Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol.

28
© 2003 Elsevier Science B.V. All rights reserved. 257

PLANT POLYPHENOLS:
STRUCTURE, OCCURRENCE AND BIOACTIVITY

PffiRGIORGIO PIETTA, MARKUS MINOGGIO, LORENZO

BRAMATI

ITB-CNR, Via FJli Cervi, 93- 20090 (MI), Italy

ABSTRACT: Main dietary plant polyphenols are grouped into structural types and their
occurrence in most common foods and beverages is briefly described. The major groups
of plant polyphenols which are examined include flavonols, flavones, flavanones,
isoflavones, anthocyanins, proanthocyanidins and flavanols. The current evidence on the
absorption and the metabolism of each group is discussed. The biochemical and
pharmacological activities of plant polyphenols are summarized, including antioxidant
and anti-radical activity, chelation of metal ions, modulation of some enzymes activity,
anticarcinogenic, antiatherosclerotic, anti-inflammatory, spasmolytic, hepatoprotective,
antiviral, antimicrobial and oestrogenic activity, and inhibition of histamine release. An
overview on the epidemiological evidence linking the intake of plant polyphenols and
diminished risk of chronic diseases is also included.

INTRODUCTION

Polyphenols constitute one of the most and widely distributed groups of

substances in the plant kingdom, with more than 8000 phenoUc structures
currently knovra. They can be divided into at least 10 different classes
based upon their chemical structure, ranging from simple molecules, such
as phenolic acids, to highly polymerized compounds, such as taimins.
Flavonoids constitute the most important group with a common
structure of diphenylpropanes (C6-C3-C6), consisting of two aromatic
rings linked through three carbons that usually form an oxygenated
heterocycle. Based upon the variations in the heterocyclic ring ,
flavonoids can be subdivided into eight major subclasses, including
flavonols, flavones, flavanones, isoflavones, flavanols, anthocyanins,
proanthocyanidins and tannins.
Plant polyphenols have been of interest for long time owing to their
role in plant pigmentation, reproduction and protection against predators
and pathogens. Recently, interest in the biological effects of plant
polyphenols (particularly flavonoids) has increased, because of the the
potential health benefits associated with some dietary polyphenols. This
258

contribution provides a brief description of the chemistry of plant

polyphenols, their occurrence, bioavailability and bioactivity.

CLASSES OF PLANT POLYPHENOLS

I. Hydroxy benzoic acids derivatives

The hydroxybenzoic acid derivatives (HBAs) are phenolic compounds

with a general structure Ce-Ci. The related C6-C2 acids (phenylacetic
acids) occur occasionally as minor components of foods.
Variations in the basic structure of HBAs include hydroxylation and
methoxylation of the aromatic ring, Fig. (1), Table 1.

Fig. (1). Basic structure of hydroxybenzoic acids

Although HBAs can be detected as free acids in some fruits (e.g.,

gallic acid in persimmons) or after being released during fruit and
vegetable processing, they occur mainly as conjugates. For example,
gallic acid and its dimer ellagic acid may be esterified with a sugar,
usually glucose to produce the so-called hydrolysable tannins. In addition,
gallic acid may esterify condensed tannins (i.e., derivatives of flavanols
like those present in tea) or quinic acid [1,2]. Four HBAs, namely 4-
hydroxybenzoic acid (4-HBA), vanillic acid (3-methoxy-4-hydroxy),
syringic acid (3,5-dimethoxy-4-hydroxy) and protocatechuic acid (3,4-
dihydroxy), are constituents of lignin [3]. These acids occur also as esters
of glucose.
 259

Table 1. Structure of the most common hydroxybenzoic acids

Trivialname MW Position of Position of

(Da) OH groups OCH3 groups

Benzoic acid 122 /

Salicylic acid 138 2
(2-Hydroxybenzoic acid)

4-Hydroxybenzoic acid 138 3

Protocatecliuic acid 154 3,4

(3,4-Dihydroxybenzoic)

Gallic acid 170 3,4,5

(3,4,5-Trihydroxy benzoic)

Vanillic acid 168 4 3

(4-Hydroxy-3-methoxybenzoic

Isovanillic acid 168 3 4

(3-Hydroxy-4-methoxybenzoic)

Syringic acid 198 4 3,5

(4-Hydroxy-3,5-dimethoxybenzoic)

Dietary occurrence

Some herbs and spices are comparatively rich in various HBAs. After
hydrolysis, protocatechuic acid is the dominant HBA in cinnamon bark
(23-27 mg/kg), accompanied by saUcylic acid (7 mg/kg) and syringic acid
(8 mg/kg). Gallic acid dominates in clove buds (175 mg/kg) and is
accompanied by protocatechuic acid (10 mg/kg), genistic acid/4-HBA (7
mg/kg) and syringic acid (8 mg/kg) [4]. The fruit of anise {Pimpinella
anisum) contains 730-1080 mg/kg of the glucoside of 4-HBA [3].
The skin of potato tubers contains protocatechuic acid (100-400 mg/kg
FW) and vanillic acid (20-200 mg/kg) along with up to 30 mg/kg of
gallic, syringic and salicylic acids [5].
Cereals contain also different HBAs. Canadian wheat flours were
found to contain vanillic acid (up to 16 mg/kg) and syringic acid (up to 7
mg/kg). Oats contain vanillic acid, 4-HBA and salicylic acid, particularly
in the hulls [6].
Alcoholic beverages (wine and beer) have a different content of HBAs.
The gaUic acid content of French wines and spirits can reach 31-38 mg/1
260

[7]. White wines contain less HBAs than red wines, namely 16-46 and
65-126 mg/1 for white and red Califomian wines [8]. Barley contains
vanillic acid (6-17 mg/kg) and syringic acid (1-22 mg/kg), and both are
found in malt (12 mg/kg each) and hops (59 and 30 mg/kg). These two
acids are foimd in stout, ale and lager beers in the range from 0-2 mg/1)
accompanied by gaUic, protocatechuic and 4-HBA (0.1-1.8 mg/1 each)
[9].
Table 2 gives an overview of the occurrence of some HBAs in foods
[10,11] and Table 3 shows the content of the major HBAs in selected
foods [12].
Table 2. Occurrence of some HBAs in different dietary sources (10,11]

HBAs Dietary source

Benzoic acid universal component of angiosperms, csp. of berries
Salicylic acid anise, dill, white mustard, allspice,rosemary,thyme, majoram
4-Hydroxy benzoic acid raspbcny, gooseberry, pecans, anise, fennel
Vanillic acid vanilla, garden cress, paprika
Syringic acid rosemary, basil, thyme, garden cress
Protocatechuic acid tarragon, clove, anise, cinnamon, blackberry, blueberry
Gallic acid tea, nuts, olive oil

Table 3. Content of some HBAs in fruits (mg/kg) [12]

4-Hydroxy benzoic acid Protocatechuic Gallic acid

Food acid
Blackberry 6-16 68-189 8-67
Blackcurrant 0-6 10-52 30-62
Raspberry 15-27 25-37 19-38
Redcurrant 10-23 3-8 -
Strawberry 10-36 - 11-44
White currant 5-19 - 3-38

2. Hydroxycinnamic acid derivatives

Cinnamic acids are ^ra«5'-phenyl-3-propenoic acids differing in their ring

substitution, Fig. (2).
COOH

I II
%^
Fig. (2). Basic structure of cinnamic acids
 261

The most common cimiamic acids are caffeic (3,4-dihydroxycimiamic

acid), ferulic (3-methoxy-4-hydroxy), sinapic (3,5-dimethoxy-4-hydroxy)
and p-coimiaric (4-hydroxy) acid, Table 4 [13].
These compoimds are widely distributed as conjugates, mainly as
esters of quinic acid (chlorogenic acids, CGA).
Table 4. Structure of the most common cinnamic acids

Trivialname MW Position of Position of

(Da) OH groups OCH3 groups

Cinnamic acid 148 /

p-Coumaric acid 164 4

(4-Hydroxycinnamic)

Cafleic acid 180 3,4

(3,4-Dihydroxycinnamic)

Ferulic acid 194 4 3

(4-Hydroxy-3-metlioxycinnamic)

Sinapic acid 224 4 3,5

(3,5-Dimetlioxy-4-iiydroxycinnamic)

Depending on the identity, number and position of the acyl residues,

these acids may be divided into the following groups: mono-esters of
caffeic, p-coumaric and ferulic acid; di-, tri- and tetra-esters of caffeic
acid [14,15]; mixed di-esters of caffeic and ferulic acid or caffeic and
sinapic acid [16]; mixed esters of caffeic acid with dibasic aliphatic acids
(e.g., oxalic, succinic) [17].
Cinnamic acids may condense with molecules other than quinic acid,
including rosmarinic, malic and tartaric acid, aromatic amino acids,
choline, mono- and polysaccharides, glycerol, myo-inositol, and different
glycosides (anthocyanins, flavonols and diterpenes) [13].

Dietary occurrence

There is no doubt that caffeic acid is the cinnamate that occurs most
extensively, and the various caffeoylquinic acids (CQA) and
dicaffeoylqumic acids (diCQA) are the most ubiquitous conjugates.
Usually the 5-isomers dominates, but in some fruit and brassicas the 3-
isomer is prevalent. Because of the quantity commonly consumed, coffee
262

beverage must top the list, with 200 ml instant brew (2% w/v) supplying
50-150 mg CQA, mg. Blueberries, aubergines, apples, cider and green
mate are good sources in some populations [13].
However, in view of quantities consumed by other populations, wines
could make a significant contribution for tartaric acid conjugates and
grapes and grape juice for caftaric acid, respectively. Lettuce is the major
source of chicoric (dicaffeoyltartaric) and caffeoylmalic acid (up to 3
mg/100 g), but endive may have twice the concentration. Spinach is
ahnost certainly therichestsource of conjugated p-coumaric acid at some
30-35 mg/100 g [18].
Broccoli florets and leafy cruciferous vegetables will be the major
source of sugar esters and of conjugated sinapic acid (10 mg/100 g).
Tomato and tomato products are likely to be the major source of
glucosides at up to 13 mg/100 g in total, and possibly the second richest
source of conjugate/7-coumaric acid (3 mg/100 g).
Cereal bran and bran-enriched products are the most important source
of wall-bound cinnamates with up to 30 and 7 mg ferulate/10 g in maize
and wheat bran, respectively. This would make these products the richest
dietary source of ferulic acid. However, coffee brew could supply up to
10 mg ferulate (as feruloylquinic acid, FQA) per 200 ml cup [13], and it
is the first for conjugated ferulic acid , followed by Citrus juices.
Table 5. Dietary sources of individual cinnamates and each major class of conjugate [13,18]

Name Dietary source

Cinnamates
CafTeic acid Coffee beverage, blueberries, apples, ciders
p-Coumaric acid Spinach, sugar beet fibre, cereals brans
Ferulic acid Coffee, citrus juices, sugar beet fibre, cereal brans
Sinapic acid Broccoli, kale, other leafy brassicas, citrus juices
Conjugates
Caffeoylquinic acids Coffee beverages, blueberries, apples, ciders
p-Coumaroylquinic acids Sweet cherries
Tartaric conjugates Coffee
Malic conjugates Spinach, lettuce, grapes, wines
Rosmarinic acid 1 Culinary herbs, mixed herbs, possibly stuffing
Cell wall conjugates Spinach, sugar beet fibre, cereal brans
 263

3. 4-oxo-flavonoids: flavonols, flavones, isoflavones, flavanones,

chalcones, dihydrochalcones

The term 4-oxo-flavonoids includes a group of complex polyphenols that

share a common structure of diphenylpropanes (C6-C3-C6) characterized
by two aromatic rings and an oxygenated heterocycle, Fig. (3). The three
rings are referred to as the A, B, and C (or pyrane) rings.

J'

11 B 1
.<^\ ^?\,/^\e^^
1 A c II
% / ^
II
0
Fig. (3). Basic structure of 4-oxo-flavonoids

Their biosynthesis derives from the condensation of three acetyl units

and of a derivative of hydroxycinnamic acid leading to the formation of a
conmion intermediate, tetrahydroxychalcone. This chalcone is precursor
of several compounds, the most important being the 4-oxo-flavonoids
[19].
The 4-oxo-flavonoids can be distinguished based upon the
modifications of the nucleus, including the pyronic cycle saturation, the
number and the position of hydroxyl groups and the degree of
methylation and glycosylation. In plants,flavonoidsoccasionally occur as
aglycones, the most commonly forms being 0-glycoside derivatives.
Flavones may also occur as C-glycosides. The bond between the aglycone
and sugar moieties is generally located at 7- (flavone, flavanone), 3-
(flavonols), or 4'-position. The sugars are mono-, di-, tri- and even
tetrasaccharides, being D-glucose, L-rhamnose, glucorhamnose,
galactose, and arabinose the most frequent [19,20].
Depending on their aglycone structure, at least five different groups of
4-oxo-flavonoids are distinguishable: flavonols, flavones, flavanones,
isoflavones and chalcones.
264

Dietary occurrence

The concentration of 4-oxo-flavonoids depends on the plant,

environmental conditions, the part of the plant consumed, the degree of
ripeness as well as on the food processing. Flavonoids are preferentially
located in the epidermis: solubilized in the vacuolar sap (especially
flavones and flavonols glycosides) or in the epicuticular zone [20]. More
detailed information about the occurrence of the different compounds is
given in each subchapter.

3.1 Flavonols

Flavonols (about 380 aglycones) are characterized by the presence of a

hydroxyl group at position 3, Fig. (4). About 90% of the flavonols have
additional hydroxyl at positions 5 and 7.

r^'^4/^
II B 1
l < ^ \ /^^/^'^^K
1 M1 ^ 1
% / ^ S " " ^OH
1 0II
Fig. (4). Basic structure of Flavonols

Flavonols occur mainly as O-glycosides and the diversity of the

glycoside moiety in this group is noteworthy with about 200 different
quercetin and kaempferol glycosides described to date [19]. Table 6
reports the most common flavonols.
 265

Table 6. Structure of the most connnon flavonols

MW Position of Substituents Position of tiie
Trivialname (Da) OH groups substituents
Aglycones
Fisetin 286.24 3,7,3',4'
Galangin 270.24 3,5,7
Kaempferol 286.24 3,5,7,4'
Morin 302.24 3,5,7,2\4'
Myricetin 318.24 3,5,7,3\4\5'
Quercetin 302.24 3,5,7,3',4'
Rhamnetin 316.27 3,5,3',4' OCHj 7
Glycosides
Quercltrin 448.38 5,7,3\4' 0-Rh 3
Rutin 610.53 5,7,3',4' 0-Ru 3
Rh = rhamnose = 6-dcoxy-L-mannose (C6H12O5); Ru = nitinose == 6-0-D-glucose

Dietary occurrence

Flavonols are present in plant foods mainly in the leaves and in the outer
parts of plants with quercetin and kaempferol the most common ones.
Quercetin and its glycoside are ubiquitous in fruits and vegetables.
Conversely, kaempferol and myricetin are less distributed (Table 7) [21-
23].
Specific quercetin glycosides have been detected in onions (quercetin-
4'-glucoside and quercetin-3,4'-diglucoside), broccoli (quercetin-3-0-
sophoroside, kaempferol-3-(9-sophoroside), green beans (quercetin-3-0-
glucuronide) and tomatoes (rutin = quercetin-3-O-rhamnosyl-glucoside)
[24,25], red wine (rutin) and tea (rutin, quercetin-3-(9-glucoside and
quercetin-3-O-galactoside) [26].
Preparation of fruits and vegetables for consumption (for example
peeling, skinning and cooking) can decrease quercetin and kaempferol
content significantly. For example, boiling, microwave cooking and
frying of onions or tomatoes involves a decrease in the content of
flavonols by 30 to 80% [2].
266

Table 7. Content of flavonois (expressed in mg/kg or mg/1) in foods determined by HPLC metliods
after liydrolysis of tlieir glycosides [21-23]

Food Quercetin Kaempferol Myricetin

Apple 20-36 - -
Apricot 25 - -
Bean, French 39 <12 -
French processed 17 <3.8 -
Green 16 - -
Broccoli 30-37 60-72 -
Currrant, black 37 1 -
Red 8-13 - -
Endive <1.3 46 -
Grape, black 15-37 - 4.5
White 2-12 - 4.5
Grape juice 4.4 - 6.2
Grape fruit juice (fresh) 4.4 - -
Kale 110-120 210-470 -
Leek - 30 -
Lettuce 14-79 - -
Onion 340-347 -
Orange juice 5.7 - -
Strawberry 6-8.6 5-12 -
Tea, black 14-17 14-16 3.0
Tomato 2-14 - -
Cherry tomato 63 -
Wine, red i 8.3 7.9

3.2 Flavones

Flavones differ from flavonois since the hyciroxyl group at position 3 of

the C-ring is missing, Fig. (5).

Fig. (5). Basic structure of flavones

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About 300 different aglycones have been identified, and the most
frequently are luteolin, apigenin (especially in parsley) and diosmetin (in
Citrusfruits).Among glycosides, the 7-0- and C-forais are very conunon,
and are characterized by a carbon-carbon bond between the anomeric
carbon of a sugar molecule and the Ce or Cg carbon of the flavone
nucleus. Table 8 describes the most common flavones [19].

Table 8. Structure of the most common flavones

MW Position of Substituents Position of the

Trivialname (Da) OH groups substituents
Aglycones
Apigenin 270.24 5,7,4'
Chrysin 254.24 5,7
Diosmetin 300.27 5,7,3* OCH3 4'
Luteolin 286.24 5,7,3\4'
Pinocembrin 256.24 5,7
Glycosides
Isoorientin 448.36 5,7,3',4' Glue 6
Isovitexin 432.36 5,7,4' Glue 6
Orientin 448.36 5,7,3\4' Glue 8
Vitexin 432.36 5.7,4' Glue 8
Glue = glueosc

Flavones contribute to plant tissue color provided that they occur in

high concentrations or are complexed with metal ions. Some flavones
participate in taste; for example, the highly methoxylated aglycones
nobiletin, sinensetin and tangeretin are responsible for the bitter taste of
citrus peel. On the other hand, some glycosylated flavones (for instance
neodiosmin and rhoifolin) reduce the bittemess of some substances
(limonin, naringin, caffeine, quinine) [2].

Dietary occurrence

Flavones are found mainly in grains and herbs and not frequently in fruits.
Apigenin and chrysoeriol have been detected in parsley, while cereal
grains and herbs contain apigenin and its glycosides as well as luteolin
[21,27].
268

Table 9. Content offlavones(expressed in mg/kg or mg/1) in foods determined by HPLC metliods after
hydrolysis of tiieir glycosides [21,28]

Food Luteolin Apigenin

Celery leaf 200 750
Stalk 5-20 16-61
Sweet peper, red 5-11 -

3.3 Isoflavones

Isoflavones are a distinct class of flavonoids which stracturally differ

from the commonflavonoidsin B-ring orientation, Fig.(6).

Fig. (6). Basic structure of isoflavones

The isoflavones have a chemical stracture similar to that of

mammalian oestrogen 17P-estradiol [28]: the phenoUc ring and a pair of
hydroxyl groups separated by a distance comparable to that occurring in
mammalian oestrogens are key structural elements of most compounds
that bind to oestrogen receptors [29,30]. Thus, isoflavones have recently
become best known for their oestrogenic activity, hence the name
"phytoestrogens". However, small differences in structures of the
individual phytoestrogens can dramatically alter their activity. For
instance, daidzein and genistein share identical structures except for an
additional hydroxyl group on the A-ring of genistein, but this favours up
to five- or six-fold oestrogenic activity of genistein in some assay systems
[31].
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Table 10. Structure of the most common isoflavones

MW Position of Substituents Position of the
Trivialname (Da) OH groups substituents
Aglycones
Daidzein 254.23 7,4' / /
Genistein 270.23 5,7,4' / /
Glycosides
Acetyldaidzein 430.23 7,4' Acetyl-gluc 6"
Acetylgeiiistin 446.23 5,7,4' Acetyl-gluc 6"
Acetyl-gluc = Acetyl-glucoside

Dietary occurrence

Isoflavones are present in plant foods either as aglycones (genistein or

daidzein) or - predominantly - as different highly polar and water-soluble
glycosides, including acetyl and malonyl glucosides and p-glucosides of
daidzein and genistein [32]. Legumes are the main source of isoflavones.
Soybeans are particularly rich in daidzein and genistein. Table 11 shows
the wide range in total isoflavone concentrations in soya products and
other legumes [32,33]. The concentration of isoflavones in soy foods is in
the range of 0.1-3.0 mg/g. However, theseflavonoidshave been detected
also in black beans, green split peas and clover [34]. Otherflavonoidsof
this group, including biochanin A and formononetin, have been found in
chick peas, green beans, and sunflower seeds.

Table 11. Content of isoflavones (expressed in ^g/g) in foods [3233]

Isoflavones (total) (fig/g) |xg per average portion size (g)

Food
Soya bean 579-3812 34740-228720 (60)
Tofu 79-674 10270-87620(130)
Soy flour 833-1778 16660-35560(20)
Textured soya protein 701-1184 28040-47360 (40)
Soya milic 34-175 3400-17500(100)
Miso 256-890 4608-16020(18)
Soy clieese 34-47 1360-1880(40)
Tofu yoghurt 151 18120(120)
Soy sauce 13-75 65-375 (5)
Green split peas 73 2920 (40)

3.4 Flavanones, chalcones, dihydrochalcones

270

Flavanones arise from flavones after reduction of the double bond in the
heterocycle (position C2/C3), Fig. (7).

Fig. (7). Basic structure of flavanones

Among aglycones, the best known are naringenin and hesperidin. Their
glycosylated forms occur commonly as O- or C-glycosides, usually as
rutinosides (6-0-a-L-rhamnosyl-D-glucosides) and neohesperidosides (2-
0-a-L-rhamnosyl-D-glucosides) attached at position 7. Flavanones
contribute to the flavour of citrus [19]. Table 12 reports the structures of
some common flavanones.
Table 12. Structure of some common flavanones

MW Position of Substituents Position of the

Trivialname (Da) OH groups substituents
Aglycones
Eriodictyol 288.26 5,7,3*,4'
Hesperetin 302.28 5,7,3' OCH3 4'
Naringenin 272.26 5,7,4'

Glycosides
Hesperidin 610.57 5,3' Rli-Gluc; OCH3 7;4'
Naringin 580.54 5,4' O-Rh-Gluc 7
Rh = rhamnose = 6-dcoxy-L-mannosc (CeHnOs); Glue = glucose

Flavanones can be easily converted to isomeric chalcones in alkali (or

vice versa in acidic media) provided that there is a hydroxyl substituent at
position 2' (or 6') of the chalcone.
Chalcones are unsaturated and, along with dihydrochalcones, contain
an open pyronic cycle and a carbon skeleton numbered in a way different
from other flavonoids, Fig. (8, 9). Native chalcone glycosides tend to
transform into flavanone glycosides during extraction procedures.
Chalcones per se are therefore of restricted occurence in foods [35].
 271

OH OH
, ^ ^ ^ 4 ^ rf^^^y

^==^,

OH OH

Fig. (8). Isosaiipiupurin (Chalcone) Fig, (9). Phloretin (Dihydrochalconc)

Dietary occurrence

Flavanones are found in a small number of foods. Chick peas (with the
flavanone garbanzol), cimiin, pepperaiint (both with hesperidin),
hawthom berry, licorice, rowanberry and citrus fruits are among those
fews containing molecules of this group. Naringenin and narirutin
glycosides are present in hawthomberry and rowanberry; liquoritigenin in
licorice roots. Flavanones neohesperidose (such as naringin) are found in
grapefruit and are usually bitter; the tasteless flavanone rutinosides (such
as hesperidin) are present in oranges [35,36]. Flavanone glucosides are
comparatively rare in species but are found for instance in different herbs
[37]. Hesperidin and the aglycones naringenin, eriodictyol and hesperitin
have been reported in the herbal tea (Honeybush tea) prepared from the
legume Cyclopia intermedia [38]. Naringenin and eriodictyol have been
reported in potato [5].
Citrusfiruitsand associated products (fiuit juices, peeled freshfruit)are
a major dietary source of flavanones (Table 13) [35]. However, the
distribution is quite scattered, and much higher concentrations are found
in the solid tissues compared to the juice. For example, an individual
drinking orange juice (250 ml) will have a daily flavone intake (as
aglycones) in the range of 25-60 mg; eating the flesh of a whole orange
(200 g) will provide about 125-375 mg.
Chalcones are comparatively rare in foods. Naringenin chalcone is
present in tomato skin and may be present in juice, paste and ketchup.
Acid hydrolysis, commonly applied prior to HPLC, converts the chalcone
to the corresponding flavanone (naringenin), which is naturally present
only in trace amounts (2-15 mg/kg) in the tomato [23].
Dihydrochalcones (DHCs) are characteristic of apples and derived
products (apple juice, cider, pomace etc.), and their content depends on
272

the cultivar. Phloretin 2'-glucoside (phloridzin), phloretin 2'(2"-xylosyl-

glucoside) and 3-hydroxyphloridzin have been identified unequivocally
and some investigators have reported phloretin 2' (2"-xylosyl-
galactoside). They are present in the skin, pulp and especially in tiie
seeds, where they account for up to 60% of the total phenols (compared
with less than 3% in the epidermis and parenchyma zones). When eating
an apple, the seeds and core of the fhiit are usually discarded and thus
some of the apple dihydrochalcones are not mgested. Whole apple fruits
are processed industrially to produce juices and ciders, and therefore the
contribution of these processed products to the intake of DHCs can be
higher than that of fresh apples (250 ml of apple juice or cider supply
about 1-5 mg phloretin. By contrast, a dessert apple of about 100 g
supplies less than 1 mg [39,40].
The dihydrochalcones aspalathin and nothofagin have been identified
as the main flavonoids in the South-Afiican Rooibos tea {Aspalathus
linearis) [41].

Table 13. Flavanones characteristic of common citrus fruits

Sweet orange Sour orange Lemon Grapefruit Lime Mandarin

(Citrus (Citrus (Citrus (Citrus (Citrus (Citrus
Flavanones sinensis) aurantium) limon) paradisi) aurantifolia) reticulata)
Eriocitrin - - ++ - - -
Narirutin + - - •H- - ++
Hesperidin +++ - +++ Trace +++ +++
Naringin - -H^ - 4-H- - -
Neohesperidin - ++ - Trace - -

4. Flavanols (FIavan-3-ols)

Flavanols have a C-ring structure similar to that of 4-oxo flavonoids, but

they are characterized by the lack of the double bond at the 2-3 position
and of the 4-oxo-group, Fig. (10) [19].

Fig. (10). Basic structure of flavanols

 273

The flavan-3-ols most occurring in nature are (-f-)-catechin and (-)-

epicatechin (EC), although gallocatechin and epigallocatechin have also
been identified [42], Proanthocyanidins (or condensed tannins) include
oligo- and polymeric forms of the monomeric flavanols and will be
examined later. Polymerization of monomeric flavanols can occur as a
result of auto-oxidation, but more often it is catalyzed by
polyphenoloxidase (PPO), an enzyme that is present in most plant tissues
[43].

4.1 Catechins - Dietary occurence

Catechins are widely distributed in plants; however, they are rich only in
tea leaves, where catechins may constitute up to 25% of dry leaf weight.
Catechins of green tea include the flavanols epicatechin, epigallocatechin,
and their gallate esters (Table 14).
Table 14. Structure of the most common catechins

MW Position of Substituents Position of the

Trivialname OH groups substituents
(+).Catechin (C) 290.3 3(^-0H)
(-)-Epicatechin (EC) (cis form) 290.3 3 ( ^»M^»NOH\

Epigallocatechin (EGC) 306.4 3 ( VWSAAVOH)^ 5 »

Epicatechin-gallate (ECg) 442.4 3

OH

Epigallocatechin-gallate (EGCg) 458.4 5' 3

OH

The most abundant monomeric flavanols of black tea are (-)-

epicatechin gallate (ECg) and (-)-epigallocatechin (EGC) and (-)-
epigallocatechin gallate (EGCg) [44]. Indeed, quantitative analyses of
black tea reports levels of 31-79 mg/1 for EC, 5-91 mg/1 for EGC, 18-229
mg/1 for EGCg and 8-110 mg/1 for ECg; for green tea levels from 10-94
274

mg/1 for EC, 20-287 mg/1 for EGC and 60-408 mg/1 for EGCg are found
[45].
During fermentation in the preparation of black tea, oxidative
polymerization of flavanols occurs with the formation of theaflavin,
theaflavingallates, thearubigins, and epitheaflavic acid [44].
Flavanols have been determined in apples, apricots, pears, cherries,
peaches and plums [47,48].
The contents of (+)-catechin and (-)-epicatechin in red wine are
relatively high (up to 208 mg/1 for catechin and 90 mg/1 for EC) [49].
Low levels of (+)-catechin (-5 mg/1) and (-)-epicatechin (-1 mg/1) have
been reported for lager beers [50].
Data on grapes are limited; qualitative studies show the presence of
(+)-catechin, (-)-epicatechin and ECg in black and white grape seeds and
skins [51].
Recently, chocolate and cocoa have gained interest because of their
contents of catechins and related polymers (procyanidin oligomers) [52].
Fruit juices processing may seriously affect flavanol content. For
example, the preparation of commercial apple juice decreased the flavanol
content in a stepwise manner. In particular, crushing and pressing, storage
of the concentrated juice at room temperature and decolorization by
treatment with activated carbon destroy theflavanolsalmost entirely [46].

5. Anthocyanidins, anthocyanins, proanthocyanidins, tannins

5.1 Anthocyanidins and anthocyanins

The fundamental nucleus in anthocyanidins (aglycones) is flavylium

chloride. Most of the anthocyanidins are derivatives of 3,5,7-
trihydroxyflavylium chloride. Thus, the hydroxylation pattems in the
natural anthocyanidins fall into the three basic groups of pelargonidin,
cyanidin and delphinidin. Anthocyanidins are rarely found in fresh plant
material because of their instability [19].
On the other hand, anthocyanins, i.e. the glycosylated anthocyanidins,
are an important group of water-soluble pigments occuring in 27 families
of food plants (mainly red fruits and vegetables). Fig. (11) [53]. Table 15
shows the most common anthocyanidins and anthocyanins.
 275

Fig. (11) Basic structure of anthocyanidins and anthocyanins

Table 15. Structure of the most common anthocyanidins and anthocyanins

MW Position of Substituents Position of the

Trivialname OH substituents
Anthocyanidins
JM
Pelargonidin 271.70 3,5,7,4'
Cyanidin 287.70 3,5,7,3*,4'
Delphinidin 303.70 3,5,7,3\4',5'
Malvidin 331.75 3,5,7,4* OCH3 3*,5'
Anthocyanins
Pelargonidin-3-glucoside 432.70 5,7,4' OGluc 3
Cyanidin-3-glucoside 448.70 5,7,3\4' OGluc 3
DeIphinidin-3-glucoside 499.70 5,7,3',4',5' OGluc 3
Malvidin-3-glucoside 492.70 5,7,4' OGluc; OCH3, 3;3',5'

The natural anthocyanins vary in:

• the basic anthocyanidin skeleton, i.e. the number and position of
hydroxyl and methoxyl substituents;
• the identity, number and positions(s) at which sugars are
attached to the skeleton; the most common sugars are glucose,
galactose, rhamnose and arabinose ( as 3-glycosides or 3,5-
diglycosides).
• the extent of sugar acylation and the identity of the acylating
agent(s); the most common acylating agents include ciimamic
acids (caffeic, p-coumaric, ferulic and sinapic), which may
themselves be glycosylated, and a range of aliphatic (for
example acetic, malic, malonic, oxalic and succinic acid) and
aromatic acids.

The anthocyanins occur in the vacuole as an equilibrium of four

molecular species: the coloured basic flavylium cation and three
276

secondary structures (the quinoidal base, the carinol pseuodobase and the
chalcone pseudobase) [54]. The pH changes the colour intensity of the
anthocyanins (for example, by the addition of vinegar or other acids while
cooking or processing). Commonly, anthocyanins are red in acid, violet in
neutral, and blue in alkaline solution. In fact, when cooking a food that is
red, such as red cabbage, it may be helpful to add an acidic substance
such as vinegar (or tomato juice or lemon juice) to prevent the food from
tuming purple. Anthocyanins contribute significantly to the red purple,
and blue color of flowers, many fruits of higher plants, vegetables and
associated products, beverages and preserves. Anthocyanins and
polymeric pigments derived from anthocyanins by condensation with
other flavonoids, are responsible for the color of red wine. It has been
recognized that anthocyanin-rich extracts might be used as food additives.
Many factors influence the stability of anthocyanins. Heat and light can
destroy sensitive anthocyanins during processing of fruits and vegetables.
In particular, anthocyanins are rapidly destroyed in the presence of a high
sugar concentration; thus processed foods containing large amounts of
sugar or syrup would not have the same amount of anthocyanins as their
improcessed counterparts [55],

Dietary occurrence

Anthocyanins are widespread in food plants, with an estimated worldwide

consumption of 10000 tonnes from black grapes alone [53]. The
anthocyanin content of many fruits and vegetables has been estimated by
various methods (Table 16) [56-58]. The main sources of these plant
pigments are fresh fruits such as cherries, plums, strawberries,
raspberries, blackberries, grapes, red currants and black currants.
 277

Table 16. Content of anthocyanins in foods (expressed in mg/l or mg/lcg) [56-58]

Food Anthocyanins
Blackberry 1150
Blueberry 825-4200
Cherry 20-4500
Cliolceberry 5060-10000
Cranberry 600-2000
Currant (blacic) 1300-4000
Grape (red) 300-7500
Orange, Blood O'uice) 2000
Raspberry, black 1700-4200
Raspberry, red 100-600
Strawberry 150-350
Cabbage, red 250
Onion up to 250
Wines, red 240-350
Wines, Porto + Marsala 140-1100

5.2 Proanthocyanidins

Proanthocyanidins (PAs, syn condensed tannins) are polymeric flavan-3-

ols whose elementary units are linked by C-C and occasionally C-O-C
bonds (polymerization degree between 3 and 11), Fig. (12) [19].
Oxidative condensation occurs usually between carbon C4 of the
heterocycle and carbons Ce or Cg [59].
A characteristic of PAs is that they yield anthocyanins upon heating in
acidic media, hence their name, they yield anthocyanidins (hence their
name).
Two main types of PAs can be distinguished according to the
substitution pattern of their B-ring:
• Procyanidins: main constituents are catechin and epicatechin,
and are characterized by the presence of two hydroxyl groups
(3\ 4') in the B-ring.
• Prodelphinidins: main constituten is epigallocatechin, which has
three hydroxyl groups (3', A\ 5') in the B-ring.
278

^<^°" r
YY^^K'^^^^OH HO^ ^«^ .0^ ^1* ^^5-^
" \ ^ - \ / ° ^ ^ v ^ V , OH
./'x
OH OH

»°\,^'-v^?\^'v^'
.1
% 4^ ^OH

Dimers:
Procyanidin B3: R3' = H Trimer.

Prodelphinidin B3: RS' = OH

Fig. (12). Basic structure of proanthocyanidins

Dietary occurence

Common sources of PAs are fruits, such as apple, strawberry, pear and
grape, beverages such as red wme and tea, and chocolate (Table 17) [59].
PAs complex protems, and are responsible for the astringency of foods
and beverages (e.g. grape skin and seeds, cider, wine) [19].
 279

Table 17. Content of proanthocyanidins in foods (expressed in mg/1 or mg/lOOg) [59]

Food Proanthocyanidins
Apple 17-50
Apple juice nd-298
Barley 64-126
Beer 3.5-19,5
Blackberry 9-11
Cacao bean 260-1200
Cherry 10-23
Grape 1-160
Grape juice 3.546
Lentil 316-1040
Pear 0.7-12
Pear juice 11-74
Raspberry, red 2-48
Strawberry 2-50
Wines, red nd-500

5.3 Tannins

Tannins are compounds of intermediate to high molecular weight that

distinguish them from the groups of low molecular weight plant
phenolics. Tannins with a molecular weight up to 30000 DA have been
found in certain Leguminosae. One of their main characteristics of tannins
is the formation of insoluble complexes with proteins leading to the
astringency of taimin-rich foods (for instance tea) because of the
precipitation of salivary proteins [59].
Plant tannins are subdivided into two major groups (Table 18) [60]:
1. Hydrolyzable tannins: they consist of a central glucose molecule
linked to molecules of gallic acid (gallotannins) or
hexahydroxydiphenic acid (ellagitannins), Fig (13). They are
readily hydrolyzed, hence their name. The most common
hydrolyzable tannin is tannic acid, Fig. (14), which is a
gallotannin formed by a pentagalloyl glucose molecule esterified
by five gallic acid units.
280

OH

HO. OH

COOH O OH

Fig. (13). Structures of gallic (a) and ellagic (b) acid

OH

HO. X. .OH

CO
/
OH2C

OH

oc—o I
oc

HO" ^ "OH

OH

Fig. (14). Structure of tannic acid

Condensed tannins (= proanthocyanidins): unlike hydrolysable

tannins, condensed tannins are polymeric flavans that are not
readily hydrolysable. They often consist of molecules of catechin
and epicatechin joined by carbon-carbon bonds. Hence catechin
and epicatechin are referred to as monomers; oligomers
containing 2-4 (epi)catechin units are referred to as oligomeric
procyanidins (OPC).
 281

Table 18. Classification of tannins

Type of tannin Example

1. Hydrolyzable tannins:
• Gallotannins Pentagalloylglucose
• EUagitannins and metabolites Geranin, Corilagin
• Ellagitannin oligomers Agrimoniin
2. Condensed tannins:
• Proanthocyanidlns: Procyanidlns Epicatechin oligomers
Prodelphinidins Epigallocatechin oligomers
• Galloylated proanthocyanidlns

ABSORPTION AND METABOLISM OF SELECTED PLANT

POLYPHENOLS IN HUMANS

Gut absorption

The absorption and the metabolism of dietary polyphenols arte

determined primarily by their chemical structure, with glycosylation
playing an important role. In fact, glycosylation influences the
bioavailability of the polyphenols. It is generally stated that flavonoid
glycosides are hydrolyzed before being absorbed [61]. Therefore, the first
step of metabolism should involve the removal of the sugar moiety by
enzymes-(glycosidases). Glycosidases activity can occur in the food itself
(endogenous or added during process) or in the cells of the
gastrointestinal mucosa or can be secreted by the colon microflora. Non-
enzymatic deglycosylation in the hiraian body, such as in the acid
conditions of the stomach, does not occur [62]. The absorption of
polyphenols should therefore be controlled by enzyme specificity and
distribution.
Polyphenols with attached glucose are potential substrates for
endogenous human enzymes, while attached rhamnose is not a substrate
for human p-glucosidases and so is only cleaved by colon microflora a-
rhamnosidases [63].

Deconjugation and reconjugation reactions in metabolism

After the hydrolysis of a polyphenol glycoside to the free aglycone,

polyphenols are conjugated by methylation, sulfation, glucuronidation or
a combination. However, there are exceptions to this sequence, as
282

supported by different studies [64-68] reporting the absorption of intact

polyphenols glycosides. This is a critical point, since the formation of
conjugates dramatically alters the biological properties of the circulating
metabolites. Furthemiore, it should be reminded that significant
differences between the administration of drugs (usually in himdreds of
milligrams in one concentrated dose) and the consumption of dietary
polyphenols (usually <100 mg in a diluted dose) exist. These differences
imply that drugs can readily saturate the metabolic pathways that rely on
the supply of cofactors such as UDP-glucuronic acid. Hence,
unconjugated drugs are often found in the blood. On the other hand,
polyphenols found in food are not expected to saturate the metabolic
pathways, therefore being in circulation in the conjugated forms [63].

Metabolism by the gut flora

Polyphenols that are not absorbed in the stomach or small bowel will be
carried to the colon. Polyphenols that are absorbed, metabolized in the
liver and secreted with bile back to the small intestine will also reach the
colon. Here, microorganisms degrade both unabsorbed and absorbed
flavonoids. Indeed, colonic bacteria produce glycosidases,
glucuronidases, sulfatases that can strip flavonoid conjugates of their
sugar moieties, glucuronic acids and sulfates [61]. Human intestinal
bacteria are able to hydrolyse 6>-glycosides [69] as well as C-glycosides
[70]. In addition, the degradation involves the splitting of the heterocyclic
oxygen-containing C-ring. Degradation products can be absorbed [71],
and subsequently metabolized by enzymes present mainly in the liver,
where 3'-0-methylation by catechol-0-methyltransferase,
dehydroxylation, p-oxidation, and conjugation with glucuronic acid,
sulfate, and glycine occurs [72]. These metabolites are considered to
contribute to the biological effects of dietary flavonoids (antioxidants).

In general, the metabolism of dietary flavonoids may be sununarized as

shown in Fig. (15).
 283

INTESTINE

Flavoaoid (agUcones
and glycosilated
fonns)
and products of the
microbic metabolism

FECES

Fig. (15). Metabolism of dietary flavonoids. GlcA = glucuronic acid; UGT = uridine 5'-
diphospoglucuronosyl transferase; Met = methyl; Sulf = sulfate; COMT = catechol-O-methyl
transferase; PST = phenol sulfo transferase

1. Flavonols

Absorption of flavonols from the diet was long considered to be

negligible, because flavonols are present in plants bound to sugars as >8-
glycosides. Only aglycones were supposed to be absorbable, whereas
glycosides were thought to be non- or only marginally absorbable [61].
One of the main flavonols studied is quercetin. Indirect evidence for
the presence of quercetin conjugates in humans was obtained by Manach
et al [73], who found the presence of quercetin in plasma after the
consumption of a complex meal rich in plant products only after p-
glucuronidase and sulfatase treatment. Furthermore, the authors reported
284

the presence of a methylated derivative of quercetin, isorhamnetin, in

three out of the ten subjects. These results are in good accordance with
data obtained by Conquer et al [74], where the concentration of quercetin
in fasting plasma of a quercetin-supplemented group was 23-fold higher
than that of the placebo-group.
Urinary excretion of quercetin increased significantly with dose and
time after the consimiption of fruit juice (blackcurrant and apple juice in a
1:1 mixture) in humans [75]. Ranges from 0.29-0.47% of ingested
quercetin were found in the urine. The presence of quercetin in urine
shows that it was absorbed by the gut, but the urinary content does not
necessarily reflect absolute absorptive efficiency because absorbed
quercetin may be metabolized (conjugated), stored and excreted through
other routes such as the biliary tract. However, since quercetin is present
in a variety of fruit and vegetables, plasma concentrations or urinary
excretion of quercetin may potentially be useful as biomarkers of habitual
intake of these foods.
The absorption of intact quercetin glycosides has been demonstrated
by some authors [64,65,76]. Holhnann demonstrated in ileostomy
subjects (who lack colon with the bacterial flora, thus circumventing the
problem of microbial degradation), that the quercetin glycosides from
regular foods (onions, tea) were far better absorbed than pure aglycone
(52%vs24%).
LC-ESI-MS analyses allowed the detection of intact flavonol
glucosides (rutin) in plasma of healthy volunteers after the consumption
of tomato extract [65].
Glycosides of flavonols from onions, such as quercetin-4'-0-glucoside
and quercetin-3'-0-methyl-4'-0-glucoside, have been found in the
plasma of volunteers with a peak of absorption of 0.5 - 4 h [64].
On the other hand, a diet-controlled cross-over-study [77] with
supplementation of quercetin and its glycoside rutin showed that rutin
occurred only as conjugated form (with glucuronic acid and/or sulfate
groups) in plasma suggesting that this glycoside was not absorbed in its
original form. Conversely, quercetin was detected in conjugated as well
as unconjugated (aglycone) forms. Interindividual differences in the
pharmacokinetics of both compounds were considerable. Sesnik et al [78]
found also no intact quercetin glucosides and only traces of aglycone in
human plasma after the administration of quercetin-3-glucoside or
quercetin-4'-glucoside as an oral solution, while quercetin glucuronides
were the major metabolites in plasma.
 285

Interestingly, different studies demonstrated that the sugar moiety of

quercetin glycosides is an important detemiinant of their absorption and
bioavailibility [79-82]. Quercetin-3-rutinoside and quercetin-4'-glucoside
are important forms of quercetin in foods. The first one accounts for about
40% of quercetin in black tea [83] and the second one for about 45% of
quercetin in onions. [84]. Although the intake of quercetin-3-rutinoside is
twice that of quercetin-4'-glucoside, the absorption of quercetin-3-
rutinoside is only 17% of ingested dose, whereas the absorption of
quercetin-4'-glucoside is 52% of ingested dose [76]. Furthermore the
bioavailibilty of quercetin-3-rutinoside is only 20% of that of quercetin-
4'-glucoside [81].
Olthof et al [82] have tested the bioavailibilty of quercetin-3-glucoside
in comparison to that of quercetin-4'-glucoside and concluded that both
quercetin glucosides are rapidly absorbed in humans, irrespective of the
position of the glucose moiety. In addition, this study provided
information on the metabohsm of quercetin into isorhamnetin (3'-
methoxyquercetin). Of the ingested quercetin glucosides, --50% is
absorbed in the small intestine and subsequently converted into
isorhanmetin, in the liver and in other organs. The 50% of ingested
quercetin that is not absorbed in the small intestine is metabolized by the
colonic microflora into quercetin aglycone and phenolic acids, which
might be absorbed from the colon [73].
The bioavailibility of quercetin-glycosides from onions, containing
mainly quercetin-p-glucosides, was superior to that of various quercetin
glycosides from apples (containing a mixture of quercetin-p-galactosides
and p-xylosides) and of pure quercetin-3-rutinoside (major species in tea).
The possible matrix effect of the foods remains unclear.
It seems that overall percentage of absorption, determined by
measuring plasma levels of flavonols after enzymatic hydrolysis, does not
exceed 2-3% of the ingested dose. It is also likely that, as with other
micronutrients, the existence of a steady-state concentration of these
compounds could result in diminished absorption. Thus, it is conceivable
that the major parts of these flavonoids are either degraded to phenolic
acids in the large intestine or excreted in the faeces [72].

2. Flavones

Data on the absorption of flavones (namely luteolin) are limited. Shimoi

286

et al [85] investigated the intestinal absorption of iuteolin and iuteolin-7-

0-P-glucoside in rats and humans. The absorption analysis using the rat-
everted small intestine demonstrated that Iuteolin was converted to
glucuronides during passing through the intestinal mucosa and that
luteolin-7-O-P-glucoside was absorbed after hydrolysis to Iuteolin. In
plasma, either free Iuteolin as well as its monoglucuronide and methylated
conjugates were present, while luteolin-7-O-p-glucoside was not detected.
This indicates that glucosides may be first hydrolyzed to Iuteolin by the
microbacteria. The same authors reported that in humans free Iuteolin and
its monoglucuronide have been detected in plasma after oral
administration of Iuteolin.

3. Isoflavones

Intestinal microflora plays a key role in the metabolism and

bioavailibility of isoflavones [86]. After ingestion, soybean isoflavones
are hydrolyzed by intestinal glucosidases, which release the aglycones,
daidzein and genistein, Fig. (16).

demethylation
dehydroxylation
intestinal glucosidases reduction
ring cleavage

equol
malonylglucosides daidzein dihydrodaidzein
acetylglucosides genistein 0-desmethylangolensin
p-glucosides p-ethylphenol

absorption

hepatic conjugation-enterohepatic cycling

urinary excretion

Fig. (16). Metabolic fate of soybean isoflavones in humans (Setchell 1999)

 287

These aglycones may be absorbed or further metabolized to different

metaboUtes, including equol, 0-<iesmethylangolensin, and p-ethylphenol
[87]. This metabolism seems to be highly variable in individuals. Setchell
et al [88] showed that after ingestion of 40 g/day soya meal over 5 days,
only four of six subjects excreted equol in urine (3-7 mg/day). After the
consumption of 40 g of whole soya flour for 2 days higher levels of
daidzein compared with genistein were recovered in the urine despite the
soya contained higher levels of genistein. Equol and O-
desmethylangolensin were detected in all individuals; in all cases, the
levels peaked on the first day post-challenge and then fell slowly
remaining above pre-challenge levels on the third day post-challenge [89].
Furthermore, tiie metabolism of isoflavones is influenced by different
components of the diet. A high fiber diet may increase the growth and/or
activity of bacteria responsible for equol production in the colon [90].
This is relevant since equol has an oestrogenic potency higher than the
precursor daidzein [91].
According to Rowland et al [92] dietary fat decreases the capacity of
gut microbioflora to synthesize equol. Additionally, the subjects may be
poor or good equol excretors, confirming that extensive interindividual
variation in isoflavone metabolism exists.
Knowledge of the pharmacokinetics of isoflavones in soy foods is
essential for making recommendations regarding efficacy in clinical
studies, rhe plasma half-life of daidzein and genistein, measured from
their plasma appearance and disappearance curves, is 7.9 h in adults; peak
concentrations occur 6-8 h after administration of the pure compounds
[93] with peak concentrations ranging firom 80 to 800 ng/ml (after a
single oral dose of 50 mg of pure isoflavones).
Similar plasma concentrations were detected for daidzein, genistein
and equol in adults consuming modest quantities of soy foods containing
about 50 mg/d of total isoflavones [91].
Few studies have been carried out in infants fed on soya formula.
Absorption of isoflavones by the infant was demonstrated firom the
appearance of daidzein and genistein in the urine of 4-month-old infants
fed soy formulas. Equol was not detected in the urine [94]. A later study
by Setchell et al [95] did not confrnn these results because equol was not
detectable or present only in traces in the serum of 4-month-old infants
fed soya infant formulas. Isoflavone concentration in human breast milk
increased after the consumption of a soya-rich diet, but their contribution
seems trivial in comparison to that from soy infant formulas [93].
288

4. Flavanones, chalcones, dihydrochalcones

The flavanones have received less attention in comparison to flavonols

and isoflavones, although their intake from the diet can be high and they
exhibit promising biological activity. Little infomiation is available about
the absorption or the kinetic behavior of the flavanones naringenm,
hesperetin and their glycosylated fomis naringin, hesperidin, and
narirutin.
Studies conceming urinary excretion of these compounds have
confimied their bioavailibility from fruits and that they are excreted, at
least to some extent, into the urine. The renal excretion of naringin,
naringenin and its glucuronides after the consumption of grapefruit juice
(20 ml/kg body weight) was investigated by Fuhr et al [96]. Only
naringenin and its glucuronides appeared in urine after an average lag-
time of 2 h. Neither naringin nor its glucuronides were found. The data
suggest that cleavage of the sugar moiety, presumably by intestinal
bacteria, is the first step of naringin metabolism. These data were
confirmed by Lee et al [97] who detected naringenin glucuronide in urine
samples after the administration of grapefruit juice (containing about 214
mg naringin).
However, a recent study reported that the glycoside naringin was
recovered (0.02% of the administered dose) in urine as unchanged
molecule, hence confirming those glycosides are absorbable [66].
The influence of glycosylation on the metabolism of naringenin-7-
glucoside and its aglycone in the conscious rat model was examined by
Choudhury et al [98]. It resulted that via oral route the glycoside group is
cleaved by an intestinal enzyme and then the aglycone is glucuronated
within the epithelium. By contrast, after intravenous dosing the majority
was detected as native glucoside in the urine.
Bioavailibility and kinetics of naringenin and hesperetin from orange
and grapefiiiit juices was also investigated by Erlimd et al [99]. Both
flavonoids were absorbed from the juices with great interindividual
variations. The authors hypothesized that these variations were caused by
differences in gastrointestinal microflora. Peak plasma concentrations of
naringenin and hesperetin were reached between 4.8 and 5.5 h, indicating
that an absorption takes place in the distal parts of the small intestine or
the colon (where enzymes capable of cleaving theflavonoidglycosides in
question are present).
 289

5. Flavanols - Catechins

Early studies (in the 1970s) on the pharmacokinetics of (+)-catechin

revealed that tiiis flavanol is absorbed from the gastromtestinal tract
following administration to healthy volunteers (4.2 g in the form of
gelatin capsules) [100]. (•f)-Catechin was excreted in the urine together
with several imidentified metabolites, and the amount excreted within 24
h was about 7.5% of the administered dose.
Other authors suggested that catechins are converted to glucuronyl
derivatives in the intestinal mucosa and are further metabolized by
methylation, sulfation and conjugation with glucuronic acid, sulfate and
glycine [101].
Indeed, Lee et al [102] determined flavanol conjugates in human
plasma after the ingestion of green tea. EGCg was mainly present as a
sulfate conjugate (65%), followed by the free form (20%) and the
glucuronide (15%). EGC on the other hand was mainly present as
glucuronide (60%), followed by sulfate (30%). About 10% was detected
as imconjugated EGC aglycone.
More recently. Da Silva et al [103] detected the presence of
glucuronides, O-methylglucuronide sulfate, and glucuronide sulfate of
epicatechin (EC) in plasma of rats fed epicatechin.
Absorption of (-)-epicatechin from chocolate has been studied by
different authors [104-106]. Baba et al [104] found maximum levels of
total EC metabolites in plasma after 2 h of chocolate or cocoa intake.
Sulfate, glucuronide and sulfoglucuronide conjugates of non-methylated
EC were the main metabolites present rather than methylated forms. In
urine samples, excretion of total EC metabolites within 24 h was about
30% of total EC intake after chocolate and 25 % after cocoa consumption.
A 12-fold increase in plasma epicatechin concentration from 22 to 257
nmol/L was reported by Rein et al [105] after consmnption of 80 g
semisweet (procyanidin rich) chocolate within 2 h after ingestion. The
total antioxidant capacity of plasma increases of 31% within the same
time, and plasma 2-thiobarbituric acid reactive substances decreased up to
40%. These data support that consimiption of chocolate increases plasma
epicatechin concentrations and decreases plasma baseline oxidation
products. These results have been confirmed in another study by Wang et
al [106].
The bioavailability and metabolism of catechins was studied in humans
after consumption of black tea containing 15.48 mg of EGC, 36.54 mg of
290

EC, 16.74 mg of EGCg and 31.1 mg of ECg [107]. Plasma concentrations

of EGC, EC and EGCg increased significantly reaching peak plateau
between 5 and 8 h (peak levels of 145,174 and 20.1 nmol/L respectively).
ECg on the other hand increased linearly over the 24h-period, peaking at
50.6 nmol/L. Urinary excretion of EGC and EC paralleled the rise in
plasma levels. EGC, EC and ECg peaked at 5 h whereas EGCg at Ih.
Fecal catechin excretion varied widely from subject to subject, but it was
significantly different from baseline for all catechins. Furthermore the
authors calculated the percentage of ingested catechins. Only 1.68% of
the total catechins consumed (400 mg) was found in the plasma, urine and
feces, providing evidence that catechins undergo considerable metabolism
and/or degradation either in the gastrointestinal tract or in the body after
absorption.
After ingestion of green tea infiision (400 mg of total catechins),
epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) were
detected in human plasma with an significant increase of these two free
catechins after enzymatic hydrolysis with glucuronidase/sulfatase,
indicating their presence in plasma mainly in the conjugated form [108].
At the same time, detectable amounts of final 60 mg catechin metabolites
were found in plasma and urine, including 4-hydroxybenzoic acid, 3,4-
dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3-
methoxy-4-hydroxybenzoic acid.
LC/ESI-MS analyses were applied to determine urinary glucuronidated
and sulfated tea catechins after the administration of green tea to humans,
mouse and rats [109]. The major conjugates were identified as
monoglucuronides and monosulfates of EGC and EC. Besides these
metabolites, also 0-methyl-EGC-O-glucuronides, 0-sulfates and O-
methyl-EC-0-sulfates in human urine were detected. Furthermore, the
ring-fission metabolites of EGC and (-)-epicatechin, 5-(3',4',5'-
trihydroxyphenyl)-x-valerolactone and 5-(3 ',4'-dihydroxyphenyl)-y-
valerolactone respectively, have been detected in the monoglucuronide
and monosulfate forms.
It is not known whether tea catechin conjugates possess any biological
activities. In a study by Manach et al [110], the glucuronic/sulfate
conjugates were shown to have the same electrochemical behaviour as the
parent drug. Considering that the oxidation potential of chemicals may
represent their antioxidant capacity, the electrochemical behaviour of the
conjugates suggests that they are effective antioxidants. Nevertheless,
because the glucuronic acid/sulfate conjugates are generally more
 291

hydrophilic than the parent compound, the tissue distributions of these

metabolites are likely to be more limited than those of the parent
catechins [111].

6. Hydroxycinnamates, hydroxybenzoic acids

Non-ruminants possess several intestinal Na'^^-dependent saturable

transport systems. These include the well-known sodium-glucose co-
transporter (SGLTl), responsible for the active uptake of glucose, and it
appears to be specific for cinnamic and ferulic acid and possibly for other
hydroxy-cinammic acids [112].
Healthy volunteers have shown to excrete caffeic, p-coumaric and
ferulic acid in the urine after the consumption of various fruits [113]. The
excretion of free ferulic acid in urine peaked after 7 h at a concentration
of-'T jiM after the consumption of tomatoes (36-73 g containing 21-44
mg ferulic acid). The concentration of free ferulic acid plus glucuronide
(and possibly sulfate) conjugates exceeded 20 |aM and accounted for
some 11-25% of the dose [114].
Simonetti et al [115] studied the plasma levels of caffeic acid after
consumption of 100,200, 300 ml of red wine (caffeic acid content of 9.01
mg/L) that provided about 0.9, 1.8, and 2.7 mg of caffeic acid,
respectively. The highest plasma levels of caffeic acid was reached 60
min after ingestion and decreased to basal values within 180 min (for 100
and 200 ml) and within 240 min (for 300 ml). Both, the absence of caffeic
acid in plasma before the trial and its significant, dose-dependent
increment after red wine ingestion suggest that it may be a possible
marker of consimiption of beverages containing this acid. Furthermore,
200 and 300 ml red wine intake produced a significant increase in plasma
total antioxidant capacity (TRAP).
Conceming HBAs, their metabolism involves conjugation with sulfate,
glucuronate and glycine. Methylation may also occur, as may
demethylation, dehydroxylation and decarboxylation (this only if there is
a 4-hydroxyl) [3].
292

7. Anthocyanins, proanthocyanidins

a. Anthocyanins

Dietary anthocyanins have gained much attention based on the

recognition of the "French paradox'* which led to the suggestion that
some components of red wine (in particular anthocyanins) may protect
against coronary heart disease.
Limited evidence on the absorption of intact anthocyanins exist until
today.
There are reports, based upon spectral properties from DAD-HPLC of
anthocyanin-like substances in plasma [116] and urine after acidification
[117]. However, the ^max observed for the components in urine was at 430
nm. Anthocyanins should not have an absorption peak around this
wavelength. Thus, the detected compounds appeared to be anthocyanin
metabolites [68].
These spectroscopic data do not provide conclusive evidence for the
absorption of intact anthocyanins. Conversely, anthocyanins from
elderberry were detected in human plasma using a more selective
approach based on HPLC coupled to UV detection (512 nm) [67]. These
findings have been confirmed by a recent study on the detection of
anthocyanins in their unchanged glycosylated form in urine and plasma
after ingestion of 720 mg anthocyanins [68].

b. Proanthocyanidins (PA)

The complexity and the lack of commercial pure standards of PA make

their analysis difficult. Their absorption depends on their degree of
polymerization. In some in vitro experiments, only PA dimers and
trimers, but not polymers with an average polymerization degree of 7,
were absorbed through an intestinal epithelium cell monolayer [118].
Experiments in chicken and sheep showed that polymeric PAs were not
absorbed through gut barrier [119,120].
Evidence occurred that polymeric proanthocyanidins could be
degraded by the colonic microflora into low-molecular-weight
compounds, which would be subsequently absorbed. The group of Deprez
[118] investigated their metabolism by human colonic microflora
incubated in vitro in anoxic conditions using non-labeled and ^"^C-labeled
 293

purified proanthocyanidin polymers. Degradation occurred almost totally

after 48 h of incubation; metabolites identified were low-molecular-
weight phenolic acids: phenylacetic, phenylpropionic and phenylvaleric
acids, monohydroxylated mainly in meta or para-position.
It is supposed that, once fermentation products have crossed the
intestinal barrier, they reach the liver through the portal vein, where they
are further metabolized by dehydroxylation, methylation or conjugation to
sulfate esters or glucuronides as it has been shown for other flavonoids
[59].

POLYPHENOLS: BIOCHEMICAL AND PHARMACOLOGICAL

PROPERTIES

Polyphenols are endowed with different biological activities, including:

1. Antioxidant/anti-radical activity and chelation of metal ions

2. Modulation of some enzymes activity
3. Anticarcinogenic activity
4. Antiatherosclerotic activity
5. Anti-inflammatory activity
6. Inhibition of histamine release and spasmolytic activity
7. Hepatoprotective activity
8. Antiviral and antimicrobial activity
9. Oestrogenic activity

1. Antioxidant and anti-radical activity, chelation of metal ion

Polyphenols can act as antioxidants by a number of potential pathways.

The most important is likely to be by free radical scavenging, in which
the polyphenol can break the radical chain reaction. Polyphenols are
effective antioxidants in a wide range of chemical oxidation systems,
being capable of scavenging peroxyl radicals, alkyl peroxyl radicals,
superoxide, hydroxyl radicals, nitric oxide and peroxynitrate in aqueous
and organic environments [121]. This activity is due to the ability of
donating an H atom from an aromatic hydroxyl group to a free radical,
and the major ability of an aromatic structure to support an unpaired
electron by delocalization around the 7i-electron system. Phenolic acids
294

and flavonoids may be good antioxidants, particularly those possessing

the catechol-type structure [122-125].
Phenolic acids and flavonoids (PP-H) can also act as free radicalchain
(ROO*) reaction terminator, as follow:

ROO* + PP-H -> ROO-H -f PP*

The PP* radical is relatively stable and could react in another reaction
as terminator, as follow:

ROOVPP*->ROO-PP

The interaction between flavonoids and phenolic acids with other

physiologic antioxidants, such as ascorbate or tocopherol, is another
possible antioxidant pathway for these compounds [72,126,127].
Nevertheless, like must other antioxidants, flavonoids may also act as
prooxidant in particular circumstances [128,129].
Phenolic acids and flavonoids can also act as chelating agents,
complexing transition metals that are responsible of the initiation of
peroxidative processes (Fenton and Haber-Weiss reactions). This property
is much stronger in phenolics having a catechol, pyrogallol, or 3-hydroxy-
4-carbonyl group [130].

2. Modulation of some enzymes activity

Interaction of low molecular weight molecules, such as phenolic acids

andflavonoids,with macromolecules can modify their chemical-physical
properties. Different studies confirm the ability of phenolics in
modulating some enzymes, such as hydrolases, transferases, kinases,
oxidases, hydroxylases, glutathione S-transferase, nitric-oxide synthase,
cytochrome P450 systems, ATPases, lipases, phospholipases, adenylate
cyclase, RNA and DNA polymerase, human DNA ligase I, ribonuclease,
reverse transcriptase, topoisomerase, aromatase, hyaluronidase, elastase,
HIV-1 proteinase and integrase, aldose reductase [131,132]. Specifically,
enzymes such as xantine-oxidase, nitric oxide synthase, phospholipase,
cyclooxygenase and lipoxygenase, involved in the production of free
radicals in biologic systems, are also inhibited, in vitro, from different
polyphenols [132,133].
 295

Overall, these activities may explain the correlation between

polyphenols and their anticancer, antithrombotic, anti-atherogenetic and
anti-inflammatory effects. However, more research is needed to determine
which of these activities can realistically be translated into clinical effects.

3. Anticarcinogenic activity

The antioxidant effect, the modulation of some enzymes and induction of

apoptosis are at the root of the anticancer mechanism. Carcinogenesis is a
multi stage process of genetic change that may be initiated by increased
and persistent damage to DNA causing permanent alterations in the
genetic message when the cell replicates its DNA and divides. ROS
(Reactive Oxygen Species) are potential carcinogens because they can
induce structural damage to DNA by oxidation, methylation, depurination
and deamination reactions.
The ability of some polyphenols to reduce or inhibit the oxidative
damage to DNA is well documented. For example chlorogenic acid
inhibits DNA damage in vitro caused by peroxynitrite [134], coumaric
acids are good free radical scavengers [135], caffeic and
dihydroxybenzoic acids are able to inhibit iron induced DNA damage
[135,136]. Chlorogenic acid can inhibit DNA damage caused by
monochloramine [137]. Many kind of flavonoids are able to scavenge free
radicals protecting DNA from oxidation, including the isoflavone
genistein [138,139], theflavonesluteolin and apigenin [140], the flavanol
epigallocatechin gallate [141], the flavonols myricetin and kaempferol
[140], quercetin [140-142], and its glycosylated derivatives, quercetin-3-
glycoside, quercitrin and rutin [140]. DNA damage may be also reduced
by metal binding properties of flavonoids, as demonstrated for rutin and
quercetin [143].
Another mechanism in reducing carcinogenesis by polyphenols is the
modulation of the enzymatic system that is in charge of the
metabolization of carcinogenic molecules. The cytochrome P450 family
of enzymes metabolizes a large number of procarcinogens to reactive
intermediates, which bind covalently to DNA and can induce mutation.
Severalflavonoidsare able to inhibit the activity of this family enzymes,
as reported by different authors [144-149]. The inhibition of this activity
byflavonoidsis directly correlated to their antimutagenic properties.
The glutathione transferases (GST), together with the tripeptide
glutathione (GSH), conjugate the highly reactive and potentially
296

carcinogenic substances, making such molecules more polar, thereby

facilitating their excretion. Flavanones, flavones, flavonols increase
hepatic GST levels and activity in rats [148,150].
Furthermore, by modification of gene expression, some polyphenols
may prevent or reverse carcinogenesis, inducing apoptosis or inhibiting
neoplastic transformation. Gallic acid induces selective cell death in
cancer cell [151], and hamster fed caffeic acid with the diet were shown
to be less susceptible to the effect of methylazoxymethanol, an initiator of
colon carcinogenesis [152]. Caffeic acid phenethyl ester from propolis,
given to mice bearing a germline mutation in the Ape gene and
spontaneously developing numerous intestinal adenomas by 15 weeks of
age, decreased tumor formation by 63% at a dietary level of 0.15% and
the examination of intestinal tissue from treated animals showed that
tumor prevention was associated with increased enterocytes apoptosis
[153]. Protocatechuic acid from Hibiscus sahdariffa induced apoptosis in
human leukemia cells [154]. Green tea polyphenols and epigallocatechin
gallate increase fragmentation in several human and rodent carcinoma
cells, but not in normal epidermal keratinocytes [155]. Epigallocatechin
gallate also induces apoptosis in transformed fibroblast [156] and in
human histiolytic lymphoma U937 cells [157]; theasinensin D, theaflavin,
theaflavin digallate induced apoptosis in the same lymphoma cells [157].
Quercetin, rutin, morin, gallic acid and tannic acid inhibited the growth of
human prostate cancer cell (LNCaP) at different concentrations, and
induced apoptosis [158].

4. Antiatherosclerotic activity

Cardiovascular heart diseases (CHD) are considered as the clinical

expression of advanced atherosclerosis. One of the initial steps in
atherogenesis is the oxidative modification of LDL and the uptake of the
modified lipoprotein particles by macrophages, which in tum become
lipid laden cholesterol-rich cells, so-called foam cells [159]. An
accumulation of foam cells in the arterial wall is the fu-st visible sign of
atherosclerosis and is termed fatty streak, the precursor to the
development of the occlusive plaque [160]. It is well known that
oxidation of LDL can be initiated in vitro by incubating isolated LDL
particles with cells (macrophages, lymphocytes, smooth muscle cells, or
endothelial cells), metal ions (copper or iron), enzymes, oxygen radicals,
or UV-light. However less is known about the mechanisms by which
 297

LDL becomes oxidized in vivo. There is evidence that LDL is protected

against oxidation in plasma by water-soluble antioxidative substances,
such as ascorbic acid, uric acid, or bilirubin. Thus, it is likely that the
majority of oxidative modification of LDL occurs in the artery wall,
where LDL is largely isolated from the plasmatic antioxidants. Recent
evidence suggests that metal ions (copper or iron) and the enzymes
myeloperoxidase and lipoxygenase play major parts in the modification
of LDL [161].
In in vitro studies the oxidation of LDL by endotheUal cells,
macrophage and Cu"*^ can be inhibited by a wide range of polyphenols
and polyphenol-rich extracts [162-164]. Such effects may be due to
polyphenols by direct scavenging of the oxidizing species, by
regeneration of a-tocopherol in LDL [165], by their ability in binding
metal ion and LDL protein [166].
In addition to in vitro studies, several animal models and trials with
human subjects indicate that ingestion of polyphenols increases the
resistance of LDL oxidation ex vivo [167,168].
Some polyphenols inhibit platelet aggregation reducing the risk of
thrombosis [171-173]. This effect may be due to a series of interaction
of flavonoids in different biochemical pathways, such as by inhibition of
cyclooxygenase and lipoxygenase, that are involved in the arachidonic
acid metabolism in the platelets, or by inhibition of the formation of
tromboxane and of the receptor function of the same [173-176]. Regular
consumption of wine, tea and chocolate has been associated to the
reduction of platelet aggregation, cardio-vascular diseases and
thrombosis [171,177-179].

5. Anti-inflammatory activity

Inflammation is a highly complex biochemical protective response to

cellular injury. It is important in the maintenance of homeostasis when
the organism is challenged by noxious agents or by tissue mechanical
injury. Inflammation is associated with a drastic rise in the number of
polymorphonuclear leukocytes and monocytes in the affected tissue and
with the release of inflanunatory mediators such as prostaglandins and
cytokines. Under normal conditions, inflammation results in the
complete recovery of the integrity of the affected tissue, but if the
response to the triggering stimulus is not subjected to tight regulation.
298

cellular and extracellular components of the organism adjacent to the

inflammation site can be injured, inducing a condition known as chronic
inflammatory disease. A chronic inflammatory like environment
characterizes the pathogenesis of various diseases such as
atherosclerosis, arthritis, and Crohn's disease, and it is thought to be
among the causative factors of more than 30% of human cancers. For
these reasons it is very important to localize and reduce the
inflammatory response. In this scenario, polyphenols are involved as
immunomodulatory and anti-inflammatory agents, scavenging ROS and
modulating the activity of key enzymes of the inflammatory response
[172,180482].

6. Inhibition of histamine release and spasmolytic activity

The flavonoids quercetin, hyperoside and isoquercetin, present in the

ethanolic extract of Drosera madagascariensis, are inducers of
spasmolytic and anti-inflammatory effects in guinea-pig ileum by
affecting cholinergic M3 and histamine HI receptors [183].
In an in vitro study by Yamada et al [184], triphenols, such as
pyrogallol and gallic acid, and among flavonols, myricetin, inhibited
histamine release from rat peritonei cells. Pyrogallol and gallic acid, and
also o- and /?- diphenols, such as catechol and hydroquinone and all
flavonols tested, strongly suppressed leukotriene B4 release in the same
cells. Another in vitro study on rat basophilc leukemia cells
demonstrated that, among tea polyphenols, (-)-epigallocatechin gallate
(EGCg), (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECg)
have different inhibitory effects on histamine release induced by a
calcium ionophore, with the following magnitude: EGCg>ECg>EGC
[185]. In the same study was also demonstrated that pyrogallol and gallic
acid exert inhibitory activity and a mixture of these two compounds
inhibited histamine release as strongly as EGCg.

7. Hepatoprotective activity

Polyphenols are also endowed to have hepatoprotective effects. For

example, quercetin reduces liver oxidative damage, ductural
proliferation and fibrosis in biliary-ostructed rats, suggesting that it may
 299

be a useful liver protective agent in patients with biliary obstruction

[186].

8. Antiviral and antimicrobial activity

Polyphenols may act as antimicrobial and antiviral agents as

demonstrated by several studies in vitro. Polyphenol-rich extracts from
various plants, such as Betula pubescens^ Epilobium angustifolium,
Perillafrutescens, Pinus sylvestris, Rubus chamaemorus, Rubus idaeus.
Solarium tuberosum, propolis and pure compounds, were tested to
evaluate their antimicrobial activity against different bacteria and yeasts
species, such as Bacillus subtilis, Escherichia coli, Mycobacterium
tuberculosis H37Rv, Pseudomonas aeruginosa. Salmonella spp,
Staphylococcus aureus. Streptococcus piogenes, Aspergillus niger,
Candida albicans, Saccharomyces cerevisiae and showed growth
inhibitory and bactericidal effect at different concentrations [187-192].
Naturally occurring flavonoids with antiviral activity have been
recognized since the 1940s [193]. Quercetin, morin, rutin, taxifolin,
dihydrofisetin, leucocyanidin, pelargonidin chloride, apigenin, catechin,
hesperidin, and naringin have been reported to possess antiviral activity
against some of 11 types of viruses [193]. (-)-Epigallocatechin gallate and
theaflavin digallate inhibited the infectivity of both influenza A virus and
influenza B virus in Madin-Darby canine kidney cells in vitro [194].

9. Oestrogenic activity

Plant-derived oestrogens may exert both oestrogenic and anti-

oestrogenic effects, depending on several factors, including their
concentration, the concentrations of endogenous oestrogens, and
individual characteristics, such as gender and menopausal status
[195,196]. The anti-oestrogenic activity of phytoestrogens may be
partially explained by their competition with endogenous 17p-estradiol
for oestrogen receptors [197]. Many of the potential health benefits of
phytoestrogens may be attributable to features that do not involve
oestrogen receptors, such as their influence on enzymes, protein
synthesis, cell proliferation, angiogenesis, calcium transport, Na"^/K"*"
adenosine triphosphatase, growth factor action, vascular smooth muscle
cells, lipid oxidation, and cell differentiation. Phytoestrogens may have
300

favorable effects on the risk of cardiovascular disease and are thought to

be hypocholesterolemic, anticarcinogenic, antiproliferative,
antiosteoporotic, and hormone altering [195,196,198,199].
Finally, flavonoids can bind to structural proteins and this feature
could explain their ability to enhance the integrity of connective tissue.

EPIDEMIOLOGIC EVIDENCE OF PLANT POLYPHENOL

HEALTH BENEFITS

1. Risk of CHD diseases

Several epidemiological studies have reported inverse relation between

intakes of flavonols and flavones and cardiovascular heart diseases
(CHD).
In a prospective study of 3454 men and women (age 55 years and
older), a significant inverse association between the intake of catechin-
rich tea and radiographically quantified aortic atherosclerosis was found
[200]. Similarly, inverse association between the consumption of red wine
and CHD mortality (French paradox) have been suggested [201]. This
beneficial effect of red wine may be due to the antioxidant ability of the
wine phenolics to inhibit the oxidation of LDL to an atherogenic form
[202],
In the Zupthen Elderly Study [203] flavonol and flavone intake at
baseline in 1985 of approximately 800 men (aged 65-85 years) was
determined using the cross-check dietary history method. Men were
divided into tertiles of flavonol and flavone intake. After five years of
follow-up 43 men died from heart disease in this period. Flavonol and
flavone intake, expressed as tertiles, was inversely associated with
mortality from coronary heart disease and to a lesser extent with the
incidence of first myocardial infarction. Furthermore, the association
between long-term flavonol and flavone intake and risk of stroke in a
cohort of 552 middle-aged Dutch men free fi"om history of stroke at
baseline was also investigated within this study. Men were divided into
quartiles of flavonol and flavone intake, and followed for 15 years.
During this period 42 men had a first stroke event. Flavonol and flavone
intake was strongly inversely associated with stroke risk. In both studies,
the men in the highest category of flavonol and flavone intake
(>30mg/day) had about one-third the risk of getting the disease compared
 301

with men in the lowest category. The major sources of dietary quercetin
and other flavonols were revealed as tea and onions (fruits and vegetables
had minor importance).
The same authors [204] confirmed these results in the Seven Country
Study. The contribution of flavonols and flavones in explaining the
variance in coronary heart disease mortaUty rates across 16 cohorts from
seven countries was studied. Flavonol and flavone intake was inversely
correlated with mortality from coronary heart disease. Thesefindingare
in line with the results of a cohort study in Finnland [205], where a
significant inverse gradient was observed between dietary intake of
flavonoids and total and coronary mortality.
A modest but not significant inverse correlation between the intake of
flavonols and flavones and subsequent mortality rates was found in a
prospective cohort study of US Health Professionals by Rimm et al [206].
The authors do not exclude thatflavonoidshave a protective effect in men
with established coronary heart disease although strong evidence was
missing. Also other studies failed to demonstrate a significant statistical
association between the intake of polyphenols and CHD. In Great Britain
for instance coronary and total mortality even rose with the intake of the
majorflavonolsource, tea [207]. The most likely explanation for the latter
observation is that in this study tea consumption merely acted as a marker
for a lifestyle that favours the development of cardiovascular disease.
Indeed, men with the highest intake of tea and flavonols tended to be
manual workers, and they smoked more and ate more fat [208].

2. Risk of cancer

The epidemiological evidence for a beneficial support of polyphenols in

cancer disease is contradictory and less clear than its role in CHD.
The Zupthen Elderly Study found a weak inverse association between
flavonoid intake from fruit and vegetables sources and cancer of the
alimentary and respiratory tracts combined [209]. The same authors
observed no independent association with mortality from other causes
between flavonoid intake and cancer mortality in the Seven Country
Study [204].
ICnekt et al [210] studied the relation between the intake of flavonoids
and subsequent cancer among 9959 finnish men and women during a
follow-up in 1967-1991, An inverse association was observed between
the intake offlavonoidsand incidence of all sites of cancer combined. Of
302

the major flavonoid sources, the consumption of apples showed an

inverse association with lung cancer incidence.
The cancer protective effects of black and green tea consiraiption,
important sources of flavonol in specific countries, have been investigated
mainly in case-control studies. Kohlmeier et al [211] evaluated the
epidemiologic literature about tea and cancer prevention, concluding that
cohort studies do not suggest a protective role for tea drinking in the total
risk of cancer. Site-specific studies give a more complex picture. For
example, a protective effect of green tea on the development of colon
cancer is suggested. On the other hand, evidence for black tea is less
clear, with some indication of a risk of colon or rectal cancer associated
with regular use of black tea.
In another cohort study of a Japanese population, researcher surveyed
more than 8000 individuals over 40 years of age on their living habits,
including daily consumption of green tea. Results found a negative
association between green tea consumption and cancer incidence,
especially among females drinking more than 10 cups per day [212].

3, Vasoprotective effects (Hypertension)

Experimental studies have shown that the administration of green tea-

enriched water to laboratory animals is associated with a reduction in
blood pressure [213].
Different epidemiologic studies have suggested that drinking either
green or black tea may lower cholesterol concentration and blood pressure
[214,215].
In a epidemiological study of Japanese women, a history of stroke was
less common among those who drank more green tea. There was no
statistically significant reduction in blood pressure alone among those
women who drank more tea [206].

4. Oestrogenic effects

Phytoestrogens represent a family of plant compounds that have been

shown to have both oestrogenic and anti-oestrogenic properties.
Accumulating evidence from molecular and cellular biology experiments,
animal studies and, to a limited extent, human clinical trials suggests that
phytoestrogens may potentially confer health benefits related to
 303

cardiovascular diseases, cancer, osteoporosis, and menopausal symptoms.

These potential health benefits are consistent with the epidemiological
evidence that the risk of heart disease, various cancers, osteoporotic
fractures, and menopausal symptoms is lower among populations that
consume plant-based diets, particularly among cultures with diets that are
traditionally high in soy products.
One study over 9 months noted a significant reduction in total
cholesterol in premenopausal women when they consumed soy products
with 45 mg conjugated isoflavones/day in comparison to levels during a
control period when they were fed isoflavone-free soy products. The
treatment group difference was significant despite the small sample size
and the selection of healthy, normocholesterolemic women who had
limited room for detectable improvements [216]. The pattem of soy
intake and its association with blood lipid concentrations in the Hong
Kong Chinese population was studied in a total of 500 men and 510
women with an age range of 24-74 years by Ho et al [217]. In men, soy
intake and total plasma cholesterol were negatively correlated (r = 20.09,
P = 0.04), as were soy intake and LDL cholesterol fr = 20.11, P = 0.02).
The respective values in women <50 y old were r = 20.11, P = 0.04 and r
= 20.11,P = 0.05.
In the Framingham Offspring Study a group of 939 postmenopausal
women was studied to correlate the association between dietary
phytoestrogen intake and metabolic cardiovascular risk factors. Mean
blood pressure, waist-hip ratio (WHR) and lipoprotein levels were
determined in quartile categories of dietary phytoestrogen (isoflavones
and lignans) intake. In the highest quartile of intake of isoflavones,
plasma triglyceride levels were 0.16 mmol/L lower (95% CI, -0.30 to -
0.02) compared with the lowest quartile of isoflavones and the mean
cardiovascularriskfactor metabolic score was 0.43 points lower (95% CI,
-0.70 to -0.16) than the lowest quartile [218].
Hormone-related cancers of the breast, ovary, endometrium, and
prostate have been reported to vary by as much as 5 to 20-fold between
populations. Migrant studies indicate that the difference is largely
attributable to environmental factors rather than genetics [219,220]. The
highest rates of these cancers are typically observed in populations with
Westem lifestyles that include relatively high fat, meat-based, low fiber
diets, whereas the lowest rates are typically observed in Asian populations
with Eastem lifestyles that include plant-based diets with a high content
of phytoestrogens [219,221].
304

In a case-control study Ingram et al [222] reported a significant

reduction in breast cancer risk among both premenopausal and
postmenopausal women who consumed phytoestrogens. In a study of
Asian-Americans of Chinese, Japanese, and Filipino heritage, it was
reported that tofii consumption was significantly and inversely associated
with breast cancer [223].
Similar findings were reported from a case-control study of women in
Singapore in which soy intake was inversely, and animal products intake
was positively, associated with breast cancer, although these findings
were significant only among premenopausal, not postmenopausal women
[224]. Soy and fiber consumptions were both associated with a decreased
risk of endometrial cancer among the multiethnic population of Hawaii, a
finding that was limited to women who had never used oestrogens and
had never been pregnant [225].
In a study conducted in Boston and Helsinki, it was demonstrated that
the lowest excretion of enterolactone and equol was found in a group of
postmenopausal breast cancer patients compared to healthy omnivorous
and vegetarian women [226].
The continual loss of bone mass in the elderly is a natural process of
aging. Women have a higher incidence of osteoporotic fractures than men
due to their lower peak bone mass, but in addition, the abrupt decrease in
oestrogen secretion in postmenopausal women accelerates bone loss.
Currently, osteoporosis-related fractures are lower in Asia than in most
Westem communities, possibly due to the phytoestrogen-rich soybeans
and vegetables consumed in large quantities in the Asian diet [227].
Investigation about rates of hip fracture in Hong Kong and the U.S.
reported that for men and women 85 yr of age or more, the rates in Hong
Kong were roughly one third the rates in the U.S. [228].

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