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Polyphenols: food sources and bioavailability
1, 2
Claudine Manach, Augustin Scalbert, Christine Morand, Christian Rémésy, and Liliana Jime´nez
ABSTRACT
Polyphenols are abundant micronutrients in our diet, and evidence
for their role in the prevention of degenerative diseases such as
cancerandcardiovasculardiseasesisemerging.Thehealtheffectsof
polyphenols depend on the amount consumed and on their bioavail-
ability. In this article, the nature and contents of the various poly-
phenols present in food sources and the influence of agricultural
practicesand industrial processesare reviewed. Estimatesof dietary
intakes are given for each class of polyphenols. The bioavailability
of polyphenols is also reviewed, with particular focus on intestinal
absorption and the influence of chemical structure (eg, glycosyla-
tion, esterification, and polymerization), food matrix, and excretion
back into the intestinal lumen. Information on the role of microflora
in the catabolism of polyphenols and the production of some active
metabolites is presented. Mechanisms of intestinal and hepatic con-
jugation(methylation,glucuronidation,sulfation),plasmatransport,
andeliminationinbileandurinearealsodescribed.Pharmacokinetic
data for the various polyphenols are compared. Studies on the iden-
tification of circulating metabolites, cellular uptake, intracellular
metabolismwithpossibledeconjugation,biologicalpropertiesofthe
conjugated metabolites, and specific accumulation in some target
tissues are discussed. Finally, bioavailability appears to differ
greatly between the various polyphenols, and the most abundant
polyphenols in our diet are not necessarily those that have the best
bioavailability profile. A thorough knowledge of the bioavailability
of the hundreds of dietary polyphenols will help us to identify those
that are most likely to exert protective health effects. Am J Clin
Nutr 2004;79:727–47.
KEY WORDS Polyphenols, flavonoids, phenolic acids, food
sources, dietary intake, intestinal absorption, metabolism, bioavail-
ability
INTRODUCTION
Over the past 10 y, researchers and food manufacturers have
become increasingly interested in polyphenols. The chief reason
for this interest is the recognition of the antioxidant properties of
polyphenols,theirgreat abundance in our diet,andtheirprobable
role in the prevention of various diseases associated with oxida-
tive stress, such as cancer and cardiovascular and neurodegen-
erative diseases (Scalbert A, Manach C, Morand C, Rémésy C,
Jime´nez L. Crit Rev Food SciNutr, in press). Furthermore, poly-
phenols, which constitute the active substances found in many
medicinal plants, modulate the activity of a wide range of en-
zymes and cell receptors (1). In this way, in addition to having
antioxidant properties, polyphenols have several other specific
biologicalactions thatare as yet poorly understood. Two aimsof
research are to establish evidence for the effects of polyphenol
consumption on health and to identify which of the hundreds of
existing polyphenols are likely to provide the greatest protection
inthe context ofpreventive nutrition. Ifthese objectives areto be
attained, it is first essential to determine the nature and distribu-
tion of these compounds in our diet. Such knowledge will allow
evaluation of polyphenol intake and enable epidemiologic anal-
ysis that will in turn provide an understanding of the relation
between the intake of these substances and the risk of develop-
ment of several diseases. Furthermore, not all polyphenols are
absorbed with equal efficacy. They are extensively metabolized
by intestinal and hepatic enzymes and by the intestinal micro-
flora. Knowledge of the bioavailability and metabolism of the
various polyphenols is necessary to evaluate their biological
activity within target tissues. The types and distribution of poly-
phenols in foods and the bioavailability of polyphenols are the
topics of the present review.
TYPES AND DISTRIBUTION OF POLYPHENOLS IN
FOODS
Severalthousand molecules having apolyphenol structure (ie,
several hydroxyl groups on aromatic rings) have been identified
in higher plants, and several hundred are found in edible plants.
These molecules are secondary metabolites of plants and are
generally involved in defense against ultraviolet radiation or
aggression by pathogens. These compounds may be classified
into different groups as a function of the number of phenol rings
that they contain and of the structural elements that bind these
rings to one another. Distinctions are thus made between the
phenolicacids,flavonoids,stilbenes,andlignans(Figure1). The
flavonoids, which share a common structure consisting of 2 ar-
omaticrings (A and B)thatare bound togetherby3 carbon atoms
thatformanoxygenated heterocycle (ring C), maythemselvesbe
divided into 6 subclasses as a function of the type of heterocycle
involved: flavonols, flavones, isoflavones, flavanones, antho-
cyanidins, and flavanols (catechins and proanthocyanidins)
(Figure 2). In addition to this diversity, polyphenols may be
associatedwithvariouscarbohydratesandorganicacidsandwith
one another.
1
From the Unité des Maladies Métaboliques et Micronutriments, INRA,
Saint-Genès Champanelle, France (CM, AS, CM, and CR), and Danone
Vitapole Research, Palaiseau cedex, France (LJ).
2
Addressreprint requests to C Manach, Unité des Maladies Métaboliques
et Micronutriments, INRA, 63122 Saint-Genès Champanelle, France. E-
mail: manach@clermont.inra.fr.
Received June 3, 2003.
Accepted for publication October 17, 2003.
727Am J Clin Nutr 2004;79:727–47. Printed in USA. © 2004 American Society for Clinical Nutrition
Phenolic acids
Two classes of phenolic acids can be distinguished: deriva-
tivesof benzoic acid and derivativesof cinnamic acid(Figure 1).
The hydroxybenzoic acid content of edible plants is generally
very low, with the exception of certain red fruits, black radish,
and onions, which can have concentrations of several tens of
milligrams per kilogram fresh weight (2). Tea is an important
source of gallic acid: tea leaves may contain up to 4.5 g/kg fresh
wt (3). Furthermore, hydroxybenzoic acids are components of
complexstructures such as hydrolyzabletannins (gallotannins in
mangoesand ellagitannins in red fruitsuch as strawberries, rasp-
berries, and blackberries) (4). Because these hydroxybenzoic
acids,bothfreeandesterified,arefoundinonlyafewplantseaten
by humans, they have not been extensively studied and are not
currently considered to be of great nutritional interest.
The hydroxycinnamic acids are more common than are the
hydroxybenzoicacids and consistchieflyof p-coumaric, caffeic,
ferulic,and sinapic acids. These acidsare rarely found in the free
form, except in processed food that has undergone freezing,
sterilization, or fermentation. The bound forms are glycosylated
derivatives or esters of quinic acid, shikimic acid, and tartaric
acid. Caffeic and quinic acid combine to form chlorogenic acid,
which is found in many types of fruit and in high concentrations
in coffee: a single cup may contain 70 –350 mg chlorogenic acid
(5). The types of fruit having the highest content (blueberries,
kiwis,plums, cherries, apples)contain0.5–2 g hydroxycinnamic
acids/kg fresh wt (Table 1) (6).
Caffeic acid, both free and esterified, is generally the most
abundantphenolicacidandrepresentsbetween75%and100%of
the total hydroxycinnamic acid content of most fruit. Hydroxy-
cinnamicacids are foundin all partsof fruit, althoughthe highest
concentrations are seen in the outer parts of ripe fruit. Concen-
trations generally decrease during the course of ripening, but
total quantities increase as the fruit increases in size.
Ferulicacid is themostabundant phenolic acidfound in cereal
grains, which constitute its main dietary source. The ferulic acid
content of wheat grain is 앒0.8–2 g/kg dry wt, which may rep-
resent up to 90% of total polyphenols (28, 29). Ferulic acid is
found chiefly in the outer parts of the grain. The aleurone layer
and the pericarp of wheat grain contain 98% of the total ferulic
acid. The ferulic acid content of different wheat flours is thus
directly related to levels of sieving, and bran is the main source
ofpolyphenols(30).Riceandoatflourscontainapproximatelythe
samequantityof phenolic acids aswheatflour (63 mg/kg), although
thecontent in maizeflour is about3 times ashigh (2). Ferulicacid is
found chiefly in the trans form, which is esterified to arabinoxylans
andhemicellulosesinthealeuroneandpericarp.Only10% of ferulic
acidis found in solublefree form in wheatbran (29). Severaldimers
of ferulic acid are also found in cereals and form bridge structures
between chains of hemicellulose.
FIGURE 1. Chemical structures of polyphenols.
FIGURE 2. Chemical structures of flavonoids.
728 MANACH ET AL
TABLE 1
Polyphenols in foods
Source (serving size)
Polyphenol content
By wt or vol By serving
mg/kg fresh wt (or mg/L) mg/serving
Hydroxybenzoic acids (2, 6) Blackberry (100 g) 80–270 8–27
Protocatechuic acid Raspberry (100 g) 60–100 6–10
Gallic acid Black currant (100 g) 40–130 4–13
p-Hydroxybenzoic acid Strawberry (200 g) 20–90 4–18
Hydroxycinnamic acids (2, 5–7) Blueberry (100 g) 2000–2200 200–220
Caffeic acid Kiwi (100 g) 600–1000 60–100
Chlorogenic acid Cherry (200 g) 180–1150 36–230
Coumaric acid Plum (200 g) 140–1150 28–230
Ferulic acid Aubergine (200 g) 600–660 120–132
Sinapic acid Apple (200 g) 50–600 10–120
Pear (200 g) 15–600 3–120
Chicory (200 g) 200–500 40–100
Artichoke (100 g) 450 45
Potato (200 g) 100–190 20–38
Corn flour (75 g) 310 23
Flour: wheat, rice, oat (75 g) 70–90 5–7
Cider (200 mL) 10–500 2–100
Coffee (200 mL) 350–1750 70–350
Anthocyanins (8–10) Aubergine (200 g) 7500 1500
Cyanidin Blackberry (100 g) 1000–4000 100–400
Pelargonidin Black currant (100 g) 1300–4000 130–400
Peonidin Blueberry (100 g) 250–5000 25–500
Delphinidin Black grape (200 g) 300–7500 60–1500
Malvidin Cherry (200 g) 350–4500 70–900
Rhubarb (100 g) 2000 200
Strawberry (200 g) 150–750 30–150
Red wine (100 mL) 200–350 20–35
Plum (200 g) 20–250 4–50
Red cabbage (200 g) 250 50
Flavonols (11–18) Yellow onion (100 g) 350–1200 35–120
Quercetin Curly kale (200 g) 300–600 60–120
Kaempferol Leek (200 g) 30–225 6–45
Myricetin Cherry tomato (200 g) 15–200 3–40
Broccoli (200 g) 40–100 8–20
Blueberry (100 g) 30–160 3–16
Black currant (100 g) 30–70 3–7
Apricot (200 g) 25–50 5–10
Apple (200 g) 20–40 4–8
Beans, green or white (200 g) 10–50 2–10
Black grape (200 g) 15–40 3–8
Tomato (200 g) 2–15 0.4–3.0
Black tea infusion (200 mL) 30–45 6–9
Green tea infusion (200 mL) 20–35 4–7
Red wine (100 mL) 2–30 0.2–3
Flavones (11–12, 14, 18) Parsley (5 g) 240–1850 1.2–9.2
Apigenin Celery (200 g) 20–140 4–28
Luteolin Capsicum pepper (100 g) 5–10 0.5–1
Flavanones (19–21) Orange juice (200 mL) 215–685 40–140
Hesperetin Grapefruit juice (200 mL) 100–650 20–130
Naringenin Lemon juice (200 mL) 50–300 10–60
Eriodictyol
Isoflavones (22–25) Soy flour (75 g) 800–1800 60–135
Daidzein Soybeans, boiled (200 g) 200–900 40–180
Genistein Miso (100 g) 250–900 25–90
Glycitein Tofu (100 g) 80–700 8–70
Tempeh (100 g) 430–530 43–53
Soy milk (200 mL) 30–175 6–35
Monomeric flavanols (6, 17, 26, 27) Chocolate (50 g) 460–610 23–30
Catechin Beans (200 g) 350–550 70–110
Epicatechin Apricot (200 g) 100–250 20–50
Cherry (200 g) 50–220 10–44
Grape (200 g) 30–175 6–35
Peach (200 g) 50–140 10–28
Blackberry (100 g) 130 13
Apple (200 g) 20–120 4–24
Green tea (200 mL) 100–800 20–160
Black tea (200 mL) 60–500 12–100
Red wine (100 mL) 80–300 8–30
Cider (200 mL) 40 8
POLYPHENOLS: FOOD SOURCES AND BIOAVAILABILITY 729
Flavonoids
Flavonolsare the most ubiquitous flavonoidsin foods, and the
main representatives are quercetin and kaempferol. They are
generally present at relatively low concentrations of 앒15–
30 mg/kg fresh wt. The richest sources are onions (up to 1.2 g/kg
fresh wt), curly kale, leeks, broccoli, and blueberries (Table 1).
Red wine and tea also contain up to 45 mg flavonols/L. These
compounds are present in glycosylated forms. The associated
sugar moiety is very often glucose or rhamnose, but other sugars
may also be involved (eg, galactose, arabinose, xylose, glucu-
ronic acid). Fruit often contains between 5 and 10 different fla-
vonol glycosides (6). These flavonols accumulate in the outer
and aerial tissues (skin and leaves) because their biosynthesis is
stimulated by light. Marked differences in concentration exist
between pieces of fruit on the same tree and even between dif-
ferent sides of a single piece of fruit, depending on exposure to
sunlight (31). Similarly, in leafy vegetables such as lettuce and
cabbage, the glycoside concentration is 욷10 times as high in the
green outer leaves as in the inner light-colored leaves (14). This
phenomenon also accounts for the higher flavonol content of
cherry tomatoes than of standard tomatoes, because they have
different proportions of skin to whole fruit.
Flavones are much less common than flavonols in fruit and
vegetables.Flavonesconsist chiefly of glycosidesofluteolinand
apigenin. The only important edible sources of flavones identi-
fied to date are parsley and celery (Table 1). Cereals such as
millet and wheat contain C-glycosides of flavones (32–34). The
skin of citrus fruit contains large quantities of polymethoxylated
flavones: tangeretin, nobiletin, and sinensetin (up to 6.5 g/L of
essential oil of mandarin) (2). These polymethoxylated flavones
are the most hydrophobic flavonoids.
In human foods, flavanones are found in tomatoes and certain
aromatic plants such as mint, but they are present in high con-
centrations only in citrus fruit. The main aglycones are naringe-
nin in grapefruit, hesperetin in oranges, and eriodictyol in lem-
ons. Flavanones are generally glycosylated by a disaccharide at
position 7: either a neohesperidose, which imparts a bitter taste
(such as to naringin in grapefruit), or a rutinose, which is flavor-
less. Orange juice contains between 200 and 600 mg hesperi-
din/L and 15–85 mg narirutin/L, and a single glass of orange
juice may contain between 40 and 140 mg flavanone glycosides
(20).Becausethesolidpartsofcitrusfruit,particularlythealbedo
(the white spongy portion) and the membranes separating the
segments, have a very high flavanone content, the whole fruit
may contain up to 5 times as much as a glass of orange juice.
Isoflavones are flavonoids with structural similarities to estro-
gens. Although they are not steroids, they have hydroxyl groups in
positions 7 and 4' in a configuration analogous to that of the hy-
droxyls in the estradiol molecule. This confers pseudohormonal
properties on them, including the ability to bind to estrogen recep-
tors, and they are consequently classified as phytoestrogens. Isofla-
vones are found almost exclusively in leguminous plants. Soya and
its processed products are the main source of isoflavones in the
humandiet.Theycontain3main molecules: genistein,daidzein,and
glycitein, generally in a concentration ratio of 1:1:0.2. These isofla-
vones are found in 4 forms: aglycone, 7-O-glucoside, 6⬙-O-acetyl-
7-O-glucoside, and 6⬙-O-malonyl-7-O-glucoside (35). The 6⬙-O-
malonylglucoside derivatives have an unpleasant, bitter, and
astringent taste. They are sensitive to heat and are often hydrolyzed
to glycosides during the course of industrial processing, as in the
production of soya milk (36). The fermentation carried out during
themanufacturingofcertainfoods,suchasmiso and tempeh, results
in the hydrolysis of glycosides to aglycones. The aglycones are
highly resistant to heat. The isoflavone content of soya and its man-
ufactured products varies greatly as a function of geographic zone,
growingconditions,andprocessing.Soybeans contain between 580
and3800mgisoflavones/kgfreshwt,andsoymilkcontainsbetween
30 and 175 mg/L (25, 37).
Flavanols exist in both the monomer form (catechins) and the
polymerform (proanthocyanidins). Catechinsare found inmany
typesoffruit (apricots, which contain250mg/kgfresh wt, are the
richest source; Table 1). They are also present in red wine (up to
300 mg/L), but green tea and chocolate are by far the richest
sources.Aninfusionof green tea containsupto200 mg catechins
(38). Black tea contains fewer monomer flavanols, which are
oxidized during “fermentation”(heating) of tea leaves to more
complex condensed polyphenols known as theaflavins (dimers)
and thearubigins (polymers). Catechin and epicatechin are the
main flavanols in fruit, whereas gallocatechin, epigallocatechin,
and epigallocatechin gallate are found in certain seeds of legu-
minousplants, in grapes, and moreimportantly in tea (27, 39).In
contrast to other classes of flavonoids, flavanols are not glyco-
sylatedinfoods.Theteaepicatechinsareremarkablystablewhen
exposed to heat as long as the pH is acidic: only 앒15% of these
substances are degraded after7hinboiling water at pH 5 (40).
Proanthocyanidins, which are also known as condensed tan-
nins, are dimers, oligomers, and polymers of catechins that are
bound together by links between C4 and C8 (or C6). Their mean
degreeof polymerization in foods hasrarely been determined. In
ciderapples, the meandegree of polymerizationranges from 4to
11 (41). Through the formation of complexes with salivary pro-
teins, condensed tannins are responsible for the astringent char-
acter of fruit (grapes, peaches, kakis, apples, pears, berries, etc) and
beverages (wine, cider, tea, beer, etc) and for the bitterness of choc-
olate (42). This astringency changes over the course of maturation
andoftendisappearswhenthefruitreachesripeness;thischangehas
been well explained in the kaki fruit by polymerization reactions
with acetaldehyde (43). Such polymerization of tannins probably
accounts for the apparent reduction in tannin content that is com-
monly seen during the ripening of many types of fruit. It is difficult
to estimate the proanthocyanidin content of foods because proan-
thocyanidinshaveawiderangeofstructuresandmolecularweights.
The only available data concern dimers and trimers, which are as
abundant as the catechins themselves (26).
Anthocyaninsarepigmentsdissolvedinthevacuolarsapofthe
epidermal tissues of flowers and fruit, to which they impart a
pink, red, blue, or purple color (9). They exist in different chem-
ical forms, both colored and uncolored, according to pH. Al-
though they are highly unstable in the aglycone form (anthocya-
nidins), while they are in plants, they are resistant to light, pH,
and oxidation conditions that are likely to degrade them. Degra-
dation is prevented by glycosylation, generally with a glucose at
position 3, and esterification with various organic acids (citric
and malic acids) and phenolic acids. In addition, anthocyanins
are stabilized by the formation of complexes with other fla-
vonoids (copigmentation). In the human diet, anthocyanins are
found in red wine, certain varieties of cereals, and certain leafy
and root vegetables (aubergines, cabbage, beans, onions, rad-
ishes), but they are most abundant in fruit. Cyanidin is the most
common anthocyanidin in foods. Food contents are generally
proportional to color intensity and reach values up to 2–4 g/kg
730 MANACH ET AL
fresh wt in blackcurrants or blackberries (Table 1). These values
increaseas the fruit ripens.Anthocyanins are found mainlyin the
skin,exceptfor certain types ofredfruit, in which theyalsooccur
in the flesh (cherries and strawberries). Wine contains 앒200–
350mg anthocyanins/L, and these anthocyanins are transformed
into various complex structures as the wine ages (10, 44).
Lignans
Lignans are formed of 2 phenylpropane units (Figure 1). The
richest dietary source is linseed, which contains secoisolaricir-
esinol (up to 3.7 g/kg dry wt) and low quantities of matairesinol.
Other cereals, grains, fruit, and certain vegetables also contain
traces of these same lignans, but concentrations in linseed are
앒1000 times as high as concentrations in these other food
sources (45). Lignans are metabolized to enterodiol and en-
terolactone by the intestinal microflora. The low quantities of
secoisolariciresinol and matairesinol that are ingested as part of
our normal diet do not account for the concentrations of the
metabolites enterodiol and enterolactone that are classically
measuredin plasma andurine. Thus, there are undoubtedlyother
lignans of plant origin that are precursors of enterodiol and en-
terolactoneand that have not yetbeen identified (46). Thompson
etal (47) usedanin vitro techniqueinvolving the fermentationof
foods by human colonic microflora to quantitatively evaluate
precursors of enterodiol and enterolactone. They confirmed that
oleaginous seeds (linseed) are the richest source and identified
algae, leguminous plants (lentils), cereals (triticale and wheat),
vegetables (garlic, asparagus, carrots), and fruit (pears, prunes)
as minor sources.
Stilbenes
Stilbenes are found in only low quantities in the human diet.
Oneofthese,resveratrol,forwhichanticarcinogenic effects have
been shown during screening of medicinal plants and which has
been extensively studied, is found in low quantities in wine
(0.3–7 mg aglycones/L and 15 mg glycosides/L in red wine)
(48–50). However, because resveratrol is found in such small
quantities in the diet, any protective effect of this molecule is
unlikely at normal nutritional intakes.
VARIABILITY OF POLYPHENOL CONTENT OF FOODS
Fruit and beverages such as tea and red wine constitute the
main sources of polyphenols. Certain polyphenols such as quer-
cetin are found in all plant products (fruit, vegetables, cereals,
leguminousplants,fruitjuices,tea,wine,infusions,etc),whereas
others are specific to particular foods (flavanones in citrus fruit,
isoflavones in soya, phloridzin in apples). In most cases, foods
contain complex mixtures of polyphenols, which are often
poorly characterized. Apples, for example, contain flavanol
monomers (epicatechin mainly) or oligomers (procyanidin B2
mainly), chlorogenic acid and small quantities of other hydroxy-
cinnamic acids, 2 glycosides of phloretin, several quercetin gly-
cosides, and anthocyanins such as cyanidin 3-galactoside in the
skin of certain red varieties. Apples are one of the rare types of
foodfor which fairlyprecisedata on polyphenolcomposition are
available. Differences in polyphenol composition between vari-
eties of apples have notably been studied. The polyphenol pro-
files of all varieties of apples are practically identical, but con-
centrations may range from 0.1 to 5 g total polyphenols/kg fresh
wt and may be as high as 10 g/kg in certain varieties of cider
apples (41, 51).
Formany plant products, the polyphenolcomposition is much
less known, knowledge is often limited to one or a few varieties,
and data sometimes do not concern the edible parts. Some foods,
particularlysome exotic typesof fruit andsome cereals, havenot
been analyzed yet. Furthermore, numerous factors other than
varietymay affect thepolyphenol content of plants; thesefactors
include ripeness at the time of harvest, environmental factors,
processing, and storage.
Environmentalfactors have amajor effect on polyphenol con-
tent.These factors may be pedoclimatic(soil type, sun exposure,
rainfall) or agronomic (culture in greenhouses or fields, biolog-
ical culture, hydroponic culture, fruit yield per tree, etc). Expo-
sure to light has a considerable effect on most flavonoids. The
degree of ripeness considerably affects the concentrations and
proportions of the various polyphenols (6). In general, phenolic
acid concentrations decrease during ripening, whereas anthocy-
aninconcentrationsincrease. Many polyphenols, especiallyphe-
nolic acids, are directly involved in the response of plants to
different types of stress: they contribute to healing by lignifica-
tionofdamagedareas, they possess antimicrobial properties,and
their concentrations may increase after infection (2, 6, 52). Al-
though very few studies directly addressed this issue, the poly-
phenol content of vegetables produced by organic or sustainable
agriculture is certainly higher than that of vegetables grown
without stress, such as those grown in conventional or hydro-
ponic conditions. This was shown recently in strawberries,
blackberries,and corn(53). With the current state of knowledge,
it is extremely difficult to determine for each family of plant
products the key variables that are responsible for the variability
inthe content ofeachpolyphenol and therelative weight ofthose
variables.A huge amount ofanalysis would be requiredto obtain
this information. For example, determination of the p-coumaric
acidcontent of 쏜500 red wines showed thatgenetic factors were
more important than was exposure to light or climate (54).
Storagemayalsoaffectthecontent of polyphenols that are easily
oxidized.Oxidation reactions resultin the formation of moreor less
polymerized substances, which lead to changes in the quality of
foods, particularly in color and organoleptic characteristics. Such
changes may be beneficial (as is the case with black tea) or harmful
(browningoffruit)toconsumeracceptability. Storage ofwheatflour
results in marked loss of phenolic acids (28). After 6 mo of storage,
flours contained the same phenolic acids in qualitative terms, but
their concentrations were 70% lower. Cold storage, in contrast, did
not affect the content of polyphenols in apples (55, 56), pears (57),
or onions (58). At 25 °C, storage of apple juice for 9 mo results in
a 60% loss of quercetin and a total loss of procyanidins, despite
the fact that polyphenols are more stable in fruit juices than is vita-
min C (59, 60).
Methods of culinary preparation also have a marked effect on
the polyphenol content of foods. For example, simple peeling of
fruit and vegetables can eliminate a significant portion of poly-
phenols because these substances are often present in higher
concentrations in the outer parts than in the inner parts. Cooking
may also have a major effect. Onions and tomatoes lose between
75% and 80% of their initial quercetin content after boiling for
15 min, 65% after cooking in a microwave oven, and 30% after
frying(18).Steamcookingof vegetables, which avoids leaching,
ispreferable. Potatoes containupto 190 mgchlorogenic acid/kg,
mainly in the skin (61). Extensive loss occurs during cooking,
POLYPHENOLS: FOOD SOURCES AND BIOAVAILABILITY 731
and no remaining phenolic acids were found in French fries or
freeze-dried mashed potatoes (54).
Industrialfood processing also affects polyphenol content. As
with fruit peeling, dehulling of legume seeds and decortication
and bolting of cereals can result in a loss of some polyphenols.
Grinding of plant tissues may lead to oxidative degradation of
polyphenols as a result of cellular decompartmentation and con-
tact between cytoplasmic polyphenol oxidase and phenolic sub-
stratespresent in thevacuoles. Polyphenols arethen transformed
into brown pigments that are polymerized to different degrees.
This unwanted process can occur, for example, during the pro-
cess of making jam or compote from fruit. Production of fruit
juice often involves clarification or stabilization steps specifi-
cally aimed at removing certain flavonoids responsible for dis-
coloration and haze formation. Manufactured fruit juices thus
have low flavonoid content. The pectinolytic enzymes used dur-
ing such processing also hydrolyze the esters of hydroxycin-
namic acid (62). Conversely, maceration operations facilitate
diffusionof polyphenols in juice,as occurs during vinificationof
red wine. This maceration accounts for the fact that the polyphe-
nolcontent of red wines is10 times ashigh as that of whitewines
(63) and is also higher than that of grape juice (64).
Because of the wide range of existing polyphenols and the
considerable number of factors that can modify their concentra-
tionin foods, noreference food-composition tables(as they exist
for other micronutrients such as vitamins) have yet been drawn
up. Only partial data for certain polyphenols, such as flavonols
andflavones, catechins, and isoflavones,have been published on
the basis of direct food analysis (11, 27) or bibliographic com-
pilations (37, 65). Since March 2003, a database in which the
flavonoid contents of 225 selected foods were compiled from 97
bibliographic sources has been available on the US Department
ofAgriculture website (66).A comprehensive compositiontable
for polyphenols is essential; it should allow daily polyphenol
consumption to be calculated from dietary questionnaires. Poly-
phenol intake could then be correlated with the incidence of
certain diseases or early markers for these diseases in epidemi-
ologic studies, which would permit investigations of the protec-
tive role of these micronutrients.
DIETARY INTAKE OF POLYPHENOLS
Only partial information is available on the quantities of poly-
phenols that are consumed daily throughout the world. These
data have been obtained through analysis of the main aglycones
(after hydrolysis of their glycosides and esters) in the foods most
widely consumed by humans.
In 1976 Kuhnau (8) calculated that dietary flavonoid intake in
theUnitedStateswas앒1g/dandconsistedofthefollowing:16%
flavonols, flavones, and flavanones; 17% anthocyanins; 20%
catechins; and 45% “biflavones.”Although these figures were
obtainedunder poorly detailed conditions,they continue to serve
as reference data. Certain studies have subsequently provided
more precise individual data concerning the intake of various
classes of polyphenols. Flavonols have been more extensively
studied. Consumption of these substances has been estimated at
앒20–25 mg/d in the United States, Denmark, and Holland (67–
69). In Italy, consumption ranged from 5 to 125 mg/d, and the
meanvalue was 35 mg/d(70). The intake offlavanones is similar
to or possibly higher than that of flavonols, with a mean con-
sumptionof 28.3 mghesperetin/din Finland (71).Because citrus
fruit is practically the sole source of flavanones, ingestion of
thesesubstances is probably greater in regions wherethese fruits
are produced, such as southern Europe. Anthocyanin consump-
tion was studied only in Finland, where high amounts of berries
are eaten, and was found to be 82 mg/d on average, although
some intakes exceeded 200 mg/d (72).
Consumption of soya in the Asian countries is 앒10–35 g/d,
which is equivalent to a mean intake of 25–40 mg isoflavones/d,
witha maximum intake of 100 mg/d (23,73, 74). Americans and
Europeans,whoeat little soya, consume onlyafew milligrams of
isoflavones per day. Nevertheless, the incorporation of growing
quantities of soya extracts into manufactured food products
could result in an increase in isoflavone intake. Women under-
going phytoestrogen replacement therapy for menopause con-
sume between 30 and 70 mg isoflavones/d in the form of soya
extract capsules (75).
In Spain the total consumption of catechins and proanthocya-
nidin dimers and trimers has been estimated at 18–31 mg/d, and
the main sources are apples, pears, grapes, and red wine (76).
Consumption of monomer flavonols in Holland is significantly
higher (50 mg/d), and the principal sources are tea, chocolate,
apples, and pears (27). Ingestion of more highly polymerized
proanthocyanidins could be as high as several hundred milli-
grams per day as previously suggested (42), but there are still no
reliable data.
Consumption of hydroxycinnamic acids may vary highly ac-
cording to coffee consumption. Some persons who drink several
cups per day may ingest as much as 500–800 mg hydroxycin-
namicacids/d,whereassubjectswhodo not drink coffee and who
also eat small quantities of fruit and vegetables do not ingest
쏜25mg/d (54). AGerman study estimateddaily consumption of
hydroxycinnamic acids and hydroxybenzoic acids at 211 and
11 mg/d, respectively. Caffeic acid intake alone was 206 mg/d,
and the principal sources were coffee (which provides 92% of
caffeic acid) and fruit and fruit juices combined (source of 59%
of p-coumaric acid) (65).
Various authors have noted a high variability in polyphenol
intake. Intake of phenolic acids ranged from 6 to 987 mg/d in
Germany(65). The meanconsumption of flavonolsand flavones
inthe Dutchpopulation was 23 mg/d; values at the10th and 90th
percentiles were 4 and 46 mg/d, respectively; and some subjects
consumed up to 100 mg/d (69). The main reason for these vari-
ations is individual food preferences. When polyphenol content
isexpressedastheamountprovidedbyafoodserving,asinTable
1, the consumption of one particular food, such as berries for
anthocyanins or coffee for hydroxycinnamic acids, clearly ap-
pears to be capable of markedly changing the total polyphenol
intake. If mean values are required, the addition of the intakes of
flavonols, flavanones, flavanols (monomers, dimers, and tri-
mers), and isoflavones gives a total daily consumption of 100–
150 mg in Western populations, to which must be added the
considerably variable intake of hydroxycinnamic acids, antho-
cyanins, and proanthocyanidins. Finally, the total polyphenol
intake probably commonly reaches 1 g/d in people who eat sev-
eral servings of fruit and vegetables per day. Note that it is really
difficult to follow a diet totally free of polyphenols. Because
polyphenol intake is difficult to evaluate by using dietary ques-
tionnaires, biomarkers for polyphenol exposure would be very
useful. A few studies have tried to correlate flavonol, flavanone,
and isoflavone intakes with plasma concentrations or urinary
excretion of metabolites (77–82), but we are not yet able to
732 MANACH ET AL
propose a reliable measurement in urine or plasma samples that
could reflect the long-term intake of the various polyphenols.
BIOAVAILABILITY OF POLYPHENOLS
It is important to realize that the polyphenols that are the most
common in the human diet are not necessarily the most active
within the body, either because they have a lower intrinsic ac-
tivity or because they are poorly absorbed from the intestine,
highly metabolized, or rapidly eliminated. In addition, the me-
tabolites that are found in blood and target organs and that result
from digestive or hepatic activity may differ from the native
substances in terms of biological activity. Extensive knowledge
of the bioavailability of polyphenols is thus essential if their
health effects are to be understood.
Metabolism of polyphenols occurs via a common pathway
(83). The aglycones can be absorbed from the small intestine.
However, most polyphenols are present in food in the form of
esters, glycosides, or polymers that cannot be absorbed in their
native form. These substances must be hydrolyzed by intestinal
enzymes or by the colonic microflora before they can be ab-
sorbed. When the flora is involved, the efficiency of absorption
is often reduced because the flora also degrades the aglycones
that it releases and produces various simple aromatic acids in the
process. During the course of absorption, polyphenols are con-
jugated in the small intestine and later in the liver. This process
mainly includes methylation, sulfation, and glucuronidation.
This is a metabolic detoxication process common to many xe-
nobiotics that restricts their potential toxic effects and facilitates
their biliary and urinary elimination by increasing their hydro-
philicity. The conjugation mechanisms are highly efficient, and
aglycones are generally either absent in blood or present in low
concentrations after consumption of nutritional doses. Circulat-
ing polyphenols are conjugated derivatives that are extensively
bound to albumin. Polyphenols are able to penetrate tissues,
particularlythose in which they aremetabolized, but their ability
to accumulate within specific target tissues needs to be further
investigated. Polyphenols and their derivatives are eliminated
chiefly in urine and bile. Polyphenols are secreted via the biliary
route into the duodenum, where they are subjected to the action
of bacterial enzymes, especially

-glucuronidase, in the distal
segments of the intestine, after which they may be reabsorbed.
This enterohepatic recycling may lead to a longer presence of
polyphenols within the body.
Intestinal absorption and metabolism
Much about the intestinal mechanisms of the gastrointestinal
absorption of polyphenols remains unknown. Most polyphenols
are probably too hydrophilic to penetrate the gut wall by passive
diffusion, but the membrane carriers that could be involved in
polyphenol absorption have not been identified. To date, the
unique active transport mechanism that has been described is a
Na
ѿ
-dependent saturable transport mechanism involved in cin-
namic and ferulic acid absorption in the rat jejunum (84).
In foods, all flavonoids except flavanols are found in glyco-
sylated forms, and glycosylation influences absorption. The fate
of glycosides in the stomach is not clear. Experiments using
surgically treated rats in which absorption was restricted to the
stomachshowedthatabsorptionat the gastric level ispossiblefor
someflavonoids, such asquercetinand daidzein, butnot for their
glycosides (85, 86). Most of the glycosides probably resist acid
hydrolysis in the stomach and thus arrive intact in the duodenum
(87).Only aglycones and someglucosides can be absorbedin the
small intestine, whereas polyphenols linked to a rhamnose moi-
ety must reach the colon and be hydrolyzed by rhamnosidases of
the microflora before absorption (88, 89). The same probably
applies to polyphenols linked to arabinose or xylose, although
this question has not been specifically studied. Because absorp-
tion occurs less readily in the colon than in the small intestine
because of a smaller exchange area and a lower density of trans-
port systems, as a general rule, glycosides with rhamnose are
absorbed less rapidly and less efficiently than are aglycones and
glucosides. This has been clearly shown in humans for quercetin
glycosides: maximum absorption occurs 0.5–0.7 h after inges-
tion of quercetin 4'-glucoside and 6–9 h after ingestion of the
same quantity of rutin (quercetin-3

-rutinoside). The bioavail-
ability of rutin is only 15–20% of that of quercetin 4'-glucoside
(90, 91). Similarly, absorption of quercetin is more rapid and
efficient after ingestion of onions, which are rich in glucosides,
than after ingestion of apples containing both glucosides and
variousotherglycosides(92).Inthe case of quercetin glucosides,
absorption occurs in the small intestine, and the efficiency of
absorptionis higher thanthat for theaglycone itself (93, 94). The
underlying mechanism by which glucosylation facilitates quer-
cetin absorption has been partly elucidated. Hollman et al sug-
gested that glucosides could be transported into enterocytes by
the sodium-dependent glucose transporter SGLT1 (93). They
could then be hydrolyzed inside the cells by a cytosolic

-glucosidase (95). Another pathway involves the lactase phlo-
ridzinehydrolase,aglucosidaseofthebrushbordermembrane of
thesmallintestinethat catalyzes extracellular hydrolysis ofsome
glucosides,whichisfollowed by diffusion of theaglyconeacross
the brush border (96). Both enzymes are probably involved, but
their relative contribution for the various glucosides remains to
be clarified. Quercetin 3-glucoside, which is not a substrate for
cytosolic

-glucosidase,iscertainlyabsorbedafterhydrolysisby
lactasephloridzine hydrolase, at least in rats, whereas hydrolysis
of quercetin 4'-glucoside seems to involve both pathways (97,
98).In humans, whatever the mechanismof deglucosylation, the
kinetics of plasma concentrations are similar after ingestion of
quercetin 3-glucoside or quercetin 4'-glucoside (99). Isoflavone
glycosides present in soya products can also be deglycosylated
by

-glucosidasesfromthe human small intestine (95,96).How-
ever, the effect of glucosylation on absorption is less clear for
isoflavones than for quercetin. Aglycones present in fermented
soya products were shown to be better absorbed than were the
glucosides ingested from soybeans (100). However, a dose or
matrix effect may explain the difference in absorption observed
in this first study. Setchell et al (101) showed that when pure
daidzein, genistein, or their corresponding 7-glucosides were
administered orally to healthy volunteers, a tendency toward
greaterbioavailabilitywas observed with theglucosides,asmea-
suredfromthe area under thecurveofthe plasma concentrations:
2.94, 4.54, 4.52, and 4.95
g·h/mL for daidzein, genistein,
daidzin, and genistin, respectively. However, in another human
study, peak plasma concentrations were markedly higher after
aglycone ingestion than after glucoside ingestion, and this effect
was observed with low or high single doses and after long-term
intakes (102). In addition, hydrolysis of isoflavone glycosides
intoaglycones in a soydrinkdid not changethebioavailability of
the isoflavones in humans (103). No data are available for other
polyphenols in humans, but note that in rats, no enhancement of
POLYPHENOLS: FOOD SOURCES AND BIOAVAILABILITY 733
absorption was observed with glucosylation of naringenin and
phlorizin (104, 105). Furthermore, diglucosylation of the lignan
secoisolariciresinol decreases its absorption (106).
Glycosylation does not influence the nature of the circulating
metabolites. Intact glycosides of quercetin, daidzein, and
genisteinwere not recoveredinplasma or urineafter ingestion as
pure compounds or from complex food (107–110). For fla-
vanones, only trace amounts of glycosides have been detected in
humanurine,correspondingto0.02%oftheadministereddoseof
naringin (111). But a very high dose (500 mg) of the pure com-
pound was administered in this study, and some metabolic pro-
cesses may have been saturated by this nonnutritional intake.
Anthocyanins constitute an exception, because intact glycosides
are the major circulating forms. The explanation for this may lie
in the instability of these molecules in the aglycone form or in a
specific mechanism of absorption or metabolism for anthocya-
nins. Passamonti et al (112) have proposed that glycosides of
anthocyaninsmay betransported by bilitranslocase at the gastric
level, bacause they have been shown to be good ligands for this
carrier. They could also be directly converted into glucuronides
byaUDPglucose dehydrogenase as suggestedbyWuet al (113).
Proanthocyanidins differ from most other plant polyphenols
because of their polymeric nature and high molecular weight.
This particular feature should limit their absorption through the
gut barrier, and oligomers larger than trimers are unlikely to be
absorbed in the small intestine in their native forms. In vitro
experiments using single layers of Caco-2 cells as a model of
absorptionin the small intestine showedthat onlythe dimers and
trimers of flavanols are able to cross the intestinal epithelium
(114). Procyanidin B2 is very poorly absorbed in rats, whereas
procyanidin B3 is not absorbed (115, 116). The possibility that
procyanidin oligomers are hydrolyzed to mixtures of flavanol
monomers and dimers in acidic conditions was suggested by
Spenceret al from in vitro experiments (117).However, purified
procyanidin dimer B3, as well as grapeseed proanthocyanidins
having a higher degree of polymerization, are not degraded to
morereadily absorbablemonomers in rats (116). The stability of
proanthocyanidins was investigated in humans by regular anal-
ysis of gastric juice sampled with a gastric probe after ingestion
of a proanthocyanidin-rich cocoa beverage (118). This study
confirmed that proanthocyanidins are not degraded in the acidic
conditions of the stomach in vivo. A minor absorption of some
procyanidin dimers seems possible in humans. The procyanidin
dimerB2was detected in the plasmaofvolunteers after ingestion
of a cocoa beverage; however, the maximal plasma concentra-
tionthatwasreached2h after ingestion was much lowerthanthat
reached after a roughly equivalent intake of epicatechin (0.04
comparedwith6.0
mol/L)(119).Proanthocyanidins, which are
among the most abundant dietary polyphenols, are very poorly
absorbed and may exert only local activity in the gastrointestinal
tract or activity mediated by phenolic acids produced through
microbial degradation. Their local action may nevertheless be
importantbecause theintestine is particularly exposed to oxidiz-
ing agents and may be affected by inflammation and numerous
diseases such as cancer (120). Polyphenol concentrations in the
colon can reach several hundred micromoles per liter (83), and
together with a few carotenoids, polyphenols constitute the only
dietaryantioxidants present inthe colon, becausevitamins C and
E are absorbed in the upper segments of the intestine.
Despitethescarcityofstudiesperformedonthebioavailability
of hydroxycinnamic acids, when ingested in the free form, these
compoundsare rapidly absorbed from the small intestineand are
conjugated and, in particular, glucuronidated in the same way
that flavonoids are (54, 121). However these compounds are
naturally esterified in plant products, and this impairs their ab-
sorption.Human tissues (intestinal mucosa, liver) and biological
fluids (plasma, gastric juice, duodenal fluid) do not possess es-
terasescapableofhydrolyzingchlorogenicacidto release caffeic
acid (122–124). This has also been observed in rats (125, 126).
Onlythecolonicmicroflorawould be capable of carryingoutthis
hydrolysis, and some of the bacterial strains involved have been
identified (127). Consequently, as observed for flavonoid gly-
cosidesthat must behydrolyzed by themicroflora, the efficiency
of absorption of phenolic acids is markedly reduced when they
arepresentin the esterified form ratherthanin the free form(123,
125, 128). In patients who have undergone colonic ablation,
caffeicacidwasmuchbetterabsorbedthanwaschlorogenicacid:
11% and 0.3% of the ingested doses were excreted in urine,
respectively (123). Similarly, when chlorogenic acid was given
by gavage to rats, no intact compound was detected in plasma in
the following 6 h, and the maximum concentrations of metabo-
lites obtained after administration of caffeic acid in the same
conditions were 100-fold those of the metabolites (various glu-
curonidated or sulfated derivatives of caffeic and ferulic acids)
obtained after chlorogenic acid administration (125). Surpris-
ingly,the plasma concentrations weremaximal only 30 minafter
gavage, which may seem inconsistent with hydrolysis of chlo-
rogenic acid in the cecum. The same observation was made in a
human study. When volunteers ingested coffee containing high
amounts of esterified phenolic acids but no free caffeic acid, the
peak plasma concentration of caffeic acid was observed only 1 h
after ingestion of the coffee (129). In this study, the alkaline
hydrolysis of coffee showed that chlorogenic acid represented
only 30% of the bound caffeic acid. Thus, a possible explanation
isthatotherforms of caffeic acid presentincoffeemay have been
hydrolyzed in the upper part of the gut. Furthermore, the modes
of administration used in both studies, ie, direct stomach intuba-
tion in the rat study and ingestion of coffee alone by fasted
volunteers in the second study, might allow a rapid transit to the
colon and explain the rapid kinetics of appearance of plasma
metabolites. However, these 2 studies raise doubt about the total
inability of the tissues to hydrolyze esterified phenolic acids.
In addition to being esterified to simple acids, hydroxycin-
namic acids may be bound to polysaccharides in plant cell walls;
the main example of this is esterification of ferulic acid to arabi-
noxylans in the outer husks of cereals. Although free ferulic acid
is reported to be rapidly and efficiently absorbed (up to 25%)
from tomatoes in humans (130), its absorption after ingestion of
cereals is expected to be much lower because of this esterifica-
tion. Ferulic acid metabolites recovered in the urine of rats rep-
resentonly 3% ofthe ingested dosewhen ferulic acid is provided
aswheatbran,whereas the metabolites represent 50%ofthedose
whenferulic acid is providedas a pure compound(131). Another
study showed that feruloyl esterases are present throughout the
entiregastrointestinal tract,particularly in the intestinal mucosa,
and that some of the ester bonds between ferulic acid and poly-
saccharides in cell walls may thus be hydrolyzed in the small
intestine (126). However, the role of feruloyl esterases seems to
be very limited, and absorption occurs mainly in the colon after
hydrolysisbyenzymesofbacterial origin. Xylanases degrade the
734 MANACH ET AL
parietal polymers to small, soluble feruloyl oligosaccharides,
and then esterases release free ferulic acid. Note that diferulic
acids from cereal brans have been shown to be absorbed in rats
(132).
The effects of the food matrix on the bioavailability of poly-
phenols have not been examined in much detail. Direct interac-
tions between polyphenols and some components of food, such
as binding to proteins and polysaccharides, can occur, and these
interactions may affect absorption. Furthermore, more indirect
effects of the diet on various parameters of gut physiology (pH,
intestinal fermentations, biliary excretion, transit time, etc) may
have consequences on the absorption of polyphenols. Enzymes
and carriers involved in polyphenol absorption and metabolism
may also be induced or inhibited by the presence of some mi-
cronutrients or xenobiotics. Interactions with milk proteins were
consideredfirstbecauseSerafinietal(133)reportedthataddition
ofmilktoblackteaabolishedtheincreaseinantioxidantpotential
that was observed when tea was consumed without milk. How-
ever,subsequent studiesshowed that addition of milk to blackor
green tea had no effect on the bioavailability of catechins, quer-
cetin, or kaempferol in humans (134, 135). Some investigators
have speculated that the presence of alcohol in red wine could
improve the intestinal absorption of polyphenols by increasing
their solubility. Ethanol was shown to enhance the absorption of
quercetin in rats, but only when present at a concentration too
high to be attained in the diet (쏜30%, by vol) (136). In humans,
plasmaconcentrations ofcatechin metaboliteswere similar after
consumption of red wine or dealcoholized red wine (137). Yet,
20% more catechin metabolites were excreted in urine after red
wine intake than after dealcoholized red wine intake, which in-
dicates a possible role of ethanol in enhancing the rate of poly-
phenol elimination, perhaps by a diuretic effect (138). On the
other hand, tartaric acid, which is a major organic acid in wine,
was shown to enhance the absorption of catechin in rats (139).
Existingdata do not suggestamarked effect ofthevarious diet
components on polyphenol bioavailability. The absorption of
quercetin, catechin, and resveratrol in humans was recently
shown to be broadly equivalent when these polyphenols were
administeredin3 different matrices:whitewine, grape juice, and
vegetable juice (140). According to Hendrich (141), neither the
background diet or type of soy food nor the presence of 40 g
wheat fiber significantly alters the apparent absorption of isofla-
vones. However, more studies are needed, especially on dietary
fiber. Dietary fiber is generally associated with polyphenols in
plant foods and stimulates intestinal fermentation, which could
influencetheproduction of particular microbial metabolites.Ad-
ministration of polyphenols without a food matrix could mark-
edly affect their bioavailability. With regard to flavonols, much
higherplasmaconcentrationswereachieved when quercetin glu-
cosides were administered to fasted volunteers in the form of a
water-alcoholsolution(upto5
mol/L)(99)thanwhenanequiv-
alent quantity was ingested with foods such as onions, apples, or
a complex meal (0.3–0.75 nmol/L) (92, 107). This suggests that
the consumption of any food may limit polyphenol absorption
and that high plasma concentrations would be obtained only if
supplements were taken separately from meals.
The role of the colonic microflora
Polyphenols that are not absorbed in the small intestine reach
the colon. The microflora hydrolyzes glycosides into aglycones
andextensively metabolizesthe aglycones into various aromatic
acids (8, 142). Aglycones are split by the opening of the hetero-
cycle at different points depending on their chemical structure:
flavonols mainly produce hydroxyphenylacetic acids, flavones
and flavanones mainly produce hydroxyphenylpropionic acids,
and flavanols mainly produce phenylvalerolactones and hy-
droxyphenylpropionic acids. These acids are further metabo-
lized to derivatives of benzoic acid. The microbial metabolites
are absorbed and conjugated with glycine, glucuronic acid, or
sulfate. The cleavage and metabolic pathways are well estab-
lished in animals, and the influence of chemical structure on
degradation is known. For example, the absence of a free hy-
droxylinposition5,7,or4'protectsthe compound from cleavage
(143). However, data are still limited in humans, so it is possible
thatnew microbial metabolites willbe identified. Interindividual
variationsandthe influence ofthemicroflora composition and of
the usual diet on microbial metabolite production have to be
evaluated.Recentstudieshaveshownthatplasmaconcentrations
and urinary excretion of microbial metabolites in humans can be
higher than those of tissular metabolites, especially for polyphe-
nols such as wine polyphenols that are not easily absorbed (128,
144, 145). Thus, the identification and quantification of micro-
bial metabolites constitute an important field of research. Some
microbial metabolites may have a physiologic effect; for exam-
ple, hydroxyphenylacetic acids have been suggested to inhibit
platelet aggregation (146). Besides, among the wide array of
aromatic acids with low molecular weight, some may be used as
biomarkers for polyphenol intake. An association between poly-
phenol intake and the amount of excreted hippuric acid was
found after consumption of black tea or a crude extract from
Equisenum arvense (147, 148). However, hippuric acid is not a
degradationproduct of catechin and can be derivedfrom sources
other than polyphenols, such as quinic acid and the aromatic
amino acids; thus, it is not a suitable biomarker of polyphenol
intake (144). 3-Hydroxyhippuric acid may be a more valid
biomarker (124).
Specific active metabolites are produced by the colonic mi-
croflora. This is the case with lignans from linseed, which are
metabolizedto enterolactone andenterodiol, which haveagonis-
tic or antagonistic effects on estrogens (149, 150). Similarly,
equol produced from soya daidzein appears to have phytoestro-
genic properties equivalent to or even greater than those of the
original isoflavone (151, 152). There is a great interindividual
variability in the capacity to produce equol. Only 30-40% of the
occidental people excrete significant quantities of equol after
consumption of isoflavones, and these persons are called “equol
producers”(152, 153). The corresponding percentage among
Asian populations is unknown, but a recent study suggested that
the percentage in Japanese men could be as high as 60% (154).
The ability or inability of persons to produce equol seems to
remain the same for at least several years (152, 155). The com-
position of the intestinal flora plays a major role. Inoculation of
germ-freeratswith human florafromequol producers confers on
these rats the capacity to produce this metabolite, whereas col-
onization with flora from non–equol producers leaves the rats
incapable of producing equol (156). Equol is not recovered in
plasma from infants who are fed soy-based formulas, which
suggests that the bacteria responsible for its production are not
developed in the first months of life (157). Three strains of
bacteria are reportedly able to convert pure daidzein to equol in
vitro: Streptococcus intermedius ssp., Ruminococcus productus
spp., and Bacteroides ovatus spp. (158). The possibility of con-
POLYPHENOLS: FOOD SOURCES AND BIOAVAILABILITY 735
verting nonproducers to producers by food must be investigated.
Equolproducerstendtoconsumelessfatandmorecarbohydrates
as percentages of energy than do non–equol producers (159,
160). Consumption of dietary fiber has been suspected to affect
equol production by favoring the growth of certain bacterial
species. However, supplementation with 16 g wheat bran/d did
not increase equol production in young women (159). In mice,
equol production increased with the addition of fructooligosac-
charidesin the diet(161). But thisresult needs to be confirmed in
humans because of obvious interspecies differences, which are
shown by the fact that rats are constitutive equol producers. The
effect of adaptation of the intestinal flora to the consumption of
isoflavones is not clear. Lu et al (162) observed an increase in
equol production after 1 mo of isoflavone consumption. Some
non-equol-producing women even acquired the ability to pro-
duce equol after consuming soymilk for 2 wk (153). But Lampe
et al (163) did not observe any effect on equol production of a
1-mo adaptation in comparison with a 4-d supplementation. A
more comprehensive knowledge of the factors that may influ-
ence equol production is all the more essential because Setchell
et al (152) convincingly proposed that equol producers might
gain more benefits from soya consumption than would nonpro-
ducers.
Conjugation and nature of metabolites
Once absorbed, polyphenols are subjected to 3 main types of
conjugation: methylation, sulfation, and glucuronidation.
Catechol-O-methyltransferase catalyzes the transfer ofa methyl
group from S-adenosyl-L-methionine to polyphenols having an
o-diphenolic(catechol)moiety.Suchareactioniswellknownfor
quercetin, catechin, caffeic acid, and luteolin, and Wu et al (113)
recently showed for the first time the methylation of cyanidin to
peonidin in humans. The methylation generally occurs predom-
inantly in the 3' position of the polyphenol, but a minor propor-
tion of 4'-O-methylated product is also formed. Note that a sub-
stantial amount of 4'-methylepigallocatechin was detected in
human plasma after ingestion of tea (164, 165). Catechol-O-
methyl transferase is present in a wide range of tissues. Its ac-
tivity is highest in the liver and the kidneys (166, 167) although
significant methylation was reported for catechin in the small
intestine of rats (168). Sulfotransferases catalyze the transfer of
asulfate moiety from3'-phosphoadenosine-5'-phosphosulfate to
ahydroxylgroup on various substrates (steroids,bileacids,poly-
phenols, etc). Neither the isoforms that are specifically involved
in the conjugation of polyphenols nor the positions of sulfation
for the various polyphenols have yet been clearly identified, but
sulfation clearly occurs mainly in the liver (166, 169). UDP-
glucuronosyltransferasesare membrane-bound enzymesthatare
located in the endoplasmic reticulum in many tissues and that
catalyze the transfer of a glucuronic acid from UDP-glucuronic
acidto steroids, bile acids,polyphenols, and thousands ofdietary
constituents and xenobiotics. The presence of glucuronidated
metabolites in the mesenteric or portal blood after perfusion of
polyphenolsinthe small intestineofrats shows that glucuronida-
tion of polyphenols first occurs in the enterocytes before further
conjugation in the liver (170–172). This is probably the case in
humans as well, because in humans the in vitro glucuronidation
of quercetin and luteolin by microsomes from the intestine is
markedly higher than that by microsomes from the liver (173).
About 15 isoforms of UDP-glucuronosyltransferases have been
identified in humans, and these isoforms have broad and over-
lapping substrate specificities and different tissue distribution
(174). The subfamily of UDP-glucuronosyltransferases called
UGT1A that is localized in the intestine probably plays a major
role in the first-pass metabolism of polyphenols. These isoen-
zymes have a wide polymorphic expression pattern that could
result in a high interindividual variability in polyphenol glucu-
ronidation. The active isoenzymes of the 1A class seem to differ
according to the polyphenol considered (173, 175). In vitro glu-
curonidation of quercetin, luteolin, or isorhamnetin by rat or
human microsomes in the intestine and the liver showed that,
even if the nature of the glucuronides formed is constant, the
proportionofthevariousmetabolitesvarieswidelydepending on
the species and organ (173, 176, 177). The highest rate of con-
jugationis observed at the7-position, and the 5-positiondoes not
appear to be a site for glucuronidation. For most flavonoids, a
significant proportion of the glucuronides that are formed in the
intestinal mucosa are secreted back to the gut lumen, which
reducesnet absorption(178, 179). The transporter multiresistant
protein 2 (MRP2) or the P-glycoprotein may be involved in this
efflux (180, 181). The proportion of glucuronides that are se-
creted toward the mucosal side depends markedly on the struc-
ture of the polyphenol (0–52% of the initial dose) (182). Intes-
tinal excretion of glucuronides does not occur with catechin and
ferulic acid, which indicates that this is not a mechanism of
elimination for all polyphenols (131, 168, 182).
The metabolic fate in the liver of the conjugates that are pro-
duced in the intestine is not yet clear. After penetration into
HepG2 cells, quercetin 7-glucuronide and quercetin 3-O-
glucuronide undergo 2 types of metabolism: methylation of the
catechol and deglucuronidation followed by 3'-sulfation (183).
However,in the same conditions, quercetin 4'-glucuronide isnot
metabolized. This could result from a lower rate of penetration
into the cells or a lower affinity of the metabolizing enzymes for
thissubstrate. Acomplex set of conjugating enzymes and carrier
systems is probably involved in the regulation of uptake and the
production and release of the various polyphenol metabolites by
the hepatocytes, as shown for other conjugates (184, 185). The
activity of these enzymes and carrier systems may depend on the
nature of the polyphenol and may be influenced by genetic poly-
morphisms that lead to important interindividual differences in
the capacity to metabolize polyphenols.
The relative importance of the 3 types of conjugation (meth-
ylation, sulfation, and glucuronidation) appears to vary accord-
ing to the nature of the substrate and the dose ingested. Sulfation
is generally a higher-affinity, lower-capacity pathway than is
glucuronidation,so that when the ingested dose increases,a shift
fromsulfationtoward glucuronidation occurs (186). Thebalance
between sulfation and glucuronidation of polyphenols also
seems to be affected by species, sex, and food deprivation (187).
Moreover,inhibition of methylation by aspecific inhibitor shifts
metabolism of quercetin glucuronides toward sulfation in
HepG2 cells (183). Regardless of the respective contributions of
methylation, sulfation, and glucuronidation, in general, the ca-
pacityforconjugationishigh.Theconcentrationoffreeaglycone
isusuallyverylowin plasma after the intakeofanutritionaldose,
except for tea catechins (up to 77% for epigallocatechin gallate)
(164).Saturation of the conjugationprocesses has been observed
in rats administered high doses and rats given an acute supply of
polyphenols by gavage (166, 170). Competitive inhibition of
conjugation could also occur in the presence of various polyphe-
nolsandxenobioticsintheintestine,butithasneverbeenstudied.
736 MANACH ET AL
In these conditions, significant amounts of free aglycones could
circulateinblood,probablywithbiologicaleffectsdifferentfrom
those of conjugated metabolites.
Identification of circulating metabolites has been undertaken
for only a few polyphenols. This identification must include not
onlythenatureand number of the conjugatinggroupsbutalsothe
positions of these groups on the polyphenol structure because
these positions can affect the biological properties of the
conjugates (176). After consumption of onions containing
glucosides of quercetin, the major circulating compounds in
human plasma were identified as quercetin 3-O-glucuronide,
3'-O-methylquercetin 3-O-glucuronide, and quercetin 3'-O-
sulfate (188). However, analysis by liquid chromatography–
tandemmassspectrometryof human plasma samples obtained
in very similar conditions did not confirm the presence of
sulfated quercetin (109). For other polyphenols, only scarce
data on the proportions of the various types of conjugates and
the percentages of free forms in plasma are available (101,
164, 175, 189–192). The main circulating compounds are
generally glucuronides.
Plasma transport and partitioning into lipid structures
Polyphenol metabolites are not free in the blood. In vitro
incubation of quercetin in normal human plasma showed that
quercetin is extensively bound to plasma proteins (99% for con-
centrations up to 15
mol/L), whereas binding to VLDL is not
significant (쏝0.5%) (193). Metabolites of quercetin are also
extensively bound to plasma proteins in the plasma of rats fed a
quercetin-enriched diet (88). Albumin is the primary protein
responsible for the binding. The affinity of polyphenols for al-
bumin varies according to their chemical structure. Kaempferol
andisorhamnetin,whichdifferfromquercetin in the nature of the
B-ring substitution, have an affinity for human serum albumin
that is similar to that of quercetin (194). In contrast, substitution
of 3-OH markedly weakens binding to albumin, as shown for
rutin and isoquercitrin, the 3-O-glycosides of quercetin (195).
The effect of sulfation and glucuronidation is unknown, but it
probably depends highly on the position of substitution. Hy-
droxycinnamicacids,especiallyferulicandcoumaricacids,have
alow affinityfor bovine serum albumin but may have a different
affinityfor albuminof human origin (196). No data areavailable
for the other polyphenols. It must be kept in mind that although
the intrinsic affinity of circulating polyphenol conjugates for
albumin may be much weaker than that of quercetin itself, the
physiologic concentration of serum albumin (앒0.6 mmol/L) is
probably large enough to allow their extensive binding. The
degreeof binding toalbuminmay have consequencesfor the rate
of clearance of metabolites and for their delivery to cells and
tissues. The conventional view is that cellular uptake is propor-
tional to the unbound concentration of metabolites. Yet, varia-
tions in local pH at specific sites may induce conformational
changes in albumin, which lead to dissociation of the ligand-
albumin complex. Conformational changes in albumin have
been shown to be induced by nonspecific interactions with var-
iousmembranes(197).Whethersuchchangescouldfacilitatethe
cellular uptake of ligands such as polyphenol metabolites is un-
clear. However, incubations of quercetin in human whole blood
or in suspensions of erythrocytes in the absence of plasma pro-
teins suggests that binding to albumin could considerably de-
crease the association of quercetin with these cells (193). The
effect of albumin binding on the biological activity of polyphe-
nols is unclear. Does the bound ligand have some biological
activity, or does the polyphenol have to be in the free form to be
active? Dangles et al (195) showed that the catechol moiety of
albumin-bound quercetin remains accessible to oxidizing agents
such as periodate. If this key structural element of quercetin is
alsoaccessible tofree radicals, this suggests that quercetin could
exert its antioxidant activity even when it is bound to albumin.
However, the biological properties of polyphenols are certainly
not limited only to their antioxidant capacity; thus, binding to
albumin may have a considerable effect.
Thepartitioningof polyphenols and theirmetabolitesbetween
aqueous and lipid phases is largely in favor of the aqueous phase
because of their hydrophilicity and binding to albumin. How-
ever, in some lipophilic membrane models, some polyphenols
penetratethemembrane to variousextents(198–202).Quercetin
showedthe deepestinteraction, probably because of its ability to
assume a planar conformation (203). At physiologic pH, most
polyphenolsinteractwith the polar headgroups of phospholipids
at the membrane surface via the formation of hydrogen bonds
thatinvolvethehydroxylgroupsofthepolyphenols(204).Ahigh
number of hydroxyl groups on the polyphenol structure and an
increasein pH thatleads to deprotonationof the hydroxyl groups
would thus enhance interactions between the polyphenols and
the membrane surface. This adsorption of polyphenols probably
limits the access of aqueous oxidants to the membrane surface
and their initial attack on that surface.
LDL is made up of lipophilic structures that, once oxidized,
participate in the etiology of atherosclerosis. Many studies have
shown that various polyphenols have the ability to protect LDL
from oxidation. However, a very small proportion of plasma
polyphenols are in fact associated with the LDL fraction after
consumptionofnutritionaldoses of these compounds (205,206).
They are associated with lipoproteins only by ionic interactions
with charged residues on the surface of the particules. The low
integration of polyphenols into LDL has been confirmed by in
vitro incubation experiments (207, 208). Protection probably
occurs at the interface between lipophilic and hydrophilic
phases. However, a recent study in which [
3
H]genistein was
incubated in human plasma showed that genistein and its li-
pophilic derivatives were incorporated into HDL and, to a lesser
extent, into LDL (209). These lipophilic derivatives could result
from enzymatic esterification with fatty acids in plasma, as re-
ported for estrogens, but further characterization of these deriv-
atives is needed.
Plasma concentrations
Plasma concentrations reached after polyphenol consumption
vary highly according to the nature of the polyphenol and the
food source. They are on the order of 0.3–0.75
mol/L after
consumptionof80–100mgquercetinequivalentadministered in
theformofapples,onions,or meals rich in plant products(90,92,
107).Wheningestedintheformofgreentea(0.1–0.7
mol/Lfor
an intake of 90–150 mg), cocoa (0.25–0.7
mol/L for an intake
of 70–165 mg) (210–213), or red wine (0.09
mol/L for an
intake of 35 mg) (137), catechin and epicatechin are as effec-
tively absorbed as is quercetin. The maximum concentrations of
hesperetin metabolites determined in plasma 5–7 h after con-
sumption of orange juice were 1.3–2.2
mol/L for an intake of
130–220 mg (189, 214). Naringenin in grapefruit juice appears
to be absorbed even better: a peak plasma concentration of
6
mol/L is obtained after ingestion of 200 mg. In contrast,
POLYPHENOLS: FOOD SOURCES AND BIOAVAILABILITY 737
plasma concentrations of anthocyanins are very low: peak con-
centrations, which occur between 30 min and 2 h after consump-
tion, are on the order of a few tens of nanomoles per liter for an
intake of 앒110–200 mg anthocyanins (215–217). Similarly, the
intake of 앒25 mg secoisolariciresinol diglucoside in the form of
linseed produces only a slight increase (앒30 nmol/L) in plasma
lignan concentrations, and this increase occurs gradually be-
tween 9 and 24 h (218). Isoflavones are certainly the best ab-
sorbed flavonoids: plasma concentrations of 1.4–4
mol/L are
obtainedbetween 6 and8hinadults whoconsume relatively low
quantities of soya derivatives supplying 앒50 mg isoflavones
(219–221). This raises the question of the harmlessness of
soymilk, which is consumed in large quantities by infants who
are allergic to cow milk. In 4-mo-old infants, isoflavone intake
can reach 50 mg/d, which, when expressed relative to body
weight, is 5–10-fold the dose shown to exert a physiologic effect
on the hormonal regulation of women’s menstrual cycles (222).
Plasma concentrations of genistein and daidzein in these infants
can reach several micromoles per liter (223).
Tissue uptake
Determination of the actual bioavailability of polyphenol me-
tabolites in tissues may be much more important than is knowl-
edge of their plasma concentrations. Data are still very scarce,
even in animals.
When single doses of radiolabeled polyphenols (quercetin,
epigallocatechin gallate, quercetin 4'-glucoside, resveratrol) are
given to rats or mice killed 1–6 h later, radioactivity is mainly
recoveredinblood and intissuesof the digestive system,such as the
stomach, intestine, and liver (224–227). However, polyphenols
have been detected by HPLC analysis in a wide range of tissues in
mice and rats, including brain (228, 229), endothelial cells (230),
heart, kidney, spleen, pancreas, prostate, uterus, ovary, mammary
gland, testes, bladder, bone, and skin (225, 231–233). The concen-
trationsobtainedinthesetissuesranged from 30to3000ngaglycone
equivalents/g tissue depending on the dose administered and the
tissue considered. The time of tissue sampling may be of great
importance because we have no idea of the kinetics of penetration
and elimination of polyphenols in the tissues.
Itisstill difficult to say whethersomepolyphenolsaccumulate
inspecifictargetorgans.Afewstudiesseemtoindicatethat some
cells may readily incorporate polyphenols by specific mecha-
nisms. The endothelium is likely to be one of the primary sites of
flavonoid action. Schramm et al (234) showed that a rapid,
energy-dependenttransport system is active inaortic endothelial
cells for the uptake of morin. This system may also transport
otherhydroxylatedphenolic compounds. Microautoradiography
of mice tissues after administration of radiolabeled epigallocat-
echin gallate or resveratrol indicated that radioactivity is un-
equallyincorporatedinto the cells of organs(225,227).Regional
selectivity has also been observed in the prostate and the brain.
After9 wk of feeding isoflavoneaglycones to rats, accumulation
of isoflavones in the dorsolateral zone of the prostate was shown
tobe 4-fold thatinthe ventral zoneof this organ(235). Similarly,
after 28 d of oral administration of tangeretin, a polymethoxy-
latedflavonefromCitrus,torats, tangeretin concentrations in the
brain were 6-fold those in other tissues (heart, lung, liver, kid-
ney), and distribution of tangeretin was unequal in the various
zones of the brain: concentrations in the hypothalamus, hip-
pocampus, and striatum were 10-fold those in the brainstem and
cerebellum (228).
The nature of the tissular metabolites may be different from
that of blood metabolites because of the specific uptake or elim-
ination of some of the tissular metabolites or because of intra-
cellular metabolism. Youdim et al (236) showed that the uptake
of flavanone glucuronides by rat and mouse brain endothelial
cultured cells is much lower than that of their corresponding
aglycones.In ratsfed a genistein-supplemented diet, the fraction
of genistein present in the aglycone form was much more im-
portantin several tissues thanin blood. It accountedfor 쏜50% of
the total genistein metabolites in mammary gland (237), uterus,
and ovary and 100% in the brain (231) and prostate (238),
whereas it represented only 8% of the total plasma metabolites.
Only 2 studies reported data on polyphenol concentrations in
human tissues. The first study measured phytoestrogens in hu-
manprostate tissue. Surprisingly, the study showed significantly
lower prostatic concentrations of genistein in men with benign
prostatic hyperplasia than in those with a normal prostate,
whereas plasma genistein concentrations were higher in men
with benign prostatic hyperplasia (239). In addition, concentra-
tions of enterodiol and enterolactone were higher in prostatic
tissuethanin plasma, whereastheopposite was true fordaidzein,
genistein, and equol. In the other study, equol concentrations in
women who ingested isoflavones were found to be higher in
breast tissue than in serum, whereas genistein and daidzein were
more concentrated in serum than in breast tissue. Note that very
high equol concentrations have been obtained in breast tissue,
andtheseconcentrations are equivalentto6
mol/Lforan intake
of앒110 mg ofits precursor, daidzein(240). These initial studies
show that plasma concentrations are not directly correlated with
concentrations in target tissues and that the distribution between
blood and tissues differs between the various polyphenols. This
raisesthequestionofwhetherplasmaconcentrationsareaccurate
biomarkers of exposure.
Elimination
Metabolites of polyphenols may follow 2 pathways of excre-
tion, ie, via the biliary or the urinary route. Large, extensively
conjugated metabolites are more likely to be eliminated in the
bile, whereas small conjugates such as monosulfates are prefer-
entially excreted in urine. In laboratory animals, the relative
magnitude of urinary and biliary excretion varies from one poly-
phenol to another (182). Biliary excretion seems to be a major
pathway for the elimination of genistein, epigallocatechin gal-
late,and eriodictyol (170, 241). Biliaryexcretion of polyphenols
in humans may differ greatly from that in rats because of the
existence of the gall bladder in humans; however, this has never
beenexamined.Intestinalbacteria possess

-glucuronidasesthat
are able to release free aglycones from conjugated metabolites
secreted in bile. Aglycones can be reabsorbed, which results in
enterohepatic cycling. Pharmacokinetic studies in rats have
shown a second maximum plasma concentration 앒7 h after
genistein administration, which is consistent with enterohepatic
circulation (233). A second plasma peak was also observed in
some human volunteers 10–12 h after ingestion of hesperetin
from orange juice or of isoflavones from soya (101, 189, 221).
Urinaryexcretionhasoftenbeendeterminedinhumanstudies.
The total amount of metabolites excreted in urine is roughly
correlated with maximum plasma concentrations. It is quite high
738 MANACH ET AL
forflavanones from citrus fruit (4–30%of intake), especiallyfor
naringeninfrom grapefruit juice (111,189, 214, 242, 243),and is
even higher for isoflavones: the percentages excreted are 16–
66% for daidzein and 10 –24% for genistein (103, 192, 219, 221,
244). It may appear surprising that plasma concentrations of
genistein are generally higher than those of daidzein despite the
higherurinary excretion ofdaidzein, but thiscan be explainedby
the efficient biliary excretion of genistein. Urinary excretion
percentages may be very low for other polyphenols, such as
anthocyanins (0.005–0.1% of intake) (113, 216, 217, 245, 246),
although Lapidot et al (247) reported elevated percentages of
anthocyanin excretion (up to 5%) after red wine consumption.
Low values could be indicative of pronounced biliary excretion
or extensive metabolism. Certain metabolites of anthocyanins
may still be unidentified as a result of analytic difficulties with
these unstable compounds. Felgines et al (248) reported that the
majormetabolitesof pelargonidin 3-glucoside thatarerecovered
in human urine after strawberry intake are glucurono- and sul-
foconjugates of pelargonidin that are extensively degraded by
simple freezing of the urine samples. Urinary excretion of fla-
vonols accounts for 0.3–1.4% of the ingested dose for quercetin
and its glycosides (90, 92, 93) but reaches 3.6% when purified
glucosides are given in hydroalcoholic solution to fasted volun-
teers (99). Urinary recovery is 0.5–6% for some tea catechins
(210,249),2–10% for redwinecatechin (138), and upto30% for
cocoa epicatechin (190). For caffeic and ferulic acids, relative
urinary excretion ranges from 5.9% to 27% (124, 130, 250)
Theexacthalf-lives of polyphenols in plasmahaverarelybeen
calculated with great precision but are on the order of2hfor
compounds such as anthocyanins (217) and 2–3 h for flavanols
(17, 164, 251, 252), except for epigallocatechin gallate, which is
eliminated more slowly probably because of higher biliary ex-
cretionor greater complexing with plasmaproteins, as described
for galloylated compounds (253, 254). The half-lives of isofla-
vones and quercetin are on the order of 4–8 (25, 244, 255) and
11–28(90, 92) h, respectively.This suggests that maintenanceof
high plasma concentrations of flavonoid metabolites could be
achieved with regular and frequent consumption of plant prod-
ucts. For instance, consumption of onions 3 times/d favors ac-
cumulationofquercetininplasma(256).Forcompounds,suchas
teacatechins,withrapidabsorptionand a short half-life, repeated
intakes must be very close together in time to obtain an accumu-
lationof metabolites inplasma (257); otherwise,plasma concen-
trationsregularly fluctuate afterrepeated ingestions, andno final
accumulation occurs (205).
Biological effects of polyphenol metabolites
The biological activities of polyphenols have often been eval-
uated in vitro on pure enzymes, cultured cells, or isolated tissues
by using polyphenol aglycones or some glycosides that are
present in food. Very little is known about the biological prop-
erties of the conjugated derivatives present in plasma or tissues
because of the lack of precise identification and commercial
standards. However, reflection on the antioxidative activity of
polyphenols suggests that the metabolism of polyphenols may
have a considerable effect. For example, the hydrophobicity of
polyphenols is intermediate between that of vitamin C (highly
hydrophilic) and that of vitamin E (highly hydrophobic). Poly-
phenolsare thus expected toactat water-lipid interfacesandmay
be involved in oxidation regeneration pathways with vitamins C
and E. Glucuronidation and sulfation render polyphenols more
hydrophilic and can affect their site of action and their interac-
tions with other antioxidants. Furthermore, their intrinsic reduc-
tive capacity may be changed. The antioxidant effect of conju-
gated derivatives of quercetin on copper ion–induced LDL
oxidation in vitro is about one-half that of the aglycone and is
dependent on the binding site of the glucuronic acid (107, 176,
258). Cren-Olive et al (259) also reported that the capacity of
3'-O-methylcatechin and 4'-O-methylcatechin to protect LDL
from in vitro oxidation is lower than that of catechin. However,
an increase in the antioxidant capacity of plasma was observed
after the consumption of various polyphenol-rich foods, which
indirectlyshows that at least some of thepolyphenol metabolites
retain antioxidant activity (212, 260–264). Conjugation might
enhance certain specific biological activities, as shown for some
xenobiotics (265). Koga and Meydani (266) showed that plasma
metabolites of catechin have an inhibitory effect on monocyte
adhesion to interleukin 1

–stimulated human aortic endothelial
cells, whereas catechin and metabolites of quercetin had no ef-
fect. In another in vitro study, quercetin 3-O-glucuronide pre-
vented vascular smooth muscle cell hypertrophy by angiotensin
II (267). However, conjugation seems instead to decrease the
specific activities of polyphenols. The affinities for estrogenic
receptors of the aglycones of daidzein and genistein are 10- and
40-fold, respectively, those of the respective glucuronides, but
the glucuronides still show weak estrogenic activity at physio-
logic concentrations (268). Spencer et al (269) showed the in-
ability of 5- and 7-O-glucuronides of epicatechin to protect fi-
broblasts and neuronal cells from oxidative stress in vitro,
whereas epicatechin and methylepicatechin were protective.
Nevertheless,it isstill difficult to draw any conclusions fromthe
fewexisting studies regardingtheeffects of thetype and position
of conjugation on the various potential activities of polyphenols.
Polyphenol metabolites could also exert biological activities
afterdeconjugation at the cellularlevel. This possibility hasbeen
shown for sulfates and glucuronides of endogenous estrogens
(270,271). Quercetin glucuronides were hydrolyzedby cell-free
extracts of human neutrophils, liver, and small intestine (272).
However,the possibility of hydrolysisof flavonoid glucuronides
inside cells has not been studied. We have seen above that the
proportion of free aglycone in some tissues may be higher than
that in blood, especially in the case of genistein in rats. This may
be explained by specific uptake of the aglycone or intracellular
deconjugation. This last hypothesis implies that anionic conju-
gates could be transported across plasma membranes via carrier
systems, as shown for other glucuronides (184, 273). Further-
more,

-glucuronidaseislocatedinthelumenoftheendoplasmic
reticulumin various organs andwouldalso have tobereached by
polyphenol glucuronides. Note that carrier-mediated bidirec-
tional transport across the membrane of the endoplasmic reticu-
lumin rat hepatocytes hasbeen described for otherglucuronides,
such as estrogen glucuronides (274). Inside the endoplasmic
reticulum,UDP-glucuronosyltransferasesare present alongwith

-glucuronidase. The respective K
m
values of these enzymes
toward flavonoids and their glucuronides seem to be in favor of
glucuronidationrather than deglucuronidation atphysiologic pH
values(176,272). These resultsarenot consistent enough togive
aclearviewofwhat occurs inside the cells, andadditionalstudies
are certainly needed.

-Glucuronidase is also present in the ly-
sosomes of various cells, from which it can be released in some
particular situations such as oxidative stress. Its activity in-
creases in some physiopathologic states, such as inflammation,
POLYPHENOLS: FOOD SOURCES AND BIOAVAILABILITY 739
cancer, and AIDS (275, 276). Luteolin 7-O-glucuronide is hy-
drolyzed into aglycone by lysosomal

-glucuronidase released
from neutrophils after induction of inflammation and in the
plasma of rats treated with lipopolysaccharide (277). In situ de-
conjugation of polyphenol metabolites might occur only in par-
ticular places, such as at inflammation sites or in tumors. Situa-
tions that decrease the pH would favor deglucuronidation
becausethe activity of

-glucuronidaseis optimal at pH 4–5and
is reduced 9-fold at neutral pH (275).
Polyphenolsmayalsohaveanindirecteffectonhealthbecause
they are metabolized by the same pathways as various xenobi-
oticsor endogenous hormones. Flavonoidssuch as quercetin and
fisetin are better substrates for catechol-O-methyl transferase
than are its endogenous substrates, catecholamines and catechol
estrogens. Deregulation of the O-methylation metabolism of
neurotransmitters and hormones in humans is an important risk
factor for the development of some neurodegenerative diseases,
cardiovascular diseases, and hormone-dependent cancers (278).
Thus, if confirmed in humans, the potential competitive inhibi-
tion of the catechol-O-methyl transferase–catalyzed
O-methylation of endogenous catecholamines and catechol es-
trogens by polyphenols with catechol groups may have a bene-
ficial effect on these pathologies. Some polyphenols, such as
quercetin and daidzein sulfoconjugates, are also efficient inhib-
itors of sulfotransferases (279 –282) and thus may have an effect
on the function of thyroid hormones, steroids, and cat-
echolamines(283).WhetherUDP-glucuronosyltransferaseis in-
duced or inhibited by polyphenols needs further investigation
(284–286). Interactions with drug transporters should also be
considered (287). Isoflavones were shown to interact with trans-
porters such as P-glycoprotein and canalicular multispecific or-
ganic anion transporter (288). Furthermore, some flavonoids
could act as cytochrome P450 inhibitors and enhance drug bio-
availability. Increased concentrations of many drugs have been
shown with coadministrated grapefruit juice, and the effect was
attributed in part to inhibition of the intestinal cytochrome P450
isoform 3A4 by naringenin (289). These data suggest that poly-
phenols could affect the bioavailability of many carcinogens,
other toxic chemicals, and therapeutic drugs by affecting the
activities of various enzymes involved in their own metabolism.
CONCLUSION
The many analytic studies of polyphenols in foods that have
been conducted to date provide a good indication of polyphenol
distribution.Fruitandbeverages such as tea, redwine,andcoffee
constitute the principal sources of polyphenols, but vegetables,
leguminousplants,andcerealsarealsogoodsources.Polyphenol
concentrations in foods vary according to numerous genetic,
environmental, and technologic factors, some of which may be
controlledtooptimize the polyphenol contentoffoods. The main
tasks ahead are identifying the plant varieties that are the richest
in the polyphenols of interest, improving growing methods, and
limiting losses during the course of industrial processing and
domestic cooking.
Thehealth effects ofpolyphenolsdepend on boththeir respec-
tive intakes and their bioavailability, which can vary greatly.
Althoughvery abundant in ourdiet, proanthocyanidins are either
very poorly absorbed or not absorbed at all, and their action is
thus restricted to the intestine. The same appears to be true for
anthocyanins, unless some of their metabolites are not yet iden-
tified but are well absorbed. Intakes of monomeric flavonols,
flavones, and flavanols are relatively low, and plasma concen-
trations rarely exceed 1
mol/L because of limited absorption
and rapid elimination. Flavanones and isoflavones are the fla-
vonoids with the best bioavailability profiles, and plasma con-
centrations may reach 5
mol/L. However, the distribution of
these substances is restricted to citrus fruit and soya. Finally,
hydroxycinnamicacidsarefoundinawidevarietyoffoods,often
at high concentrations, but esterification decreases their intesti-
nal absorption. As a general rule, the metabolites of polyphenols
are rapidly eliminated from plasma, which indicates that con-
sumption of plant products on a daily basis is necessary to main-
tain high concentrations of metabolites in the blood.
Recent studies have greatly increased our knowledge of the
plasma concentrations and urinary excretion of polyphenol me-
tabolites in humans. However, values for these variables do not
seem to be well correlated with concentrations measured in tis-
sues. Available data, essentially those obtained from animal
studies,indicate that somepolyphenol metabolites mayaccumu-
late in certain target tissues rather than just equilibrate between
blood and tissues. The metabolites present may differ between
tissues and plasma, and the nature of these metabolites needs to
be further elucidated. More animal studies are needed to inves-
tigateintracellularmetabolism and the accumulationofpolyphe-
nol metabolites in specific organs. However, some important
differences may exist between animals and humans in some
metabolic processes, especially the conjugation process.
Thenotion of bioavailability integratesseveral variables, such
as intestinal absorption, excretion of glucuronides toward the
intestinal lumen, metabolism by the microflora, intestinal and
hepatic metabolism, plasma kinetics, the nature of circulating
metabolites, binding to albumin, cellular uptake, intracellular
metabolism, accumulation in tissues, and biliary and urinary
excretion. The difficulty lies in integrating all the information
and relating the variables to health effects at the organ level.
These tasks are made all the more difficult because the relative
weight of each variable may depend on the polyphenol consid-
ered.Somepolyphenolsmaybelessefficientlyabsorbedthan are
others but nevertheless reach equivalent plasma concentrations
becauseof lower secretiontowardthe intestinal lumenand lower
metabolism and elimination.
Better knowledge of bioavailability is essential for investigat-
ing the health effects of polyphenols, whatever the approach
used. The fact that aglycones are not important metabolites in
bloodbecauseofextensiveintestinalandhepaticconjugation has
thus far been largely ignored, and many in vitro studies on the
mechanisms of action of polyphenols continue to concentrate on
aglyconesorglycosides rather than ontheidentifiedmetabolites,
often at concentrations that cannot realistically be attained in the
body. It is thus essential to confirm the effects observed with
aglycones through studies using physiologic concentrations of
the metabolites actually found in the body. In addition, the ac-
tivities of microbial metabolites must be examined in further
studies to determine active structures, available concentrations,
and potential modulation of the capacity of the microflora to
producesuchmetabolites. Clinical studies willbeof great help in
investigating the health effect of polyphenols, provided that
markers of effects that are reliable and related to the prevention
of diseases are available. Better knowledge of some variables of
polyphenol bioavailability, such as the kinetics of absorption,
accumulation, and elimination, will facilitate the design of such
740 MANACH ET AL
studies. Besides, more precise data on the nature of the circulat-
ingmetabolites and on metabolism bythe microfloracan now be
used for interpretations. For example, taking into account
whether subjects are equol producers or non–equol producers
seems particularly judicious in evaluating the health effects of
soya isoflavone consumption.
Research on polyphenol bioavailability must finally allow us
tocorrelatepolyphenol intakes withoneor several accurate mea-
sures of bioavailability (such as concentrations of key bioactive
metabolites in plasma and tissues) and with potential health ef-
fects in epidemiologic studies. Knowledge of these correlations
must be attained despite the difficulties linked to the high diver-
sity of polyphenols, their different bioavailabilities, and the high
interindividual variability observed in some metabolic pro-
cesses, especially those in which the microflora is involved.
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