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Molecular Aspects of Medicine 31 (2010) 446467

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Molecular Aspects of Medicine


journal homepage: www.elsevier.com/locate/mam

Review

Bioavailability of dietary avonoids and phenolic compounds


Alan Crozier a,, Daniele Del Rio b, Michael N. Clifford c
a
b
c

Plant Products and Human Nutrition Group, Graham Kerr Building, School of Medicine, University of Glasgow, Glasgow G12 8QQ, UK
Human Nutrition Unit, Department of Public Health, University of Parma, Via Volturno 39, 43100 Parma, Italy
Food Safety Research Group, Centre for Nutrition and Food Safety, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK

a r t i c l e

i n f o

Keywords:
Flavonoids
Chlorogenic acids
Ellagitannins
Bioavailability
Metabolism
Small intestine absorption
Colonic degradation

a b s t r a c t
This paper reviews recent human studies on the bioavailability of dietary avonoids and
related compounds, including chlorogenic acids and ellagitannins, in which the identication of metabolites, catabolites and parent compounds in plasma, urine and ileal uid was
based on mass spectrometric methodology. Compounds absorbed in the small intestine
appear in the circulatory system predominantly as glucuronide, sulfate and methylated
metabolites which seemingly are treated by the body as xenobiotics as they are rapidly
removed from the bloodstream. As a consequence, while analysis of plasma provides valuable information on the identity and pharmacokinetic proles of circulating metabolites
after acute supplementation, it does not provide accurate quantitative assessments of
uptake from the gastrointestinal tract. Urinary excretion, of which there are great variations with different classes of avonoids, provides a more realistic gure but, as this does
not include the possibility of metabolites being sequestered in body tissues, this too is an
under estimate of absorption, but to what degree remains to be determined. Even when
absorption occurs in the small intestine, feeding studies with ileostomists reveal that substantial amounts of the parent compounds and some of their metabolites appear in ileal
uid indicating that in volunteers with a functioning colon these compounds will pass to
the large intestine where they are subjected to the action of the colonic microora. A diversity of colonic-derived catabolites is absorbed into the bloodstream and passes through the
body prior to excretion in urine. There is growing evidence that these compounds, which
were little investigated until recently, are produced in quantity in the colon and form a
key part of the bioavailability equation of dietary avonoids and related phenolic
compounds.
2010 Elsevier Ltd. All rights reserved.

Contents
1.
2.

3.

4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Flavan-3-ols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Green tea flavan-3-ol monomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Proanthocyanidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Flavonols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Onion quercetin-O-glucosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Tomato juice quercetin-3-O-rutinoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Orange juice flavanones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isoflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author.
E-mail address: Alan.Crozier@glasgow.ac.uk (A. Crozier).
0098-2997/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mam.2010.09.007

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6.

7.
8.
9.

Anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Strawberries and blackberries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Blueberries and blackcurrants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.
Accumulation of anthocyanins in body tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.
Stability of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ellagitannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chlorogenic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
The protective effects of diets rich in fruits, vegetables and derived beverages are due not only to ber, vitamins and minerals, but also to a diversity of plant secondary metabolites, in particular phenolic compounds and avonoids. The bioavailability of these compounds after dietary intake has been a topic of increasing research in recent years. Following the
ingestion of dietary avonoids which, with the notable exception of avan-3-ols and proanthocyanidins, exist in planta predominantly as glycoside conjugates, absorption of some but not all components into the circulatory system occurs in the
small intestine (Donovan et al., 2006b). Typically, the absorption at this site is associated with hydrolysis, with release of
the aglycone as a result of the action of lactase phloridzin hydrolase (LPH) in the brush-border of the small intestine epithelial cells. LPH exhibits broad substrate specicity for avonoid-O-b-D-glucosides and the released aglycone may then enter
the epithelial cells by passive diffusion as a result of its increased lipophilicity and its proximity to the cellular membrane
(Day et al., 2000). An alternative hydrolytic step is mediated by a cytosolic b-glucosidase (CBG) within the epithelial cells. In
order for CBG-catalysed hydrolysis to occur, the polar glucosides must be transported into the epithelial cells, possibly with
the involvement of the active sodium-dependent glucose transporter SGLT1 (Gee et al., 2000). Thus, it has been accepted that
there are two possible routes by which the glucoside conjugates are hydrolysed and the resultant aglycones appear in the
epithelial cells, namely LPH/diffusion and transport/CBG. However, an investigation, in which SGLT1 was expressed in
Xenopus laevis oocytes, has indicated that SLGT1 does not transport avonoids and that glycosylated avonoids, and some
aglycones, have the capability to inhibit the glucose transporter (Kottra and Daniel, 2007). Using Caco-2 cells Johnson
et al. (2005) found that glucose uptake into cells under sodium-dependent conditions was inhibited by avonoid glycosides
and non-glycosylated polyphenols whereas aglycones and phenolic acids were without effect.
Prior to passage into the blood stream the aglycones undergo metabolism forming sulfate, glucuronide and/or methylated
metabolites through the respective action of sulfotransferases (SULT), uridine-50 -diphosphate glucuronosyltransferases
(UGTs) and catechol-O-methyltransferases (COMT). There is also efux of at least some of the metabolites back into the lumen of the small intestine and this is thought to involve members of the adenosine triphosphate (ATP)-binding cassette
(ABC) family of transporters, including multidrug resistance protein (MRP) and P-glycoprotein (P-gp). Once in the portal
bloodstream, metabolites rapidly reach the liver, where they can be subjected to phase II metabolism with further conversions and enterohepatic recirculation may result in some recycling back to the small intestine through bile excretion (Donovan et al., 2006b). Analysis of ileal uid collected from ileostomists after the ingestion of various foodstuffs indicate that even
when dietary avonoids are absorbed in the small intestine, substantial quantities none-the-less pass from the small to the
large intestine (Kahle et. al., 2005, 2006; Jaganath et al., 2006; Marks et al., 2009) where the colonic microbiota will cleave
conjugating moieties and the resultant aglycones will undergo ring ssion leading to the production of smaller molecules,
including phenolic acids and hydroxycinnamates. These can be absorbed and may be subjected to phase II metabolism in
the liver before being excreted in urine in substantial quantities that, in most instances, are well in excess of the avonoid
metabolites that enter the circulatory system via the small intestine (Jaganath et al., 2006; Roowi et al., 2009, 2010; Stalmach
et al., 2010a,b).
Manach et al. (2005) published a detailed and subsequently much quoted review on the bioavailability of polyphenols in
humans. Much of the research covered involved feeding volunteers a single supplement and monitoring the levels of avonoids in plasma and urine over a 24 h period. As avonoid metabolites were and, indeed, still are rarely available, analysis
almost invariably involved treatment of samples with mollusc glucuronidase/sulfatase preparations and the subsequent
quantication of the released aglycones by HPLC using either absorbance, uorescence or electrochemical detection. Some
more recent bioavailability studies have analysed samples directly by HPLC with tandem mass spectrometric (MS) detection without recourse to enzyme hydrolysis. The availability of reference compounds enables specic metabolites to be
identied by HPLCMS2 and MS3 (Mullen et al., 2006). In the absence of standards it is not possible to distinguish between
isomers and ascertain the position of conjugating groups on the avonoid skeleton. None-the-less, a metabolite that in reality is pelargonidin-3-O-glucuronide can be partially identied as a pelargonidin-O-glucuronide on the basis of its MS fragmentation pattern (Mullen et al., 2008b). The use of MS in this way represents a powerful HPLC detection system as, with
low ng quantities of sample, it provides structural information on analytes of interest that is not obtained with other
detectors.
Quantication of partially identied metabolites by MS using consecutive reaction monitoring (CRM) or selected ion
monitoring (SIM) is, of necessity, based on a calibration curve of a related compound, which in the instance cited above could

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A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) 446467

Fig. 1. Concentrations of (epi)gallocatechin-O-glucuronide (EGC-GlcUA), 40 -O-methyl-(epi)gallocatechin-O-glucuronide (40 -Me-EGC-GlcUA), 40 -O-methyl(epi)gallocatechin-O-sulfates (40 -Me-EGC-S), ()-epicatechin-30 -O-glucuronide (EC-30 -GlcUA), 30 - and 40 -O-methyl-(epi)catechin-O-sulfates (Me-EC-S),
()-epigallocatechin-3-O-gallate (EGCg) and ()-epicatechin-3-O-gallate (ECg) in the plasma of human subjects 08 h after the ingestion of 500 ml of green
tea. Data expressed as mean values with their standard errors (n = 10) depicted by vertical bars. Note that no avan-3-ols or their metabolites were detected
in plasma collected 24 h after ingestion of the green tea.

be pelargonidin-3-O-glucoside as it can be purchased from commercial sources. In such circumstances, as the slopes of the
glucoside and glucuronide SIM doseresponse curves will not necessarily be identical there is a potential source of error in
the quantitative estimates and there is a view that quantitative estimates based on enzyme hydrolysis are, therefore, much
more accurate. We do not share this opinion. The glucuronidase/sulfatase preparations are characterised by various enzyme
activities and there can be substantial batch-to-batch variation in their specicity (Donovan et al., 2006b). There are no reports of avonoid bioavailability studies using glucuronidase/sulfatase preparations where information on the identity,
number and quantity of the individual sulfate and glucuronide conjugates in the samples of interest has been obtained.
As a consequence, there are no direct data on the efciency with which the enzymes hydrolyse the individual metabolites
and release the aglycone. This introduces a varying, unmeasured error factor. The accuracy of quantitative estimates based
on the use of glucuronidase/sulfatase preparations are, therefore, probably no better than those based on HPLCCRM/SIM.
The fact that enzyme hydrolysis results in very reproducible data does not reect the validity of the method as it is a measure
of precision not accuracy (Reeve and Crozier, 1980). These shortcomings of analyses based on enzyme hydrolysis apply to
bioavailability studies with all dietary avonoids and it is interesting to note that the one publication on the subject to date
reports that the use of enzyme hydrolysis results in an underestimation of isoavone metabolites (Gu et al., 2005). Even if MS
is used only to locate and characterise the conjugate and quantication is performed using the UV or visible absorbance an
error can still occur when an aglycone is used for calibration because the relevant conjugate is not available.
This review concentrates principally on post-2005 human bioavailability studies where metabolites and related compounds were identied by mass spectrometric-based methods without recourse to the use of enzyme hydrolysis prior to
analysis.
2. Flavan-3-ols
2.1. Green tea avan-3-ol monomers
Green tea, produced by aqueous infusion of young leaves of Camellia sinensis, is a rich source of several avan-3-ol monomers, typically, with ()-epigallocatechin-3-O-gallate, ()-epigallocatechin and ()-epicatechin predominating (Del Rio

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A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) 446467


Table 1
Pharmacokinetic analysis of avan-3-ols and their metabolites detected in plasma of healthy volunteers following the
ingestion of 500 mL of green tea.*

Flavan-3-ols (number of isomers)

Cmax (nmol/L)

Tmax (h)

(Epi)gallocatechin-O-glucuronide (1)
40 -O-Methyl-(epi)gallocatechin-O-glucuronide (1)
40 -O-Methyl-(epi)gallocatechin-O-sulfates (2)
(Epi)catechin-O-glucuronide (1)
(Epi)catechin-O-sulfates (2)
O-Methyl-(epi)catechin-O-sulfates (5)
()-Epigallocatechin-3-O-gallate (1)
()-Epicatechin-3-O-gallate (1)

126 19
46 6.3
79 12
29 4.7
89 15
90 15
55 12
25 3.0

2.2 0.2
2.3 0.3
2.2 0.2
1.7 0.2
1.6 0.2
1.7 0.2
1.9 0.1
1.6 0.2

Data expressed as mean values SE (n = 10).

Table 2
Quantication of the major groups of avan-3-ol metabolites excreted in urine 024 h after the
ingestion of 500 mL of green tea by ten human volunteers.*
Flavan-3-ol metabolites (number of isomers)

024 h excretion (lmol)

(Epi)gallocatechin-O-glucuronide (1)
40 -O-Methyl-(epi)gallocatechin-O-glucuronide (1)
40 -O-Methyl-(epi)gallocatechin-O-sulfates (2)
(Epi)gallocatechin-O-sulfates (3)
Total (epi)gallocatechin metabolites
(Epi)catechin-O-glucuronide (1)
(Epi)catechin-O-sulfates (2)
O-Methyl-(epi)catechin-O-sulfates (5)
Total (epi)catechin metabolites
Total avan-3-ol metabolites

6.5
4.4
19.8
2.6
33.3 (11.4%)
1.5 0.3
6.7 0.7
10.9 1.2
19.1 (28.5%)
52.4 (8.1%)

Data expressed as mean values in lmol standard error (n = 10). Italicised gures in parentheses
indicate amount excreted as a percentage of intake.

et al., 2004). In a recent study, ten healthy human subjects, who had been on a low avonoid diet for two days, consumed
500 mL of green tea containing 648 lmol of avan-3-ols after which plasma and urine were collected over a 24 h period and
analysed by HPLCMS2 (Stalmach et al., 2009b). Plasma contained a total of twelve metabolites, in the form of O-methylated,
sulfated and glucuronide conjugates of (epi)catechin and (epi)gallocatechin along with the native green tea avan-3-ols ()epigallocatechin-3-O-gallate and ()-epicatechin-3-O-gallate.1 The concentrations of these compounds in plasma after green
tea intake are presented in Fig. 1 and a pharmacokinetic analysis of the proles is presented in Table 1. None of the avan-3-ols
were present in the circulatory system before tea intake, but they were present in detectable quantities 30 min after. The main
component to accumulate was an (epi)gallocatechin-O-glucuronide with Cmax of 126 nmol/L and a Tmax of 2.2 h while an
(epi)catechin-O-glucuronide, probably the 30 -O-conjugate, attained a Cmax of 29 nmol/L with a 1.7 h Tmax. The unmetabolised
avan-3-ols, ()-epigallocatechin-3-O-gallate and ()-epicatechin-3-O-gallate, have Cmax values of 55 and 25 nmol/L after 1.6
and 2.3 h, respectively. The Tmax values ranged from 1.6 to 2.3 h (Table 1) and all the avan-3-ols and their metabolites were
present only in trace quantities after 8 h and were not detected in the 24 h plasma. This indicates a small intestine absorption,
a fact conrmed when a similar avan-3-ol metabolite plasma prole was obtained after the ingestion of green tea by humans
subjects with an ileostomy, having had their colon removed surgically (Stalmach et al., 2010a).
Urine collected 024 h after green tea consumption by healthy subjects with a functioning colon contained a similar spectrum metabolites of (epi)catechin and (epi)gallocatechin to plasma except for the appearance of two (epi)catechin-O-sulfates
and an absence of unmetabolised avan-3-ols (Table 2). The overall metabolite excretion was equivalent to 8.1% of the
648 lmol avan-3-ol intake. However, there was notable distinction between the excretion of (epi)catechin and (epi)gallocatechin metabolites. The recovery of (epi)gallocatechin metabolites was 11.4% while that of (epi)catechin metabolites was
28.5% of the ()-epicatechin and (+)-catechin intake (Table 2). These high levels of excretion are also in keeping with recoveries obtained in other studies with green tea, cocoa and related products (Baba et al., 2000; Auger et al., 2008; Mullen et al.,
2009) conrming that ()-epicatechin and (+)-catechin, in particular, are highly bioavailable being absorbed and excreted to

1
Analysis of avan-3-ols and their metabolites is somewhat more subtle than is generally appreciated. For instance, without reference compounds which
can be separated by reversed phase HPLC, MS is unable to distinguish between epicatechin and catechin metabolites and also epigallocatechin and
gallocatechin metabolites. We, therefore refer to metabolites as (epi)catechins or (epi)gallocatechins. To complicate matters further, although chiral
chromatography, using a mobile phase that is not compatible with on-line MS, can separate (+) and () avan-3-ol enantiomers, they co-chromatograph when
analysed by reversed phase HPLC (Donovan et al. 2006a). Although, some degree of interconversion may occur between optical isomers with, for instance ()epicatechin forming (+)-epicatechin (Gotti et al. 2006), for simplicity we have assumed that unmetabolised green tea avon-3-ols detected in teas, plasma and
ileal uid have not undergone such a change.

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A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) 446467

OH

OH

OH
OH

OH
HO

HO

OH
O

OH

OH

OH
HO

OH

OH

OH

OH

OH

( )-epigallocatechin (IF)

OH
OH
( )-epigallocatechin gallate (IF)

( )-epicatechin (IF)
OH

OH
OH
OH

HO

OH

OH
OH

HO

OH

OH

OH

OH

OH

OH

1-(3',4',5')-trihydroxyphenyl)3-(2'',4'',6''-trihydroxy)propan-2-ol*

OH

1-(3',4')-dihydroxyphenyl)3-(2'',4'',6''-trihydroxy)propan-2-ol*

pyrogallol (F,U)

OH

OH

OH
OH

OH

OH

( )-5-(3',4',5')-trihydroxyphenyl
- -valerolactone (F,U)

pyrocatechol (F,U)

OH
O

O
( )-5-(3',4')-dihydroxyphenyl
- valerolactone (F)

OH

OH
OH

OH
HOOC

OH

5-(3,4,5)-trihydroxyphenyl- -valeric acid*

HOOC
5-(3,4)-dihydroxyphenyl- -valeric acid (F)

OH
OH
HOOC

HOOC

4-hydroxyphenylacetic acid (F,U)

OH

OCH3
OH
HOOC
3-methoxy-4-hydroxyphenylacetic acid (U)

3-(3-hydroxyphenyl)propionic acid (F,U)

OH
HOOC

HOOC
4-hydroxybenzoic acid (U)

HOOC

H
N

OH

3-hydroxyphenylhydracrylic acid (U)

hippuric acid (U)


Fig. 2. Proposed pathways involved in the colonic catabolism and urinary excretion of green tea avan-3-ols. Following consumption of green tea more
than 50% of the ingested ()-epicatechin, ()-epigallocatechin and ()-epigallocatechin-3-O-gallate (blue structures) pass into the large intestine. When
incubated with fecal slurries these compounds are catabolised by the colonic microora probably via the pathways illustrated with red structures. Analysis
of urine after green tea consumption indicates that some of the colonic catabolites enter the circulatory and undergo further metabolism before being
excreted in urine. Green structures indicate catabolites detected in urine but not produced by fecal fermentation of ()-epicatechin, ()-epigallocatechin or
()-epigallocatechin-3-O-gallate. The dotted arrow between pyrogallol and pyrocatechol indicate this is a minor conversion. Double arrows indicate
conversions where the intermediate(s) did not accumulate and are unknown, although metabolism of 4-hydroxyphenylacetic acid to 3-methoxy-4hydroxyphenylacetic acid probably proceeds via 3,4-dihydroxyphenylacetic acid. Compounds detected in ileal uid after green tea consumption (IF);
catabolites detected in fecal slurries (F) and in urine (U); potential intermediates that did not accumulate in detectable quantities in fecal slurries(*).

A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) 446467

451

a much greater extent than other avonoids with the possible exception of isoavones (Donovan et al., 2006b; Crozier et al.,
2009; Manach et al., 2005).
Despite the relatively high absorption of avan-3-ols in the small intestine, Stalmach et al. (2010a) report that after ileostomists drank green tea containing 634 lmol of avan-3-ols, 69% of the intake was recovered in 024 h ileal uid as a mixture
of native compounds and metabolites. Thus, in volunteers with a functioning colon most of the ingested avan-3-ols will
pass from the small to the large intestine where their fate is a key part of the overall bioavailability equation. To mimic these
events two sets of experiments were carried out (Roowi et al., 2010). Firstly, 50 lmol of ()-epicatechin, ()-epigallocatechin and ()-epigallocatechin-3-O-gallate were incubated under anaerobic conditions in vitro with fecal slurries and their
degradation to phenolic acid by the microbiota monitored. A limitation of in vitro fermentation models is that it may not
fully depict the in vivo conditions. The use of fecal material may alter the bacterial composition and, thus, may not fully represent the microbiota present in the colonic lumen and mucosa, where catabolism occurs in vivo. Obviously, the accumulation and retention of the degradation products in the fermentation vessel makes collection, identication and quantication
of the metabolites easier but is not necessarily representative of the events that occur in vivo where the actual concentration
of a metabolite at any time interval is dependent on the combined rates of catabolism and absorption. However, the use of an
in vitro model provides information on the types of breakdown products formed, helps elucidate the pathways involved, and
the rate of catabolism can be determined.
To complement the in vitro incubations, phenolic acids excreted in urine 024 h after (i) the ingestion of green tea and
water by healthy subjects in a cross-over study, and (ii) the consumption of green tea by ileostomists, was also investigated
(Roowi et al., 2010). The data obtained in these studies provided the basis for the operation of the catabolic pathways that are
illustrated in Fig. 2. Some of these catabolites, such as 4-hydroxyphenylacetic acid and hippuric acid, were detected in urine
from subjects with an ileostomy indicating that they are produced in the body by additional routes unrelated to colonic degradation of avan-3-ols. It is, for instance, well known that there are pathways to hippuric acid from compounds such as
benzoic acid, quinic acid (Clifford et al., 2000), tryptophan, tyrosine and phenylalanine (Self et al., 1960; Grumer, 1961;
Bridges et al., 1970). None-the-less, the elevated urinary excretion of hippuric acid and 4-hydroxyphenylacetic acid, occurring after green tea consumption, is likely to be partially derived from avan-3-ol degradation. Earlier research showing statistically signicant increases in urinary excretion of hippuric acid after consumption of both green and black tea by human
subjects (Clifford et al., 2000; Mulder et al., 2005) supports this hypothesis.
Quantitative estimates of the extent of ring ssion of the avan-3-ol skeleton are difcult to assess because, as discussed
above, the production of some of the urinary phenolic acids was not exclusive to colonic degradation of avan-3-ols. If these
compounds, along with pyrogallol and pyrocatechol which are derived from cleavage of the gallate moiety from ()-epigallocatechin-3-O-gallate rather than ring ssion, are excluded, the excretion of the remaining urinary phenolic acids, namely
4-hydroxybenzoic acid, 3-methoxy-4-hydroxyphenylacetic acid, 3-hydroxyphenylhydracrylic acid and 5-(30 ,40 ,50 -trihydroxyphenyl)-c-valerolactone, after ingestion of green tea was 210 lmol compared to 38 lmol after drinking water. The 172 lmol
difference between these gures corresponds to a 39% of the 439 lmol of avan-3-ols entering the colon after consumption
of green tea. Added to this is the ca. 8% excretion of glucuronide, sulfate and methylated avan-3-ols originating from absorption in the small intestine. This estimate of a 47% recovery is nonetheless a minimum value because with the analytical methodology used some urinary catabolites will have escaped detection (Roowi et al., 2010). This will include glucuronide and
sulfate metabolites of ()-5-(30 ,40 ,50 -trihydroxyphenyl)-c-valerolactone, ()-5-(30 ,40 -dihydroxyphenyl)-c-valerolactone and
()-5-(30 ,50 -dihydroxyphenyl)-c-valerolactone which were detected after green tea consumption with a cumulative 0
24 h excretion corresponding to 16% of avan-3-ol intake (Li et al., 2000; Meng et al., 2002; Sang et al., 2008). More recently,
in a similar study in which urine was collected for 24 h after green tea intake, valerolactone metabolites were excreted in
quantities equivalent to 36% of intake (Del Rio et al., 2010). When added to the 47% recovery of Roowi et al. (2010) this gives
a total of 83% of intake. While this gure is obviously an approximation because of factors such as different volunteers, avan-3-ol intakes and analytical methodology, it does demonstrate that, despite substantial modication as they pass through
the body, there is a very high urinary recovery of avan-3-ols, principally in the form of colon- derived catabolites.
2.2. Proanthocyanidins
Proanthocyanidins (condensed tannins), the oligomeric forms of avan-3-ols, are among the most widespread polyphenols in plants (Ferreira and Slade, 2002) and also in the human diet (Gu et al., 2004) occurring at signicant levels in
foods including cocoa, grapes, apples, strawberries, and red wine. The average dietary intake of proanthocyanidins in the
United States has been estimated at 58 mg/day (Gu et al., 2004), but there is good evidence that this might be an underestimate because of problems associated with extraction from the food matrix prior to quantication (Tarascou et al., 2010).
Procyanidins are the commonest type of proanthocyanidin with (+)-catechin and ()-epicatechin their main constituent
units (Ferreira and Slade, 2002; Gu et al., 2004), but (epi)gallocatchin and (epi)afzelchin units are also found.
There are numerous feeding studies with animals and humans indicating that the oligomeric and polymeric avan-3-ols,
the proanthocyanidins, are not absorbed. Most pass unaltered to the large intestine where they are catabolised by the colonic
microora yielding a diversity of phenolic acids (Selma et al., 2009) including 3-hydroxyphenylhydracrylic acid and 4-Omethyl-gallic acid (Dprez et al., 2000; Gonthier et al., 2003; Ward et al., 2004) which are absorbed into the circulatory system and excreted in urine. There is one report based on data obtained in an in vitro model of gastrointestinal conditions, that
procyanidins degrade yielding more readily absorbable avan-3-ol monomers (Spencer et al., 2000) Subsequent studies have

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not supported this conclusion (Donovan et al., 2002; Rios et al., 2002; Tsang et al., 2005). There are two reports of minor
quantities of procyanidin dimers B1 and B2 being detected in human plasma after the respective consumption of a grape
seed extract (Sano et al., 2003) and a avan-3-ol-rich cocoa (Holt et al., 2002). In the latter study, the levels of the B2 dimer
in plasma were ca. 100-fold lower than those of avan-3-ol monomers. There is also evidence after oral dosing of dimethylated procyanidin dimers in the plasma of volunteers (Duweler and Rohdewald, 2000).
Recent studies using pure synthetic procyanidin B2 and [14C]procyanidin-B2, the commonest dimer consisting of two
()-epicatechin units with a 48 linkage, have helped clarify their in vitro gut ora catabolism (Appeldoom et al., 2009; Stoupi et al., 2010a,b) and rodent pharmacokinetics (Stoupi et al., 2010c). After oral dosing approximately 60% of the radioactivity was excreted in the urine after 96 h. Comparison of the total clearance and volume of distribution following oral and
i.v. doses has established in rodents that the vast majority of the radioactivity absorbed after oral dosing is in a form(s) very
different from the intact procyanidin dosed. This observation is consistent with the in vitro studies that show extensive
catabolism by the gut microora. The scission of the interavan bond represents a minor route (Appeldoom et al., 2009; Stoupi et al., 2010a), and the dominant products are a series of phenolic acids having one or two phenolic hydroxyls and between one and ve aliphatic carbons in the side chain. There are, in addition, some C6C5 catabolites with a side chain
hydroxyl, and associated lactones, and several diaryl-propan-2-ols, most of which are also produced from the avan-3-ol
monomers.
In vitro, procyanidin B2 also yields 24 dimeric catabolites, i.e. having a mass greater than the constituent monomer
()-epicatechin (290 a.m.u), which early in the incubation collectively accounted for some 20% of the substrate. Clearly,
these catabolites retain the interavan bond. One was identied tentatively as either 6- or 8-hydroxy-procyanidin B2. Thirteen were characterised as having been microbially reduced in at least one of the epicatechin units. Five contained an
apparently unmodied epicatechin unit but in at least one case this was shown to consist of the B-ring of the upper epi-

Fig. 3. Concentration of (A) quercetin-30 -O-sulfate, quercetin-3-O-glucuronide (B) a quercetin-O-glucuronide-O-sulfate, isorhamnetin-3-O-glucuronide and
a quercetin-O-diglucuronide in plasma from six human volunteers collected 06 h after the ingestion of onions containing 275 lmol of avonol glucosides.
Data expressed as mean values in nmol/L standard error (n = 6). Note that no quercetin metabolites were present in detectable amounts in plasma
collected 24 h after supplementation.

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catechin unit and the A-ring of the lower. It is not known whether these unique catabolites are produced in vivo, and if so,
whether they are absorbed (Stoupi et al., 2010c).
The biological effects of procyanidins are generally attributed to their more readily absorbed colonic breakdown products,
the phenolic acids, although there is a lack of detailed study in this area. There is, however, a dissenting view as trace levels
of procyanidins, in contrast to ()-catechin and (+)-epicatechin, inhibit platelet aggregation in vitro and suppress the synthesis of the vasoconstriction peptide, endothelin-1 by cultured endothelial cells (Corder, 2008). Supporting this view is a
study in which individual procyanidins were fed to rats after which dimers through to pentamers were detected in plasma
which was extracted with 8 mol/L urea, rather than the more traditional methanol/acetonitrile, which it was proposed prevented the irreversible binding of procyanidins to plasma proteins (Shoji et al., 2006). The procyanidins were, however,
administered by gavage at an extremely high dose, 1 g/kg body weight and it remains to be determined if procyanidins
can be similarly detected in urea-extracted plasma after the ingestion of more nutritionally relevant quantities by humans.
3. Flavonols
3.1. Onion quercetin-O-glucosides
Onions are a rich source of quercetin-40 -O-glucoside and quercetin-3,40 -O-diglucoside and Mullen et al. (2006) reported
on an acute human feeding study with 270 g of lightly fried onions containing a total of 275 lmol of avonol glucosides with
the main constituents being 143 lmol of the 40 -O-glucoside and 107 lmol of the 3,40 -O-diglucoside. Plasma and urine
collected over 24 h post-supplementation were analysed by HPLCMS2. Five principal quercetin metabolites, quercetin30 -O-sulfate, quercetin-3-O-glucuronide, isorhamnetin-3-O-glucuronide, a quercetin-O-glucuronide-O-sulfate, and a quercetin-O-diglucuronide were detected in plasma and their 06 h concentration proles are illustrated in Fig. 3. No quercetin
metabolites were detected in plasma collected either prior to consumption or 24 h after supplementation. A pharmacokinetic analysis of the ve plasma metabolites is summarised in Table 3 with information on maximum post-ingestion plasma
Cmax, Tmax and the elimination half-life (T1/2).
The two major metabolites, quercetin-30 -O-sulfate (Cmax 665 nmol/L) and quercetin-3-O-glucuronide (Cmax 351 nmol/L)
appeared in plasma within 30 min of the ingestion of onions, both had Tmax values lower than 1 h and T1/2 values of 1.71 and
2.33 h, respectively (Fig. 3, Table 3) (Mullen et al., 2006). The quercetin-O-diglucuronide had a lower Cmax and similar Tmax
and T1/2 values. The pharmacokinetic proles of isorhamnetin-3-O-glucuronide and the quercetin-O-glucuronide-O-sulfate
were somewhat different in that both had a much longer T1/2 and the glucuronide sulfate also had a much delayed Tmax
(Table 3). However, the total contribution of these two compounds to the overall absorption prole was minimal, having
no effect on the Tmax and only extending the T1/2 to 2.61 h. This T1/2 is much shorter than values obtained in earlier avonol
absorption studies (Hollman et al., 1996), almost certainly a consequence of the enhanced accuracy of analytical data available with improved chromatographic resolution and the application of HPLCMS2.
In keeping with the rapid Tmax and short plasma T1/2 values, most urinary excretion of quercetin metabolites occurred
within the rst 8 h after ingestion of the onions and over the 024 h collection period a total of 12.9 lmol of metabolites
were excreted which corresponds to 4.7% of intake (Mullen et al., 2006). The urinary metabolite prole was very different
from that of the plasma as shown in Table 4. The main plasma metabolite, quercetin-30 -O-sulfate was excreted only in trace
quantities while isorhanmetin-3-O-glucuronide and quercetin-O-diglucuronide that were minor components in plasma
were major urinary metabolites. Several other metabolites, including quercetin-30 -O-glucuronide and isorhamnetin-40 -Oglucuronide, which were present in trace quantities or absent from plasma were excreted in urine in substantial amounts
(Table 4).
The data obtained by Mullen et al. (2006) indicate absorption in the proximal part of the small intestine but provide no
information on the mechanisms involved or the efciency with which quercetin-40 -O-glucoside and quercetin-3,40 -O-diglucoside are hydrolysed and the aglycone enters the enterocyte. On the basis of the plasma metabolite prole, it is evident
that, following the release of the aglycone, quercetin is subjected to sulfation, glucuronidation and methylation. Arguably,
the short Tmax times of quercetin-30 -O-sulfate, quercetin-3-O-glucuronide and the quercetin-O-diglucuronide may be indicative of sulfation and glucuronidation occurring in the enterocyte prior to passage of the metabolites into the circulatory system. The longer plasma T1/2 value of isorhamnetin-3-O-glucuronide could reect post-absorption 30 -O-methylation of
Table 3
Pharmacokinetic analysis of quercetin metabolites in the plasma of volunteers after the consumption of 270 g of fried
onions containing 275 lmol of avonol glucosides.*

Metabolites

Cmax (nmol/L)

Tmax (h)

T1/2 (h)

Quercetin-30 -O-sulfate
Quercetin-3-O-glucuronide
Isorhamnetin-3-O-glucuronide
Quercetin-O-diglucuronide
Quercetin-O-glucuronide-O-sulfate

665 82
351 27
112 18
62 12
123 26

0.75 0.12
0.60 0.10
0.60 0.10
0.80 0.12
2.5 0.22

1.71
2.33
5.34
1.76
4.54

Data presented as mean values standard error (n = 6).

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Table 4
Quercetin metabolites detected in plasma and urine after the consumption of 270 g of fried onions containing 275 lmol of
avonol glucosides by six human volunteers.*
Metabolites

Plasma Cmax (nmol/L)

024 h Urinary excretion (nmol)

Quercetin-3-O-glucuronide
Quercetin-30 -O-sulfate
Isorhamnetin-3-O-glucuronide
Quercetin-O-glucuronide-O-sulfate
Quercetin-O-diglucuronide
Quercetin-30 -O-glucuronide
Isorhamnetin-40 -O-glucuronide
Quercetin-O-glucuronide-O-sulfate
Quercetin-O-glucoside-O-sulfates
Quercetin-O-glucoside-O-glucuronide
Methylquercetin-O-diglucuronides

351 27
665 82
112 18
123 26
51 13
Trace
Trace
n.d.
n.d.
n.d.
n.d.

912 149
Trace
1789 27
1229 190
2223 417
1845 193
700 11
1384 163
1214 156
163 23
1429 156

Data presented as mean values standard error (n = 6) n.d. not detected. Trace compound detected but not in sufcient
amounts for routine quantication.

Fig. 4. Concentration of quercetin-3-O-glucuronide and isorhamnetin-3-O-glucuronide in the plasma of six healthy human subjects 08 h after the
consumption of tomato juice containing 176 lmol of quercetin-3-O-rutinoside. Data expressed as mean values in nmol/L standard error (n = 6). Note
neither metabolite was present in detectable amounts in plasma collected 24 h after supplementation.

Table 5
Pharmacokinetic analysis of quercetin metabolites in the plasma of six human volunteers after the
consumption of 250 mL of tomato juice containing 176 lmol of quercetin-3-O-rutinoside.*

Metabolites

Cmax (nmol/L)

Tmax (h)

Quercetin-3-O-glucuronide
Isorhamnetin-3-O-glucuronide

12 2
4.3 1.5

4.7 0.3
5.4 0.2

Data presented as mean values standard error (n = 6).

quercetin-3-O-glucuronide in the liver. Likewise, the delayed T1/2 of the quercetin-O-glucuronide-O-sulfate could be a consequence of post-absorption sulfation of quercetin-3-O-glucuronide and/or glucuronidation of quercetin-30 -O-sulfate. The
unusually marked differences in the plasma and urinary metabolite proles are suggestive of extensive phase II metabolism
and rapid turnover and removal from the circulatory system via the kidneys, but where in the body these conversions occur
remains to be determined.

3.2. Tomato juice quercetin-3-O-rutinoside


In a human study parallel to that carried out with quercetin glucosides in onions, the bioavailability of quercetin-3-Orutinoside was investigated by feeding 250 mL of tomato juice containing 176 lmol of the quercetin rhamnoseglucose
disaccharide (Jaganath et al., 2006). In this instance only two metabolites were detected in plasma, quercetin-3-O-glucuronide and isorhamnetin-3-O-glucuronide (Fig. 4). They were present in ca. 25-fold lower quantities than in the onion study
with respective Cmax values of 12 and 4.3 nmol/L. The Tmax times were extended to ca. 5 h (Table 5) indicating absorption in

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455

Fig. 5. Proposed pathway for colon bacteria-mediated catabolism of quercetin-3-O-rutinoside in the large intestine resulting in the production of 3,4dihydroxyphenylacetic acid and smaller quantities of 3-hydroxphenylacetic acid with the subsequent hepatic conversion of 3,4-dihydroxyphenylacetic acid
to 3-methoxy-4-hydroxyphenylacetic acid prior to urinary excretion. Dotted arrow indicates a minor pathway.

the large rather than the small intestine. A total of nine methylated and glucuronidated quercetin metabolites were detected
in urine but some volunteers excreted only 34 metabolites. The overall level of metabolite excretion ranged from 0.02% to
2.8% of quercetin-3-O-rutinoside intake, which is probably a reection of variations in the colonic microora of the individual volunteers. Conrmation of large intestine absorption was obtained in a separate feeding study using subjects with an
ileostomy. Unlike the subjects with a functioning colon, neither plasma nor urinary metabolites were detected and ileal uid
collected post-tomato juice consumption contained 86% of the ingested quercetin-3-O-rutinoside. The urine collected from
the ileostomy volunteers as well as not containing quercetin metabolites, also lacked the phenolic acid catabolites 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylacetic acid. These catabolites were
present in the urine of the volunteers with a functioning colon in quantities corresponding to 22% of quercetin-3-O-rutinoside intake (Jaganath et al., 2006).
It was proposed that the rutinose sugar moiety of quercetin-3-O-rutinoside is not cleaved by the action of either LPH or
CBG during passage through the small intestine and that, as a consequence, following the release of quercetin through the
action of colonic bacterial enzymes, low level production and absorption of methylated and glucuronidated quercetin metabolites takes place in the large intestine. However, most of the quercetin undergoes ring ssion, releasing substantial quantities of 3,4-dihydroxyphenylacetic acid and smaller quantities of 3-hydroxphenylacetic acid. Subsequent methylation of
3,4-dihydroxyphenylacetic acid yields 3-methoxy-4-hydroxyphenylacetic acid (Fig. 5), with all three catabolites being excreted in urine. Most of these steps probably occur in the large intestine mediated by local colonic microbes. Enterobacter
species are among the colonic bacteria that are able to hydrolyse a rhamnosyl moiety by releasing a- and b-rhamnosidases
to cleave the sugar moiety (Braune et al., 2005). However, some post-absorption metabolism may also occur as a result of the
involvement of hepatic enzymes, such as O-methyltransferases.
It is of interest to note that after feeding tomato juice containing quercetin-3-O-rutinoside, where absorption is restricted
to the large intestine, no quercetin sulfates were detected either in plasma or urine. In marked contrast, after feeding onions
containing quercetin-O-glucosides that are transformed in the small intestine, quercetin-30 -O-sulfate was the major plasma
metabolite and other sulfated metabolites accumulated in urine, as described in Section 3.1. This indicates that sulfation of
quercetin occurs exclusively in the wall of the small intestine and that data obtained in ex vivo studies in which quercetin3-O-glucuronide was converted to quercetin-3-O-sulfate by liver cell-free preparations (OLeary et al., 2003) may not accurately reect in vivo sulfation. It also suggests that formation of mixed conjugates such as quercetin-O-glucuronide-O-sulfate
might occur following glucuronide excretion in bile and re-absorption in the large intestine, and this would be consistent
with Tmax values of <1 h for quercetin-3-O-glucuronide compared with ca. 3 h for the mixed conjugate (Mullen et al., 2006).

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Table 6
Quantities of hesperetin and naringenin metabolites excreted in the
urine of eight human volunteers 024 h after the consumption of
250 mL of orange juice containing 168 lmol of hesperetin-7-O-rutinoside and 12 lmol of naringenin-7-O-rutinoside.*
Metabolites

nmol

Hesperetin-7-O-glucuronide
Hesperetin-O-glucuronide
Hesperetin-O-glucuronide
Hesperetin-O-diglucuronide
Hesperetin-O-glucuronide-O-sulfates

1373 471
3662 1483
2319 420
767 361
2841 699

Total hesperetin metabolites


Naringenin-7-O-glucuronide
Naringenin-40 -O-glucuronide
Naringenin-O-diglucuronide

10,962 (6.5%)
1001 344
976 389
98 46

Total naringenin metabolites

2075 (17.3%)

Data expressed as mean values standard error (n = 8). Italicised


gures in parentheses indicate excretion of hesperetin and naringenin
metabolites as a percentage on intake.

4. Orange juice avanones


Several early studies, where analyses involved the use of enzyme hydrolysis, have shown that orange juice avanone rutinosides are absorbed in the large intestine (Erlund et al., 2001; Manach et al., 2003). In a more recent study metabolites were
analysed by HPLCMS2 after volunteers consumed 250 mL of orange juice containing 168 lmol of hesperetin-7-O-rutinoside
and 12 lmol of naringenin-7-O-rutinoside (Mullen et al., 2008). The hesperetin-7-O-rutinoside dose was therefore very similar to that of quercetin-3-O-rutinoside in the study outlined in Section 3. Plasma contained hesperetin-7-O-glucuronide and
a second unassigned hesperetin-O-glucuronide and the combined Cmax for both metabolites was 922 nmol/L at a Tmax of 4.4 h.
The two hesperetin metabolites were also excreted in urine along with a third hesperetin-O-glucuronide, two hesperetin-Oglucuronide-O-sulfates and a hesperetin-O-diglucuronide. These marked differences in the plasma and urinary hesperetin
metabolite proles demonstrate that substantial post-absorption phase II metabolism is occurring. The quantities of these
metabolites excreted 024 h after ingestion corresponded to 6.5% of hesperetin-7-O-rutinoside intake. Although no naringenin metabolites were detected in plasma, urine contained naringenin-7-O-glucuronide, narigenin-40 -O-glucuronide and a
naringenin-O-diglucuronide in amounts equivalent to 17.3% of the ingested naringenin-7-O-rutinoside (Table 6) (Mullen
et al., 2008a). The differing levels of excretion of hesperetin and naringenin metabolites, relative to the amounts ingested,
is a trend that has been observed in some but not all avanone feeding studies (Manach et al., 2005). While it could be a
dose effect reecting the higher intake of the hesperetin conjugate, it is more likely to be due to naringenin-7-O-rutinoside
being more bioavailable than hesperetin-7-O-rutinoside indicating that the 30 and 40 substituents impact on absorption.
Although both are absorbed in the large intestine, the 922 nmol/L Cmax of the hesperetin-O-glucuronides is more than
50-fold higher than that of the quercetin-3-O-rutinoside metabolites (Table 5), and the amount fed were extremely similar.
This, coupled with the higher level of excretion of the orange juice metabolites, indicates that hesperetin-7-O-rutinoside is
absorbed from the large intestine much more effectively than quercetin-3-O-rutinoside. This may be a consequence of the
hesperetin-7-O-rutinoside being converted to glucuronides in the large intestine more efciently than quercetin-3-O-rutinoside, perhaps because it is less prone to degradation by colonic bacteria. Among the avanone metabolites excreted in quantity were two hesperetin-O-glucuronide-O-sulfates (Table 6). This contrasts with the absence of sulfated naringenin
metabolites and with metabolites derived from large intestine absorption of quercetin-3-O-rutinoside in the tomato juice
feed (Section 3.2). Thus, there appears to be clear differences in the substrate specicity of avonoid SLTs in the large intestine and/or the liver.
Analysis of phenolic acids excreted in urine after the ingestion of orange juice indicates that the hesperetin, released
through colonic bacteria-mediated deglycosylation, as well as being glucuronidated, undergoes ring ssion and is catabo-

Table 7
Quantities of key phenolic acids excreted in human urine 024 h after drinking 250 mL of water and 250 mL of orange juice, containing 168 lmol hesperetin-7O-rutinoside and 12 lmol naringenin 7-O-rutinoside, with and without yoghurt.*

Water
Orange juice
Orange juice with yoghurt
*

02 h

25 h

510 h

1024 h

Total (024 h)

1.8 0.4
0.5 0.0
0.5 0.0

1.1 0.2
1.7 0.2
1.7 0.2

1.2 0.2a
26 2b
26 2b

2.7 0.5a
34 12b
34 12b

6.7 1.8a
62 18b
62 18b

Data were expressed in lmol as mean values standard error (n = 5). Quantications based on the combined levels of 3-hydroxyphenylacetic acid,
3-hydroxyphenylhydracrylic acid, dihydroferulic acid and 3-methoxy-4-hydroxyphenylhydracrylic acid and 3-hydroxyhippuric acid presented in Table 2.
Means in columns followed by different superscript letters are signicantly different at p < 0.05.

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OH
OCH3
O

O
O

HO
HO

HO
HO
OH
HO

OH O

hesperetin-7-O-rutinoside

OH
OCH3
HO

OH O

O-demethylation

hesperetin
H2O

O-demethylation
O-methylation
H2O

OH

O-demethylation

H2O

OCH3
OH

H2O

OH

HO

HO

HOOC

HOOC
3-methoxy-4-hydroxyphenyl
hydracrylic acid

COOH

3-hydroxyphenyl
hydracrylic acid

3-hydroxyphenyl
acetic acid

O-demethylation
conjugation
conjugation

H2O

H2O

OH

OCH3
OH
O
NH
HOOC

HOOC
4-hydroxy-3-methoxyphenylpropionic acid
(dihydroferulic acid)

3-hydroxyhippuric acid

Fig. 6. Proposed catabolism of hesperetin-7-O-rutinoside in humans.

lised producing 3-hydroxyphenylhydracrylic acid of undetermined chirality, 3-hydroxyphenylacetic acid, 3-methoxy-4hydroxyphenylhydracrylic acid, dihydroferulic acid and 3-hydroxyhippuric acid. (Roowi et al., 2009). The overall level of
the ve phenolic acids excreted 024 h after drinking water was 6.7 lmol, and this rose to 62 lmol, equivalent to 37% of
the ingested avanones, following orange juice consumption (Table 7). When the 250 mL of orange juice was ingested with
150 mL of full-fat natural yoghurt, phenolic acid excretion fell back markedly to 9.3 lmol. This did not appear to be due to a

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difference in mouth to caecum transit time of the head of the meal, as measured with breath hydrogen production. Arguably,
there may have been a slowing of the bulk of the meal reaching the large intestine, which may then have altered the catabolism of the avanones to phenolic acids by the colonic microbiota. Exactly how this is brought about is a topic that requires
further investigation, especially as it is an event that is not exclusive to avanones and yoghurt, as Urpi-Sarda et al. (2010)
have reported when cocoa was consumed with milk rather than water there was reduced excretion of nine out of fteen
phenolic acids derived from colonic degradation of avan-3-ols.
The phenolic acids that accumulated in urine after orange juice consumption may have originated from the breakdown of
hesperetin-3-O-rutinoside via the pathways illustrated in Fig. 6 (Roowi et al., 2009). In this scheme, degradation of hesperetin-7-O-rutinoside starts with deglycosylation to form hesperetin (Schoefer et al., 2003). The C-ring is then opened by
cleavage of the ether-O-linkage followed by dehydrogenation resulting in the formation of 3-methoxy-4-hydroxyphenylhydracrylic acid. This CC cleavage probably occurs between the ether-O-linkage and the A-ring and between C4
and the A-ring as illustrated in Fig. 6. As no phloroglucinol (1,3,5-trihydroxybenzene) was detected in urine, cleavage of
the ether-O-linkage and C2, and of C4 and the A-ring, are unlikely. 3-Hydroxyphenylhydracrylic acid may be produced from
the same CC cleavage of the hesperetin C-ring followed by O-demethylation. Alternatively, it could also arise from Odemethylation of 3-methoxy-4-hydroxyphenylhydracrylic acid. These two compounds may then link to dihydroferulic acid,
3-hydroxyhippuric acid and 3-hydroxyphenylacetic acid via the routes indicated in Fig. 6. Most of these steps probably occur
in the large intestine mediated by the colonic microora where Enterobacteriaceae, along with a number of other human
intestinal bacteria, including Eubacterium limosum and Escherichia coli, possess O-demethylase activity (Grbic-Galic, 1986;
DeWeerd et al., 1988; Hur and Rai, 2000). However, some post-absorption metabolism may also occur as a result of the
involvement of hepatic enzymes, such as O-methyltransferases.
5. Isoavones
Isoavones, though not a major component of the European diet, have always been considered as the better absorbed
dietary avonoids, with urinary excretion of metabolites typically being 2050% of intake (Manach et al., 2005; Donovan
et al., 2006b). A study by Rfer et al. (2008) in which seven male volunteers were given either pure daidzein or pure daidzein-7-O-glucoside, both at a dose of 3.9 lmol/kg body weight, has demonstrated that the plasma Cmax, at ca. 89 h, was three
to six times greater after consumption of the glucoside, which is dominant in soya compared with the aglycone, the main
component in fermented soya products. The metabolites, quantied after deconjugation, included dihydrodaidzein, O-desmethylangolensin, 6-hydroxy-daidzein, 8-hydroxy-daidzein and 30 -hydroxy-daidzein. One of the seven volunteers also produced equol. The bioavailability reported in this study contrasts markedly with the results obtained when tablets containing
a crude preparation of soya saponins and either daidzein and genistein aglycone or their mixed glycosides was given to eight
volunteers at doses of 0.11 and 1.7 mmol (Izumi et al., 2000). At the higher dose, the isoavone aglycone mixture produced
plasma Cmax concentrations up to ve times higher than the preparation containing the daidzein and genistein glycosides.
The Tmax in this study was ca. 4 h which is much earlier than the 89 h reported by Rfer et al. (2008). These differences
are not easily explained but a possible role for the saponins is suggested.
A study in which two volunteers consumed 50 g of kinako (baked soya bean powder) containing 66 lmol of daidzein,
106 lmol of genistein, 120 lmol of diadzein-7-glucoside and 205 lmol of genistein-7-O-glucoside suspended in 300 mL
of cows milk, used HPLCMS to establish the presence of daidzein, genistein, daidzein-40 -O-glucuronide, genistein-40 -O-glucuronide, daidzein-7-O-glucuronide, genistein-7-O-glucuronide, daidzein-40 -O-sulfate, genistein-40 -O-sulfate, daidzein-7-Osulfate and genistein-7-O-sulfate in plasma in the 17 h period post-consumption (Hosoda et al., 2008). Traces of the glucosides of genistein and daidzein were also detected in plasma 1 h post-consumption. The short duration of the study prevented determination of Tmax, Cmax and T1/2. The aglycone concentration never exceeded ca. 200 nmol/L with genistein
exceeding daidzein for one volunteer but the reverse for the other. Within the period studied the isoavone metabolites
never exceeded ca. 3 lmol/L in total, and no single metabolite exceeded 0.8 lmol/L. Conjugation for both isoavones occurred preferentially at the C7 position, but the ratio of glucuronides to sulfates varied with time.
Although, as mentioned above, traces of the glucosides have been detected in plasma most absorption occurs after deconjugation. The rst phase of absorption, up to one hour, is impaired in lactose malabsorbers, suggesting a role for LPH, but
overall this is compensated by microbial hydrolysis, and total absorption was not signicantly affected by lactose malabsorption (Tamura et al., 2008). The ability of ileostomists to absorb isoavone glycosides, not signicantly different from volunteers with an intact colon, conrms that absorption occurs in the upper gastrointestinal tract. However, urinary excretion of
the microbial metabolites equol, dihydrodaidzein and dihydrogenistein by ileostomists was lower than that of healthy subjects, and the ileostomy group contained fewer equol-producers. Equol was characterised as the S-enantiomer (Wang et al.,
2005; Walsh et al., 2007).
6. Anthocyanins
Anthocyanins, for people who eat berries and drink red wine on a routine basis, are major dietary components. Although
there are exceptions, unlike other avonoids that are absorbed and excreted, most anthocyanins do not appear to undergo
extensive metabolism of the parent glycosides to glucurono, sulfo or methyl derivatives (McGhie et al., 2003; Miyazawa

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459

et al., 1999; Milbury et al., 2002; Cooney et al., 2004; Ichiyanagi et al., 2005). In feeding studies with animals and humans,
typically ca. 0.1% of the quantities ingested, and sometimes much less, has been detected in urine. The available data imply
that the determinants of absorption and excretion are inuenced by not only the nature of the sugar moiety but also the
structure of the anthocyanidin aglycone (McGhie et al., 2003; Wu et al., 2005).
The complex array of information on anthocyanin bioavailability obtained with human and animal test systems has been
reviewed by Prior and Wu (2006). One of the reasons for the complicated picture that has emerged is that many feeds have
involved berry or fruit supplements containing several structurally diverse anthocyanins. For instance, black raspberries contain ve cyanidin-3-O-sugar conjugates ranging from mono to trisaccharides, while blueberries contain 12 anthocyanins,
principally 3-O-glucosides, galactosides and arabinosides of cyanidin, delphinidin, petunidin and malvidin (McGhie et al.,
2003; Cho et al., 2004). This makes the complex anthocyanin content of plasma and urine exceedingly difcult, if not impossible to assess, in terms of absorption, excretion and potential phase I and phase II metabolism, especially when 30 -O-methylation can convert cyanidin to peonidin, and delphinidin to petunidin, and 50 -O-methylation converts petunidin to malvidin.
Much simpler anthocyanin proles are found in strawberries and blackberries, both of which contain one predominant
anthocyanin, pelargonidin-3-O-glucoside in the former and cyanidin-3-O-glucoside in the latter (Wu et al., 2006) As a consequence, data on anthocyanins bioavailability after ingestion of these berries by humans are potentially more straight forward to interpret.
6.1. Strawberries and blackberries
In a recent human study 200 g of strawberries containing 222 lmol of pelargonidin-3-O-glucoside and trace quantities of
pelargonidin-3-O-rutinoside (13 lmol) and cyanidin-3-O-glucoside (6 lmol) were consumed by six subjects after which
plasma and urine were collected over a 24 h period (Mullen et al., 2008b). The plasma contained a pelargonidin-O-glucuronide in substantial quantities along with non-quantiable amounts of three other pelargonidin-O-glucuronides and pelargonidin-3-O-glucoside, the latter perhaps derived from removal of the 600 -rhamnose moiety from pelargonidin-3-Orutinoside. The main pelargonidin-O-glucuronide had a Cmax of 274 24 nmol/L, a Tmax of 1.1 0.4 h, in keeping with small
intestine absorption, and T1/2 of 2.1 0.7 h. All the plasma anthocyanins also appeared in urine along with small quantities of
pelargonidin aglycone and a pelargonidin-O-sulfate. The pelargonidin-3-O-glucuronide that was the main metabolite in plasma was by far the predominant component in urine accounting, over 024 h, for 1498 nmol of a total of 1672 nmol of anthocyanins excreted. This corresponds to 0.75% of pelargonidin-3-O-glucoside intake. There is, therefore, no evidence of
substantial post-absorption metabolism prior to excretion.
In an earlier feeding study with strawberries, Felgines et al. (2003) reported a urinary excretion equivalent to 1.8% of the
179 lmol of ingested pelargonidin-3-O-glucoside and this is also similar to values obtained in a 1560 lmol dose study with
strawberries (Carkeet et al., 2008). These urinary recoveries are high for anthocyanins and suggest that pelargonidin-3-Oglucoside is absorbed more readily that other anthocyanins. In a separate human feeding study with 200 g of blackberries
containing 960 lmol of cyanidin-3-O-glucoside, 12 anthocyanins were excreted including unmetabolised cyanidin-3-Oglucoside, a cyanidin-O-glucuronide and a peonidin-O-glucuronide in quantities equivalent to 0.16% of intake (Felgines
et al., 2005). This suggests that pelargonidin-3-O-glucoside, while it is metabolised to fewer products, may be absorbed more
readily than cyanidin-3-O-glucoside. However, the high cyanidin-3-O-glucoside content of the blackberry supplement may
have had an impact on absorption and/or excretion. In the circumstances, it would be of interest to carry out a feeding study
and to determine not only urinary but also plasma anthocyanin prole after ingestion of blackberries and strawberries containing similar quantities of anthocyanins.
6.2. Blueberries and blackcurrants
Ingestion of a blueberry extract, containing 1.2 g of a complex array of anthocyanins, with a high-fat meal resulted in an
increase in serum ORAC, but not TEAC, antioxidant capacity 1 and 4 h after intake by human volunteers. This was associated
with the appearance of trace levels of blueberry anthocyanins in serum, but as they accounted for only 0.0020.003% of intake, it appears unlikely that the anthocyanins themselves were directly responsible for the increase in antioxidant status
(Kay and Holub, 2002; Mazza et al., 2002). In a separate human study, following consumption of a blueberry extract containing 439 mg of anthocyanins, trace quantities of unmetabolised anthocyanins corresponding to 0.02% of intake were detected
in urine collected over a 7 h post-ingestion period (McGhie et al., 2003). In another study, Wu et al. (2002) fed 690 mg of
blueberry anthocyanins to 6070 year old women and total urinary excretion during the rst 6 h after consumption was
23.2 lg, which is equivalent to 0.004% of intake.
Human urinary excretion of blackcurrant anthocyanins, like those from blueberry, is low with ca. 0.06% of intake being
recovered as unmetabolised native anthocyanins (McGhie et al., 2003). A similar recovery level in urine was reported with
weanling pigs but in this case ca. 25% of the anthocyanins were methyl and/or glucuronide metabolites (Wu et al., 2005).
6.3. Accumulation of anthocyanins in body tissues
There is evidence from a number of sources that consumption of berry extracts can delay the decline of various aspects of
cognitive function in elderly rats (Willis et al., 2009). There is, however, contradictory evidence as to whether avonoids

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themselves cross the bloodbrain barrier. In one of the early studies, Andrs-Lacueva et al. (2005) detected trace levels of
several anthocyanins in brains of rats that had received a diet supplemented with a blueberry extract, containing unspecied
amounts of anthocyanins, for a period of 10 weeks.
In a further study, 18 h after feeding pelargonidin to rats by gavage at a dose of 50 mg/kg, the unmetabolised anthocyanidin was detected in brains at a concentration of ca. 0.2 nmol/g (fresh weight) (El Mohsen et al., 2006). In contrast, anthocyanins did not accumulate in detectable amounts in the brains of rats obtained up to 24 h after acute supplementation by
gavage with 2.8 mL of raspberry juice, which is a nutritional relevant dose as it is equivalent to a 70 kg human drinking
700 mL of juice (Borges et al., 2007). In contrast, following a 4-week supplementation of pigs with a blueberry extract, containing undened amounts of anthocyanins, 300 pg of anthocyanins/g was detected in cerebellum tissue and 700 pg/g in eye
tissue. Anthocyanins, however, were also found in the tissues of pigs that did not receive the blueberry supplement (Kalt
et al., 2008). In another study, oral ingestion of 100 mg/kg of blackcurrant anthocyanins by rats resulted in a plasma anthocyanin Cmax of 1.9 lg/ml 30 min after ingestion and a maximum concentration of anthocyanins in the whole eye of 115 ng/g,
also after 30 min (Matsumoto et al., 2006). However, in a study with male mice, feeding a bilberry extract for two weeks
resulted in the accumulation of anthocyanins in detectable amounts in plasma, liver, kidney, testes and lungs but not in other
tissues including the brain and eyes (Sakakibara et al., 2009).
One of the possible reasons for the seemingly contradictory data obtained in these studies could be the use of extracts
containing very high amounts of anthocyanins that could not possibly be ingested as part of a normal berry-based diet.
In a recent study, where this was not the case, greennches consumed one blackberry per day for a period of 14 days
and, ca. 18 h after the last feed, the birds were sacriced, the brain removed and extracted. Analysis of the extracts by HPLC
with high resolution MS revealed the presence of unmetabolised cyandin-3-O-glucoside in amounts ranging from
12148 pmol/brain with an average of 40 16 pmol (Mullen et al., 2010).
6.4. Stability of anthocyanins
A point of note is that anthocyanins are readily distinguished from other avonoids as they undergo re-arrangements in
response to pH. The red avylium cation predominates at pH 13 but as the pH increases to 4 and above the colourless carbinol pseudobase is the major component along with smaller amounts of the colourless chalcone pseudobase and the blue
quinoidal base (Clifford, 2000). Anthocyanins are traditionally extracted and analysed in acidic medium as the red avylium
cation is the most stable form. However, it is not known what forms predominate in vivo. The limited available experimental
evidence indicates that, in the acidic conditions that prevail in the stomach, anthocyanins are in the red avylium form but
once they enter more basic conditions in the small intestine the carbinol pseudobase is likely to predominate. It could be that
the colourless carbinol pseudobase is the main form in the small intestine where it undergoes limited absorption, possibly
being metabolised to conjugates that are overlooked because they cannot be converted to red avylium forms prior to the
eventual analysis. Recent data, however, have shown anthocyanins may be more stable in the upper gastrointestinal tract
than previously envisaged. Following acute ingestion of 300 g of raspberries containing 204 lmol of anthocyanins, principally in the form of cyanidin-3-O-glucoside and cyanidin-3-O-(200 -O-glucosyl)rutinoside, by ileostomists there was a 40%
recovery in ileal uid (Gonzlez-Barrio et al., 2010). In a similar study with blueberries there were anthocyanin recoveries
of up to 85%, depending upon the sugar moiety, in ileal uid (Kahle et al. 2006). Thus, in healthy subjects with an intact colon,
substantial quantities of anthocyanins are likely to pass from the small to the large intestine. There are reports that the microbiota result in cyanidin-based anthocyanins undergoing cleavage of the sugar moiety followed by ring ssion of the released cyanidin which produces 3,4-dihydroxybenzoic acid (protocatechuic acid) (Aura et al., 2005; Vitaglione et al.,
2007; Galvano et al., 2008).
7. Ellagitannins
Studies into the bioavailability of ellagitannins following ingestion by humans have been carried out mainly with pomegranate which contains punicalin and punicalagins (Cerd et al., 2004; Seeram et al., 2006; Mertens-Talcott et al., 2006) but
the fate of ellagitannins in strawberries, raspberries, walnuts and oak-aged wines has also been investigated (Cerd et al.,
2005).

Fig. 7. Proposed colonic degradation of pomegranate ellagitannins to urolithins.

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461

After drinking a pomegranate juice containing 318 mg of punicalagins, ellagic acid was detected in plasma with Cmax of
60 nmol/L at a Tmax of 0.98 h suggesting acid hydrolysis of at least some of the ellagitannins releasing free ellagic acid, which
is absorbed directly from the stomach or the proximal small intestine (Seeram et al., 2006). Also detected in the plasma of
some but not all volunteers, mainly 6 h after supplementation, were urolithin A, urolithin A-3-O-glucuronide, urolithin B
and a methylated urolithin B. Urinary metabolites which began to appear after 12 h included urolithin A-3-O-glucuronide,
urolithin B-3-O-glucuronide and 3,8-O-dimethylellagic acid-2-O-glucuronide, and excretion continued for up to a further
36 h. None of these compounds were quantied and there was much subject-to-subject variation in the spectrum of metabolites produced. This implies that when the ellagitannins and/or ellagic acid reach the distal part of the small intestine and the

Fig. 8. Plasma pharmacokinetic proles of circulating chlorogenic acids and metabolites, following the ingestion of 200 mL of coffee containing 412 lmol of
chlorogenic acids by healthy human subjects.

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colon they are metabolised by the gut microora producing urolithins A and B which are then absorbed along with ellagic acid
and subjected to the action of Phase II UGTs and/or methyltransferases before being excreted in urine (Seeram et al., 2006).
In a further study in which volunteers ingested one litre of pomegranate juice containing 4.37 g of punicalagins on a daily
basis for ve days, circulating urolithin levels reached a concentration of 18.6 lmol/L (Cerd et al., 2004). Feeding human
subjects a single dose of strawberries, raspberries, walnuts and oak-aged red wine, all of which contain ellagitannins, resulted in excretion of urolithin A-3-O-glucuronide in quantities equivalent to 2.8% (strawberries), 3.4% (raspberries), 6.5%
(oak-aged red wine) and 16.6% (walnuts) of intake (Cerd et al., 2005).
Following the ingestion of a raspberry supplement, containing 123 lmol of ellagitannins, mainly as sanguiin H-6, by
healthy human subjects, urolithin-A-O-glucuronide, two of its isomers and urolithin B-O-glucuronide were excreted in
748 h urine (Gonzlez-Barrio et al., 2010). There was marked variation in the urolithin prole of individual volunteers with
recoveries relative to the ingested ellagitannins ranging from 08.2%, indicative of differences in the colonic microora
responsible for ellagitannin degradation. The 08.2% conversion of ellagitannins to urolithins may well be an overestimate
as in theory 1 mol of sanguiin H-6 can produce 4 mol of ellagic acid, each of which can be metabolised to urolithins. To what
extent ellagitannins are also converted to phenolic acids in the colon remains to be investigated.
In time course incubations of pomegranate punicalagins with fecal slurries carried out by Bialonska et al. (2010), the
appearance of urolithins, but not phenolic acids, was monitored. The tetrahydroxylated urolithin D was the initial product
of microbial transformation, followed by urolithin C and urolithin A. Although no urolithin B was detected, these observations suggest that colonic degradation of pomegranate ellagitannins follows the pathway illustrated in Fig. 7. The absence
of glucuronidated urolithins in this in vitro system implies that in vivo glucuronidation of urolithins takes place in the wall
of the large intestine or is a post-absorption event occurring prior to urinary excretion.
The most detailed study on ellagitannins to date has been carried out with Iberian pigs which in their natural habit feed
on oak acorns which are a further source of ellagitannins (Espn et al., 2007). The pigs were given an average of 4.04 kg of
acorns on a daily basis for 117 days after which tissues and body uids were processed and analysed by HPLCMS3. A total
of 31 ellagitannin-derived metabolites were detected, including 25 urolithin and six ellagic acid derivatives. A summary of
the complex picture that emerges is that in the jejunum the acorn ellagitannins release ellagic acid which the intestinal
microora metabolise sequentially producing urolithin D, urolithin C, urolithin A and urolithin B, as illustrated in Fig. 7.
These urolithins are absorbed preferentially as their lipophilicity increases with plasma containing mainly urolithin A-3O-glucuronide and urolithin B-3-O-glucuronide with traces of a urolithin C-O-glucuronide and 3,8-O-dimethylellagic acid2-O-glucuronide. The urolithin A and C glucuronides were the major components in urine. Among the 26 conjugated metabolites detected in bile were glucuronides and methyl glucuronides of ellagic acid and substantial quantities of urolithin A, C
and D derivatives. This indicates substantial hepatic metabolism and active enterohepatic circulation, and also explains the
persistence of urinary urolithin metabolites observed in the human studies. No ellagitannins or their metabolites were detected in body tissues outside the gastrointestinal tract (Espn et al., 2007).
8. Chlorogenic acids
Coffee, in different preparations, is widely consumed throughout the world, and contains high levels of chlorogenic acids
with a single serving providing between 20 and 675 mg depending on the type of roast and the volume consumed. Regular
consumers easily have an intake in excess of 1 g per day (Clifford, 1999; Stalmach et al., 2006). Chlorogenic acids are a group
of compounds comprising hydroxycinnamates, such as caffeic acid, ferulic acid, and p-coumaric acid, linked to quinic acid to
form a range of conjugated structures known as caffeoylquinic acids (CQA), feruloylquinic acids (FQA), and p-coumaroylquinic acids all of which exist in several isomeric forms (Clifford, 1999). As well as these compounds coffee also contains
dicaffeoylquinic acids and caffeoylquinic acid lactones (CQAL).
The literature describing the catabolism of coffee chlorogenic acids in human subjects is scarce and, in some instances,
contradictory. A study by Nardini et al. (2002) observed an increase of conjugated caffeic acid in plasma after the ingestion
of 200 mL of coffee, while Rechner et al. (2001) detected ferulic acid, isoferulic acid, dihydroferulic acid, 3-methoxy-4hydroxybenzoic acid, hippuric acid and 3-hydroxyhippuric acid in urine from ve human subjects after three ingestions
of two cups of coffee at 4-h intervals. Monteiro et al. (2007) reported the presence of unmetabolised CQAs in human plasma
at a Cmax of 7.7 lmol/L 2.3 h (Tmax) after acute ingestion of coffee containing 3395 lmol of CQAs. Despite the high Cmax of the
CQAs, chlorogenic acids were not detected in urine collected 024 h after coffee intake. However, in a subsequent study by
the same group, in which human volunteers consumed a coffee containing a much lower 451 lmol of chlorogenic acids,
4- and 5-O-CQAs were detected in sulfatase/glucuronidase-treated urine from some, but not all, subjects (Farah et al., 2008).
The most recent and detailed research on the fate of chlorogenic acids after the ingestion of coffee is that of Stalmach et al.
(2009a, 2010b) who in studies with healthy humans and ileostomists, in which analysis comprised HPLCMS2-based methodology, noted that during passage through the body extensive metabolism of chlorogenic acids occurs with some compounds being absorbed in the small intestine and others in the colon. The plasma pharmacokinetic proles of circulating
chlorogenic acids and their metabolites observed with healthy subjects with a functioning colon, after the ingestion of
412 lmol of chlorogenic acids are illustrated in Fig. 8. Cmax values ranged from 6 nmol/L for 5-FQA to 385 nmol/L for dihydroferulic acid, with the Tmax extending from 0.6 h (ferulic acid-4-O-sulfate, 3-CQLA-O-sulfate) to 5.2 h (dihydroferulic acid).
The compounds detected in highest concentrations in plasma were free and sulfated conjugates of dihydroferulic acid and
dihydrocaffeic acid with Cmax values ranging from 41 to 385 nmol/L. The Tmax for these compounds was in a narrow range

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Fig. 9. Proposed metabolism of chlorogenic acids following the ingestion of coffee by human volunteers. 5-O-CQA and 5-O-FQA are illustrated structures
but their respective 3- and 4-isomers would be metabolized in a similar manner and likewise with 4-O-CQAL and 3-O-CQAL. COMT, catechol-Omethyltransferase; ET, esterase; RA, reductase; GT, UDP-glucuronyltransferase; ST, sulfuryltransferase; Co-A, co-enzyme A. Bold arrows indicate major
routes, dotted arrows minor pathways. Steps blocked in subjects with an ileostomy and hence occurring in the colon are indicated.

Table 8
Urinary excretion of chlorogenic acid metabolites in 024 h urine of healthy subjects (n = 11) and ileostomists (n = 5)
following the ingestion of 200 mL of coffee.*
Chlorogenic acid and metabolites

Subjects without a colon


(385 lmol ingested)

Subjects with a colon


(412 lmol ingested)

3-O-Caffeoylquinic acid lactone-O-sulfate


4-O-Caffeoylquinic acid lactone-O-sulfate
3-O-Feruloylquinic acid
4-O-Feruloylquinic acid
5-O-Feruloylquinic acid
Ferulic acid-4-O-sulfate
Feruloylglycine
Dihydroferulic acid
Dihydroferulic acid-4-O-sulfate
Dihydroferulic acid-4-O-glucuronide
Isoferulic acid-3-O-sulfate
Isoferulic acid-3-O-glucuronide
Dihydro-isoferulic acid-3-O-glucuronide
Caffeic acid-3-O-sulfate
Caffeic acid-4-O-sulfate
Dihydrocaffeic acid-3-O-sulfate
Dihydrocaffeic acid-3-O-glucuronide
Total

0.6 0.1
0.4 0.1
0.9 0.2
0.9 0.2
1.1 0.2
9.9 1.9
2.1 0.3a
n.d.a
0.8 0.2
n.d.
0.2 0.0
3.9 0.8
n.d.a
6.2 1.2
0.6 0.1
3.2 0.9a
n.da
30.8 4.3 (8.0%)a

1.1 0.1
1.0 0.1
1.2 0.1
1.1 0.1
1.0 0.2
11.1 1.6
20.7 3.9b
9.7 2.0b
12.4 3.4
8.4 1.9
0.4 0.1
4.8 0.5
2.5 0.4b
6.4 0.8
0.6 0.1
37.1 8.2b
0.7 0.2b
120.2 17.0 (29.2%)

*
Data represent mean values in mmol SE. n.d. not detected. Different superscripts within rows indicate a statistical
difference between the two sets of volunteers (Two-sample T-test, P-value <0.05). Figures in bold italicised parentheses indicate excretion as a percentage of chlorogenic acid intake.

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from 4.7 to 5.2 h, indicating absorption in the large intestine. Much shorter Tmax values of 0.6 to 1.0 h, indicative of small
intestine absorption, were obtained with 5-CQA, CQLA-O-sulfates and FQAs, all of which had relatively low Cmax values
(Fig. 8). As noted by Stalmach et al. (2009a) most of the chlorogenic-derived compounds were rapidly removed from the circulatory system with T1/2 values ranging from 0.3 to 1.9 h. The only compounds with an extended T1/2 were dihydroferulic
acid-4-O-sulfate (4.7 h), dihydroferulic acid-3-O-sulfate (3.1 h) and ferulic acid-4-O-sulfate which had an unusual biphasic
plasma prole with dual Tmax values at 0.6 h and 4.3 h. The only unmetabolised compounds detected in plasma were three
FQAs and trace concentrations of 5-CQA (Cmax 2.2 nmol/L; Tmax 1.0 h). It is also of note that the free acid, dihydroferulic
acid, as opposed to the more typical glucuronide and sulfate metabolites, was the principal component accumulating in plasma, which also contained dihydrocaffeic acid in a lower concentration (Fig. 9).
The ileostomists drank a coffee with a very similar 385 lmol chlorogenic acid prole to that ingested by the health subjects (Stalmach et al., 2010b). Plasma was not investigated but analysis of the 024 h ileal uid revealed the presence of
275 lmol of chlorogenic acids mainly, but not exclusively, as unmetabolised compounds. This indicates that ca. 30% of
the chlorogenic acids were absorbed in the small intestine of the ileostomists and that in subjects with a functioning colon
ca. 70% of intake will pass from the small to the large intestine.
The quantities of chlorogenic acids and their metabolites excreted in urine by healthy subjects and ileostomists over a
24 h period post-ingestion of coffee are summarised in Table 8. The healthy volunteers excreted a total of 120.2 lmol, which
corresponds to 29.2% of intake while urine from ileostomists contained 30.8 lmol which equates with only 8.0% of the ingested chlorogenic acids. This highlights the importance of the colon in the bioavailability of dietary chlorogenic acids.
The data presented in Table 8 show that absence of a colon had minimal impact of the excretion of CQAL-O-sulfates and
FQAs, as well as caffeic, ferulic and isoferulic acid-O-sulfates. Furthermore, the data indicate that the small intestine is most
probably the site for (i) cleavage of quinic acid from CQAs and FQAs releasing caffeic acid and ferulic acid, (ii) metabolism of
caffeic acid to its 3- and 4-O-sulfates, and ferulic acid to ferulic acid-4-O-sulfate and (iii) the methylation of caffeic acid to
form isoferulic acid and its subsequent 3-O-sulfation and glucuronidation. In contrast, there were major reductions in the
excretion of dihydrocaffeic acid-3-O-sulfate, dihydrocaffeic acid-3-O-glucuronide, dihydroferulic acid and its glucuronide
and sulfated derivatives, dihydro-isoferulic acid-3-O-glucuronide and feruloylglycine by the ileostomists. This demonstrates
that the colon is the site for (i) the conversion of ferulic acid to feruloylglycine and dihydroferulic acid and (ii) metabolism of
caffeic acid to dihydrocaffeic acid which is further metabolised to dihydro-isoferulic acid. Despite its dual plasma Tmax at 0.6
and 4.3 h in healthy subjects (Fig. 9), urinary excretion of ferulic acid-4-O-sulfate was unaffected by the absence of a colon
(Table 8) indicating that its secondary plasma Tmax is not a consequence of colonic absorption. Stalmach et al. (2009a, 2010b)
proposed the data obtained in their coffee feeding studies with healthy volunteers and ileostomists are in keeping with the
metabolic routes illustrated in Fig. 9.
9. Conclusions
The recent publications that are reviewed in this paper established that both avan-3-ols in green tea and chlorogenic
acids in coffee undergo extensive metabolism prior to absorption, initially in the small intestine prior to passage to the large
intestine where the colonic microbiota-mediated production of phenolic acids occurs. The chlorogenic acids and the avan3-ols produce their own unique spectrum of colonic catabolites which are excreted in urine in substantial amounts corresponding to 29% of intake after coffee consumption and ca. 83% after drinking green tea. Some, but far from all of the metabolites and colonic breakdown products appear transitorily in plasma, but seemingly are treated by the body as xenobiotics
and are rapidly removed from the bloodstream. As a consequence, while analysis of plasma provides valuable information on
the identity, Cmax and Tmax values of circulating metabolites after acute supplementation, estimates of area under the curve
values do not provide accurate quantitative assessments of uptake from the gastrointestinal tract. Urinary excretion provides
a more realistic gure but, as this does not include the possibility of metabolites being sequestered in body tissues, this too is
an under estimate of absorption, but to what degree remains to be determined.
There is a growing realisation that the colon plays an important role in the bioavailability of dietary phenolic and polyphenolic compounds with the studies discussed in this review showing that even when absorption occurs in the small intestine, substantial quantities pass to the large intestine where the parent compounds and their catabolites can impact on both
colonic health and the colonic microbiota. The level of urinary excretion indicates that substantial quantities of the colonic
catabolites are absorbed into the portal vein and pass through the body in the circulatory system prior to excretion. Some of
these compounds may play a key role in the protective effects of a fruit and vegetable-rich diet as there is evidence that they
have anti-inammatory effects in experimental models (Larrosa et al., 2009). Moreover, consumption of a diet rich in polyphenols, with particular emphasis towards those that are less absorbable in the small intestine, is accompanied by a significant increase in stool bulk, a factor considered to be of potential relevance for bowel health (Bianchi et al., 2010).
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