzein Genistein Soy proteins, 60 g/d for 28 d Gooderham et al Daidzein Genistein Soy proteins, 20 g/d for 9 d Karr et al Daidzein Flavanones Naringin Grapefruit juice, 120 ml Fuhr and Kummert 1995 Naringin Grapefruit and orange juice, Ameer et al ml each Hesperidin Naringin Pure compounds Ameer et al Hesperidin Anthocyanins Anthocyanins Red wine, 300 ml Lapidot et al Polyphenols, principally in the form of conjugated metabolites, as sulfate esters or glucuronides, in plasma and urine, were hydrolyzed by acid or enzymes before chromatrographic or colorimetric analysis. ND, not detected. glucose or rhamnose but can also be galactose, arabinose, xylose, glucuronic acid or other sugars (Harborne 1994). The number of sugars is most commonly one but can be two or three, and there are several possible positions of substitution on the polyphenol. The sugars can be further substituted, for example, with a malonic acid group. The glycosylation influences chemical, physical and biological properties of the polyphenol. For example, partition coefficients measure the relative affinity of a compound for aqueous and organic phases and are important in determining whether a compound will passively diffuse across a biological membrane and how they might partition in a cell. The flavonol quercetin has a partition coefficient (log octanol/water) of , whereas for a glycoside, quercetin-3-o-rhamnoglucoside, the value is lower ( ), showing greater hydrophilicity (Brown et al. 1998). For glycosylated polyphenols, the predicted effect of the attached moiety on passive diffusion across biological membranes suggests that removal of the hydrophilic moiety will usually be necessary for passive diffusion across the small intestine brush border to occur. Therefore, the first step of metabolism should be removal of the sugar by enzymes (gly-

7 DIETARY POLYPHENOL INTAKE AND BIOAVAILABILITY 2079S TABLE 3 Comparison of substrate specificity of human and rat tissue cell-free extracts Initial rate of deglycosylation Substrate Human small intestine1 Human liver1 Rat duodenum2 Rat jejunum2 Rat ileum2 nmol min 1 mg 1 Quercetin-4 -O-glucoside Quercetin-3-O-glucoside ND Quercetin-3-O-rhamnoglucoside ND ND Quercetin-3,4 -O-diglucoside ND ND NM NM NM Genistein-7-O-glucoside NM NM NM Daidzein-7-O-glucoside NM NM NM 1 Day et al Ioku et al ND, not detected; NM, not measured. cosidases). Glycosidase activities can occur in the food itself (endogenous or added during processing) or in the cells of the gastrointestinal mucosa or can be secreted by the colon microflora. Nonenzymatic deglycosylation in the human body, such as in the acid conditions of the stomach, does not occur (Gee et al. 1998). The absorption of polyphenols should therefore be controlled by enzyme specificity and distribution. Human cells express some -glucosidases, but the expression pattern is tissue specific and often regulated during development. Polyphenols with attached glucose (or possibly arabinose or xylose) are potential substrates for endogenous human enzymes. Attached rhamnose is not a substrate for human -glucosidases and so is only cleaved by colon microflora -rhamnosidases. Table 3 shows the activity of cell-free extracts from human and rat intestine and from human liver on various polyphenol glycosides. In humans, there is no hydrolysis of rhamnosides, suggesting that rhamnosides are not metabolized or absorbed in these tissues. In marked contrast, there is hydrolysis of rhamnosides in rat tissues, albeit to a small extent. This difference is important to note when interpreting uptake and metabolism data. Furthermore, the human small intestine cellfree extract hydrolyzes quercetin-3-o-glucoside, whereas the liver does not. This is ascribed to the presence of lactase phlorizin hydrolase (LPH), 3 a -glucosidase, on the outside of the brush border membrane (Day et al. 2000). Importantly, activity of this enzyme does not require prior absorption of the polyphenol glycoside into the small intestine epithelial cells. The activity of the liver extracts and of some of the small intestine is due to cytosolic -glucosidase (CBG), a soluble enzyme found in many tissues. The CBG purified from pig liver exhibited the same specificity as predicted from these studies on human tissue (Lambert et al. 1999) (i.e., no activity on quercetin-3-o-glucoside). The enzyme data above are in excellent agreement with those from a study on human volunteers in which pure quercetin derivatives were administered to volunteers. Quercetin- 3-O-rhamnoglucoside was absorbed more slowly than quercetin-4 -O-glucoside (maximum concentration at 6 and 0.5 h, respectively) as expected if the glucoside is absorbed in the small intestine after hydrolysis by CBG or LPH and the 3 Abbreviations used: LPH, lactase phlorizin hydrolase; CBG, -glucosidase; COMT, catechol-o-methyltransferase; SULT, phenol sulfotransferases; UGT, UDP glucuronosyl transferase. rhamnoglucoside is absorbed only after hydrolysis later in the colon by microflora. In addition, the amount of the metabolites found reflects a much smaller uptake of the rhamnoglucoside compared with the glucoside. The maximum concentration in plasma was 3.5 and 0.2 M, respectively, with the area under the curve also showing large differences (19 M h 1 compared with 3.7, respectively) (Hollman et al. 1999). However, the half-life of each compound was similar (20 30 h) and much longer than the half-life of commonly administered drugs. A longer half-life is generally predicted to be the reason that some drugs exhibit a greater efficacy than others with a shorter clearance time. Acylated flavonoids. Flavanols such as ( )-epicatechin are often acylated, especially by gallic acid. Galloyl substitutions result in only a small change in the partition coefficients of flavanols and do not influence the bioavailability of polyphenols as dramatically as glycosylation. Flavanols appear to pass through biological membranes and to be absorbed without deconjugation or hydrolysis. There is some evidence for degalloylation of the flavanol ( )-epigallocatechin gallate in saliva in humans (Yang et al. 1999). However, in humans given green tea, plasma levels of epigallocatechin and epigallocatechin gallate were 0.2 2% of the ingested amount (2 3 cups of tea) depending on the human subjects but showed no difference between galloylated and nongalloylated polyphenols (Nakagawa et al. 1997). Phenolic acid esters. Hydroxycinnamates such as ferulic acid and caffeic acid are also commonly esterified to sugars, organic acids and lipids. As an example, chlorogenic acid is caffeic acid ester linked to quinic acid, and this compound is found at very high levels in coffee (Clifford 1999). These ester-linked substitutions have a marked effect on the chemical, physical and biological properties of the polyphenols. There are no esterases in human tissues able to release caffeic acid from chlorogenic acid (Plumb et al. 1999). In agreement with this, rat small intestine takes up only a very small amount of chlorogenic acid and does not metabolize it (Spencer et al. 1999). Consequently, the only significant site for chlorogenic acid metabolism is the colonic microflora. Similarly, ferulic or other hydroxycinnamic acids bound to plant cell walls are also not released by mammalian endogenous enzymes but require release by enzymes such as xylanases and esterases of the colonic microflora (Kroon et al. 1996). Ellagitannins are also hydrolyzed. Ellagic acid was found in urine and lungs of mice fed raspberry and pomegranate ellagi-

8 2080S SUPPLEMENT tannins (Boukharta et al. 1992). However, it is not certain whether this results from acid hydrolysis in the stomach or from the action of the gut microflora (Daniel et al. 1991). Proanthocyanidins. The absorption of polyphenols also depends on the molecular weight. Because of their large molecular weight, proanthocyanidin polymers are likely not as easily absorbed in the small intestine. Evidence showing the absorption of proanthocyanidins through the gut barrier is still scarce (Santos-Buelga and Scalbert 2000). Experiments on the in vitro absorption through a cell monolayer derived from the human intestinal cell line Caco-2 showed that radiolabeled procyanidin dimer and trimer were absorbed in contrast to procyanidin polymers having an average degree of polymerization of 7 (Déprez 1999). The dimer and the trimer were absorbed to a similar extent as ( )-catechin. This must be confirmed with in vivo studies. Deconjugation and reconjugation reactions in metabolism. After hydrolysis of a polyphenol derivative to the free aglycone, polyphenols are conjugated by methylation, sulfation, glucuronidation or a combination. The steps are controlled by the specificity and distribution of the enzymes that catalyze the reactions. The pathway followed is common to drug metabolism, and much of the information available on the metabolism of polyphenols derives from comparisons with drug metabolism. The formation of conjugates can dramatically alter the biological properties of the circulating metabolites. However, there are significant differences between the administration of drugs (usually in hundreds of milligrams in one concentrated dose) and the consumption of dietary polyphenols (usually 100 mg in a diluted dose). These differences imply that drugs can readily saturate the metabolic pathways that rely on the supply of cofactors such as UDP-glucuronic acid. Consequently, unconjugated drugs are often found in the blood. On the other hand, polyphenols in food would not be expected to saturate the metabolic pathways, and hence the circulating species would be expected to be conjugated. When food polyphenols are administered at pharmacological doses, they are found in the free form in the blood (Hackett et al. 1983). After the intake of a large dose (2 g) of ( )-catechin, free ( )-catechin was detected in the plasma after 30 min. After 2 h, traces of methyl-catechin were detected, and after 8 h, 40% of the urinary catechin was methylated, sulfated and glucuronidated. However, after the consumption of a few milligrams of ( )-catechin, normally present in (reconstituted) red wine, all of the circulating catechin was conjugated and no free polyphenol was detected (Bell et al. 2000). The dose will also determine the primary site of metabolism. Large doses are metabolized primarily in the liver. Small doses may be metabolized by the intestinal mucosa, with the liver playing a secondary role to further modify the polyphenol conjugates from the small intestine. For example, in rats, after an oral administration of 10 mg ( )-epicatechin, the polyphenol was first glucuronidated during intestinal absorption, followed by hepatic sulfation and methylation, with possible further methylation in the kidneys before excretion (Piskula and Terao 1998). This implies that the intestine is an important site for metabolism of food-derived polyphenols. Most bioavailability studies on polyphenols have measured total polyphenols in blood after treatment with deconjugating enzymes. However, in studies using rats or isolated rat intestine as models, attempts to measure some conjugates have been made. In these studies, most polyphenol glycosides are first deglycosylated and then converted to glucuronides or sulfates with or without methylation as has been shown for phloridzin (Mizuma and Awazu 1998), luteolin-7-o-glucoside (Shimoi et al. 1998), quercetin glycosides (Crespy et al. 1999), kaempferol-3-o-glucoside (Spencer et al. 1999), genistin and daidzin (Piskula et al. 1999). A very limited number of human studies have been carried out in which the nature of the conjugates has been established, but studies on quercetin and kaempferol (Erlund et al. 1999, Watson and Oliveira 1999) and naringin (naringenin-7-rhamnoglucoside) (Furuta and Kasuya 1997) are in support of the animal data. However, there are some exceptions to the deconjugationreconjugation sequence. After feeding red fruit anthocyanins, cyanidin-3-glucoside and cyanidin-3,5-diglucoside, glucosides were found in human plasma (Miyazawa et al. 1999). Quercetin-3-O-rhamnoglucoside was detected in the plasma of volunteers fed tomatoes (Mauri et al. 1999). There is some evidence that flavonoids from parsley are absorbed by the rat stomach without deglycosylation (Pforte et al. 1999). Glycosides of flavonols in onions, such as quercetin-4 -O-glucoside and quercetin-3 -O-methyl-4 -O-glucoside, may be found in the plasma of volunteers with a peak of absorption of h (Aziz et al. 1998). In a study using the isolated rat intestine and several flavonoids and flavonoid glycosides, only quercetin-3-o-glucoside was not fully deglycosylated and glucuronidated, because some unchanged glycoside passed across the rat small intestine (Spencer et al. 1999). The interpretation of these data is complicated by analytical difficulties in measuring polyphenol metabolites in plasma. The existence of glycosides in plasma needs to be clarified, because their fate within our body should substantially differ from that of aglycones. Glucosides would be slowly deconjugated by liver -glucosidases, provided that the glucoside could enter hepatocytes. Rutinosides would be difficult to metabolize, because there are no human -rhamnosidases and this activity occurs only in the colon as a product of microflora. This would mean that quercetin-3-o-rhamnoglucoside, once absorbed, could have a long half-life. The data available on polyphenol bioavailability are still limited compared with that available for other components of the diet or for drugs. Nevertheless, there is now enough information to develop a working hypothesis which allows the prediction of uptake of polyphenols from the diet. This hypothesis is shown in Figure 2 and facilitates the future design of experiments to test polyphenol uptake. The model is based on data from human interventions in vivo together with knowledge gained from enzyme specificity studies and, when human data are missing, from animal models. The enzymes involved in polyphenol metabolism. The metabolic steps in polyphenol metabolism are shown in Figure 3. The individual reactions are catalyzed by enzymes, some of which show genetic polymorphisms and are also inducible by diet. The levels and sites of expression in human tissues determine the metabolic fate and the pharmacokinetics of ingested polyphenols. The conjugating enzymes have been intensely studied owing to their role in drug metabolism. The distribution, inducibility and polymorphisms are summarized below for each enzyme. It is important to take these factors into account in the interpretation of future human intervention studies. CBG (EC ) is found in a wide variety of tissues but especially in liver. It is thought to catalyze the hydrolysis of a wide variety of xenobiotic glycosides (Gopalan et al. 1992, Lamarco and Glew 1986). For example, a high specificity was shown toward -glucose residues on steroids such as estradiol by rabbit -glucosidase (Mellor and Layne 1971, 1974). LPH (EC ) is found only in the small intestine. The physiological substrates of this mixed function -glycosidase are glucosylceramides and lactosylceramides (in milk fat globules) (Leese and Semenza 1973). Lactose is also a substrate,

9 DIETARY POLYPHENOL INTAKE AND BIOAVAILABILITY 2081S FIGURE 2 A hypothesis for prediction of the absorption of polyphenols in humans based on evidence from in vivo and in vitro studies. and hydrolysis by LPH is required before absorption. Five percent of Europeans and 90% of Africans and Asians have LPH deficiency in adulthood. (Anonymous 1991). LPH has recently been suggested to play an important role in metabolism of polyphenol glucosides, because it catalyzes the hydrolysis of a wide range of polyphenol glucosides, including quercetin-3-o-glucoside, which is not a substrate for CBG (Day et al. 2000). After deconjugation, polyphenols are conjugated (Fig. 3). Catechol-O-methyltransferase (COMT; EC ) plays a crucial role in the metabolism of dopamine. There is a common functional genetic polymorphism in the COMT gene, which results in a threefold to fourfold difference in COMT enzyme activity in humans (Tiihonen et al. 1999). This enzyme methylates polyphenols and occurs in a wide range of tissues. The specificity for polyphenols will determine which hydroxyl groups on the polyphenol ring are methylated. However, cytochrome P450 demethylates flavonols at the 4 position and not at the 3 position; therefore, specificity of methylation of quercetin could be defined by specificity of demethylation by cytochrome P450, not methylation by COMT (Nielsen et al. 1998). UDP glucuronosyl transferase (UDPGT, UGT; EC ) catalyzes the conjugation of polyphenols to glucuronic acid. It is situated in the endoplasmic reticulum and exists as a large family of related enzymes. Glucuronidation of polyphenols is predominantly by the UGT1A family, which occurs in intestine, liver and kidney. UGT1A1, -1A3, -1A4, -1A6 and -1A9 are found in human liver; UGT1A1, -1A3, -1A4, -1A6, -1A8, -1A9 and -1A10 are expressed in human colon; and kidney is high in UGT1A9. Human gastric epithelium expressed UGT1A7 and -1A10, but UGT1A1 shows polymorphism and is expressed only in 29% of samples. Of all tissues, liver has the greatest capacity for glucuronidation (Mojarrabi and MacKenzie 1998, Strassburg et al. 1998, 1999). In Gilbert s syndrome patients, glucuronidation is reduced to 35% of normal (mild unconjugated hyperbilirubinemia); this is found in 5% of the population. Drugs, alcohol and smoking induce UGT1A, which creates latent Gilbert s syndrome patients. Furthermore, diet affects the levels of UGT (Bu-Abbas et al. 1995, Burchell et al. 1995). Clearly, glucuronidation is modified by environment, diet and genetic polymorphisms, which could explain interindividual differences observed in the glucuronidation of catechin (Yang et al. 1998). The effect of glucuronidation at exact locations on most polyphenols is not known. The biological activities of metabolites is an important area for further research. Phenol sulfotransferases (P-PST, SULT; EC ) are a small group of cytosolic enzymes that are widely distributed. The endogenous substrate are iodothyronines, although other substrates include 4-nitrophenol, phenols and hydroxyarylamines (Coughtrie et al. 1998). Several forms have been described. For example, SULT1A1 is high in liver, whereas SULT1A3, with a high activity on catechol groups found in many polyphenols, is high in colon. Generally, sulfotransferases are not induced by diet, xenobiotics or environment (Burchell et al. 1995). Some sulfotransferases are inhibited by polyphenols. Quercetin inhibited human SULT1A1 with a noncompetitive K i value of 0.10 M, which was three to four orders of magnitude more potent than its inhibition of other human sulfotransferases (Walle et al. 1995). Dealcoholized red wine (2000-fold dilution) or a 10,000-fold dilution of strong coffee inhibited SULT1A1 by 50% (Burchell and Coughtrie 1997). There is no specific genetic disease associated with sulfotransferase deficiency, although there is extreme variation within the human population. N-Acetyl transferase catalyzes acetylation of amines but is unimportant for polyphenol metabolism. Some glutathione transferases, cytochrome P450s and epoxide hydrolases show genetic polymorphisms but generally are thought to play a minor role in polyphenol metabolism (Wormhoudt et al. 1999). Metabolism by the gut microflora. Polyphenols that are not absorbed in the stomach or small bowel will be carried to the colon (Fig. 4). In addition, polyphenols that are absorbed, metabolized in the liver and excreted in the bile or directly from the enterocyte back to the small intestine will also reach

10 2082S SUPPLEMENT Simplified scheme showing the metabolism of poly- FIGURE 3 phenols. humans in vivo, unchanged quercetin-3-o-rhamnoglucoside and quercetin were not found in human urine after the administration of the parent compounds, but metabolites from breakdown by colonic microflora were 3-hydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxytoluene and -m-hydroxyphenylhydracrylic acid (Baba et al. 1983). In rats, 33 44% of the dose of labeled catechin was excreted in the bile as glucuronide conjugates, but other metabolites [m- and p-hydroxyphenylpropionic acid, -(3-hydroxyphenyl)- -valerolactone and -(3,4-dihydroxyphenyl)- -valerolactone] were from gut microflora metabolism (Das and Griffiths 1969). A procyanidin polymer was also shown to be degraded by a human colonic microflora grown in vitro and anaerobically into lowmolecular-weight phenolic acids that might well be absorbed in vivo through the colon (Déprez et al. 2000). Bioavailability and plasma antioxidant capacity. Plasma concentrations of the intact parent polyphenols in plasma are often low (Table 2) and do not account on their own for the increase in the antioxidant capacity of the plasma. Metabolites also contribute to increase this antioxidant capacity. Polyphenols can be partially O-methylated in the liver: 20% of catechin present in plasma 1 h after consumption of red wine was O-methylated (Donovan et al. 1999), and quercetin was partially methylated in humans consuming a quercetin-rich meal (Manach et al. 1998). Microbial metabolites formed in the colon are also important. Equol may be three to four times more abundant in plasma than the parent isoflavones (Cassidy et al. 1994). Many of the aromatic acids formed in the colon still bear free phenolic groups and retain part of the reducing capacity of the parent molecule. Measurement of the total antioxidant capacity of plasma after the consumption of polyphenol-rich food allows a comparison of their contribution to the total plasma antioxidant capacity with that of ascorbic acid, the other main aqueous soluble dietary antioxidant found in our diets. The consumption of 300 ml of red wine (containing 500 mg of polyphenols; Table 1) was reported to induce an increase of the plasma antioxidant capacity similar to that of 1 g of ascorbic acid (Whitehead et al. 1995). The plasma concentration of ascorbic acid after the consumption of 1gofvitamin C is 75 M the colon but in a different chemical form, such as a glucuronide (Fig. 4). The colon contains microorganisms/cm 3 and has enormous catalytic and hydrolytic potential. Deconjugation reactions readily occur. For example, quercetin-3-orhamnoglucoside and quercetin-3-o-rhamnoside are not hydrolyzed by endogenous human enzymes but are readily hydrolyzed by gut microflora to quercetin by organisms such as Bacteroides distasonis ( -rhamnosidase and -glucosidase), B. uniformis ( -glucosidase) and B. ovatus ( -glucosidase) (Bokkenheuser et al. 1987). Enterococcus casseliflavus utilizes the sugar moiety of quercetin-3-o-glucoside to give formate, acetate and lactate but does not further metabolize the aglycone. Quercetin-3-O-glucoside is transformed to 3,4-dihydroxyphenylacetic acid, acetate and butyrate by Eubacterium ramulus from the human colon. The number of bacteria able to use quercetin-3-o-glucoside was estimated to be 10 7 to 10 9 /g dry mass (Schneider et al. 1999). Unlike enzymes in human tissues, colonic microflora catalyze the breakdown of the polyphenol itself to more simple compounds, such as phenolic acids. For example, when quercetin-3-o-rhamnoside was incubated anaerobically with human intestinal bacteria, quercetin, 3,4-dihydroxyphenylacetic acid and 4-hydroxybenzoic acid were found as metabolites. In FIGURE 4 Possible routes for consumed polyphenols in humans.