molecules

Review
Role of Intestinal Microbiota in the Bioavailability
and Physiological Functions of Dietary Polyphenols
Kyuichi Kawabata , Yasukiyo Yoshioka and Junji Terao *
Faculty of Clinical Nutrition and Dietetics, Konan Women’s University, 6-2-23 Morikita-machi, Higashinada-ku,
Kobe City, Hyogo 658-0001, Japan; [email protected] (K.K.); [email protected] (Y.Y.)
* Correspondence: [email protected]; Tel.: +81-78-413-3114




Received: 14 December 2018; Accepted: 17 January 2019; Published: 21 January 2019


Abstract: Polyphenols are categorized as plant secondary metabolites, and they have attracted
much attention in relation to human health and the prevention of chronic diseases. In recent years,
a considerable number of studies have been published concerning their physiological function in the
digestive tract, such as their prebiotic properties and their modification of intestinal microbiota.
It has also been suggested that several hydrolyzed and/or fission products, derived from the
catabolism of polyphenols by intestinal bacteria, exert their physiological functions in target sites
after transportation into the body. Thus, this review article focuses on the role of intestinal microbiota
in the bioavailability and physiological function of dietary polyphenols. Monomeric polyphenols,
such as flavonoids and oligomeric polyphenols, such as proanthocyanidins, are usually catabolized
to chain fission products by intestinal bacteria in the colon. Gallic acid and ellagic acid derived
from the hydrolysis of gallotannin, and ellagitannin are also subjected to intestinal catabolism.
These catabolites may play a large role in the physiological functions of dietary polyphenols.
They may also affect the microbiome, resulting in health promotion by the activation of short
chain fatty acids (SCFA) excretion and intestinal immune function. The intestinal microbiota is a key
factor in mediating the physiological functions of dietary polyphenols.

Keywords: polyphenol; tannin; intestinal microbiota; bioavailability; microbial catabolite; physiological

function; prebiotics

1. Introduction

1.1. Classification
Polyphenols are plant secondary metabolites ubiquitously present in many parts of the plant,
including flowers, leaves, pulp, stems, and roots. They are not directly responsible for the growth and
development of plants, but are necessary for the plant to survive in its environment. They are produced
from primary metabolites and intermediates through unique biosynthetic pathways. In recent years,
much attention has been paid to their potential role as functional food ingredients. A variety of
polyphenols and their derivatives are currently expected to be used as dietary factors to prevent
chronic diseases, such as diabetes, cancer, and stroke [1–5]. In 2013, Del Rio et al. [6] reviewed more
than 500 publications focusing on the bioavailability of dietary polyphenols and their protective effects
against chronic diseases.
Approximately 8000 compounds of polyphenols are found in the plant kingdom. These can
be separated into high molecular weight tannins and low molecular weight polyphenols. Tannins
consist of hydrolysable tannins (gallotannins and ellagitannins, Figure 1) and non-hydrolysable,
condensed tannins (proanthocyanidins, Figure 2) [7]. Low molecular weight polyphenols can
be categorized into several subgroups: phenolic acid derivatives, flavonoids, lignans, stilbenes,

Molecules 2019, 24, 370; doi:10.3390/molecules24020370 www.mdpi.com/journal/molecules

 Molecules 2019, 24, 370 2 of 25
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and curcumins.
physiological For many
function years,
of low large numbers
molecular of studies have
weight polyphenols, been undertaken
in particular, to discover
flavonoids; the
carboxylic
physiological function
acids, such asfunction of
caffeic acid low molecular
andmolecular
ferulic acid; weight polyphenols,
and resveratrol [8]. inin particular,
Flavonoids flavonoids; carboxylic
physiological of low weight polyphenols, particular,are typical lowcarboxylic
flavonoids; molecular
acids,
weight such as caffeic acid
polyphenols and ferulic
present in acid; and
plant resveratrol
foods, and [8]. Flavonoids
they are are typicalbylowa molecular
characterized coplanar
acids, such as caffeic acid and ferulic acid; and resveratrol [8]. Flavonoids are typical low molecular
weight polyphenols
diphenylpropane present
structure in plant foods, and they are characterized by a coplanar diphenylpropane
weight polyphenols present(Figure
in plant3). Among
foods, them,
and catechins
they are (flavan-3-ols)
characterizedarebyalso a present
coplanaras
structure
molecular (Figure
units 3). Amongof
consisting them, catechins (flavan-3-ols) are also present as molecular units consisting
proanthocyanidins.
diphenylpropane structure (Figure 3). Among them, catechins (flavan-3-ols) are also present as
of proanthocyanidins.
molecular units consisting of proanthocyanidins.

Figure 1. Structures of hydrolysable tannins. (A) Molecules making up tannins, (B) examples of
Figure 1. Structures
gallotannins, of hydrolysable
(C) examples tannins. (A) Molecules making up tannins, (B) examples of
of ellagitannins.
Figure 1. Structures of hydrolysable tannins. (A) Molecules making up tannins, (B) examples of
gallotannins, (C) examples of ellagitannins.
gallotannins, (C) examples of ellagitannins.

Figure 2. Structures of non-hydrolysable condensed tannins (proanthocyanidins). (A) Molecules making

Figure 2. Structures of non-hydrolysable condensed tannins (proanthocyanidins). (A) Molecules
up proanthocyanidins, (B) Examples of proanthocyanidins.
making
Figure 2. up proanthocyanidins,
Structures (B) Examples
of non-hydrolysable of proanthocyanidins.
condensed tannins (proanthocyanidins). (A) Molecules
1.2. making
Background of Polyphenol Research
up proanthocyanidins, (B) Examples of proanthocyanidins.
1.2. Background of Polyphenol Research
In 1936, Bentsáth et al. [9] first reported that citrus flavonoids (hesperidin and rutin) reduced
1.2. Background
In 1936,
capillary of Polyphenol
Bentsáth
fragility al.Research
and etpermeability
[9] first reported
in human that citrusvessels.
blood flavonoids (hesperidin
Thereafter, the and rutin)effects
vascular reducedof
capillary fragility
plant-derived
In 1936, Bentsáth andetpermeability
flavonoids were
al. [9] first in human
gradually
reported blood
explored
that vessels.
[10].
citrus In Thereafter,
1993, the vascular
an epidemiological
flavonoids (hesperidin effects
and study
rutin) of plant-
inreduced
a Dutch
derived fragility
population
capillary flavonoids andwere
first revealed gradually
that the high
permeability explored
intake of
in human [10].vessels.
In 1993,
flavonoids
blood an epidemiological
(mainly flavonol-type
Thereafter, study
effectsinof
flavonoids)
the vascular aplant-
Dutch
correlates
population
with a decrease
derived first
flavonoids revealed
in death
weredue that the high
to atherosclerotic
gradually intake
explored vascularof flavonoids
[10]. In disease
1993, an (mainly
[11]. flavonol-type
This study encouraged
epidemiological flavonoids)
study inresearchers
a Dutch
correlates
investigatingwith a decrease
dietary in
flavonoidsdeathanddue to atherosclerotic
triggered human vascular
intervention disease
studies
population first revealed that the high intake of flavonoids (mainly flavonol-type flavonoids) [11].
of This
their study encouraged
bioavailability and
researchers
bio-efficacy
correlates with investigating
[12]. dietary flavonoids and triggered human intervention
a decrease in death due to atherosclerotic vascular disease [11]. This study encouraged studies of their
bioavailability
researchers and bio-efficacy
investigating dietary[12].
flavonoids and triggered human intervention studies of their
bioavailability and bio-efficacy [12].
Molecules 2019, 24, 370 3 of 25
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Figure 3. Generic structures of major flavonoids.

Figure 3. Generic structures of major flavonoids.
Polyphenols, including flavonoids, can act as antioxidants because they possess electron-donating
Polyphenols,
phenolic groups inincluding flavonoids,
their structures. can act
Therefore, as antioxidants
several studies havebecause they possess
investigated electron-
their antioxidant
donatinginphenolic
function groupsofinoxidative
the prevention their structures. Therefore,
stress-related cellularseveral studies have
and extracellular investigated
damage. Antioxidanttheir
antioxidant function in the prevention of oxidative stress-related cellular and
function has long been suggested to have a major role in promoting vascular health for the prevention extracellular damage.
Antioxidant
of function
atherosclerosis [13].has long been suggested
Nevertheless, to have a of
the bioavailability major role in is
flavonoids promoting
significantlyvascular
lowerhealth
than thefor
the prevention of
bioavailability of antioxidant
atherosclerosis [13]. Nevertheless,
vitamins and pro-vitamins the bioavailability of flavonoids
(vitamin E, vitamin C, andis carotenoids).
significantly
lower than the bioavailability of antioxidant vitamins and pro-vitamins
Hollman et al. [14] suggested that antioxidant function may not explain the major vascular effects (vitamin E, vitamin C, andof
carotenoids). Hollman et al. [14] suggested that antioxidant function may
dietary flavonoids. The cellular targets of flavonoids may be kinases involved in signal transduction not explain the major
vascular effects
pathways of dietary
(Raf, Fyn, MEK, flavonoids.
and PI3K) [15]. The The
cellular targetsmodulation
resulting of flavonoids may be
of these kinases seems
pathways involved in
to be
signal transduction pathways (Raf, Fyn, MEK, and PI3K) [15]. The resulting
a major component of the physiological role of flavonoids after their absorption into the body [16]. modulation of these
pathways
They seems
are able to be
to act asamodulators
major component of the
of cellular physiological
signaling role ofprovided
pathways, flavonoids after
that they their
areabsorption
efficiently
into the body [16]. They are able to act as modulators of cellular signaling
absorbed into the body and accumulate in target cells or target fluids [10,17]. The bioavailability pathways, provided that
they are efficiently absorbed into the body and accumulate in target cells or
of dietary flavonoids is, in general, very low. Therefore, they are mostly excreted into feces after target fluids [10,17]. The
bioavailability into
transportation of dietary flavonoids
the gut. Furthermore, is, inhigh
general, very low.
molecular weightTherefore,
tannins they are mostly
are scarcely excreted
absorbed into
in their
feces after transportation into the gut. Furthermore, high molecular weight
original form because of their relatively large molecular size. Investigations into their physiological tannins are scarcely
absorbedhas
function in their original
long been form because
neglected or limited of to
their relatively
their role in the large molecular
digestive tract.size.
The Investigations
antioxidant effects into
their physiological function has long been neglected or limited to their role
of dietary polyphenols may only be exerted in the digestive tract, because of their high concentrationin the digestive tract. The
antioxidant effects of dietary polyphenols may only be exerted in the digestive
in the gut. However, recent studies on the relationship between intestinal microbiota and dietary tract, because of their
high concentration
polyphenols in the gut.
have provoked new However,
ideas on therecent studies function
physiological on the relationship
of non-absorbable between intestinal
polyphenols,
microbiota and dietary polyphenols have
such as non-hydrolysable and hydrolysable tannins [18]. provoked new ideas on the physiological function of non-
absorbable polyphenols, such as non-hydrolysable and hydrolysable tannins [18].
1.3. Interest in the Actions of Intestinal Microbiota
1.3. Interest in the Actions of Intestinal Microbiota
The gut microbiota is strongly associated with the occurrence of obesity by increasing the capacity
The gut
for energy microbiota
harvest [19]. Itis is,
strongly
therefore,associated withanthe
likely that occurrence
alteration of intestinal
of the obesity bymicrobiota
increasingmay the
capacity for energy harvest [19]. It is, therefore, likely that an alteration
contribute to the improvement of human health. In fact, recent studies have demonstrated that of the intestinal microbiota
may contribute to
non-absorbable the improvement
polyphenols act in theof human
intestinehealth. In fact,
to alter the gutrecent studiescommunity,
microbial have demonstrated resultingthat in
non-absorbable
lower polyphenols and
systemic inflammation act in the intestine
improved to alter
metabolic the gut[20–22].
outcomes microbial community,
Polyphenols may resulting
also exert in
lower systemic inflammation and improved metabolic
beneficial effects on microbiota by acting as prebiotics [23]. outcomes [20–22]. Polyphenols may also exert
beneficial effects on microbiota by acting as prebiotics [23].
In addition, a large new area of study involves investigating the effect of the gut microbiota on
the brain and behavioral traits [24,25]. However, the association between behavior and the action of
Molecules 2019, 24, 370 4 of 25

In addition, a large new area of study involves investigating the effect of the gut microbiota
on the brain and behavioral traits [24,25]. However, the association between behavior and the
action of dietary polyphenols in the digestive tract is not well understood. On the other hand,
the bioconversion of dietary polyphenols by gut microbiota has recently been shown to affect the
nutritional phenotype of humans [26]. Enterobacteria-dependent catabolites of polyphenols may
exert their effects in the body by transferring into the blood circulation. For example, urolithin or
its glucuronide conjugate, an ellagitannin catabolized by intestinal bacteria, has been reported to
increase muscle function in rodents [27], inhibit metastasis of cancer cell lines [28], and protect the
impairment of cardiomyocytes [29]. Intestinal polyphenol catabolites may also exert their effects
within the intestinal tract and intestinal walls. These may include inhibitory effects on colorectal cancer
and suppressive effects on inflammatory bowel diseases [30–33].
The purpose of this review is to focus on the physiological effects of non-absorbable monomeric
and polymeric polyphenols on the intestinal microbiota. Colonic bacteria-dependent catabolic
pathways, bioavailability, and the function of their catabolites in the digestive tract and circulatory
system is also discussed. Finally, we will discuss the mechanism by which plant polyphenols contribute
to human health through the intestinal microbiota. In this paper, we will review flavonoids, tannins,
curcumins, and resveratrol, but not isoflavones.

2. Hydrolysis and Absorption of Polyphenols in the Stomach and Small Intestine

2.1. Stability of Polyphenols in the Stomach

In general, low molecular weight polyphenols are partially absorbed into the body directly or
after phase II enzyme-dependent metabolic conversion in small intestinal cells, as described above [34].
High molecular weight tannins and even low molecular weight polyphenols are transported into the
large intestine in their original form. They are then excreted with feces without intestinal absorption,
or they are catabolized by enterobacteria [35,36]. Therefore, the stomach is the minor location of
absorption of polyphenols. In fact, low molecular weight polyphenols are stable, and glucose-bound
polyphenols are only partially hydrolyzed, despite the low pH in the stomach [37]. It should be noted
that anthocyanins (anthocyanidin glucosides) can be absorbed from the stomach via an active transport
pathway involving bilitranslocase, an organic anion membrane carrier [38]. The stability of tannins
in the stomach is similar to that of low molecular weight polyphenols [39]. However, the data for
proanthocyanidins are more conflicting. An in vitro study suggests that procyanidins are hydrolyzed
to bioavailable flavan-3-ol monomers [40], but an in vivo study claims a lack of depolymerization [41].

2.2. Intestinal Absorption of Sugar-Bound Polyphenols

In the small intestine, glucose-bound polyphenols are deglucosylated to aglycone by
lactase-phlorizin hydrolase (LPH) or cytosolic β-glucosidase (CBG) of intestinal epithelial cells and
then converted into O-glucuronide/O-sulfate conjugates by phase II enzymes in intestinal cells [34].
These conjugated metabolites are transported into the liver, where they are further metabolized,
after which they either enter into the blood circulation or return to the digestive tract through the
enterohepatic circulation (Figure 4). The total concentration of conjugated metabolites may reach
micromolar levels within a few hours and then almost disappear after 24 h [42].
Other glycosides that are bound to sugars other than glucose are not substrates for these hydrolytic
enzymes that release aglycone in the small intestine [34]. The time to reach Cmax (the compound’s
maximum concentration in the blood) is significantly longer after rutin (quercetin-3-O-β-rutinoside)
supplementation than after quercetin aglycone supplementation [43]. In contrast to quercetin aglycone
and its glucosides, rutin is barely absorbed in the small intestine [44,45]. Most dietary rutin enters into
the large intestine, where the colonic microbiota liberates quercetin aglycone from rutin. Quercetin
aglycone can then be absorbed or further degraded to produce various ring-fission products by the
action of enterobacteria (Figure 4) [46,47].
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Figure 4. Pathway of absorption and metabolism of isoquercitrin (quercetin 3-O-glucoside) and rutin
(quercetin 3-O-β-rutinoside)
3-O-β-rutinoside) in the digestive system [34,46,48]. CBG, cytosolic β-glucosidase; LPH,
β-glucosidase; LPH,
lactose phlorizin hydrolase; Glc, glucose; Rha,
Rha, rhamnose;
rhamnose; GlcA,
GlcA, glucuronic
glucuronic acid.
acid.

2.3. Intestinal Absorption of Monomeric Epicatechin and Its Related Monomeric Flavan-3-ols
2.3. Intestinal Absorption of Monomeric Epicatechin and Its Related Monomeric Flavan-3-ols
In the case of flavan-3-ols, monomeric epicatechin is partly absorbed in the small intestine
In the case of flavan-3-ols, monomeric epicatechin is partly absorbed in the small intestine in its
in its original form and is then subjected to glucuronidation, sulfation and/or O-methylation [49].
original form and is then subjected to glucuronidation, sulfation and/or O-methylation [49]. Natsume
Natsume et al. [48] identified (−)-epicatechin-30 -O-glucuronide, 40 -O-methyl-(−)-epicatechin-30 -O-
et al. [48] identified (−)-epicatechin-3’-O-glucuronide, 4’-O-methyl-(−)-epicatechin-3’-O-glucuronide,
glucuronide, and 40 -O-methyl-(−)-epicatechin-5 or 7-O-glucuronide as metabolites of (−)-epicatechin
and 4’-O-methyl-(−)-epicatechin-5 or 7-O-glucuronide as metabolites of (−)-epicatechin in human
in human urine. Ottaviani et al. [50] demonstrated that 82 ± 5% of ingested (−)-epicatechin is
urine. Ottaviani et al. [50] demonstrated that 82 ± 5% of ingested (−)-epicatechin is absorbed, and more
absorbed, and more than 20 different metabolites are present in human plasma14 after ingestion of
than14
20 different metabolites are present in human plasma after ingestion of [2- C](−)-epicatechin.
[2-
TheyC]( −)-epicatechin.
also suggested that They
thealso
gut suggested
microbiotathatis athe
keygut microbiota
driver is a key driver
of (−)-epicatechin of (−)-epicatechin
metabolism, because
metabolism,
the concentration of its metabolites in plasma shows a biphasic pattern as a function of time. Inas
because the concentration of its metabolites in plasma shows a biphasic pattern thea
function of time. In the case of green tea flavan-3-ols, overall flavan-3-ol metabolite excretion
case of green tea flavan-3-ols, overall flavan-3-ol metabolite excretion is estimated to be 8.1% of intake is
estimated to be 8.1%un-metabolized
[51]. Interestingly, of intake [51]. Interestingly, un-metabolized (−
(−)-epigallocatechin-3-gallate )-epigallocatechin-3-gallate
and (−)-epicatechin-3-gallate and
are
−)-epicatechin-3-gallate
(also detected in the plasma, aretogether
also detected in the plasma,O-sulfated,
with O-methylated, together with and O-methylated,
O-glucuronide O-sulfated,
conjugates
and O-glucuronide
of (−)-epicatechin andconjugates of (−)-epicatechin
(−)-epigallocatechin [52]. and (−)-epigallocatechin [52].
2.4. Intestinal Absorption of Oligomeric Procyanidins
2.4. Intestinal Absorption of Oligomeric Procyanidins
It is generally accepted that procyanidin polymers and oligomers with a degree of polymerization
It is generally accepted that procyanidin polymers and oligomers with a degree of
(DP) > 4 are not directly absorbed from the small intestine, although dimers and trimers can
polymerization (DP) > 4 are not directly absorbed from the small intestine, although dimers and
be detected in the plasma [53]. A-type procyanidin dimers, trimers, and tetramers can be
trimers can be detected in the plasma [53]. A-type procyanidin dimers, trimers, and tetramers can be
transported across human intestinal epithelial Caco-2 cells [54]. Shoji et al. [55] found that apple
transported across human intestinal epithelial Caco-2 cells [54]. Shoji et al. [55] found that apple
procyanidins of each group, from dimers to pentamers, are present in rat plasma. Donovan et al. [39]
procyanidins of each group, from dimers to pentamers, are present in rat plasma. Donovan et al. [39]
claimed that neither monomers nor oligomers existed at detectable concentrations in vivo when
claimed that neither monomers nor oligomers existed at detectable concentrations in vivo when
grapeseed procyanidins were administered to rats. However, Serra et al. [56] reported free forms
grapeseed procyanidins were administered to rats. However, Serra et al. [56] reported free forms of
of dimers and trimers in rat plasma after oral intake of a grapeseed extract. The degree of
dimers and trimers in rat plasma after oral intake of a grapeseed extract. The degree of
polymerization has a major impact on the fate of procyanidins in the body, with greater degrees
polymerization has a major impact on the fate of procyanidins in the body, with greater degrees of
of polymerization showing poorer absorption through the gut barrier [57]. Baba et al. [58] found
polymerization showing poorer absorption through the gut barrier [57]. Baba et al. [58] found that
that the procyanidin dimer, B2 [epicatechin-(4-8)-epicatechin], is absorbed and a portion of B2 is
the procyanidin dimer, B2 [epicatechin-(4-8)-epicatechin], is absorbed and a portion of B2 is degraded
degraded to monomeric epicatechin after B2 administration to rats. Interestingly, another study in
to monomeric epicatechin after B2 administration to rats. Interestingly, another study in rats showed
rats showed that the procyanidin dimer, A1 [epicatechin-(2-O-7,4-8)-catechin] and the procyanidin
that the procyanidin dimer, A1 [epicatechin-(2-O-7,4-8)-catechin] and the procyanidin dimer, A2
dimer, A2 [epicatechin-(2-O-7, 4-8)-epicatechin] are better absorbed than the procyanidin dimer,
[epicatechin-(2-O-7, 4-8)-epicatechin] are better absorbed than the procyanidin dimer, B2
[epicatechin-(4-8)-epicatechin], although absorption of the A-type dimers was only 5–10% of
monomeric epicatechin absorption [59].
Molecules 2019, 24, 370 6 of 25

B2 [epicatechin-(4-8)-epicatechin], although absorption of the A-type dimers was only 5–10% of

monomeric epicatechin absorption [59].

2.5. Hydrolysis of Tannins in the Digestive Tract

A human pilot trial using gallotannin-rich mango pulp (Mangifera indica L. cv. Keitt) strongly
suggested that gallotannins release free gallic acid in the gastrointestinal tract, indicating that
gallotannins may serve as a pool of pro-gallic acid compounds that can be absorbed or can undergo
microbial metabolism [60]. In humans, ellagic acid can be released by the hydrolysis of ellagitannins
after the consumption of pomegranate juice. This is facilitated by physiological pH and/or gut
microbiota actions [61]. A study of ileostomists consuming raspberries indicated that the hydrolysis
of ellagitannins occurs in the stomach or small intestine [62]. Therefore, hydrolysable tannins can be
hydrolyzed in the digestive tract before absorption or microbial catabolism.

3. Decomposition and Metabolism by Intestinal Bacteria

The gut microbiota influences polyphenol bioavailability by modifying the structure of aglycones,
glycosides, and conjugates, such as O-glucuronides and O-sulfates. A large proportion of O-glycosides
are converted into their corresponding aglycones by the acidic conditions of the stomach, by mucosal
enzymes in the small intestine, or by enzymes in the gut microbiota. Aglycones are subjected to
the activity of phase II enzymes during the absorption process and after their delivery to the liver,
resulting in O-glucuronides and/or O-sulfates. These polyphenol conjugates are then excreted in the
bile and re-enter the intestinal tract. In addition to enzymatic activity to deconjugate O-glycosides
and O-glucuronides, gut microbes have catabolic capacity to perform carbon-carbon cleavage of
heterocyclic and aromatic rings, dehydroxylation, decarboxylation, and hydrogenation of alkene
moieties. These catabolic transformations follow a procedure of reactions that conform to the basic
principles of chemistry. In this section, we outline the mechanism of the gut microbial transformation
of major dietary polyphenols.

3.1. Flavonoid Quercetin

Quercetin, one of the major flavonols, is ubiquitously distributed in plants. It is mainly present as
O-glycosides in various fruits and vegetables. The bacterial catabolites of rutin have been identified
as 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3-(3-hydroxyphenyl)propionic
acid [44,47,63]. Several studies on the catabolism of quercetin or its glycosides by fecal microbiota have
demonstrated an alternative conversion that generates 3,4-dihydroxybenzoic acid (i.e., protocatechuic
acid) and 2,4,6-trihydroxybenzoic acid as the major catabolites (Figure 5) [64–67]. These catabolites are
then further transformed into their O-methyl derivatives [68]. The enzymatic cleavage of quercetin
to 3,4-dihydroxybenzoic acid is mediated by fungal and bacterial quercetin dioxygenases, known as
quercetinases [69,70]. These enzymes contain divalent metal ions, such as iron, copper, or manganese,
in their catalytic domain [71]. A reaction involving the chelation of oxygen atoms at positions 3 and
4 and the generation of superoxide or peroxyl radical species is proposed as the mechanism for
producing 3,4-dihydroxybenzoic acid [69,72]. That is, quercetin degradation by quercetinase is initiated
by chelation of the 4-keto/2,3-enol moiety with a divalent cation, resulting in the 3,4-diketo tautomer.
The metal ion-bound flavonol complex places the oxygen atoms at position 2 via semi-quinone
radicals, derived from phenoxy radicals. Arrangement of the endo-peroxide, based on the conversion
of the 3-keto group into an acylium group, results in an ester, 2-protocatechuoyl-phloroglucinol
carboxylic acid, linking the two aromatic rings and the release of carbon monoxide. While quercetin
dioxygenases yield 2-protocatechuoyl-phloroglucinol carboxylic acid after incubation of quercetin
with the microbiota, no catabolite of quercetin formed by hydrolytic cleavage of the ester, that is,
3,4-dihydroxybenzoic acid or 2,4,6-trihydroxybenzoic acid, is found in either animals or humans.
On the other hand, there are no reports of 2,4,6-trihydroxybenzoic acid as a catabolite of quercetin.
Molecules 2019, 24, 370 7 of 25

This may be because a large portion of 2,4,6-trihydroxybenzoic acid may be modified to phloroglucinol,
Molecules
as 2019, 24gut
the known FORmicrobial
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catabolite of quercetin [73]. 7

Figure 5. A mechanism for quercetinase-mediated conversion of quercetin into protocatechuic acid,

Figure 5. A mechanism for quercetinase-mediated conversion of quercetin into protocatechuic acid,
2,4,6-trihydroxybenzoic acid and phloroglucinol [64–67].
2,4,6-trihydroxybenzoic acid and phloroglucinol [64–67].

Quercetinase- and other dioxygenase-mediated catabolic conversions depend on oxygen

Quercetinase- and other dioxygenase-mediated catabolic conversions depend on oxygen
molecules. However, these microbial reactions can occur in the anoxic environment of the colon.
molecules. However, these microbial reactions can occur in the anoxic environment of the colon. The
The gastrointestinal mucosa separates the highly vascularized and oxygen-rich epithelium from the
gastrointestinal mucosa separates the highly vascularized and oxygen-rich epithelium from the
anoxic gut lumen, and the colon, which is relatively anoxic compared with the small intestine, is helpful
anoxic gut lumen, and the colon, which is relatively anoxic compared with the small intestine, is
for intestinal bacteria to form the more complex community [74,75]. Blood vessels in the sub-epithelial
helpful for intestinal bacteria to form the more complex community [74,75]. Blood vessels in the sub-
tissue of the intestine accelerate efficient nutrient uptake and supply oxygen to the epithelium and then,
epithelial tissue of the intestine accelerate efficient nutrient uptake and supply oxygen to the
indirectly, to the gut lumen by diffusion. The resulting steep oxygen gradient across the epithelial cell
epithelium and then, indirectly, to the gut lumen by diffusion. The resulting steep oxygen gradient
layer affords stress on the epithelium and on the absolute anaerobic micro-organisms in the gut lumen.
across the epithelial cell layer affords stress on the epithelium and on the absolute anaerobic micro-
Facultative anaerobic micro-organisms may survive and thrive at this oxic/anoxic interface without
organisms in the gut lumen. Facultative anaerobic micro-organisms may survive and thrive at this
interference from absolute anaerobes. Bacillus subtilis has been documented as an aerobic soil microbe
oxic/anoxic interface without interference from absolute anaerobes. Bacillus subtilis has been
that can be a facultative anaerobe [76]. Moreover, its isolation and behavior in the human intestinal
documented as an aerobic soil microbe that can be a facultative anaerobe [76]. Moreover, its isolation
tract has led to its recognition as a normal gut commensal bacterium in humans [77]. Quercetinase from
and behavior in the human intestinal tract has led to its recognition as a normal gut commensal
B. subtilis has been overexpressed in E. coli, isolated, and biochemically characterized [70,78]. Based on
bacterium in humans [77]. Quercetinase from B. subtilis has been overexpressed in E. coli, isolated,
these studies, B. subtilis is proposed to be a quercetinase-dependent producer of protocatechuic
and biochemically characterized [70,78]. Based on these studies, B. subtilis is proposed to be a
acid from dietary quercetin in the human gut. As a major gut microbial metabolite of quercetin,
quercetinase-dependent producer of protocatechuic acid from dietary quercetin in the human gut.
protocatechuic acid contributes to the health effects of fruits and vegetables rich in these flavonoids.
As a major gut microbial metabolite of quercetin, protocatechuic acid contributes to the health effects
of fruits
3.2. and vegetables rich in these flavonoids.
Proanthocyanidin
Dietary proanthocyanidins, which are formed by the condensation of single or multi-component
3.2. Proanthocyanidin
of flavan-3-ols, are found in apples, chocolate, and grapes [79]. While monomeric, dimeric,
Dietary proanthocyanidins,
and trimeric which to
catechins are absorbed, aresome
formed by thefrom
extent, condensation
the smallofintestine,
single or multi-component
larger oligomers
of flavan-3-ols, are found in apples, chocolate, and grapes [79]. While
have very poor bioavailability [80]. It has been demonstrated that proanthocyanidins monomeric, dimeric, and
undergo
trimericacid-catalyzed
partial catechins are absorbed,
cleavage to some
into extent, from
monomeric the smallunits
flavan-3-ol intestine,
in thelarger oligomers
gastric have very
environment [40].
poor bioavailability [80]. It has been demonstrated that proanthocyanidins
The microbial catabolites of monomeric and oligomeric catechins in the large intestine appear undergo partial acid-
to
catalyzed
be cleavage into monomeric
3-hydroxyphenylacetic flavan-3-ol units in theacid,
acid, 3,4-dihydroxyphenylacetic gastric environment [40]. The microbial
3-(3-hydroxyphenyl)propionic acid,
catabolites
and of monomeric and oligomeric
5-(30 -hydroxyphenyl)-γ-valerolactone [81]. catechins in the large
The latter catabolite is the intestine appear
result of A-ring to be by
cleavages 3-
hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, 3-(3-hydroxyphenyl)propionic
two consecutive reverse Claisen reactions (Figure 6). Cleavage of the C-ring of a methylated quinone acid, and
5-(3’-hydroxyphenyl)-γ-valerolactone
intermediate prepares the molecule for [81]. The latter
retro-Claisen catabolite
cleavage. is the
There areresult of A-ring
two possible cleavages for
mechanisms by
twocatabolism
the consecutive of reverse
catechin.Claisen reactions
One possible (Figure 6).
mechanism is Cleavage
a reduction ofof
the
theC-ring of acarbonyl,
quinone methylated quinone
yielding the
intermediate prepares the molecule for retro-Claisen cleavage. There are two possible mechanisms
for the catabolism of catechin. One possible mechanism is a reduction of the quinone carbonyl,
yielding the corresponding p-hydroxy catabolite, which is then subjected to dehydroxylation. The
other one is that the carbonyl quinone hydride attacks at the methylated carbon, yielding 3,4-
dihydroxyphenyl catabolites, which then eventually give rise to 5-(3’,4’-dihydroxyphenyl)-γ-
Molecules 2019, 24, 370 8 of 25

corresponding p-hydroxy catabolite, which is then subjected to dehydroxylation. The other one is
Molecules
that the 2019, 24 FORquinone
carbonyl PEER REVIEW
hydride attacks at the methylated carbon, yielding 3,4-dihydroxyphenyl8
catabolites, which then eventually give rise to 5-(30 ,40 -dihydroxyphenyl)-γ-valerolactone. The latter
valerolactone. The latter compound has been reported to be a product of gut microbial catabolism
compound has been reported to be a product of gut microbial catabolism [82–87].
[82–87].

Figure 6. Gut microbial conversion of procyanidin [81].

Figure 6. Gut microbial conversion of procyanidin [81].
3.3. Anthocyanidins
3.3. Anthocyanidins
Anthocyanidins, a subclass of flavonoid plant pigments, are responsible for red, purple, and blue
colorsAnthocyanidins,
of flower petals, a subclass
vegetables, of flavonoid
and fruits.plant pigments, are
Anthocyanins are responsible
the glycosylatedfor red, purple,
forms and
of their
blue colors of flower
corresponding petals,While
aglycones. vegetables, and fruits.
the glycosides areAnthocyanins
bioavailable, are theythe areglycosylated
susceptible forms of their
to hydrolytic
corresponding aglycones. While the glycosides are bioavailable, they
conversion into their corresponding anthocyanidins [88–92]. The glycosides and aglycones are are susceptible to hydrolytic
conversion
both found into their corresponding
as glucuronides anthocyanidins
in the urine of animals [88–92].
and humans The glycosides
[93,94]. The andanthocyanidins
aglycones are both are
found as glucuronides in the urine of animals and humans [93,94]. The anthocyanidins
frequently metabolized into protocatechuic acid [95]. Using cyanidin as the prototypic anthocyanidin, are frequently
metabolized
its metabolisminto protocatechuic
is initiated by cleavageacid of[95]. Using cyanidin
the heterocyclic as thering
flavylium prototypic
at neutral anthocyanidin,
or slightly basic its
metabolismofisthe
conditions initiated by cleavage
small intestine. of the heterocyclic
Subsequent attack of the flavylium ring at at
flavylium carbon neutral
position or 2slightly
producesbasic
an
conditions of the small intestine. Subsequent attack of the flavylium carbon
unstable hemi-ketal that rapidly forms a ketone. Through keto-enol tautomerism of the neighboring at position 2 produces an
unstable hemi-ketal that rapidly forms a ketone. Through keto-enol tautomerism
enol functionality, the resulting α-diketo group is cleaved by gut microbiota to form protocatechuic acid of the neighboring
enol2,4,6-trihydroxyphenylacetic
and functionality, the resulting α-diketo
acid (Figuregroup7). is cleaved the
Although by gut microbiota
cleavage reactionto form protocatechuic
mechanism remains
acid and 2,4,6-trihydroxyphenylacetic acid (Figure 7). Although the
to be elucidated, it seems reasonable to propose that the C-C cleavage involves the attack cleavage reaction mechanism
of either
remains to
carbonyl bybe elucidated,
a peroxyl anion it species,
seems reasonable
similar to theto propose thatinthe
initial step theC-C cleavage involves the
dioxygenase-mediated attack of
conversion
either
of carbonyl byinto
α-ketoglutarate a peroxyl
succinateanion species,
[96]. similar
Insertion of thetoresulting
the initial step in
alkoxy the dioxygenase-mediated
oxygen between the original
conversion of α-ketoglutarate into succinate [96]. Insertion
carbonyl carbons yields an anhydride, which forms two phenolic acids upon of the resulting alkoxy oxygen
hydrolysis between
(Figure 7).
the original
This proposed carbonyl carbons
mechanism yields
is also an anhydride,
similar to that ofwhich
oxidativeformsconversion
two phenolic acids upon
of benzoins hydrolysis
into benzoic
(Figure
acids 7). After
[97]. This proposed
absorptionmechanism is also similar
from the intestinal tract andto that of oxidative
hepatic phase II conversion
metabolism,ofthese benzoins into
phenolic
benzoic acids [97]. After absorption from the intestinal tract and hepatic phase
acids are frequently detected in the urine, either in their original form or as their O-glucuronide or II metabolism, these
phenolic derivatives
O-methyl acids are frequently
[98–100]. detected in the urine, either in their original form or as their O-
glucuronide or O-methyl derivatives [98–100].

3.4. Curcumin
In animals and humans, curcumin, a yellow pigment from turmeric, is catabolized to its
hydrogenated (dihydro-, tetrahydro-, hexahydro-, and octahydro-), desmethyl, O-glucuronide, and
O-sulfate metabolites [101,102]. E. coli NADPH-dependent reductase, the enzyme responsible for the
stepwise reduction of curcumin, has been isolated from human feces (Figure 8A) [103]. While the
enzyme, NADPH-dependent curcumin/dihydrocurcumin reductase (CurA), catalyzes curcumin
 Resveratrol is a stilbene-type polyphenol characteristically present in red wine and grapes. The
main gut microbial catabolites of resveratrol are dihydroresveratrol and the m-deoxy metabolites of
both resveratrol
Molecules 2019, 24 FORand dihydroresveratrol
PEER REVIEW (Figure 8B) [104]. Using a panel of gut microbes, Slackia9
equolifaciens and Adlercreutzia equolifaciens were identified as dihydroresveratrol producers. Upon
Molecules 2019, 24, 370 9 of 25
incubation
selectively, of resveratrol
it does withtetrahydrocurcumin
not reduce fecal samples, bothtodihydroresveratrol
yield correspondingand 3,4′-dihydroxystilbene
secondary alcohols. CurA
were observed
has been as intermediates
identified as a memberin ofthe
theformation of lunularin
medium-chain [104].
dehydrogenase/reductase superfamily [103].

3.5. Resveratrol
Resveratrol is a stilbene-type polyphenol characteristically present in red wine and grapes. The
main gut microbial catabolites of resveratrol are dihydroresveratrol and the m-deoxy metabolites of
both resveratrol and dihydroresveratrol (Figure 8B) [104]. Using a panel of gut microbes, Slackia
equolifaciens and Adlercreutzia equolifaciens were identified as dihydroresveratrol producers. Upon
incubation of resveratrol with fecal samples, both dihydroresveratrol and 3,4′-dihydroxystilbene
were observed as intermediates in the formation of lunularin [104].

Figure 7.
Figure Proposed mechanism
7. Proposed mechanism for
for the
the microbial
microbial conversion
conversion of
of anthocyanidin
anthocyanidin into
into protocatechuic
protocatechuic acid
acid
and 2,4,6-trihydroxyphenylacetic acid [96].
and 2,4,6-trihydroxyphenylacetic acid [96].
3.4. Curcumin
In animals and humans, curcumin, a yellow pigment from turmeric, is catabolized to its
hydrogenated (dihydro-, tetrahydro-, hexahydro-, and octahydro-), desmethyl, O-glucuronide,
and O-sulfate metabolites [101,102]. E. coli NADPH-dependent reductase, the enzyme responsible for
the stepwise reduction of curcumin, has been isolated from human feces (Figure 8A) [103]. While the
enzyme, NADPH-dependent curcumin/dihydrocurcumin reductase (CurA), catalyzes curcumin
Figure it
selectively, 7.does
Proposed mechanism
not reduce for the microbial conversion
tetrahydrocurcumin of anthocyanidin
to yield corresponding into protocatechuic
secondary acid has
alcohols. CurA
been and 2,4,6-trihydroxyphenylacetic
identified acid [96].
as a member of the medium-chain dehydrogenase/reductase superfamily [103].

Figure 8. Gut microbial metabolism of (A) curcumin by curcumin reductase CurA and (B) resveratrol
into lunularin [101,102,104].

3.6. Ellagitannin
Ellagitannins, including ellagic acid, punicalin and punicalagin, are known to be present in
pomegranates, raspberries, strawberries, and walnuts [105,106]. Acid hydrolysis of ellagitannins
releases free ellagic acid [107]. The process of gut microbial conversion of ellagic acid into urolithins
can be explained by the carboxyl group-driven dehydroxylation of polyphenols. Strawberries,
Figure 8. Gut
pomegranate microbial
juice, metabolism
and walnuts of
are of (A) curcumin
good by curcumin
sources of reductase CurA and (B)formed
resveratrol
Figure 8. Gut microbial metabolism (A) curcumin by dietary
curcuminellagic acid,
reductase which
CurA and is by C-C
(B) resveratrol
into
coupling lunularin
of two [101,102,104].
molecules of gallic acid, followed by intramolecular condensation to form a di-
into lunularin [101,102,104].
3.5. Resveratrol
3.6. Ellagitannin
Resveratrol is a stilbene-type polyphenol characteristically present in red wine and grapes.
Ellagitannins,
The main including
gut microbial ellagic
catabolites acid, punicalin
of resveratrol and punicalagin, and
are dihydroresveratrol are known to bemetabolites
the m-deoxy present in
pomegranates, raspberries, strawberries, and walnuts [105,106]. Acid hydrolysis
of both resveratrol and dihydroresveratrol (Figure 8B) [104]. Using a panel of gut microbes, of ellagitannins
releasesequolifaciens
Slackia free ellagic and
acid Adlercreutzia
[107]. The process of gut microbial
equolifaciens conversion
were identified of ellagic acid intoproducers.
as dihydroresveratrol urolithins
can beincubation
Upon explainedof by the carboxyl
resveratrol group-driven
with fecal dehydroxylation
samples, both of polyphenols.
dihydroresveratrol Strawberries,
and 3,40 -dihydroxystilbene
pomegranate juice, and walnuts are good sources of dietary
were observed as intermediates in the formation of lunularin [104]. ellagic acid, which is formed by C-C
coupling of two molecules of gallic acid, followed by intramolecular condensation to form a di-
Molecules 2019, 24, 370 10 of 25

3.6. Ellagitannin
Ellagitannins, including ellagic acid, punicalin and punicalagin, are known to be present in
pomegranates, raspberries, strawberries, and walnuts [105,106]. Acid hydrolysis of ellagitannins
releases free ellagic acid [107]. The process of gut microbial conversion of ellagic acid into urolithins
can be explained by the carboxyl group-driven dehydroxylation of polyphenols. Strawberries,
pomegranate juice, and walnuts are good sources of dietary ellagic acid, which is formed by
Molecules 2019, 24 FOR PEER REVIEW 10
C-C coupling of two molecules of gallic acid, followed by intramolecular condensation to form
a di-lactone [108–111]. Urolithins are, arguably, the main urinary biomarkers of nut consumption [112].
lactone [108–111]. Urolithins are, arguably, the main urinary biomarkers of nut consumption [112].
In the catabolic pathway from ellagic acid to urolithins, one of the two lactone moieties undergo
In the catabolic pathway from ellagic acid to urolithins, one of the two lactone moieties undergo
hydrolysis and the methylated quinone tautomer of the resulting carboxylic acid is then reduced
hydrolysis and the methylated quinone tautomer of the resulting carboxylic acid is then reduced in
in a manner similar to the ferredoxin-mediated reduction of 4-hydroxybenzoic acid (Figure 9).
a manner similar to the ferredoxin-mediated reduction of 4-hydroxybenzoic acid (Figure 9). This
This results in a semi-hydroquinone, from which the p-hydroxy group leaves as a water molecule
results in a semi-hydroquinone, from which the p-hydroxy group leaves as a water molecule
following decarboxylation. Subsequent dehydroxylation can occur via reduction of quinone-methide
following decarboxylation. Subsequent dehydroxylation can occur via reduction of quinone-methide
tautomers to form urolithins A, B, and C. In the microbial pathway from ellagic acid to urolithins,
tautomers to form urolithins A, B, and C. In the microbial pathway from ellagic acid to urolithins,
only the first dehydroxylation is driven by decarboxylation. Subsequent dehydroxylations involve
only the first dehydroxylation is driven by decarboxylation. Subsequent dehydroxylations involve a
a step-by-step reduction of a quinone, in which keto-enol tautomerism first produces a secondary
step-by-step reduction of a quinone, in which keto-enol tautomerism first produces a secondary
alcohol and a subsequent hydride attack of the quinone leads to the dehydration step. Analogous
alcohol and a subsequent hydride attack of the quinone leads to the dehydration step. Analogous
dehydration is found in the dehydroxylation of catechin (Figure 6), which leads to the formation of
dehydration is found in the dehydroxylation of catechin (Figure 6), which leads to the formation of
5-(30 -hydroxyphenyl)-γ-valerolactone. In addition, the pathway described for the dehydroxylation of
5-(3’-hydroxyphenyl)-γ-valerolactone. In addition, the pathway described for the dehydroxylation of
4-hydroxybenzoic acid may explain the formation of 3-hydroxybenzoic acid from protocatechuic acid
4-hydroxybenzoic acid may explain the formation of 3-hydroxybenzoic acid from protocatechuic acid
after C-ring cleavage of cyanidin or quercetin (Figure 5).
after C-ring cleavage of cyanidin or quercetin (Figure 5).

Figure 9. Gut microbial conversion of ellagitannin into urolithins A, B, and C [108–111].

Figure 9. Gut microbial conversion of ellagitannin into urolithins A, B, and C [108–111].
4. Physiological Functions Mediated by Intestinal Microbiota
4. Physiological Functions Mediated by Intestinal Microbiota
4.1. Bioavailability and Action of Polyphenol Catabolites
4.1. Bioavailability and Action of Polyphenol Catabolites
The total intake of dietary polyphenol at the small intestine is estimated at around 10% [113–115].
Hence,Thea large proportion
total intake of ingested
of dietary polyphenols
polyphenol are transported
at the small intestine istoestimated
the large intestine,
at aroundwhere they
10% [113–
are
115].catabolized to phenolic
Hence, a large acids
proportion ofby the intestinal
ingested bacteria.
polyphenols Recently, much
are transported attention
to the has beenwhere
large intestine, paid
to theare
they bioavailability
catabolized toand physiological
phenolic actions
acids by of their catabolites
the intestinal and to the
bacteria. Recently, muchfunctional interaction
attention has been
between
paid to polyphenols and intestinal
the bioavailability bacteria (Figure
and physiological 10). of their catabolites and to the functional
actions
interaction between polyphenols and intestinal bacteria (Figure 10).
Cyanidin glucosides are mainly converted into 3,4-dihydroxybenzoic acid in the human large
intestine and are then absorbed into the bloodstream [116]. In the plasma, it is estimated that 44%
and 0.02% of ingested cyanidin glucoside is in the form of 3,4-dihydroxybenzoic acid and cyanidin-
3-glucoside, respectively [116], suggesting that 3,4-dihydroxybenzoic acid is a key compound for the
vascular effects of cyanidin glucosides (Table 1). The intake of a cranberry juice cocktail containing
mainly peonidin and cyanidin glycosides, hyperoside, and quercein, increases the plasma levels of
these flavonoids and phenolic acids (Table 1) [117]. This study also showed a higher concentration of
methylated quercetin conjugates in rat plasma compared with the plasma concentrations in rats
treated with quercetin-3-glucoside or α-glucosyl rutin alone [118,119]. This suggests that FOS may
decrease the degradation of quercetin via the modification of the intestinal microbiota and therefore,
increase the bioavailability of the parent compound, quercetin. Dietary fiber shows promise in
Molecules 2019, 24, 370 11 of 25
modifying the bioavailability and physiological functions of polyphenols by changing components
of the microbiota.

Figure 10. Pathways of absorption, bioconversion, and physiological action of non-absorbable

Figure 10. Pathways of absorption, bioconversion, and physiological action of non-absorbable
polyphenols in the intestinal tract.
polyphenols in the intestinal tract.
Cyanidin glucosides are mainly converted into 3,4-dihydroxybenzoic acid in the human large
intestine and are then absorbed into the bloodstream [116]. In the plasma, it is estimated that
44% and 0.02% of ingested cyanidin glucoside is in the form of 3,4-dihydroxybenzoic acid and
cyanidin-3-glucoside, respectively [116], suggesting that 3,4-dihydroxybenzoic acid is a key compound
for the vascular effects of cyanidin glucosides (Table 1). The intake of a cranberry juice cocktail
containing mainly peonidin and cyanidin glycosides, hyperoside, and quercein, increases the plasma
levels of these flavonoids and phenolic acids (Table 1) [117]. This study also showed a higher
concentration of 3,4-dihydroxybenzoic acid than quercetin in human plasma. Plasma antioxidant
capacity, estimated by the Oxygen Radical Absorption Capacity (ORAC) assay, correlates with the
plasma levels of quercetin, epicatechin, 3,4-dihydroxybenzoic acid, 4-hydroxy-3-methoxy-benzoic acid,
and 3,4-dihydroxyphenylacetic acid. 3,4-Dihydroxybenzoic acid exhibits the strongest correlation
with plasma ORAC activity (r = 0.440) and also correlates with plasma TAP activity (r = 0.233)
and protective activity against LDL oxidation (r = 0.503). Epicatechin conjugates, including O-sulfate
and O-glucuronide, in human plasma after oral intake of 207 µmol of epicatechin, showed higher
Cmax and faster Tmax than those of their conjugated catabolites (Table 1) [50]. However, the AUC
for 0–24 h of the conjugated catabolites was higher than that of the epicatechin conjugates due to a
longer elimination half-life. These pharmacokinetic data show that the increase in catabolites and
their conjugates in plasma occurs around 7 h after detection of the parent flavonoids, due to colonic
microbiota-dependent catabolism. Additionally, the bimodal peak of the original flavonoids suggests
that they may be absorbed from the large intestine. A large proportion of ingested flavonoids seem
to be transported into the large intestine, where they are degraded into their catabolites, including
phenolic acids. The absorption level of the resulting catabolites is, consequently, high enough to exert
greater physiological actions than the parent flavonoids. The combination of quercetin-3-glucoside
or α-glucosyl rutin with fructooligosaccharide (FOS) increases the concentration of quercetin and
O-methylated quercetin conjugates in rat plasma compared with the plasma concentrations in rats
treated with quercetin-3-glucoside or α-glucosyl rutin alone [118,119]. This suggests that FOS may
decrease the degradation of quercetin via the modification of the intestinal microbiota and therefore,
increase the bioavailability of the parent compound, quercetin. Dietary fiber shows promise in
modifying the bioavailability and physiological functions of polyphenols by changing components of
the microbiota.
Molecules 2019, 24, 370 12 of 25

Table 1. Pharmacokinetic properties of flavonoid catabolites in human trials.

Subjects Tmax1 Tmax2 T1/2

Samples Detected Compounds Cmax1 (M) Cmax2 (M) AUC0-last (M·h) Refs
(Gender; Age; BMI) (hours) (hours) (h)
Blood orange juice (71 mg Healthy men & women; Cyanidin-3-glucoside 0.0019 0.5 0.007 ac
116
cyanidin glucosides) 20–24; 21–31 3,4-Dihydroxybenzoic acid 0.492 2 11 ac
Quercetin 1.1 1.4 1.5 7.8 9.9 bc
Epicatechin 0.082 2.6 0.079 8.1 0.586 bc
Myricetin 0.068 1.7 0.05 7.8 0.319 bc
Isorhamnetin 0.099 0.5 0.153 7.3 0.267 bc
Healthy nonsmoking Peonidin glycosides 0.00017–0.0021 0.9–4.7 0.00038–0.0053 bc
Cranberry juice cocktail
men & postmenopausal Cyanidin glycosides 0.00019–0.00073 1.7–3.3 0.000092–0.0033 bc 117
(188.5 mg phenolics)
women; 50–70; 18.5–29.9 3,4-Dihydroxybenzoic acid 10 8.8 91 bc
4-Hydroxy-3-methoxybenzoic acid 12 0.7 11 6.1 16 bc
4-Hydroxybenzoic acid 1.4 0.8 1.4 7.2 9.9 bc
4-Hydroxyphenylacetic acid 0.613 1.5 0.783 7.8 2.3 bc
3,4-Dihydroxyphenylacetic acid 0.083 8.4 0.348 bc
Epicatechin conjugates g 1.223 1 4.943 b 1.9
0.272 d 6.4 7.595 bd 6.3
[2-14 C](−)-Epicatechin (207 5-(30 ,40 -Dihydroxyphenyl)-γ-
Healthy men; 31 ± 3; 0.177 e 6.1 3.237 be 4.4
valerolactone 50
mmoles) 24.5 ± 3.3 0.039 f 5.5 1.017 bf 6.4
5-(3-Hydroxyphenyl)-4-
0.056 d 5.9 1.492 bd 7.6
hydroxyvaleric acid
5-(3,4-Dihydroxyphenyl)-4-
0.054 e 4.9 0.835 be 6.5
hydroxyvaleric acid
Pomegranate juice (318 mg
Healthy men & women;
of punicalagin; 12 mg of Ellagic acid 0.06 0.98 0.17 a 0.71 107
32.6 ± 10.2; 21.3 ± 1.4
ellagic acid)
Pomegranate juice (857 mg Healthy men & women;
Ellagic acid 0.06 0.65 0.14 a 1.14 120
of gallic acid equivalent) 29.7 ± 8.3; 24.1 ± 3.6
a6 h; b 24 h; c Unit is converted based on the data of the references; d sulfate conjugate; e glucuronide conjugates; f sulfate and glucuronide conjugate; g sum of sulfate, glucuronide, and/or
methylated conjugates.
Molecules 2019, 24, 370 13 of 25

The hydrolysable tannin, ellagitannin, is an oligomer composed of gallic acid, ellagic acid,
and glucose. Ellagic acid is detected in human plasma 40 min after pomegranate intake [107,120].
However, plasma ellagic acid is undetectable within 5 h after the intake, suggesting it is rapidly cleared.
Urolithins, which are well-known active catabolites of ellagic acid, and their conjugates can be detected
in plasma at 0.5 and 6 h (0.04 and 0.11 µM of urolithin A respectively), with continuous excretion into
the urine for 48 h after pomegranate consumption in some, but not all, volunteers [107,120]. Therefore,
ellagic acid, produced by pH-dependent hydrolysis of ellagitannins [121], may be absorbed into the
bloodstream at the upper digestive tract and may also undergo sustainable absorption as its bacterial
catabolites, urolithins, at the colon. Since urolithin A and its conjugates are detected in some mouse
tissues, including colon, prostate, liver, and kidney, at 1 to 6 h after oral administration of urolithin
A [32], the continuous circulation of urolithins may allow their accumulation in tissues, thus facilitating
their physiological functions.
The physiological functions of polyphenol catabolites, such as anti-oxidative, anti-inflammatory,
and anti-proliferation activities, may differ from the functions of their parent polyphenols.
Quercetin catabolites, such as 3,4-dihydroxybenzoic acid, 3-methyoxy-4-hydroxybenzoic acid,
3,4-dihydoxyphenylacetic acid, and 3-(3,4-dihydroxyphenyl)propionic acid, show one-half to
one-eighth lower anti-oxidative activity than that of quercetin [122].
In an in vitro proximal colon model, green tea, black tea, and citrus flavonoids containing
flavan-3-ols, theaflavin, quercetin, rutin, and hesperidin were catabolized to 4-hydroxyphenylacetic
acid, 3-methoxy-4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and other catabolites.
The anti-proliferation effect of these phenolic acids on colon cancer cell lines was one-fifth
of the effect of the corresponding parent flavonoids [123]. A mixture of phenolic acids
containing 3-(4-hydroxy-3-methoxyphenyl)propionic acid, 4-hydroxy-3-methoxybenzoic acid,
3,4-dihydroxybenzoic acid, and 3-(3-hydroxyphenyl)propanoic acid, which are detected in
human plasma after blueberry juice consumption, have no effect on Nrf2 activation in human
umbilical vein endothelial cells [124]. Furthermore, the inhibitory effect of phenolic acids,
including 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, 3-phenylpropionic acid,
3-(3,4-dihydroxyphenyl)propionic acid, resorcinol, and phloroglucinol, on nitric oxide production
in lipopolysaccharide-activated macrophages is minimal compared to the effects of their parent
compound [125]. However, procyanidin A2 and its microbial catabolite, 3-(4-hydroxyphenyl)propionic
acid, inhibit oxidized LDL-induced foam cell formation, inflammatory responses, oxidative stress,
and the expression of cholesterol efflux/influx-related genes to the same degree, when applied at
the same concentration [126]. Interestingly, urolithins show higher anti-oxidative, anti-inflammatory,
and anti-proliferation activities than ellagitannin and ellagic acid [32,127,128]. Taken together with the
pharmacokinetic data, bacterial catabolites can exert their specific functions in the large intestine and
bloodstream, although the physiological function of polyphenols may be weakened or altered by the
catabolism of parent compounds.

4.2. Action of Polyphenols on Gut Microbiota

Polyphenols are known to exhibit growth-promoting effects, namely prebiotic actions, on
intestinal bacteria. This subject has recently been summarized in other review papers [129–132].
Although the molecular mechanisms of these prebiotic effects have not been fully elucidated,
they may be associated, in part, with their selective antimicrobial activity against pathogenic
bacteria. Phenolic extracts obtained from eight berries containing anthocyanins, flavan-3-ols,
B-type proanthocyanidins, and ellagitannins inhibit the growth of food-poisoning bacteria, such
as Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus, but do not affect the
growth of the probiotic bacterium, Lactobacillus rhamnosus [133]. Pomegranate ellagitannins strongly
suppress the growth of Clostridium bacteria and S. aureus [134]. Lactic acid bacteria are also slightly
reduced by treatment with ellagitannin, while the growth of bifidobacteria is markedly enhanced.
However, in batch culture of human feces, pomegranate ellagitannin, but not punicalagin, increases
Molecules 2019, 24, 370 14 of 25

the number of Bifidobacteria spp. and Lactobacillus-Enterococcus group bacteria after 5 and 10 h
of inoculation, respectively, with no effect on Eubacterium rectale-Clostridium coccoides group and
Clostridium histolyticum group bacteria [135]. Tannic acid from grape seeds and pomace show potent
growth-promoting effects on Lactobacillus acidophilus [136]. Mango peels containing gallotannins also
show prebiotic effects on bifidobacteria and lactic acid bacteria [18,137]. However, phenolic acids
derived from the colonic bacteria-dependent catabolism of flavonoids have low selectivity against
probiotic and pathogenic bacteria [138]. An intervention trial of 10 healthy adult men (45–50 years)
investigated changes in the components of gut microbiota [139]. Drinking red wine containing
anthocyanins, flavan-3-ols, B-type proanthocyanidins, flavonols, stilbenes, and phenolic acids for
20 days was shown to increase the proportions of the phyla Proteobacteria, Actinobacteria, Fusobacteria,
Firmicutes, and Bacteroidetes in feces compared with baseline measurements. Meanwhile, de-alcoholized
red wine with the same polyphenol composition only increased the proportions of Fusobacteria
and Bacteroidetes and therefore, reduced the Firmicutes/Bacteroidetes ratio compared with subjects
drinking red wine. Subjects drinking gin for the same period, as a non-polyphenol control group,
showed a reduction in the proportions of these phyla. Curcumin and resveratrol also decreased the
Firmicutes/Bacteroidetes ratio and have anti-inflammation and anti-carcinogenesis effects by modifying
colonic microbial ecology in animal experiments [140–144]. Furthermore, resveratrol is thought to
inhibit the production of trimethylamine and hence, trimethylamine oxide, from choline via the
remodeling of the gut microbe composition [144]. Human microbiota-associated (HMA) animals
(also known as human flora-associated, HFA) are germ-free rodents inoculated with a human fecal
microbiota in order to study the human gut microbiome. Green tea polyphenols reduced blood glucose
level, lipid metabolism biomarkers, and the Firmicutes/Bacteroidetes ratio in HFA mice fed a high-fat
diet [145,146].
Akkermansia muciniphila is a gram-negative bacterium, isolated as a novel mucin-degrading
bacterium from human feces [147]. Both live and pasteurized A. muciniphila are able to improve
metabolic function in mice fed a high-fat diet [148]. This improvement is partly explained by the
fact that an outer membrane protein, Amuc_1100, enhances glucose metabolism and gut barrier
function by activating insulin and toll-like receptor 2 signaling [148]. The microbiome of mice on
a high-fat diet show that an abundance of A. muciniphila is positively correlated with fatty acid
oxidation and the browning of white adipocytes, but is negatively correlated with inflammation
and metabolic syndrome makers [149]. This finding strongly suggests that A. muciniphila is a
promising beneficial bacterium to prevent the development of metabolic syndrome. Interestingly,
Colombian type 2 diabetes patients taking metformin have a higher abundance of A. muciniphila
and SCFA-producing bacteria, such as Butyrivibri, Bifidobacteria, and Megasphaera, compared with
those who do not take this drug [150]. Concord grape polyphenols, which appear to be composed of
anthocyanidins, B-type proanthocyanidins, and flavan-3-ols, suppress inflammation in the intestinal
tract and metabolic abnormalities and increase gut barrier integrity [20]. Cranberry extracts containing
flavonols, anthocyanins, and proanthocyanidins also improve glucose and lipid homeostasis in mice
fed high-fat and high-sucrose diets [22]. Oligomeric (monomer to tetramer) and polymeric (>pentamer)
proanthocyanidins from apples suppress the abnormalities in both glucose and lipid homeostasis
by modifying the expression of genes associated with inflammation, lipid metabolism, and gut
epithelial tight junctions. However, there are some differences in efficiency between the oligomers
and polymers [21]. More interestingly, only polymeric proanthocyanidins tend to decrease the
Firmicutes/Bacteroidetes ratio and increase the relative abundance of A. muciniphila and Verrucomicrobia
in the cecum of mice on a high-fat/high-sucrose diet. Although the role of A. muciniphila and
Verrucomicrobia in the amelioration of metabolic abnormalities by these polyphenols needs to be
fully investigated, the oligomeric and polymeric proanthocyanidins may exert these effects through a
variety of molecular mechanisms.
Adhesion to the intestinal epithelia and subsequent colonization are thought to be important
criteria for probiotic bacteria to exert their physiological effects on human health [151]. Epigallocatechin
Molecules 2019, 24, 370 15 of 25

and procyanidin B1 and B2 increase the adhesion of lactic acid bacteria to intestinal epithelial
cells in vitro, while some types of flavan-3-ols inhibit this adhesion [152]. Apple extracts
containing procyanidin B2 and chlorogenic acid increase adhesion to intestinal epithelial cells in vitro.
Quercetin and its glycosides show the most potent activity to enhance bacterial adhesion [153].
SCFA including acetate, propionate, and butyrate, are important metabolites from the action
of colonic bacteria on indigestible carbohydrates. They play a role as physiological mediators
by providing an energy source and acting as immunomodulators and metabolic regulators [154].
Rutin and some phenolic acids increase total SCFA levels and the ratio of acetate among SCFA in a
multi-reactor gastrointestinal model [155]. The intake of a grapefruit extract, containing hesperidin
and naringin, increases the wet weight of cecum digesta, and hence total SCFA content, in rats,
while the combination of the extract with inulin weakened this effect [156]. Apple polyphenols
containing epicatechin, procyanidins, and chlorogenic acid also increase rat cecum size and SCFA
concentration in the cecum, and the combination of apple polyphenols with apple pectin shows an
additive effect [157]. Inoculation with pomegranate ellagitannin increases the concentration of SCFAs
in human fecal batch culture, while punicalagin has no such effect [135]. However, ellagitannin shows
contradictory results for its combinational effect with FOS. Dietary FOS shows beneficial effects on pH,
SCFA production, and bacterial enzymatic activity in the cecum. Different studies have shown that
ellagitannin either interfered with or enhanced these positive effects of FOS [158,159]. Anthocyanins
and phenolic acids may enhance SCFA production in the cecum of animals after FOS ingestion [118,159].
Flavonoids and phenolic acids, which promote the growth of SCFA-producing bacteria, including
Bifidobacterium spp. and A. muciniphilla as mentioned above, may enhance the production of SCFAs.
In addition, epigallocatechin-3-gallate, but not quercetin, stimulates the in vitro production of acetate
and lactate by Bifidobacterium adolescentis, without growth-promoting effects [160], suggesting that
flavonoids may be able to induce the production of SCFAs by colonic bacteria via activation of their
metabolic function.
Culture supernatants of bifidobacteria and lactic acid bacteria are known to have
anti-inflammatory activity [161–163], but both the active compounds and the mechanism of
this action has not been fully demonstrated. When probiotic bacteria and polyphenols were
anaerobically incubated for 3 h, a culture supernatant showed increased anti-inflammatory activity
against lipopolysaccharide-stimulated macrophages [125]. Among 60 phytochemicals, quercetin,
epigallocatechin-3-gallate, and some flavonoids increased the anti-inflammatory effects of B. adolescentis
culture supernatant on nitric oxide production in activated macrophages in vitro [125,160]. On the other
hand, these polyphenols were not able to potentiate the anti-inflammatory activity of lactic acid bacteria.
Phenolic acids, which are assumed to be catabolites of these polyphenols, and the bifidobacteria
metabolites, acetate and lactate, have no such effect [125,164], suggesting that these polyphenols
may make B. adolescentis produce unknown active compounds. Interestingly, stearic acid has been
tentatively identified as the anti-inflammatory compound obtained from the culture supernatant of
quercetin-treated bifidobacteria [164]. Taken together, these findings suggest that polyphenols reaching
the large intestine may not only be catabolized to small phenolic acids, but also elicit potentially
beneficial effects of intestinal probiotic bacteria.

5. Future Directions
In recent years, metagenomic analysis of intestinal microbiota have provided a breakthrough
in our understanding of the etiology of digestive diseases. Moreover, it is now recognized
that changes in the intestinal microbiota affect the onset of not only digestive diseases, but also
diabetes, atherosclerosis, neuropsychiatric diseases, and other systemic diseases [165]. However,
the gut is an immune organ, in which more than half of all immune cells within the body are
concentrated. The immune system in the gut affects the progression of obesity, diabetes, food allergies,
and inflammatory bowel disease [166,167]. It is, therefore, of much interest to understand the
relationship between the intestinal immune system and the intestinal microbiota. In this context,
Molecules 2019, 24, 370 16 of 25

it is essential to focus research on elucidating the physiological function of polyphenols in the

gut and their effects on the intestinal microbiota. In other words, microbiota should be taken into
consideration when discussing the health effects of dietary polyphenols. It is, therefore, necessary
to fully clarify the process of microbiota-dependent catabolism of dietary polyphenols in the large
intestine. The effect of polyphenols on microbial behavior should also be evaluated in detail. As a
whole, the relationship between dietary polyphenols and the intestinal microbiota is still obscure and
unexplored. The intestinal microbiota is quite diverse among individuals within and between different
ethnic groups. Therefore, the relationship between dietary polyphenols and microbiota is also diverse
and profoundly complicated. To clarify the function of polyphenols in the absence of this confounding
diversity, innovative in vitro experimental systems need to be established to replace rodent models,
which have different microbiota to humans.
Finally, it should be noted that the role of microbiota in the brain–gut interaction is a future trend of
polyphenol research. The intestinal microbiota is known to contribute to the development of reactions
of the hypothalamic-pituitary-adrenal axis, which comprises major emotional reactions [25,168].
Microbiota is apparently recognized as a key factor in determining the stress responses and behavioral
characteristics of the host. Successive intake of polyphenols may affect mental stress and behavior
through the modification of intestinal microbiota. This is a subject on which future research
should focus.

6. Conclusions
Polyphenols are composed of various subgroups whose basic structures differ for individual
compounds. They can be divided into monomeric polyphenols and oligomeric tannins. After oral
ingestion, some of the monomeric polyphenols and oligomeric tannins are absorbed, with or without
deglucosylation, and enter into the blood circulation by crossing the epithelial cells in the small
intestine. However, most polyphenols are transported into the large intestine, where intestinal bacteria
catabolize them to chain fission products and/or monomeric catechins. Hydrolysable tannins, such
as gallotannins and ellagitannins, generate gallic acid, ellagic acid, and their catabolites in the gut.
Thereafter, these catabolites are excreted into the feces, but some of may be absorbed into the body
through epithelial cells in the bowel. Polyphenols can be converted to various catabolites by the action
of hydrolase and dioxygenase present in the microbiota. These catabolites may be non-negligible
contributors to the health effects of dietary polyphenols. Furthermore, it is apparent that polyphenols,
especially proanthocyanidins, have suppressive effects on the progress of life-style related diseases,
such as diabetes, by modifying the patterns of the intestinal microbiota.
The intake of polyphenols improves the health effects of the intestinal microbiota by
activating SCFA excretion, intestinal immune function, and other physiological processes.
The microbiota-dependent effects of polyphenols may to be applied practically to the health food
or supplement industries. For this purpose, further research is necessary to analyze the catabolic
reactions of polyphenols and their reaction products, and to determine the mechanisms of action of
these compounds on the intestinal microbiota.

Author Contributions: Conceptualization, J.T.; Writing—original draft preparation, K.K., Y.Y., and J.T.;
writing—review and editing, K.K., Y.Y., and J.T.; Funding acquisition, J.T.
Funding: This work is supported by JSPS Kakenhi (Grant No. 16K00815 for J.T).
Conflicts of Interest: No potential conflicts were disclosed.

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