Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology

Nutrition Research 24 (2004) 851 – 874 www.elsevier.com/locate/nutres

Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology Iris Erlund* Biomarker Laboratory, Department of Health and Functional Capacity, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland Received 15 October 2003; revised 7 June 2004; accepted 16 July 2004

Abstract The bioactivities, dietary sources, bioavailability, metabolism, and epidemiology of 3 flavonoids, quercetin, hesperetin, and naringenin, are reviewed. The use of their plasma concentrations as biomarkers of dietary intake is also discussed. The compounds were chosen because of their significant dietary intakes and promising bioactivities, and in the case of quercetin, because epidemiological studies suggest protection against cardiovascular disease. D 2004 Elsevier Inc. All rights reserved. Keywords: Flavonoids; Quercetin; Hesperetin; Naringenin; Bioavailability; Metabolism; Biomarker; Epidemiology

1. Introduction Flavonoids are a large group of phenolic plant constituents. To date, more than 6000 flavonoids have been identified [1], although a much smaller number is important from a dietary point of view. That flavonoids possess bioactive potential has been recognized for long, but until recently, data about their bioavailability, metabolic fate, and health effects were limited. In the 1990s, interest in these compounds truly commenced and has been growing ever since. Flavonoids are potent antioxidants in vitro, and therefore one of the main interests in the compounds has involved protection against cardiovascular disease. Antioxidation is, however, only one of the many mechanisms through which flavonoids could exert their actions. * Tel.: +358 9 4744 8466; fax: +358 9 4744 8281. E-mail address: [email protected]. 0271-5317/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2004.07.005

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Flavonoids are divided to several subgroups, and it is important to keep in mind that the biological and chemical properties of flavonoids belonging to different subgroups can be quite different. This review will mainly focus on the flavonoids quercetin, hesperetin, and naringenin.

2. Flavonoids 2.1. Chemistry and classification Flavonoids consist of 2 benzene rings (A and B), which are connected by an oxygencontaining pyrene ring (C) (Fig. 1). Flavonoids containing a hydroxyl group in position C-3 of the C ring are classified as 3-hydroxyflavonoids (flavonols, anthocyanidins, leucoanthocyanidins, and catechins), and those lacking it as 3-desoxyflavonoids (flavanones and flavones). Classification within the 2 families is based on whether and how additional hydroxyl or methyl groups have been introduced to the different positions of the molecule. Isoflavonoids differ from the other groups; the B ring is bound to C-3 of ring C instead of C-2. Anthocyanidins and catechins, on the other hand, lack the carbonyl group on C-4 [2]. Flavonoids are mainly present in plants as glycosides. Aglycones (the forms lacking sugar moieties) occur less frequently. At least 8 different monosaccharides or combinations of these (di- or trisaccharides) can bind to the different hydroxyl groups of the flavonoid aglycone [3]. The large number of flavonoids is a result of the many different combinations of flavonoid aglycones and these sugars. The most common sugar moieties include d-glucose and l-rhamnose. The glycosides are usually O-glycosides, with the sugar moiety bound to the hydroxyl group at the C-3 or C-7 position. 2.2. Occurrence in food Flavonoids are present in most edible fruits and vegetables, but the type of flavonoids obtained from different dietary sources varies. The main dietary flavonoids and their sources are shown in Table 1. Intake estimates for flavonoids on a population level are only available for a few flavonoid subclasses, such as flavonols, flavanones, and isoflavones. 2.2.1. Flavonols The most common flavonol in the diet is quercetin. It is present in various fruits and vegetables, but the highest concentrations are found in onion (Table 1) [4]. The importance of different foods as quercetin sources varies between countries. Hertog et al [5] calculated flavonol intakes from the Seven Countries Study, which was started in the late 1950s, and reported that tea was the predominant source of quercetin in the Netherlands and Japan. Wine was the major source of quercetin in Italy, while onion and apples contributed most in the United States, Finland, Greece, and former Yugoslavia. More recently, H7kkinen et al [6] estimated that onions, followed by tea, apples, and berries are the major sources of quercetin in Finland. It should be noted that onion is usually not consumed in high quantities, but it is an important source because of its high quercetin content. Tea and especially wine, on the other hand, contain relatively low amounts of quercetin but are consumed, at least in some countries, in rather high quantities. The daily intake of quercetin was estimated to range between 3 and 38 mg in the Seven Countries Study [5], and in Finnish male smokers, the

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Fig. 1. Chemical structures of the flavane nucleus, quercetin (M w = 302 g/mol), hesperetin (M w = 302 g/mol), and naringenin (M w = 272 g/mol), and some of their most common glycosides.

intake estimate (from the 1980s) was 7.4 mg [7]. In the United States, the estimated intake of flavonols and flavones was 20 to 22 mg/d, of which 73% to 76% was quercetin (values are for women and men) [8]. Quercetin is present in plants in many different glycosidic forms [2], with quercetin3-rutinoside, also called quercetin-3-rhamnoglucoside or rutin, being one of the most widespread forms. In onions, quercetin is bound to 1 or 2 glucose molecules (quercetin4V-glucoside and quercetin-3,4V-glucoside). Examples of other dietary quercetin glycosides are quercetin galactosides, which are found in apples, and quercetin arabinosides, which are present in berries. Other flavonols in the diet include kaempferol (broccoli), myricetin (berries), and isorhamnetin (onion). The chemical structures of quercetin and some quercetin glycosides are shown in Fig. 1.

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Table 1 Main dietary flavonoids and their sources in the diet Flavonoid Flavonol Quercetin-3,4V-glucoside Quercetin-3-glucoside Quercetin-3-rhamnoglucoside (rutin) Quercetin-3-galactoside Quercetin-3-rhamnoside Quercetin-3-arabinoside Quercetin-3-glucoside Quercetin-3-rhamnoglucoside Quercetin-3-rhamnoside Quercetin-3-galactoside Myrisetin-3-glucoside Flavone Luteolin-7-apiosylglucoside Flavanone Hesperetin-7-rhamnoglucoside (hesperidin) Naringenin-7-rhamnoglucoside (narirutin) Naringenin-7-rhamnoglucoside (naringin) Naringenin-7-rhamnoglucoside (narirutin) Flavonols (+)-Catechin ( )-Epicatechin (+)-Catechin ( )-Epicatechin (Epi)catechin and their gallates Anthocyanins Cyanidin-3-glucoside Cyanidin-3-rutinoside Delphinidin-3-glucoside Delphinidin-3-rutinoside Isoflavones Genistein-7-glycoside Daidzein-7-glycoside

Source

Content of aglycone (mg/kg) and reference

Onion

284-486 [4]

Black tea Apple

10-25 [161] 21-72 [4]

Black currant

44 [6]

71 [6] Red pepper

7-14 [4]

Orange juice

116-201 [162] 15-42 [162] 68-302 [162]

Grapefruit juice

Apple

Black tea

4-16 [13] 67-103 [13] 16-53 [14] 9-42 [14] 102-418 [14]

Black currant

760 [163]

Red wine

590 [163]

Soybeans

480 [16] 330 [16]

2.2.2. Flavanones Flavanones occur almost exclusively in citrus fruits. The highest concentrations are found in the solid tissues, but concentrations of several hundred milligrams per liter are present in the juice as well [9]. Hesperidin (hesperetin-7-rutinoside) and narirutin (naringenin-7-rutinoside) are the major flavonoids of oranges and mandarins. The main flavonoids of grapefruit are naringin (naringenin-7-neohesperoside) (70%) and narirutin (20%) [10]. Low concentrations of naringenin are also found in tomatoes and tomato-based products. Fresh tomatoes, especially tomato skin, also contain naringenin chalcone, which is converted to naringenin during processing to tomato ketchup [11]. In Finland, the average intake of naringenin has been estimated to be 8.3 mg/d, and for hesperetin, the estimate is 28.3 mg/d [12]. The structures of hesperetin, naringenin, and their most important glycosides are shown in Fig. 1.

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2.2.3. Catechins Catechins usually occur as aglycones or are esterified with gallic acid. (+)-Catechin and ( )-epicatechin are found in various fruits and vegetables, such as apples, pears, grapes, and peaches [13]. The highest concentrations of catechins are found in tea and red wine [14]. 2.2.4. Flavones The main flavones in the diet are apigenin and luteolin. Their dietary intake is rather low because they occur in significant concentrations in few commonly consumed plants. Most important sources are red pepper [4] and celery [15]. 2.2.5. Anthocyanidins Anthocyanins (or anthocyanidin glycosides) are responsible for the red, blue, or violet color of edible fruits, such as plums, apples, eggplant, and many berries. The most common anthocyanidins include pelargonidin, cyanidin, delphinidin, and malvidin [2]. 2.2.6. Isoflavonoids The predominant isoflavonoids are the isoflavones genistein and daidzein, which occur mainly in legumes. The highest concentrations are found in soybean and soy products, and much lower concentrations are present in other legumes [16,17], not to mention other vegetables and fruit.

3. Quercetin, hesperetin, and naringenin 3.1. Biological activities In vitro studies indicate a wide range of biologic activities for different flavonoids. These studies have mainly been performed with flavonoid aglycones or glycosides. Until very recently, flavonoid metabolites were rarely used, mainly because data about their identity were scarce; moreover, chemical standards for only a few potential metabolites are commercially available. A few general reviews on flavonoids [18-20] have been published during the past few years. These reviews mainly concern quercetin, which is the most studied flavonoid. Because of the large number of studies on quercetin (over 3000 citations listed in PubMed), only representative examples of its bioactivities can be given here. Quercetin has been reported to exhibit antioxidative [21,22], anticarcinogenic [23-25], anti-inflammatory [26], anti-aggregatory [27], and vasodilating [28] effects. The mechanisms behind the effects are largely unknown, but it is possible that several different types of biochemical events precede them. Antioxidation, for instance, could be a result of metal chelation [29,30], scavenging of radicals [31,32], enzyme inhibition [33,34], and/or induction of the expression of protective enzymes [35]. Anticarcinogenesis, on the other hand, could result from enzyme inhibition [36,37], antioxidation, or effects on gene expression [38-40]. Altered gene expression could lie behind the anti-inflammatory effect as well [41]. Regarding anticarcinogenesis, it should be noted that in the 1970s, quercetin was actually considered a carcinogen because the compound showed mutagenicity in the Ames test [42]. However, a number of long-term animal studies subsequently performed with different species have indicated that this is not the case. On the contrary, quercetin has been shown to inhibit

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carcinogenesis in laboratory animals [43]. During the past few years, some reports on the biological activities of potential quercetin metabolites have been published. In these studies, it has been shown that the site and/or type of conjugation are important determinants of its antioxidant activity [44-46]. The flavanones have gained less interest than quercetin and have been covered less extensively in previous reviews. A lot of attention has been paid to their anticarcinogenic properties. Hesperetin (and orange juice) has been shown to inhibit chemically induced mammary [47], urinary bladder [48], and colon [49,50] carcinogenesis in laboratory animals. Hesperetin [51], as well as the other major citrus flavanone naringenin [52,53], also possess some antioxidant activities, although this activity is poorer compared with many other polyphenols. Other possible effects of hesperetin and naringenin are on lipid metabolism. They have been reported to regulate apolipoprotein B secretion by HepG2 cells, possibly through inhibition of cholesterol ester synthesis [54], and to inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acyl coenzyme A:cholesterol O-acyltransferase in rats [55,56]. Furthermore, a decrease in plasma low-density lipoprotein levels and hepatic cholesterol levels in rabbits fed a high-cholesterol diet has been observed [57]. An increase of high-density lipoprotein levels in hypercholesterolemic human subjects after consumption of orange juice was also reported [58]. According to a literature search in PubMed, these results had not been confirmed or refuted in other human studies. Other biological activities attributed to naringenin include antiinflammatory [59] actions and different types of effects on sex hormone metabolism [60-63]. The compound has, for instance, been shown to bind to estrogen receptors [64]. Most publications on naringenin concern its possible role in grapefruit juice–drug interactions [65,66]. The considerable increase in plasma concentrations of many drugs metabolized by intestinal cytochrome P-450 IIIA (CYP3A4) when administered concominantly with grapefruit juice is well documented and is of clinical relevance [67,68]. Naringenin is an inhibitor of the enzyme [69] and could be one of the compounds causing the interaction. However, other grapefruit constituents seem to be more important in this phenomenon. In any case, the grapefruit juice–drug interaction is a very interesting indication of how dietary components may influence our health (ie, by modulation of the biotransformation system). 3.2. Metabolism Important sites of flavonoid metabolism are the gastrointestinal lumen, cells of the intestinal wall, and the liver. The metabolism of flavonoids is a matter of interest because metabolism often affects the biological activity of a compound and its ability to enter cells. One common characteristic of flavonoids is that they occur as glucuronide and sulfate conjugates in the bloodstream. 3.2.1. Metabolism before absorption The mechanisms of and the events preceding flavonoid absorption have been a matter of much debate. Previously, flavonoid uptake was thought to occur in the large intestine only, where bacterial enzymes capable of cleaving flavonoid glycosides before absorption of the aglycone are present. In the 1990s, however, quercetin or quercetin conjugates were shown to rapidly appear in plasma after consumption of foods containing quercetin glycosides,

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indicating absorption from the small intestine. The sodium-dependent d-glucose cotransporter was hypothesized to transport quercetin glucosides across the enterocytes [70]. Later, transport of quercetin-4V-glucoside was actually demonstrated in a cell model of human intestinal absorption (Caco-2 cell line), but the compound was effluxed from the cells by the secretory protein multidrug resistance–associated protein 2 (MRP2), and transcellular absorption did not occur [71]. In fact, quercetin glycosides have not been reliably shown to occur in plasma. Attempts have been made to analyze them in plasma; however, this is a difficult task because the retention times of flavonoid glucuronides and glycosides are very similar and their ultraviolet spectra are not very specific [44]. Others [72-74] and I (unpublished data) have not been able to find quercetin glucosides in human plasma using mass spectrometry or high-performance liquid chromatography with electrochemical detection. The presence of enzymes capable of cleaving flavonoid glycosides in the small intestine has now been demonstrated, and it appears that as a general rule, flavonoid glycosides are cleaved, either in the lumen or in the cells of the gut, before absorption. Anthocyanins are an exception to this rule; they are found in urine as glycosides (and glucuronides) [75,76]. This has added to the confusion; however, there is a good explanation. The aglycons of anthocyanins are very unstable in the pH range of the intestines and are likely degraded before absorption can take place. Therefore, even a small amount of glycoside present in urine, in relation to other forms, will be important. Small intestinal enzymes capable of cleaving flavonoid glycosides are lactase-phlorizin hydrolase, also called lactase [77], and another, less well-characterized b-glycosidase with a broad substrate specificity [78]. In vitro, lactase has been shown to cleave quercetin4V-glucoside, quercetin-3-glucoside, quercetin-3,4V-glucoside, 3V-methylquercetin-3-glucoside, genistein-7-glucoside, and daidzein-7-glucoside. Quercetin-3-rhamnoglucoside (rutin) and naringenin-7-rhamnoglucoside (naringin) were not substrates for the enzyme. Cell-free extracts of human small intestine and liver, containing the latter enzyme, hydrolyzed several flavonoid glucosides with the sugar moiety attached to the 4V-OH or 7-OH moieties. Compounds such as quercetin-3,4V-glucoside, quercetin-3-glucoside, quercetin-3-rhamnoglucoside (rutin), and naringenin-7-rhamnoglucoside (naringin) were not hydrolyzed by it. Which enzymes in the large intestine are responsible for the hydrolysis of flavonoid glycosides remains to be elucidated. Enzymes (b-glucosidase, a-rhamnosidase) produced by gastrointestinal bacteria, such as Bacteroides JY-6 [79], Streptococcus faecium VGH-1, and Streptococcus sp. strain FRP-17 [80], and Escherichia coli HGH21 and HGH6 [81], hydrolyze some flavonoid glycosides, but other, yet unidentified bacteria, may also be important. Enzymes from at least Eubacterium ramulus [82,83] and Clostridium orbiscindens [84], also cleave the flavonoid ring itself, which results in the formation of ring fission products, such as hydroxyphenylacetic acids and hydroxyphenylpropionic acids [85-89]. Some of these ring fission products are likely absorbed, and could, at least in theory, exert effects both systemically and in the gastrointestinal tract. 3.2.2. Conjugation and methylation in the intestinal wall and the liver? Incubation of human plasma with a mixture of b-glucuronidase/sulfatase releases quercetin aglycone, which shows indirectly that quercetin is present in plasma at least as glucuronides, sulfates, or mixed sulfoglucuronides. Perfusion studies performed with rat intestines indicate

858

Study design and reference

Source

Dosea (mg/d)

Healthy subjects (n = 9)b [98], random crossover, single ingestion

Onion

68

1.4 F 0.5

AUC(0-36

Apple sauce Q-3-rutinoside Q-3-rutinoside

98 100 94

0.4 F 0.2 0.3 F 0.4

AUC(0-36 h) = 3.5; C max = 305 AUC(0-36 h) = 3.3; C max = 298 AUC(0-l) = 3.7; C max = 179

Q-4V-glucoside Q aglycone

94 8 20 50

AUC(0-l) = 18.8; C max = 3500 AUC(0-32 h) = 2.1; C max = 136 AUC(0-32 h) = 3.5; C max = 219 AUC(0-32 h) = 4.4; C max = 285

Q-3-rutinoside

8 20 50 98

3.0 F 0.3

AUC(0-32 AUC(0-32 AUC(0-32 AUC(0-36

2.6 F 0.4

AUC(0-36

Healthy subjects (n = 9)c [70], random crossover, single ingestion Healthy subjects (n = 16) [104], random crossover, single ingestion, 3 doses per treatment

Healthy subjects (n = 9)c [164], random crossover, single ingestion Healthy subjects (n = 27)c [165], randomized parallel, 28 d, 4 ingestions per day Healthy subjects (n = 5) [139], random crossover (3 times for 1 week), several ingestions per day

Q-3-glucoside Q-4V-glucoside Q-aglycone + Q-3-rutinoside Fruit juiced (750 mL) Fruit juice (1000 mL) Fruit juice (1500 mL)

100 1000 100 4.8 6.4 9.6

Fasting plasma Urinary Bioavailability (lmol/L or nmol/L) (nmol/L) recovery (%)

1414 F 295

50 106 30

0.3-0.5 0.3-0.5 0.3-0.5

h)

= 7.7; C max = 742

h)

= = = =

h)

= 17.5; C max = 4454

h) h) h)

1.6; C max = 79 2.6; C max = 159 4.0; C max = 298 19.1; C max = 5053

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Table 2 Selected bioavailability studies on quercetin (Q)

Diabetic subjects (n = 10)c [136], random crossover (2 times for 2 weeks), three servings per day

Healthy subjects (n = 12)b,e [100]

Healthy subject (n = 19) [166] Healthy subject (n = 16) [167], random crossover

11 + 90

288 F 89

0.26

Tea (1500 mL) + onion (400 g)

11 + 57

159 F 40

0.27

Red wine (750 mL) Tea (375 mL) Onion (50 g) Habitual diet

14 14 16 6 F 0.6

26 26 53 46

Habitual diet + bilberries, black currants, and lingonberries (100 g/d)

12 F 1

70 F 7

Various fruits and vegetables Low-phenolic diet Organically produced food Conventionally produced food

21 0.2 4.2 2.6

F F F F

10 13 17 7

0.8 0.6 1

2.1 0.5 F 0.08 0.6 F 0.07

C max = maximum plasma concentration. a Quercetin equivalents ingested during one day. b Mean F SD. c Mean F SEM. d Apple juice and black currant juice (1:1). e Tea and red wine were divided evenly among lunch, dinner, and 2200 and 2400 h. Onions were consumed at lunch and dinner (median F SEM).

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Healthy subjects (n = 40)c [102], randomized parallel, 6 weeks

Tea (1500 mL) + onion (400 g)

859

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that at least a part of the formation of quercetin glucuronides and sulfates occurs in the intestinal wall [90,91]. High concentrations of methylated quercetin, such as 3V-methylquercetin (isorhamnetin) and 4V-methylquercetin (tamarixetin), have been measured in plasma, urine, and bile of rats kept on a high-quercetin diet [92] and in a human hepatoma cell line (HepG2) [93]. In human beings, they occur in plasma at very low concentrations, if any [44] (Erlund et al, unpublished data). In rats, they appear to be formed mainly in the liver, not the intestinal wall [90]. Whether the formation of methylated quercetin metabolites is species-dependent or whether a concentration threshold exists is not known. In the studies where they have been found, rather high amounts of quercetin, typically 0.25% of the diet, have been given to laboratory animals. Something that should be kept in mind when studying quercetin methylation in human beings after consumption of quercetin-containing food is that isorhamnetin is not only a metabolite of quercetin but is also obtained in small amounts from onions [94]. 3.3. Bioavailability The bioavailability and metabolism of flavonoids, especially quercetin and (+)-catechin, were investigated by several groups in the 1960s and 1970s [85,86]. In most of these studies, rather high doses of flavonoids were given to laboratory animals and the main focus was on the bioavailability of flavonoid degradation products produced by the gastrointestinal microflora. Today, sensitive methods allowing serum and urine analyses are available for many flavonoids. These methods allow estimations of flavonoid bioavailability in human beings at appropriate dietary intake levels. It is evident that the bioavailability of flavonoids varies greatly between different subgroups and compounds. This is hardly surprising, considering the differences in their chemical properties (eg, polarity). 3.3.1. Quercetin Gugler et al [95] failed to detect quercetin in plasma and urine of subjects receiving 4 g of quercetin aglycone orally, which indicated that quercetin is not absorbed in human beings. Hollman et al [96-99] and de Vries et al [100], however, showed that the compound is bioavailable from onions, tea, apples, red wine, and supplements containing quercetin glycosides. In their studies, bioavailability was examined after ingestion of relatively high amounts of pure compounds or food containing them. Ingestion was either once or over a few days. In Table 2, results from selected bioavailability studies with quercetin are shown. Relatively few studies have measured plasma quercetin levels after ingestion of quercetin in amounts comparable with those attainable from a normal diet. Recently, we published results from 3 such studies. In these studies, the mean plasma values ranged between 15 and 24 lg/L (50-80 nmol/L) in subjects consuming their habitual diets [101-103] and was 42 lg/L (140 nmol/L) after a diet containing 815 g/d of vegetables, fruits, and berries [103]. Consumption of 100 g/d of berries (lingonberries, bilberries, and black currants) in addition to a normal diet resulted in a mean plasma level of 21 lg/L (70 nmol/L) [102]. Studies with pure compounds have given more precise information about the absorption and kinetics of quercetin. These studies indicate that quercetin aglycone and quercetin glucosides are absorbed from the upper parts of the gastrointestinal tract, probably the

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duodenum, while quercetin-3-rutinoside is absorbed from the distal parts, probably the colon [104,105]. In one study [104], we gave low doses of quercetin aglycone and quercetin3-rutinoside to healthy volunteers in a crossover setting. Mean bioavailability of quercetin was similar from the 2 sources, but there was marked interindividual variation in bioavailability from quercetin-3-rutinoside, in particular. A rather interesting finding was that quercetin from quercetin-3-rutinoside was more bioavailable in women compared with men (Fig. 2), and plasma levels were highest in women using oral contraceptives. These results suggest interindividual variation, possibly gender specific, in gastrointestinal microflora, or absorption or biotransformation mechanisms [106]. Furthermore, the study showed that

Fig. 2. Body weight-adjusted relative bioavailability of quercetin for men and women after ingestion of equivalent doses (8, 20, and 50 mg) of quercetin aglycone or rutin [104]. Sixteen healthy volunteers received single doses of the compounds in a diet-controlled, 2-period, crossover setting. The lowest dose was given on the 6th day, the second dose on the 9th day, and the highest dose on the 13th day of a low-quercetin diet period lasting 14 days. AUC values were calculated from plasma samples obtained up to 32 h.

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despite previous speculations, quercetin is bioavailable when given as aglycone. This finding was confirmed by Walle et al [107] in a study in which radiolabeled quercetin aglycone was used. The results indicated absolute bioavailability of 36% to 53% for quercetin aglycone and that a substantial portion of quercetin is excreted by the lungs as CO2. It should be kept in mind that the results were based on recovery of radioactivity, and therefore, the findings may reflect bioavailability of degradation products, which could have been formed, at least partly, before absorption. The urinary excretion of quercetin has been investigated in several studies. In these studies, urinary recovery, as a percentage of the ingested dose, ranged between 0.07% and 3%. Furthermore, it was lower after ingestion of quercetin-3-rutinoside than after ingestion of onion. From the urinary excretion data, it cannot be concluded that b3% of quercetin is bioavailable. Biliary excretion cannot be ruled out and has been shown to be a major route of quercetin elimination in rats [92,108]). In rats fed a diet containing 0.25% quercetin, the concentrations of quercetin and methylated metabolites were approximately 3-fold in bile compared with urine. The high molecular weight of quercetin glucuronides and sulfates and their extensive binding to protein [109-111] could favor their biliary excretion [112]. 3.3.2. Hesperetin and naringenin Analytical methods allowing the analysis of flavanones in plasma only became available recently [113]. Until then, knowledge about their bioavailability relied on animal studies and human urinary excretion data. The results concerning urinary excretion have varied in different studies. For hesperetin, the urinary recovery was 3% in a subject ingesting 500 mg of naringin and 500 mg of hesperidin once and 24% in 5 subjects ingesting 1250 mL of grapefruit juice and 1250 mL of orange juice daily for 4 weeks [114]. For naringenin, individual urinary recoveries of 5% to 59% (6 subjects) [115], 5% (1 subject) [114], 14% to 15% (2 subjects) [116], and 1% to 6% (6 subjects) [117] were reported after single ingestion of 214 to 700 mg of naringin as a supplement or in juice. The half-life for naringenin conjugates in urine was estimated as 2.6 h [115]. Erlund et al [113] studied the bioavailability and pharmacokinetics of flavanones after single ingestion of 400 to 760 mL of orange juice or grapefruit juice. The resulting plasma concentrations were comparatively high (up to 4 mg/L or 15 lmol/L), which is not surprising, considering that citrus fruits and juices contain quite high concentrations of the compounds (several hundred milligrams per liter). The plasma half-lives of flavanones were relatively short (1-2 h). Furthermore, renal clearance of naringenin appeared to be dependent on dose. Similar plasma levels were reported after consumption of 0.5 or 1 L of orange juice [118]. We also showed that the compounds are bioavailable when citrus fruits and juices are consumed as part of a normal diet (1 glass of orange juice, one half orange, and one half mandarin per day) [101]. Hesperetin, and especially naringenin, levels were, however, below the limit of detection of the analytical method in a large part of the plasma samples, which were collected after an overnight fasting. Bugianesi et al [119] recently made the interesting finding that naringenin is bioavailable from tomato paste, which is a notable source because of its widespread use, despite its low naringenin content.

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3.3.3. Interindividual variation in bioavailability Several studies indicate marked individual variation in the bioavailability of flavonoids [104,105,113]. Such variation is because of both physiological (differences in body weight, body composition, and gastric motility) and molecular factors (differences in the activity or synthesis of transporters or enzymes involved in biotransformation). Individual variation has been reported to occur for secretory transporters, such as P-glycoprotein [120,121] and MRPs [122], and biotransformation enzymes, such as CYP3A4 [123,124], uridine diphosphate glucuronosyltransferases [125], and sulfotransferases [126]. All of these proteins have been associated with flavonoids; quercetin interacts in vitro with P-glycoprotein [127], MRP1 [128], and MRP2 [71] and is a substrate for uridine diphosphate glucuronosyltransferases [129-131] and sulfotransferases. In addition, the composition and metabolic activity of the gastrointestinal microflora are likely determinants of the bioavailability of flavonoids absorbed from the distal parts of the gastrointestinal tract. To date, knowledge about the factors affecting the processes involved in the absorption and gastrointestinal metabolism of flavonoids has been rather fragmentary [132,133]. However, recent advances in molecular methods are expected to result in new information about the influence of environmental and genetic factors on the activity and expression of biotranformation enzymes [134,135] and the composition of the microflora. This will almost certainly, in the near future, improve our understanding about the bioavailability of specific compounds such as flavonoids. 3.4. Quercetin, hesperetin, and naringenin as a biomarker of intake Few studies have attempted to assess the use of plasma or urine quercetin levels as biomarkers of intake. Noroozi et al [136] studied the effect of 2 high-flavonol diets on plasma quercetin concentrations in 10 diabetic subjects receiving daily an onion dish containing either 90 or 57 mg of quercetin (both prepared from 400 g of white onions). After the 2-week study period, the mean F SD fasting plasma concentrations were 87 F 27 lg/L (n = 5) and 48 F 12 lg/L (n = 5), respectively. The mean baseline value was 23 F 4 lg/L, whereas during a 2-week low-flavonoid diet, when no food known or suspected to contain flavonoids were consumed, the concentration was 6 F 3 lg/L. These findings indicate that plasma quercetin concentrations increase with increasing intake. Radtke et al [137] estimated the intake of several flavonoids from 1- or 7-day dietary records obtained from 48 female students. Intake data were correlated with fasting plasma flavonoid concentrations. For 1-day dietary records (collected on the last day before blood sampling), Spearman correlations were 0.42, 0.64, and 0.47 for quercetin, hesperetin, and naringenin, respectively. For the 7-day dietary records, the corresponding values were 0.30, 0.32, and 0.35, respectively. These correlations are similar to what has been reported for many other nutrients, the plasma concentrations of which are used as biomarkers of intake. Results from our laboratory indicate that plasma quercetin is a good biomarker of intake because it responds to changes in intake levels. In a pharmacokinetics study, the mean plasma quercetin concentration increased with increasing dose after ingestion of both quercetin aglycone and quercetin rutinoside. In another study, middle-aged men consumed either their habitual diets or 100 g/d of berries in addition to their habitual diets. In this study,

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plasma quercetin was 30% to 50% higher in the subjects consuming berries, compared with the control group [102]. In a strictly controlled dietary intervention study, 77 healthy men and women consumed either 170 or 850 g of fruits, vegetables, and berries daily. Quercetin intake was calculated to be 3 and 24 mg/d on the respective diets. The mean F SD plasma quercetin concentration was 78 F 56 nmol/L during the habitual diet, and it decreased to 70% during the low-vegetable diet and increased to 170% during the high-vegetable diet (changes statistically significant) [103]. In addition to blood samples, 24-h urine samples were also collected from the study and analyzed in Denmark [138]. Urinary quercetin was clearly higher after the high-vegetable diet compared with the low-vegetable diet and decreased during the low-vegetable diet. In another study from Denmark [139], consumption of rather low amounts of quercetin in 3 increasing doses of fruit juice resulted in a significant increase in urinary quercetin with both dose and time. The results of the 2 studies indicate that the urinary recovery of quercetin in 24-h urine samples also respond to changes in dietary intake. The use of plasma flavanone levels as biomarkers of intake were investigated in 2 studies in our laboratory. Bioavailability was studied after both single ingestion [113] and long-term consumption [101]. According to the results of the first study, flavanones are clearly bioavailable, but the plasma half-lives are short (1-2 h). Urinary excretion appeared to be dependent on dose [113]. In the second study, 37 Finnish volunteers consumed their habitual diets followed by a diet containing on average 211 g of orange juice, one half orange, and one half mandarin per day for 5 weeks. During the habitual diets, flavanones were detectable in few samples. After the consumption of citrus, hesperetin was detectable in half of the fasting plasma samples and naringenin in 20% of them [101]. The results indicate that fasting plasma flavanone concentrations are problematic as biomarkers of intake. In the controlled dietary intervention study mentioned in the previous paragraph [138], urinary flavanones clearly increased after the high-vegetable diet compared with the low-vegetable diet, which suggests that pooled 24-h urinary samples may be useful as biomarkers of flavanone intake in intervention studies. Repeated plasma measurements combined with urine measurements would probably give the most accurate results. Plasma or serum quercetin appears to be a good biomarker of intake and can be used for this purpose in epidemiological studies. We do not usually obtain good correlations between calculated intake of the compound and plasma levels, but this likely reflects the incapacity to estimate quercetin intake correctly. It should be noted that an accurate assessment of the intake of onion, qualitatively and quantitatively, the most important source of quercetin, is problematic, because onion is a commonly used bhidden ingredientQ of many homemade and processed food. For flavanones, on the other hand, the situation is quite different. The errors in estimating their intake are probably relatively small because they are mainly obtained from citrus fruits and juices. Therefore, in epidemiological studies, a more sensible approach would be to assess their intake from food records or questionnaires (if citrus consumption was asked). In general, the question about which type of data should be used for the estimation of intake is complicated. For compounds with short half-lives, dietary intake data or 24-h urinary excretion data may be a good alternative. However, dietary intake assessment has its problems (underestimation, incomplete food composition data, etc). Urinary data, on the

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other hand, may be misleading for compounds mainly using other routes of excretion, for instance, biliary excretion. In intervention studies, the best way to assess bioavailability would be to compare values of area under curve (AUC) obtained after multiple blood samplings. If the percentage of the compound excreted into the urine does not vary too much between individuals, urinary data may yield good results. In epidemiological studies, however, 24-h urine samples have rarely been collected, and therefore dietary intake or serum data are usually used. In most situations, the ideal would be to combine dietary intake with plasma and urine data. 3.5. Association between flavonoid intake and risk of chronic diseases The association between flavonoid intake and the risk of cardiovascular disease and cancer has been investigated in several epidemiological studies. In most studies, the term flavonoid refers to flavonols and flavones, with quercetin being quantitatively the most important flavonoid. Most, but not all, prospective cohort studies have indicated some degree of inverse association (from borderline to modest) between flavonoid intake and coronary heart disease. An inverse association was found in the Zutphen Elderly Study [140], the Finnish Mobile Clinic Study [141,142], the Iowa Women’s Health Study [143], the Alpha-Tocopherol, BetaCarotene Cancer Prevention (ATBC) Study [144], and the Rotterdam Study [145]. No association was found between flavonoid intake and risk of coronary heart disease in subjects free of disease at baseline in the Health Professionals Follow-up Study [146] and in the Women’s Health Study [147]. Interestingly, in the Caerphilly Study [148], flavonol intake in 1900 men was directly associated with the risk of ischemic heart disease and all-cause mortality. The result may be explained by tea being a very important source of flavonoids in Scotland; its consumption was associated with lower social class, smoking, and higher fat intake [148]. In the above-mentioned ATBC Study, no association was found between flavonoid intake and risk of stroke [149]. The epidemiological evidence regarding the cancer-protecting effects of flavonoids is conflicting. Some of the case-control studies have indicated an inverse association between intake of flavonoids and cancer risk (lung cancer [150,151], upper digestive tract cancer [152], and gastric cancer [153]). Other case-control studies found no associations (lung cancer [154] and bladder cancer [155]). In 2 cohort studies, no association between intake of flavonoids and risk of several cancer types was present [5,156], but in 2 other cohort studies, an inverse association was shown for lung cancer [157,158]. Knekt et al [142] studied the association between the intake of flavonoids and the risk of several chronic diseases in 10 054 participants of the Finnish Mobile Clinic Health Examination Survey. Higher quercetin intakes were associated with lower risk of asthma and lung cancer, and there was a trend toward a decrease in the risk of type 2 diabetes. Persons with higher quercetin intakes had lower mortality from ischemic heart disease. The incidence of cerebrovascular disease and asthma was lower with higher intakes of hesperetin and naringenin. Overall, the epidemiological evidence concerning flavonoids and chronic diseases is difficult to interpret. Often, the reported associations have not been very strong. Furthermore,

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confounding factors (a common problem of epidemiological studies) probably affect the outcome of the studies. In many countries, drinking tea and consuming high quantities of vegetables and fruit are merely indicators of a generally healthy lifestyle or a high level of education. This is not the case for onion, which is obtained from so-called unhealthy food as well, but still, this source of flavonoids may be problematic in epidemiological studies. Onion is qualitatively, and in many countries, quantitatively, the most important source of quercetin [6,146,159,160]. The accurate assessment of an individual’s onion consumption is quite difficult with dietary survey methods because it is a commonly used hidden ingredient of many homemade and processed food, such as soups, salads, sausages, hamburger meat, and others. Thus, it is likely that intake estimates for onions contain a large margin of error. Assessment of tea intake, on the other hand, another important source of quercetin in many countries, is probably accurate. However, the bioavailability of quercetin from tea is poorer than from onion. Considering the possible impact on the results of epidemiological studies, it is rather surprising that these problems have not been discussed in the reports. In summary, the epidemiological evidence concerning the association between flavonoid intake and the risk of chronic diseases is conflicting. Whether flavonoids protect against chronic diseases may be difficult to show using traditional epidemiological methods. An alternative, which may help to overcome some of the problems associated with intake assessment, is to use serum concentrations as biomarkers of intake. This approach is not faultless, but the sources of error are different. 4. Conclusion Most human beings are exposed to flavonoids daily, and therefore, their impact on human health is of relevance. The health effects of flavonoids are, however, still largely unknown. Interpretation of the results from studies performed so far is problematic, because several different mechanisms and complex pathways may be involved. Several types of studies could yield more information on the issue. For instance, studies are needed on the metabolism of flavonoids to enable in vitro studies to be performed with metabolites present in human tissues. In epidemiology, serum biomarkers, in addition to dietary intake data, could be used when studying the associations between flavonoids and risk of diseases. Most importantly, clinical studies on the health effects of both high and low doses of flavonoids are warranted. In these studies, bioavailability should be monitored, because marked interindividual variation could confound the results. Finally, it is emphasized that any bioactive compound could, at least in theory, also possess adverse effects or exhibit different effects at varying doses. This should be kept in mind when planning long-term human studies.

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