Bioavailability of Phenolic Compounds

Critical Reviews in Food Science and Nutrition

ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: http://www.tandfonline.com/loi/bfsn20

SIBEL KARAKAYA

To cite this article: SIBEL KARAKAYA (2004) Bioavailability of Phenolic Compounds, Critical Reviews in Food Science and Nutrition, 44:6, 453-464, DOI: 10.1080/10408690490886683

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Download by: [Chinese Academy of Agricultural Sciences] Date: 19 January 2017, At: 01:10 Critical Reviews in Food Science and Nutrition, 44:453–464 (2004) Copyright C Taylor and Francis Inc. ISSN: 1040-8398 DOI: 10.1080/10408690490886683

Bioavailability of Phenolic Compounds

SIBEL KARAKAYA Department of Food Engineering, Faculty of Engineering, Ege University, Izmir, Turkey

Phenolic compounds in foods originate from one of the main classes of secondary metabolites in plants. They are essential for the growth and reproduction of plants, and are produced as a response for defending injured plants against pathogens. In recent years, there is a growing interest in phenolic compounds and their presumed role in the prevention of various degenerative diseases, such as cancer and cardiovascular diseases. The importance of antioxidant activities of phenolic compounds and their possible usage in processed foods as a natural antioxidant have reached a new high in recent years. The absorption and bioavailability of phenolics in humans are also controversial. Data on these aspects of phenolics are scarce and merely highlight the need for extensive investigations of the handling of phenolics by the gastrointestinal tract and their subsequent absorption and metabolism. In this article, absorption, metabolism, and the bioavailability of phenolic compounds are reviewed.

Keywords absorption, bioavailability, ﬂavonoids, phenolic acids, phenolic compounds

INTRODUCTION of phenolic compounds extracted from, especially, tea (Vinson et al., 1995a; Cao et al., 1996; Karakaya et al., 2000), fruits The term phenolic compounds refers to the main classes of and vegetables (Vinson et al., 1995b; Wang et al., 1996; Miller secondary metabolites in plants. Several thousand molecules and Rice-Evans, 1997; Prior et al., 1998; Karakaya et al., 2000), have been identified in various plant species. They are closely and red and white wines (Kanner et al., 1994; Vinson and Hontz, related to the sensory and nutritional quality of foods derived 1995; Hurtado et al., 1997; Soleas et al., 1997; Ghiselli et al., from plant sources. Phenolic compounds, at low concentration, 1998; Gündü¸c and El, 2001) have been intensively studied using may act as an antioxidant and protect foods from oxidative de- in vitro methods. The results of these studies showed that pheno- terioration. However, at high concentrations, they or their oxi- lic compounds are powerful antioxidants. However, there is still dation products may interact with proteins, carbohydrates, and a controversy whether in vitro similar effects can be obtained minerals. Phenols are important compounds because of their in vivo, because the lack of knowledge concerning whether phe- contribution to human health and their multiple biological ef- nolic compounds can stay at sufficient time with efficient chem- fects, such as antioxidant activity, antimutagenic and/or anti- ical forms in human body. Phenolic molecule is characteristic of carcinogenic activities, and antiinflammatory action (Grimmer a plant species or even of a particular organ or tissue of that plant. et al., 1992; Xie et al., 1993; Stavric, 1994; Shahidi and Naczk, Therefore, to know all of the phenolic compounds we ingest is 1995; Hollman and Katan, 1997; Ek¸si, 1998; Acar, 1998; Parr impossible. Scalbert and Williamson, (2000) suggested that it and Bolwell, 2000). would be desirable to know the nature of main phenolic com- The results of epidemiologic studies showed the inverse re- pounds consumed. The main classes of phenolic compounds lationship between coronary heart diseases and flavonoid con- according to the nature of their carbon skeleton are mostly sumption (Hertog et al., 1993; Hertog et al., 1995; Knekt et al., phenolic acids, flavonoids and the less common stilbenes and 1996; Rimm et al., 1996). In recent years, the number of stud- lignans. Therefore studies in the field of phenolic compounds ies that are conducted to the determine antioxidant activity of bioavailability are discussed in two parts, phenolic acids and phenolic compounds has increased due to the possible role of flavonoids. reactive oxygen species in the pathogenesis of degenerative diseases, such as atherosclerosis, cancer, and chronic inflamma- tion (Halliwell, 1994). In this respect, the antoxidant activities PHENOLIC ACIDS

Address correspondence to Sibel Karakaya, Ph.D. Department of Food Engineering, Faculty of Engineering, Ege University, Izmir 35100, Turkey. Hydroxycinnamic acids, such as ferulic, sinapic, caffeic, and E-mail: [email protected] p-coumaric acid, are among the most widely distrubuted in plants 453 454 S. KARAKAYA and grains. They occur in most tissues in a variety of conjugated enriched with ferulic acid at a dose of 5.15 mgkg−1 body weight. forms, but seldom as the free acids. Esters and amides are the However, ferulic acid and conjugated forms were not detected most frequently reported types of conjugates, whereas glyco- in plasmas of the control group. The main forms in plasma were sides rarely occur. These include low molecular weight, water- sulfated metabolites (58% of the total ferulic acid), followed by soluble compounds present in cytasol, lipid-soluble forms asso- free ferulic acid (24%) and glucuronidated metabolites (18%). ciated with waxes at the plant surface, and bound forms esterified The maximum ferulic acid concentration in the urine obtained or etherified to cell wall polymers. The most abundant member 1.5 h after ingestion was 38.8–48% of total ferulic acid. The of dietary hydroxycinnamates are ferulic acid and caffeic acid. recovered ferulic acid was mainly in the conjugated form (84% Ferulic acid is covalently linked to plant cell walls and is es- of total excreted ferulic acid). This result suggested that ferulic pecially abundant in an insoluble form in cereal brans. Caffeic acid rapidly metabolized and excreted after ingestion. The dif- acid is ester linked to quinic acid (chlorogenic acid) and found ference between the proportion of conjugated forms detected in at high levels in coffee (Kroon and Williamson, 1999; Chesson urine (84%) and plasma (76%) was explained by the possible et al., 1999; Faulds and Williamson, 1999). conjugation in kidney. According to the researchers the renal In a study, when rats were fed with 14C-labeled hydroxycinna- pathway could be an efficient way in the metabolism of ferulic mates obtained from cultured spinach cell walls, approximately acid. Rondini et al. (2002) concluded that the correlation be- 25% label was found in body tissues after 2 h. Since no label was tween the decrease of glucuronidated conjugates with time, and found in hindgut within this period, researchers concluded that the increase of sulfoglucuronidated or sulfated forms in bladder hydroxycinnamates were absorbed from the foregut (Chesson urine could be accepted as an indicator of the conjugation in the et al., 1999). An in situ model of intestinal perfusion was used kidney by the action of phase II enzymes, such as sulfatese or to determine the bioavaiability of ferulic acid, which was given to β-glucuronidase. However, in the study of Bourne and Rice Wistar rats. Adam et al. (2002) reported that the directly propor- Evans (1998), the excretion of ferulic acid in humans was found tional absorption of ferulic acid to the perfusion concentration to be slower (7–9 h after ingestion) than those of rats. Cereal suggests that ferulic acid could be absorbed by passive diffu- brans contain significant amounts of the ferulic acid, and of its sion or by facilitated transport that appears not to be saturated, oxidatively coupled products, the diferulic acids are ester-linked even at a luminal concentration of 50 µmol L−1.Itwas found to the cell wall polymers. The analysis of plasma samples of male that absorption of ferulic acid and free cinnamic acid were con- Wistar rats showed that diferulic acids were present in the plas- trolled by the Na+/dependent carrier-mediated transport process mas of rats. Researchers concluded that these results provide the in rat jejunal segments (Wolffram et al., 1995; Chesson et al., first evidence for the absorption of diferulic acids through the 1999). When Wistar rats were fed a diet enriched with ferulic gastrointestinal barrier into the vascular system of mammals. acid (10–50 µmol l−1), 56% of the perfused ferulic acid was In this study, it was shown that human small intestinal mucosal found to be absorbed through the small intestine, and 5–7% cells and nonfecal extracts were able to convert diferulic acids of perfused dose was determined in bile conjugated with glu- to monoesters, and fecal extract was able to hydrolyze diferulic curonic acid or sulfate. The level of ferulic acid in feces was acids to monoesters and free acids. This result was supported the reported to be negligible, regardless of the dose of ferulic acid claim that the colon was the most effective area in the degrada- ingested. The estimated conjugated form of ferulic acid in pe- tion of diferulic acids (Andreasen et al., 2001). Chlorogenic acid ripheral tissues showed that 45–53% of the perfused dose was is formed by the esterification of caffeic acid with quinic acid. available for peripheral tissues. Since there was a proportion be- It is found in a wide range of fruits and vegetables, but partic- tween ferulic acid urinary excretion and ingested dose of ferulic ularly in high concentration in coffee. The cells obtained from acid, researchers agree that this compound was highly absorbed. human plasma, liver, small intestine, and colon incubated with Also, researches concluded that absorbed ferulic acid was read- chlorogenic acid and the amount of chlorogenic acid remained ily eliminated in urine, because the plasma concentrations of in the medium was determined to explain the chlorogenic acid ferulic acid 18 h after the meal were low or even undetectable. metabolism in human body. The form of chlorogenic acid was When rats were fed Duriac wheat enriched with ferulic acid, determined as unchanged in the extracts obtained from small in- fecal excretion of ferulic acid was significantly increased. This testine, liver, and plasma, whereas chlorogenic acid, caffeic acid, implies that the absorption of ferulic acid is strongly related and caffeic acid metabolites were found in fecal extract. These with its position. When it is present in a complex matrix, such results were explained by the enzymatic degradation of chloro- as cereals, the absorption of this compound is depressed. This genic acid to caffeic acid and quinic acid, with the estereases observation supported the finding related to urinary excretion produced by the colonic microflora. Researchers stated that caf- of ferulic acid in foods that was lower than those of rats fed a feic acid obtained from the degradation of chlorogenic acid could diet enriched with ferulic acid. Also, plasma ferulic acid con- be metabolised further either by colonic microflora or in the liver centration was found to be undetectable (Adam et al., 2002). according to data based on previous studies (Plumb et al., 1999). Rondini et al. (2002) reported that plasma concentration of fer- The urine samples of five healthy male volunteers with the mean ulic acid reached a maximum 30 min after ingestion and de- age of 30.8 years were collected after the coffee consumption creased quickly between 0.5 and 1.5 h and then decreased more containing 4 g of instant coffee powder and was continued on two slowly until 4.5 h, when Sprague-Dawley rats were fed a diet further occasions, 4 and8hafter the first coffee consumption. BIOAVAILABILITY OF PHENOLIC COMPOUNDS 455

Caffeic acid and caffeic acid metabolites were investigated in the Table 1 The subgroups of flavonoids (Robards et al., 1999; Morton et al., urine samples. Ferulic acid, a 3-O-methylated product of caffeic 2000; Aherne and O’Brien, 2002) acid, and isoferulic acid, a 4-O-methylated product of caffeic Main acid, were found in higher amounts in the urine post-coffee con- structure Flavonoids Chemical structure Samples sumption after β-glucuronidase treatment of the urine. The hy- C6C3C6 Flavones Cinencetin, nobiletin, pothetical absorption rate for caffeic acid was approximately tangeritin, 5.9%. The determination of ferulic and isoferulic acid in the isocinencitin, luteolin, highest amount, 1–3 h after the first coffee consumption, was apigenin evaluated as absorption and metabolism, and elimination of the caffeic acid metabolites were relatively fast. The amounts of dihydroferulic acid excreted in the individual samples reached a Flavonols Quercetin, kaempferol maximum 8 to 12 h after the first coffee consumption. Dihy- droferulic acid was found to be a nonconjugated form; this was the crucial difference from ferulic acid, mainly excreted as glucuronide. The amounts of hippuric acid and 3-hydroxyhippuric acid were reached at the maximum 12 h after the first coffee Flavanones Hesperidin, naringenin consumption for all volunteers. This finding supported the phenomena that proposes the degradation of caffeoyl quinic acid metabolites in the colon. Another finding related to the role of colonic microflora in the metabolism of caffeic acid was individual variations in the urinary excretions of ferulic, isoferulic, Flavanols (+)-catechin, and dihydroferulic acid (Rechner et al., 2001). Following the ap- (catechins) (−)-epicatechin, ple wine consumption, the amount of caffeic acid in the plasma (+)-gallocatechin, samples of 6 healthy volunteers was increased rapidly and de- (−)-epigallocatechin creased to undetectable limit at the end of the 90 min. However, no chlorogenic acid and ferulic acid were detected in the plasmas Anthocyanins Peonidin, delfinidin, (DuPont et al., 2002). The metabolism of chlorogenic acid and petunidin, cyanidin caffeic acid was investigated in the ileostomy fluid and in the urine obtained from subjects without a colon. Absorption was measured as the difference between the amount of supplement ingested and the amount of supplement excreted in ileostomy fluid. Chlorogenic and caffeic acid were also incubated in vitro Isoflavones Daidzein, genistein in gastric juice and duedonal fluid, and were incubated ex vivo in ileostomy fluid to determine their degradation in gastrointestinal fluids. The excretion rate of chlorogenic acid and caffeic acid were 67% and 5% in ileostomy fluid respectively. Of the ingested caffeic acid, 11% was excreted in urine, whereas the Chalcones Phloretin, arbutin, excretion of chlorogenic acid was negligible. Chlorogenic acid chalconaringenin and caffeic acid were almost completely recovered following in vitro incubation with gastric juice and duedonal fluid, and ex vivo incubation in ileostomy fluid. Therefore, researchers concluded that the absorption rates of chlorogenic acid and caffeic acid were 33% and 95%, respectively (Olthof et al., 2001). In another study, caffeic acid was determined as a conjugated form in plasma and urine 2 h after the consumption of dried plum alkylation and/or glycosylation of these groups. The hydroxy- (Cremin et al., 2001). lation degree of flavonoids is a determinant for their tendency to degradation in the colon and their degradation products produced by colonic microflora (Rice-Evans et al., 1996; Hollman FLAVONOIDS and Katan, 1997; Karakaya and El, 1997; Acar, 1998; Robards et al., 1999). Flavonoids are polyphenolic compounds that have the While the absence of the hydroxyl group in the molecule pre- diphenyl propane (C6-C3-C6)skeleton. The individual flavonoid vents the degradation of ring structure, the degree of hydroxy- subgroups are shown in Table 1. Individual differences within lation, such as 5,7-dihydroxylation and/or 4-hydroxylation, en- each group result from the variation in number and arrangement hances the tendency to degradation. The absence of the methyl of the hydroxyl groups, as well as from the nature and extent of group in the molecule causes the decrease in the tendency to 456 S. KARAKAYA degradation (Rice-Evans et al., 1996; Hollman and Katan, 1997; immediately after absorption (Olthof et al., 2000). Sesink et al. Karakaya and El, 1997; Acar, 1998; Robards et al., 1999). (2001) found trace amounts of quercetin aglycone in the plas- Flavonoids that exist in foods are usually glycosylated. The mas of subjects who consumed 325 µmol quercetin-3-glucoside linked sugar is often glucose or rhamnose, but can also be galac- or quercetin-4-glucoside. However, quercetin glucoside was tose, arabinose, xylose, glucuronic acid, or other sugars. The not determined in the plasma. The main metabolite found in number of sugars is most commonly one, but can be two or plasmas was quercetin glucuronides. In contrast, Manach et al. three, and there are several possible positions of substitution on (1999a) found that all plasma metabolites of quercetin obtained the polyphenol. The sugars can be further substituted. The glyco- from rats fed with 0.25% quercetin diet were present as conju- sylation influences chemical, physical, and biological properties gated forms, mainly sulfo and glucurono-sulfo conjugates. How- of the flavonoids (Scalbert and Williamson, 2000). ever, some free aglycones were detected in the liver. Time dependent concentrations of quercetin β-glucoside and quercetin β-rutinoside were determined to understand the effect of sugar FLAVONOLS AND FLAVONES moiety on quercetin absorption in humans (Sesink et al., 2001). The maximum plasma concentration of glucoside form was Flavonols and flavones have a similar C-ring structure with 20 times greater, and the time needed to reach this concentra- a double bond at the 2–3 position. Flavones, as opposed to tion was 10 times faster than the rutinoside form. Data sup- flavonols, lack a hydroxyl group at the 3-position. The absorp- ported the phenomena that quercetin glucosides absorbed from tion of quercetin aglycone in humans was found to be poor small intestine, whereas the rutinoside form absorbed from the (<1%) in the study of Gugler and his colleagues (Hollman, colon following the deglycosylation. The absorption of gluco- 1997). In contrast, the absorption of orally administered side from the small intestine was an indicator that represented quercetin aglycone was about 20% in rats (Hollman, 1997). the transportation of intact quercetin glucoside into enterocyte. In recent years, cinnamates and flavonoids in urine (Bourne The absorption of intact quercetin glucoside might be the rea- and Rice-Evans, 1998), and phloridzin, quercetin glycosides, son that they are resistant to hydrolysis by HCl in the stomach, and rutinosides in plasma were determined (Paganga and Rice- or that β-glucosidases are not secreted into the small intestine, Evans, 1997). Hollman et al. (1995) showed that the absorption or that the broad-specificity β-glucosidases needed to hydrolyze of orally administered quercetin aglycone was 24% in ileostomy quercetin glucoside are not bound to the brush border membrane. subjects. In the study, the absorption of quercetin glycosides Therefore, glucoside form of flavonoids might be carried into the from onions (mostly consists of quercetin β-glucoside) and pure small intestine enterocyte via active transport, for example, via quercetin rutinoside, a common glycoside in foods, was 52% the intestinal Na+/glucose cotransporter. The transportation of and 17%, respectively. Researchers concluded the possibility of naphtol glucosides across the intestinal wall of rats and the trans- glycosides absorption in small intestine. The bioavailability and portation of quercetin glucosides in everted sacs of rat jejenum pharmacokinetics of various forms of quercetin were investi- were occured by the active Na+/glucose transporter. However, gated using 9 healthy volunteers in the study of Hollman et al. no evidence for active transport of quercetin glucosides was (1997). Quercetin from onions was rapidly absorbed, whereas found in human intestinal epithelial Caco-2 cells (Hollman and pure quercetin 3-rutinoside, the major species in tea, showed a Arts, 2000; Hollman, 2001). In the study of Gee et al. (2000), markedly delayed absorption. The absorption rate from apples, it was demonstrated that quercetin 3-glucoside inhibited intesti- a source of quercetin β-glucoside and β-xyloside, was found nal transport of D-galactose when both were present simultane- to be intermediate. The time needed to reach maximum peak ously in the mucosal medium of male Wistar rats. Labeled D- levels following the ingestion of onions, apples, and quercetin- glalactose released more quickly from preloaded mucosal tissue 3-rutinoside were 0.7 h, 2.5 h and 9 h, respectively. The bioavail- in the presence of quercetin 3-glucoside compared to a substrate ability of quercetin from apples and of pure quercetin rutinoside free medium. These results stated that the glucoside interacts was both 30% relative to onions. These results supported the with the Na+/D-glucose cotransporter (SGLT1), which provides predominant role of sugar moiety in the bioavailability and ab- the main route of glucose and galactose absorption. The up- sorption of dietary quercetin in the human body (Hollman et al., take of radiolabeled quercetin aglycones was compared with the 1997). The increase in the plasma quercetin concentrations of uptake of three different similarly radiolabeled quercetin glu- 4 female and 6 male volunteers was determined after the con- cosides. Significantly quick transportation rates of quercetin- sumption of a complex meal rich in plant products. The plasma 3-glucoside and quercetin-4-glucoside were determined with quercetin concentration began to decrease 7 h after the inges- respect to the transportation of quercetin aglycones. However, tion and reached basal levels 20 h after the ingestion. Detected transportation rates of quercetin-3,4-diglucoside and quercetin quercetin was in the form of 3 methyl-quercetin (isorhamnetin) aglycone were found to be similar. Researchers proposed that (Manach et al., 1998). Similarly, Dupont et al. (2002) found that glycosylation might be an important factor in the transportation low doses of quercetin were extensively methylated in humans. rate of quercetin, but more critically significant factors were the The determination of the isorhamnetin peak in plasma, shortly number and positions of the glucose moieties. Transportation after ingestion of a single dose of quercetin glucosides, indicated of intact quercetin 3-glucoside across a perfused rat gut and that quercetin glucosides can be methylated into isorhamnetin preferential uptake of quercetin 3-glucoside from rat intestine BIOAVAILABILITY OF PHENOLIC COMPOUNDS 457 in vivo were determined in two different studies. However, only in vitro using Caco-2-cell monolayers. A more efficient uptake conjugated metabolites of quercetin were found in the serum of quercetin aglycone than its glucosides into Caco-2-cells was (Manach et al., 1999a; Gee et al., 2000) In the study of Gee et al. found. Quercetin, kaempferol, luteolin, and apigeninin were (2000), there was no intact quercetin 3-glucoside in the mucosal converted to glucuronide/sulfates by Caco-2-cells (Murota et al., tissue extracts and serosal solution of everted sacs from male 2000; Murota et al., 2002). The absorption mechanisms of lute- Wistar rats. The metabolites derived from the incubation of ev- olin and luteolin 7-O-β-glucoside, using Sprague Dawley, rats, erted sacs of the small intestines with quercetin 3-glucoside or were demonstrated. Luteolin glucuronides and luteolin were quercetin were found to be identical. This finding indicated that determined in serosal solution. It was concluded that luteolin the deglycosylation had occured rapidly. To explain the differ- 7-O-β-glucoside first hydrolyzed to luteolin and then absorbed ences between deglycosylation sides and rates of quercetin 4- in the form of luteolin and glucuronides. Luteolin, luteolin con- glucoside and quercetin 3-glucoside, researchers proposed that jugates and methylated conjugates were detected in rat plasma. another enzyme, such as lactase phlorizin hydrolase (LPH), may However, luteolin 7-O-β-glucoside was not present in plasma. act in the deglycosylation of quercetin 3-glucoside. Also, in an- To explain the existence of free luteolin in plasma, researchers other study, deglycosylation activity of LPH obtained from sheep proposed that some luteolin could escape from the intestinal con- small intestines of quercetin 3-glucoside, quercetin 4-glucoside, jugation and hepatic sulfation/methylation. They also reported and quercetin 3-4-diglucoside were shown (Day et al., 2000). that luteolin was absorbed more efficiently from the duodeno- Consequently Gee et al., 2000 proposed two possible poten- jejunum than that of from the ileum. Absorbed luteolin was tial mechanisms for the transportation of quercetin glucosides converted to glucuronides passing through intestinal mucosa (Figure 1). (Shimoi et al., 1998). Crespy et al. (2002) compared the absorption and metabolism of quercetin, rutin, and isoquercitrin (quercetin 3-O-glucose) in the stomach of Wistar rats. Gastric juice showed no degradation FLAVANONES effect on quercetin aglycones and quercetin glucoside. Sixty- two percent of ingested quercetin was recovered and absorbed Two studies published in 1958 reported that the metabolism of quercetin (5.7 µmol l−1)was regained in the bile 20 min af- naringin and hesperidin in humans was similar to the metabolism ter ingestion (4.07 ± 0.10 µmol l−1). Researchers also reported in animals. Although urinary excretion of phenolic acid was that the recovery rate was not influenced by a β-glucuronidase/ detected after the ingestion of naringin, hesperedin was ab- sulfatase hydolysis. Ingested amounts of rutin and isoquercitrin sorbed without any degradation (Hollman, 1997). Following almost completely recovered in gastric content. Metabolism and the consumption of grapefruit juice, naringin content of urine uptake of quercetin and quercetin glucosides were determined was changed between 5% and 57%, depending on the personal

Figure 1 Possible mechanisms for the absorption of quercetin glycosides in the small intestine (Gee et al., 2000) Abbreviation: Q 3-glc; quercetin 3-glucoside; Q, quercetin; SGLT1, sodium-dependent glucose transporter; LPH, Lactase phlorizin hydrolase; Q-glcA, quercetin glucuronide; UDGPT, Uridine-diphospho-D- glucose glucuronosyltransferase; β-G cytosolic β-glucosidase. 458 S. KARAKAYA variations (Scalbert and Williamson, 2000). In the study of Gil- and one-third glucuronide (Hollman and Arts, 2000). In con- Izquierdo et al. (2001), the bioavailability of flavanones (mainly trast, catechin metabolites found in the plasmas of rats fed with hesperidin, narirutin, hesperitin, and visenin 2) in fresh hand- 0.25% catechin diet, were mainly in the form of glucuronidated squeezed orange juice was determined by using an in vitro derivatives. The high methylation rate of catechin in the liver method. There was no change in the content of flavanone fol- (95%) reflected the intensive catechol O-methyltransferase ac- lowing the pepsin-HCl digestion. Both hesperidin and narirutin tivity (Manach et al., 1999b). Maximum plasma epicatechin were determined in the dialyzed fraction obtained from the level (260 nmol l−1)was determined 2 h after the consumption pancreatin-bile salt digestion. The soluble dialyzed flavanone of chocolate containing 137 mg of epicatechin. The reduction fraction was 16% of the soluble fraction of the juice. However, in plasma catechin concentration was about 40%,6hafter the the dialyzation rate of visenin 2 was higher as 20% of the soluble ingestion (Rein et al., 2000). flavonoids. The nondialyzed fraction contained 50% hesperidin and 50% hesperidin chalcone. Similar results were obtained in ANTHOCYANINS commercially pasteurized orange juice for the pepsin digestion. Following pancreatin-bile digestion, the hesperidin content of Anthocyanins are the most important group of water-soluble dialyzed fraction was found to be 30% of total flavanones. As a pigments in plants. They are generally found in the form of gly- consequence, it was reported that orange juice was a very rich cosides. The aglycones are rarely found in fresh plants. Glucose, source of flavanones (400–750 mg/l), but only the soluble form galactose, rhamnose, and arabinose are the sugars most com- wasavailable for absorption under the conditions of the small in- monly encountered, usually as 3-glycosides or 3,5-diglycosides testine. Therefore, small amounts of flavanones, approximately (Acar, 1998; Clifford, 2000). 15% to 30% of the soluble flavanones, can be absorbed through The studies on the bioavailability of anthocyanins are lim- small intestine. ited in literature. In the early studies, the presence of red pigments in urine was accepted as an indicator of anthocyanins absorption. According to the results of these studies, 1–2% of FLAVANOLS (CATECHINES) 500 mg anthocyanins were absorbed after oral administration in rabbits, similarly, red pigments in the urine of rats was ob-

The study published in 1979 indicated that plasma (+) cat- served following 50 mg percutaneous injection of anthocyanins. echin concentration in humans was about 10% of the ingested However, red pigments were not detected in the urine when rats dose. In another study published in 1983, the plasma peak con- were given 100 mg by gavage. Similar controversial results were centration of catechin 3 h after the ingestion was found to be also obtained in dogs (Clifford, 2000a). It was shown that a about 0.3% of the ingested dose. In the study published in 1985, glycoside of pelargonidin (3,5,7,4-tetrahydroxyflavylium) was the peak plasma concentration of 3-O-methyl-(+)-catechin was degraded by intestinal microflora in rats after oral administra- represented as 3% of the ingested dose 2 h after the consumption, whereas cyanidin (3,5,7,3-4-pentahydroxyflavylium) was tion. Lee and colleagues, in 1995, found that urinary extraction found to be stable (Hollman, 1997). Two studies published in following a single oral dose of decaffeinated green tea in 4 hu- 1972, reported that p-hydroxy-phenyllactic acid, a metabolite man volunteers was about 5% of the ingested dose for both generated by the colon microflora, was found in the urine of (−)-epigallocatechin and (−)-epicatechin, whereas epicatechin rats fed pelargonidin-3-glucoside. However, after feeding del- gallate and epigallocatechin gallate could not be detected. Cat- phinidin or malvidin-3,5-diglucoside, no metabolites were de- echins present in plasma were mainly in conjugated form and tected. (Clifford, 2000a). The absorption and metabolism of an- total plasma concentration 1 h after administration were 50– thocyanins (cyanidin-3-glucoside and malvidin-3-glucoside) in 250 ng mL−1, corresponding to about 0.2–0.9% of the dose male Wistar rats adapted for a diet enriched with a lyophilized given (Hollman, 1997). The significant increase in plasma to- blackberry powder were studied. No anthocyanins were detected tal catechin concentration was reported 30 min after the con- in plasma samples obtained at any time. Only glucoside forms sumption of 500 mL green tea in 5 healthy volunteers. To reach were detected in urine, although there were no detectable agly- maximum plasma concentration, (2.36 µMl−1), the researchers cone or conjugated forms after the treatments of urine with waited 60 min. After that time the peak plasma concentration be- β-glucuronidase. The detection of peonidin–3-glucoside was gan to decrease, and it was reduced to 50% of the peak concentra- attributed to the methylation of hydroxyl group at 3 position tion at the end of the 4 h period. Black tea ingestion caused the of cyanidin-3-glucoside. Researchers reported that ∼0.26% of rapid increase in plasma catechin + theaflavine concentration, ingested cyanidin-3-glucoside, either in glycoside form or in which was 1.29 µMl–1at the end of the 80 min, after ingestion methylated form, and 0.67% of ingested malvidin-3-glucoside (Kivits et al., 1997). Main forms of (−)-epigallocatechin gallate were recovered in urine. According to the researchers, the pres- present in plasma were sulphate conjugate (65%), followed by ence of low amounts of glucosides and cyanidin in fecal content the free form (20%) and glucuronide (15%). (−)-Epigallocatechin and type of compounds detected in urine clearly supported the was mainly present as glucuronide (60%), followed by sulphate differences in the metabolisms of anthocyanins than those of (30%), and the unconjugated aglycone form (10%). However, other flavonoids (Felgines et al., 2002). Similarly, cyanidin 3- conjugated (−)-epicatechin was found with two-thirds sulphate O-β-D-glucoside rapidly appeared in the plasma of male Wistar BIOAVAILABILITY OF PHENOLIC COMPOUNDS 459 rats after orally administration, whereas cyanidin, the aglycone (Murkovic et al., 2000; Cao et al., 2001; Perez-Vicente et al., form, was not detected. Protocatechuic acid, which was sup- 2002). The absorption of anthocyanins without the removal of posed to be a degradation product of cyanidin, was also de- glycoside was demonstrated in the study of Milbury et al. (2002), tected in plasma. The concentration of protocatechuic acid, was who examined the bioavailability and pharmacokinetics of elder- 8-fold higher than that of cyanidin-3-O-β-D-glucoside. Again berry anthocyanins in humans. They found neither glucuronates following the enzymatic treatment of plasma, there were no de- nor sulfates of the anthocyanins in the plasma samples. In the tectable additional products. The determination of the cyanidin study of Wu et al. (2002), the amount of anthocyanins in the urine in the jejunum of rats, but not in the plasma, was attributed to the samples of women who consumed the blueberry were found to production of cyanidin from cyanidin-3-O-β-D-glucoside by β- be lower than those of women who consumed elderberry, al- glucosidase reaction in the intestines. In tissues (liver and kid- though the amount of total anthocyanins in both sources were ney), methylated form of cyanidin-3-O-β-D-glucoside and/or nearly the same. This was explained either by the differences be- cyanidin-3-O-β-D-glucoside were detected. Researchers indi- tween the individual anthocyanin contents of two samples, or by cated that this observation was completely different, with respect the possiblle lower absorption rate of anthocyanins in blueberry. to other flavonoids (Tsuda et al., 1999). Researchers also tried to conclude on the similarities and/or dif- Miyazawa et al. (1999) reported that flavylium cation struc- ferences between the absorption mechanisms of anthocyanins ture of anthocyanins could make them more stable to bacterial and other flavonoids. They suggested that the presence of the hydrolyze than those of other flavonoids. Anthocyanin content sambubioside form of cyanidin was an indicator for the role of urine samples obtained from 6 healthy volunteers after the of sugar carriers, as shown for other flavonoids, especially for consumption of white or red wine were investigated. Following quercetin glucosides absorbed via intestinal glucose transporter. red wine consumption, any pigments represented as red wine Cyanidin is an anthocyanidin with a 3,4-dihydroxylation of anthocyanins were not observed in urine. However, adjustment ring B and has a similar chemical structure to those of flavonoids of pH to 1.0 in concentrated urine samples resulted in the devel- that could be metabolized after methylation, indicated by previ- opment of a pink pigment. In contrast, there were no pigments ous studies. This result was confirmed by the study of Felgines in the urine samples after the consumption of white wine in et al. (2002), who detected intact and methylated glycoside forms similar conditions. The main anthocyanins of red wine (mal- of blackberry anthocyanins in the urine samples of rats. Also, vidin 3-glycoside and malvidin 3-glycoside-acetate) were not Miyazawa et al. (1999) showed that the concentration of methy- detected in the urine samples before incubation with HCl, but lated anthocyanin was much higher than the original form in two compounds, which were identified as anthocyanin dimers, the rat liver, but they did not find the methylated form in urine were determined. Following the incubation of HCl, several peaks and plasma. However, Wu et al. (2002) detected the methylated that showed typical spectra of anthocyanins were detected in the form of anthocyanins in the urine of humans. In addition, they urine samples. The total amount of anthocyanins recovered in detected the glucuronide conjugate of anthocyanins in the urine the urine was 1.5–5.1% of the amount of those anthocyanins of humans after the consumption of elderberry. ingested by the volunteers (Lapidot et al., 1998). Anthocyanin availability in pomegranate juice was evaluated by using in vitro simulation of human digestion and absorption. Although a slight ISOFLAVONES increase in anthocyanin concentration (10%) was observed after stomach digestion, there was a significant decrease in an- Isoflavones are naturally occuring plant components that are thocyanin concentration following small intestine digestion. A structurally similar to the mammalian oestrogen and exhibit oe- slight increase was explained by the differencies in the pH of strogenicity. They possess a diphenylpropane structure in which the stomach condition (pH 2) and pomegranate juice (pH 3.8). the B-ring is located at the 3-position. Isoflavones are also ma- After the pancreatin bile salt digestion, the total dialyzed antho- jor dietary components of soybeans and soy products. Naturally cyanin fraction represented only 2.4%, whereas the nondialyzed occurring isoflavones in soybeans and soy products are genis- fraction was 15.3%. Adjustment of pH 2.0 caused an increase in tein, daidzein, their glucosides, namely genistin and daidzin, the anthocyanin concentration, both in dialyzed (22%) and non- and their methoxylated derivatives, namely biochanin A and for- dialyzed (48%) fractions (Perez-Vicente et al., 2002). In both mononetin (Figure 2) (Ruiz-Larrea et al., 1997). Daidzein, genis- studies, the high loss of anthocyanins were detected. Still, the tein, o-desmethylangolensin, and equol are the main isoflavones reason for this loss remained unknown, although the possibility in an ordinary Japanese diet. They are mainly found in con- was considered that part of the anthocyanins is metabolized to jugated form in the plasma samples of Japanese men. All of some noncolored forms, oxidized, or degraded into other chem- them, except o-desmethylangolensin, were mainly in the form icals, which would escape from the detection (Lapidot et al., of glucuronide, whereas o-desmethylangolensin was found to be 1998; Perez-Vicente et al., 2002). The extremely low bioavail- mainly in the free plus sulfates (Adlercreutz et al., 1993). The ability of anthocyanins from elderberry observed in one healthy urinary excretion of daidzein and genistein were 21% and 9%, volunteer was attributed to the quick degradation or excretion respectively, 24 h after the ingestion of soy milk, which contains of anthocyanins (Murkovic et al., 2000). Researchers also indi- daidzein and genistein glycosides equivalent to 2 mg of agly- cated that no glycoside hydrolysis takes place during digestion cone. The plasma concentration of these compounds was found 460 S. KARAKAYA

Figure 2 Chemical structure of naturally occurred isoflavonoids in soybeans and soy products. (Joannou et al., 1995; Ruiz-Larrea et al., 1997). to be 2 µMl –1 6.5 h after consumption. It was reported that known dose of isoflavone-rich foods was reported. It was shown the differences in the urinary excretion amounts of isoflavones that consumption of textured vegetable protein, which was equal reflected the differences in their bioavailability (Hollman, 1997). to the amount of 45 mgday−1 conjugated isoflavones, over a Adlercreutz et al. (1995) determined the pattern of conju- one month period, resulted in a 1000-fold increase in isoflavone gation of the phytoestrogens in 4 urine samples from vegetar- metabolite excretion. Variations between urinary equol excre- ian or semivegetarian women and in two samples from men. tion of individuals were detected. Although only 2 of the 6 They reported that more than 60% of all detected compounds subjects excreted substantial levels of equol, the other 4 sub- occured in the monoglucuronide fraction. Daidzein, enterodiol, jects predominantly excreted daidzein and genistein. Breinholt and equol were excreted to a relatively high extent as sulfoglu- et al. (2000) determined the urinary recovery of orally admin- curonides, while genistein was excreted as diglucuronide. This istrated equol, genistein, biochanin A, and daidzein to 6-week- difference was explained by the variations in the number of hy- old female mice. The urinary recovery of equol was over 90% doxyl groups between daidzein and genistein. Genistein, which of a single administrated dose, whereas the urinary recoveries has 3 free hydroxyl groups was, therefore, more susceptible of genistein, biochanin A, and daidzein were 10.8, 16.3, and to form diconjugates than was daidzein. The mean percent- 3.3%, respectively. Urinary metabolites of genistein were in the age excretion of matairesinol, entrodiol, enterolactone, daidzein, form of hydroxylated and dihydrogenistein. Dihydrobiochanin, genistein, o-desmethylangolensin, and equol in the urine sam- dihydrogenistein, hydroxylated biochanin A, and genistein were ples of 5 females and 2 males was detected to be mainly in determined in the urinary samples of mice after the oral adminis- the monoglucuronide form. In the other studies, the determi- tration of Biochanin A. Urinary metabolites of daidzein were in nation of the conversion of daidzein to equol (70%) and to the form of dihydrodaidzein, desmethylangolensin, and equol. o-desmethlyangolensin in sheep, and the detection of equol to Piskula et al. (1999) compared the absorption of isoflavone agly- o-desmethylangolensin in human urine samples was accepted as cones and glucosides in rats. Aglycones were rapidly absorbed, an indicator showing the breakdown of daidzein via alternative and isoflavone metabolites were detected in the blood 3 min after pathways (Cassidy et al., 2000). Joannou et al. (1995) reported administration. Glucoside metabolites were found in the plasma the proposed pathway of daidzein metabolism. In this path- with a few minutes delay, as compared to aglycones. This delay way, after the conversion of daidzein to dihydrodaidzein, two wasexplained by the differences in the metabolism of aglycones paths could be possible. In the first path, dihydrodaidzein was and glucosides. Extra time was needed for glucosides to reach converted to tetrahydrodaidzein and then equol; in the second the duodenum, where they were absorbed. However, the agly- path, dihydrodaidzein was converted to 2-dehydro-o-desmethyl- cones were absorbed from the stomach, while their glucosides angolensin and then o-demethylangolensin. Conversely, genis- were not. It was also observed that genistein was better absorbed tein metabolism did not parallel to that of daidzein. With respect from the stomach than daidzein at pH 2 and pH 6.2. The average to proposed pathway, genistein first converted to dihydrogenis- level of total isoflavones in the blood of soy fed infants was re- tein and then to 6-hydoxy-o-desmethylangolensin. In addition, ported to be as 979 ngml−1 and 25000 ngml−1 to 50000 ngml−1 considerable individual variability in metabolic response to a in urine (Cassidy et al., 2000). Pharmacokinetic studies showed BIOAVAILABILITY OF PHENOLIC COMPOUNDS 461 that the peak plasma concentrations of premenopausal women take of bearberry leaf extract. Following the administration of who consumed 50 mg of pure isoflavones, or their glucosides, herbal medicinal products containing bearberry leaf extracts, were detected between 80 and 800 ngml−1. The time to reach glucuronide and sulfate conjugates of arbutin were found as main this concentration was about 6 to 8 h. In another study, the time urinary metabolites. The total concentration of both conjugates to reach maximum plasma concentration (daidzein 396 µgl−1, in urine was about half of the administered dose. Crespy et al. genistein 659 µgl−1)was6hin7menwhoconsumed 60 g (2001a) investigated the absorption and metabolism of quercetin of soy bean. (Cassidy et al., 2000). Isoflavone aglycones (genis- and isoquercitrin and compared it with those of phloretin and tein, daidzein) and isoflavone glucosides (genistin, daidzin) were phloridzin in rats using an in situ intestinal perfusion model. It incubated with Caco-2 cell monolayers. Glucuronides and sul- was shown that phloretin and phloridzin were absorbed in the fates form of aglycones appeared in the mucosal side. Although small intestine of rats. Conjugated forms of both phloretin and no changes were observed in the amount of isoflavone gluco- phloridzin were recovered in the effluent. The absorption rate sides at the end of the incubation period, isoflavıne aglycones of glucoside was found to be lower than the absorption rate of decreased significantly1hafter the incubation, and their glu- aglycone. The hydrolysis of glucose moiety was reported to be curonide/sulfates appeared. Total recovery of both intact agly- the crucial step in the intestinal metabolism. In the following cones and their glucuronide/sulfates was about 35% of initial study, Crespy et al. (2001b) compared the changes in plasma dose. For genistin and daidzin, intact glucosides, their aglycones, and urine concentrations of phloretin and phloridzin in rats. The and glucurono/sulfo conjugates were detected in the serosal so- bioavailability of both was found to be similar. However, the lution. However, their total recovery was about 1.5% of initial plasma kinetics of phloretin and phloridzin were different. The dose. The differences were also detected in the tranport of genis- appearance of phloretin in plasma was more rapid than that of tein, possesing a B-ring at the 2-position, and flavonoids pos- phloridzin. Phloretin detected in plasma was mainly in conju- sesing a B-ring at the 3-position. The intact aglycone form of gated forms (85–95%), but some unconjugated phloretin was quercetin, kaempferol, luteolin, and apigenin were found to be also determined. In contrast, intact phloridzin was not detected significantly less than their glucuronide/sulfates in serosal so- in the plasma of rats fed a phloridzin meal. However, researchers lution. Contrary to flavonols and flavones, the aglycone form stressed that the dose of phloretin added to the experimental of genistein was greater than their conjugates. Researches con- meals was relatively high (22 mg), so that the bioavailability cluded that isoflavonoid aglycones were taken up across the of phloretin should be modified by lower doses, especially by cell membranes and transported without metabolic conversion. consuming apples. Researches also stated that affinity to the cellular membrane af- fected the efficiency of cellular uptake by the passive diffusion. Increased order of affinity to liposamal membranes were re- CONCLUSION ported as genistin = daidzin < daidzein < genistein < flavonoid agkycones (Murota et al., 2002). Absorption and Bioavailability of Food Phenolics

In conclusion, the factors that effect the absorption and bio- CHALCONES availability of phenolic compounds can be summarized as follows: The most common chalcones found in foods are phloretin and its glucoside phloridzin (phloretin 2-O-glucose), chalconarin- 1. Partition coefficients seem to play an effective role in the genin, and arbutin. Phloretin and phloridzin are characteristic absorption of phenolics that have no sugar or organic acid of apples. Chalconaringenin is characteristic of tomatoes, and substitutes in their structure. Hydrophilic compounds can- arbutin is characteristic of pears. Arbutin is also found in straw- not be absorbed in the upper part of gastrointestinal tract berry and bearberry, in wheat, in wheat products, and in trace (Scalbert and Williamson, 2000). amounts in tea, coffee, red wine, and broccoli (Robards et al., 2. Partition coefficients seem to play an effective role in the 1999, Clifford 2000b). absorption of hydrophobic phenolics that have sugar or or- Studies on the bioavailability of chalcones from food sources ganic acid and ester linked substitutions in their structure, are limited. Most of the studies aimed to investigate the pharma- whereas hydrophilic phenolics having similar structure are cological effect for the evaluation of the availability of poten- degraded by the esterases produced by the colonic microflora tially bioactive compounds, to prove the efficiacy for develop- in the colon (Scalbert and Williamson, 2000; Olthof et al., ing recpective formulations, or to develop selective and sensitive 2001; Rechner et al., 2001; Adam et al., 2002; Rondini et al., method for the determination of free compounds, metabolites, 2002). and conjugates. In the study of Alvarado and Monreal (1967), 3. The number of sugar molecules seems to play an effective in vitro active transport of phenyl-β-D-glucopyranosides was role in the absorption of phenolics. If the phenolics contain the same pathway as active free sugar transport. Glöck et al. a sugar molecule, such as glucose, galactose, or xylose, they (2001) developed a validated method for the determination of will be absorbed through the small intestine by the cytoso- hydroquinone glucuronide and sulfate in urine after oral in- lic β-glucosidase/lactase phlorizin hydrolase. The absorption 462 S. KARAKAYA

is also related to the specificity of carriers (Hollman et al., 1997; Hollman and Arts, 2000; Gee et al., 2000; Scalbert and Williamson, 2000; Hollman, 2001; Muroto et al., 2002). 4. Phenolics, which have rhamnose in their molecule, cannot be absorbed through the small intestine. They are degraded by the action of rhamnosidases produced by the colonic microflora. 5. Acylated flavonoids, such as (−)-epicatechin and (−)- epigallocatechin, are absorbed without deconjugation and hydrolysis (Manach et al., 1999a; Hollman and Arts, 2000; Scalbert and Williamson, 2000). 6. Isoflavone aglycones were absorbed from the stomach, while their glycosides were absorbed from the duedonum (Murota et al., 2002). 7. Dihydrochalcones were absorbed in the small intestine of rats following the conjugation and, thus, could be recovered intact in plasma (Glöck et al., 2001; Crespy et al., 2001a, 2001b). 8. Personal variations are effective in the absorption and/or transportation of phenolics.

Metabolism of Food Phenolics

Studies showed that phenolic compounds are metabolized by

the deconjugation and reconjugation reactions. Phenolics are hydrolyzed to their free aglycones, then are conjugated by methylation, sulfation, glucuronidation, or a combination. A followed metabolic pathway is similar to that of a drug metabolism. How- ever, there are significant differences between the administration of drugs and the consumption of dietary phenolic compounds. Drugs are usually administrated in hundreds of milligrams in one concentrated dose, however, dietary phenolics are usually, consumed in lower than hundreds of milligrams in a diluted dose. Therefore, drugs can readily saturate the metabolic pathways, whereas food phenolics cannot. On the other hand, cir- Figure 3 Possible metabolic pathway of phenolics (Scalbert and Williamson, 2000). Abbreviations used: CBG, cytosolic β-glucosidase; LPH, lactase phlo- culating species of food phenolics would be expected to be rizin hydrolase; COMT, catechol-O-methyltransferase; UDPGT, glucuronosyl conjugated. When food phenolics are administered at pharma- transferase; SULT, phenol sulfotransferases. cological doses, they are found in the free form in the blood (Scalbert and Williamson, 2000). The dose will also determine that allows the prediction of uptake of phenolics from the diet the primary site of metabolism. Large doses are metabolized (Figure 3). primarily in the liver. However, small doses may be metabolized in the intestinal mucosa. The liver has a secondary role in the metabolism of small doses (Scalbert and Williamson, 2000). It was shown that phenol glycosides were first deglycosilated REFERENCES and then converted to glucuronides or sulfates, with or without methylation, in studies using rats or isolated rat intestine Acar, J. 1998. Fenolik bile¸sikler ve doˇgal renk maddeleri. In: Gıda Kimyası, pp. 435–452. Saldamlı, I.,˙ Ed., Hacettepe Universitesi¨ Yayınları, Ankara. (Hollman et al., 1997; Manach et al., 1998; Shimoi et al., 1998; Adam, A., Crespy, V., Levrat-Verny, M.-A., Leenhardt, F., Leuillet, M., Gee et al., 2000; Sesink et al., 2001; Adam et al., 2002; Rondini et Demigné, C., and Rémésy, C. 2002. The bioavailability of ferulic acid is al., 2002; Murota et al., 2002). The existence of conjugation reac- governed primarily by the food matrix rather than its metabolism in intestine tions in the metabolism of phenolics were also shown in human and liver in rats. J. Nutr., 132:1962–1968. studies (Adlercreutz et al., 1995; Manach et al., 1998; Rechner Adlercreutz, H., Markkanen, H., and Watanabe, S. 1993. Plasma concentrations of phyto-oestrogens in Japanese men. Lancet, 342:1209–1210. et al., 2001; Felgines et al., 2002; Wu et al., 2002). However, Adlercreutz, H., van der Wildt, J., kinzel, J., Attalla, H., Wähälä, K., Mäkelä, T., the data available on phenolics bioavailability are still limited, Hase, T., and Fotsis, T. 1995. Lignan and isoflavonoid conjugates in human Scalbert and Williamson (2000) proposed the possible pathway urine. J Steroid Biochem Molec Biol., 52(1):97–103. BIOAVAILABILITY OF PHENOLIC COMPOUNDS 463

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