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ISSN: 0360-2532 (print), 1097-9883 (electronic)

Drug Metab Rev, Early Online: 1–16

! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03602532.2014.1003649

REVIEW ARTICLE

Flavonoid interactions during digestion, absorption, distribution and

metabolism: a sequential structure–activity/property relationship-based
approach in the study of bioavailability and bioactivity
Gerard Bryan Gonzales1,2,3, Guy Smagghe2, Charlotte Grootaert1, Moises Zotti2,4, Katleen Raes3, and
John Van Camp1
1
Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium, 2Department of Crop Protection,
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Faculty of Bioscience Engineering, Ghent University, Gent, Belgium, 3Department of Industrial Biological Science, Faculty of Bioscience Engineering,
Ghent University, Kortrijk, Belgium, and 4Department of Crop Protection, Laboratory of ChemoGenomics and Bioinformatics, Federal University of
Santa Maria, Santa Maria, Brazil

Abstract Keywords
Flavonoids are a group of polyphenols that provide health-promoting benefits upon ABC transporters, amylase, flavonoids,
consumption. However, poor bioavailability has been a major hurdle in their use as drugs or glucuronidation, lipase, mucus layer,
nutraceuticals. Low bioavailability has been associated with flavonoid interactions at various protein–flavonoid interaction, QSAR,
stages of the digestion, absorption and distribution process, which is strongly affected by their sulfation
molecular structure. In this review, we use structure–activity/property relationship to discuss
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various flavonoid interactions with food matrices, digestive enzymes, intestinal transporters and History
blood proteins. This approach reveals specific bioactive properties of flavonoids in the
gastrointestinal tract as well as various barriers for their bioavailability. In the last part of this Received 10 October 2014
review, we use these insights to determine the effect of different structural characteristics on Revised 22 December 2014
the overall bioavailability of flavonoids. Such information is crucial when flavonoid or flavonoid Accepted 22 December 2014
derivatives are used as active ingredients in foods or drugs. Published online 30 January 2015

Introduction aspect of digestion. In this article, we will follow the fate of

flavonoids throughout the entire gastro-intestinal tract and in
Flavonoids are a large group of secondary plant metabolites.
the circulation and discuss the structure–activity relationship
They are the most widespread and diverse group of polyphe-
(SAR) at every stage of this process. By investigating the
nols and occur as either aglycones or conjugates with
stepwise interactions of flavonoids with enzymes and matrix
glycosides and acyl groups, wherein around 8000 different
compounds using qualitative and quantitative SAR (QSAR),
types have been identified so far (Gonzales et al., 2014a).
we will be able to estimate the chance of survival of the
They exert a variety of biological activities, including anti-
compounds in the circulation and the reasons for their non-
oxidative (Fiol et al., 2012; Pietta, 2000), anti-hypertensive
survival. In addition, we will compare various in vitro and
(Al Shukor et al., 2013; Balasuriya & Rupasinghe, 2012),
in vivo studies and discuss certain discrepancies in flavonoid
anti-obesity (Hsu & Yen, 2008), anti-viral, hepatoprotective
bioavailability between the two approaches.
and immune-regulatory activities (Middleton et al., 2000).
Albeit their health-promoting benefits, their poor oral
bioavailability has been regarded as a major hurdle in using Flavonoid structure
these compounds as health-promoting ingredients (Manach
et al., 2004, 2005; Scalbert & Williamson, 2000; Flavonoids consist of a 15-carbon skeleton consisting of two
benzene rings attached via a heterocyclic pyrane ring, labeled
Thilakarathna & Rupasinghe, 2013). Generally, factors such
as rings A, B and C, in a C6-C3-C6 arrangement, as depicted
as food matrix interactions, food processing, host (human)-
in Figure 1. They are divided into several groups depending
related factors (e.g. age, occurrence of certain diseases and
on the degree of hydroxylation, methoxylation, prenylation,
lifestyle) and the flavonoids’ chemical structure cause their
glycosylation or even the attachment of the B ring (in the case
poor oral bioavailability (D’Archivio et al., 2010). Many
of isoflavones). Flavonoids occur either as glycosides,
reviews discuss the relationship of flavonoid structure to their
methylated derivatives or aglycones (the basic structure).
oral bioavailability; however, they tend to focus on a single
The position of the B ring may be at the C2-position in the
case of most flavonoids or in the C3-position in the case of
Address for correspondence: Prof. John Van Camp, Department of Food isoflavones. Common hydroxylation points are at positions 5,
Safety and Food Quality, Faculty of Bioscience Engineering, Ghent
University, Gent 9000, Belgium. Tel: +32-(0)9 264 62 08. E-mail: 7 (A ring), 30 , 40 , 50 (B ring), 3 and 2 (C ring). Differences
John.VanCamp@UGent.be also depend on the presence of the C2¼C3 double bond and
 2 G. B. Gonzales et al.
Figure 1. Flavonoid basic structure
(aglycone) and its chemical classes
(Kumar & Pandey, 2013).
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C4-ketone moiety. Figure 1 shows the basic structures and
common classes of flavonoids (Dai & Mumper, 2010; Kumar
& Pandey, 2013).
SAR analyses
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SAR analysis is a technique to discern the structural or
chemical features that lead to a desired activity or property
(Gandhi & Morris, 2009). The fundamental premise of SAR
is that chemical and physical properties of molecules, when in
contact with a biological system, determine its biological/
toxicological/physical behavior (McKinney et al., 2000). SAR
is useful in ascertaining the relative importance of certain
functional groups in relation to a given biological or physical
activity. Therefore, investigators are able to examine which
constitutes a class of molecules that are active, what
determines their activity and what distinguishes them from
non-active compounds (McKinney et al., 2000). When used to
explain the presence or absence of certain functional groups,
it will be hereto referred as qualitative SAR, whereas when
predictive mathematical models are made, the term QSAR
will be used. The majority of the biological activities
presented in this review cover only qualitative SAR, which
is partly due to the lack of data regarding screening large
amounts of flavonoids. Nonetheless, such qualitative SARs,
although not mathematically predictive, are highly compre-
hensible even to those not familiar with SAR techniques.
The goal of QSAR modeling is to construct a mathematical
relationship between the molecular structural descriptors and
activity or property (Gandhi & Morris, 2009). This typically
involves four steps: extracting descriptors from the molecular
structure, choosing the appropriate descriptors that are
relevant to the property in question, using the values of the
descriptors as independent variables to explain the property
observed through the use of computational techniques and
validation of the model (Dudek et al., 2006). By far the most
common computational methods used in 2D QSAR analysis
are multiple linear regression (MLR) and partial least squares
Drug Metab Rev, Early Online: 1–16
regression (PLSR). MLR and PLSR are both modeling
methods that predict the activity or property as a linear
function of molecular descriptors (Dudek et al., 2006). MLR
models the activity to be predicted as a linear function of all
descriptors. The problem with this technique arises in the
case of large descriptors-to-compounds ratios, where multi-
collinearity occurs. This makes the model rather unstable,
especially when predicting external datasets or when com-
pounds not included in the dataset are used to develop the
model as a training set (Dudek et al., 2006). Nonetheless, as
seen later in this paper, stepwise MLR (SMLR) is a very
popular technique, perhaps due to its ease of operation and
interpretability.
PLSR on the other hand is a developed generalization of
MLR. Unlike MLR, PLSR can analyze data with strongly
collinear, noisy and numerous independent variables (Wold
et al., 2001). PLSR assumes that a large number of descriptors
can actually be explained by a small amount of latent
variables by decomposing the input matrix of descriptors into
orthogonal score vectors. These score vectors then serve as
new predictors (Dudek et al., 2006; Wold et al., 2001). For
this reason, PLS has been successfully used for analyzing
even more complex molecular descriptors such as in 3D
QSAR analysis. Techniques for 3D QSAR have been
increasingly popular over the recent years because the
descriptors used in these analyses usually comprise of
location-dependent structural characteristics (Perkins et al.,
2003), which makes it easier for non-experts to interpret
QSAR results. Comparative Molecular Field Analysis
(COMFA) is by far the most studied and applied 3D-QSAR
technique (McKinney et al., 2000). Generally, this technique
involves a crucial alignment step of molecular 3D structures.
Then steric (Lennard-Jones) and electrostatic (Coulumbic)
interactions of a probe atom within the molecule are
calculated at uniform grid points and placing these values in
a matrix. PLSR is then used to generate a model that
associates these interactions to the observed activity/property,
to verify the predictive ability of the generated model and to
 DOI: 10.3109/03602532.2014.1003649 QSAR of flavonoids during digestion, absorption, distribution and metabolism
use the model for predicting the activity of new compounds
not in the model. The beauty of this technique lies in its
ability to plot the interactions to their corresponding region
within the molecular structure, which also corresponds to a
particular molecular moiety (McKinney et al., 2000).
Somehow considered as a development of COMFA, Com-
parative Molecular Similarity Index Analysis (COMSIA)
extends to more spatial descriptors, such as hydrophobicity,
hydrogen bond acceptors and donors. It also does not involve
cut-off values, which makes the calculations outside the
molecular surface possible, thus making COMSIA a more
reliable technique (Nilewar & Kathiravan, 2014). This review
does not aim to provide an exhaustive explanation on the
modeling technique and procedures performed in various
experiments. Instead, we focus on the results of these QSAR
models and their implications to flavonoid bioavailability and
to some extent, bioactivity. This is the first time that flavonoid
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interactions to different factors at various levels of digestion,
absorption and distribution are being reviewed in the
perspective of structure–property relationship.
Upper gastrointestinal tract
Binding of flavonoids to a-amylase and a-glucosidase
and their interaction with food carbohydrates
The increased prevalence of diabetes mellitus has attracted
attention to carbohydrate digestion of in general and
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has prompted the search for post-prandial hyperglycemia-
reducing strategies. In this context, the effect of flavonoids on
the delay in glucose absorption by inhibiting carbohydrate-
hydrolyzing enzymes in the digestive tract has been
investigated (Kim et al., 2014; Wang et al., 2010). Both
a-glucosidase and a-amylase are key enzymes involved in the
digestion of carbohydrates in humans (Tadera et al., 2006).
Human a-amylase is produced in the salivary glands and
the pancreas and is responsible for rapidly converting
digestible starch into sugar monomers (Lo Piparo et al.,
2008; Xiao et al., 2013b). Human a-glucosidase is an enzyme
found in the small intestinal epithelium, which catalyzes the
hydrolysis of disaccharides, mainly sucrose and maltose, and
other oligosaccharides into simple sugars (Fatmawati et al.,
2013).
Several polyphenols, especially flavonoids, have been
reported to inhibit these enzymes. This has been extensively
reviewed (Cao & Chen, 2012; Xiao et al., 2013a,b), and based
on the results, a qualitative SAR can be summarized as
follows:
(1) Hydroxyl groups, especially at the C5 and C7 positions of
the A–C ring and C30 and C40 -positions of the B ring,
increase the inhibitory activity of flavonoids toward
a-glucosidase and a-amylase.
(2) Methylation and methoxylation, which ‘‘blocks’’ the free
hydroxyl groups, decrease inhibitory activity.
(3) Although glycosylation obviously increases the number
of free hydroxyl groups, the inhibitory activity of
flavonoid glycosides is much lower than that of their
aglycone counterparts.
(4) Planarity of the flavonoids, which is due to the unsatur-
ation of the C2–C3 bonds increases a-glucosidase and
a-amylase inhibitory activity.
3
(5) Galloylcatechins have a higher inhibition than non-
galloylated catechins, and catechins are influenced by
the presence of hydroxyl moieties in the C3 and C5
positions of the A–C ring, number of hydroxyl groups in
the B ring or C ring and the 2,3-cis/trans isomerism
(Miao et al., 2014).
(6) Increased inhibitory activity has been observed for
flavonoids having the B ring attached to the C-2 position
rather than to the C3-position (in the case of isoflavones)
(Lo Piparo et al., 2008; Tadera et al., 2006).
It has also been previously reported that an increased
number of hydrogen bond donors and acceptors is related
with a higher human a-amylase inhibition in vitro (correlation
values R2 ¼ 0.69 and 0.54, respectively). Furthermore,
XlogP3, a measure of compounds’ partitioning coefficient
and thus lipophilicity, negatively correlates with percent
inhibition. This suggests that the interaction of the flavonoids
toward the a-amylase is not governed by hydrophobic
interaction, but rather by hydrogen bonding (Xiao et al.,
2013b). This hypothesis was supported by a docking-based
approach using the published crystal structure of human
salivary a-amylase (PDB: 1SMD) using both flexible ligand-
rigid binding site (Glide) and flexible ligand-flexible binding
site (FLO+) protocols (Ramasubbu et al., 1996). Results
revealed that salivary a-amylase inhibition generally depends
on two types of interactions: hydrogen bonding formed by the
hydroxyl moieties of C7 (A ring) and C40 (B ring) with the
side chains of Asp197 and Glu233, and pi-interactions
between the indole Trp59 and the heterocyclic ring of
the flavonoid (Lo Piparo et al., 2008). The latter stresses
the importance of the C2¼C3 double bond, which keeps the
planar structure of the flavonoid molecule. Another docking
study was performed using human pancreatic a-amylase
(PDB: 1HNY) using a genetic algorithm ligand conform-
ational search (AutoDock 4.2, La Jolla, CA). Results from this
experiment confide with the earlier study on salivary amylase,
wherein both hydrogen bonding and pi-interactions play
crucial roles in the binding of flavonoids to the active site
(Madeswaran et al., 2014).
A predictive 2D-QSAR model was built for the inhibitory
activity of flavonoids against a model a-glucosidase from
yeast using MLR of molecular descriptors calculated using
DRAGON (Milan, Italy), wherein topological charge index
(JGI2), information index (CIC2) and the number of exo-
conjugated C atoms (nCconjR) were found to correlate with
inhibitory activity (R2training ¼ 0.771, R2test ¼ 0.851, Q2 ¼ 0.832)
(Rastija et al., 2012). Although the authors interpreted the
model as being related to the number of hydroxyl groups and
the presence of the C2¼C3 bond, it is hard to generate a direct
link between the molecular parameters of the model to the
actual structural properties that govern the inhibitory effect,
which is typical for QSAR models employing indices as
descriptors. In the end, it seems that both a-amylase and
a-glucosidase share the same properties in terms of structural
requirements for inhibition.
Besides altering starch digestion by inhibition of the
above-mentioned enzymes and inhibiting glucose transporters
in the brush border, flavonoids have also been shown to form
non-covalent interactions that cause structural changes to food
carbohydrates, especially starches (Bordenave et al., 2014).
 4 G. B. Gonzales et al.
However, such interactions were only significant when the
flavonoid level is above 10% of the starch content. In normal
food intake levels, inhibitions of the above-mentioned
enzymes are the prime causes for altering carbohydrate
digestibility (Bordenave et al., 2014). Although flavonoids
may exist to be attached to sugars in nature, no reports on the
SAR of flavonoid–carbohydrate interaction in the GI tract
upon co-administration exist, as far as we know.
In agreement with the (Q)SAR models, in vivo studies
confirm that flavonoid–carbohydrate interactions affect fla-
vonoid bioavailability. Human feeding studies have demon-
strated that co-administration of flavonoids with a
carbohydrate-rich food enhanced flavonoid absorption,
which was explained by their protective effect against
microbial deterioration in the intestines, the release of
bound flavonoids through fermentation and the activity of a
carbohydrate–flavonol transporter (GLUT) (Neilson et al.,
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2009; Schramm et al., 2003; Zhang et al., 2014). The latter,
however, remains questionable since it has been previously
argued that flavonoids are not transported by glucose
transporters (Kottra & Daniel, 2007). This issue therefore
needs further investigation.
Flavonoid interaction to lipase and effect of fat
consumption on bioavailability
Pancreatic lipase plays an important role in the digestion and
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absorption of triacylglycerols in the intestine after a fat-rich
diet. Dietary fat is not directly absorbed by the body unless
the fat has been subjected to hydrolysis by the lipase, more
specifically converting triacylglycerols into sn2-monoglycerol
and sn1,3-fatty acids. Pancreatic lipase constitutes 50–70%
of the lipolytic activity, whereas gastric lipase contributes to
10–30%. Lingual lipase has a minimal contribution (Birari &
Bhutani, 2007). Among many treatment approaches for
obesity, retarding lipase activity may therefore offer an
effective alternative (Yun, 2010). With the development and
success of tetrahydrolipstatin (OrlistatÕ ), a potent clinically
approved lipase inhibitor, this enzyme has been targeted as a
valid approach in the treatment of obesity by reducing the
amount of absorbed fat from food (Wilcox et al., 2014).
Consequently, lipase inhibition has been the most widely
studied mechanism for the determination of anti-obesity
potential of natural products (Birari & Bhutani, 2007).
Several papers reported the anti-obesity potential of
polyphenols through several mechanisms, which include
pancreatic lipase inhibition. In fact, several polyphenol-rich
plant extracts have been shown to inhibit pancreatic lipase
activity (Birari & Bhutani, 2007; Yun, 2010). Yet, lipase
inhibition of a wide range of isolated flavonoids or flavonoid
standards has not been found in literature (except for catechin
derivatives). Therefore, it is difficult to generate a qualitative,
even more quantitative, SAR. Papers reporting in vitro lipase
inhibitory activity of purified flavonoid standards are listed
in Table 1.
As listed in Table 1, flavan-3-ols apparently have stronger
lipase inhibitory activity compared to flavonols, isoflavones,
flavones and flavanones. This may imply that planarity, which
increases the hydrophobicity of the compounds, reduces the
lipase inhibitory activity. However, native catechin and
Drug Metab Rev, Early Online: 1–16
epicatechin are less active compared to their galloylated
forms, and an even higher lipase inhibitory activity is
observed with increasing number of ester linkages.
According to a study of the lipase inhibition of phenolic
compounds from oolong tea, it was observed that indeed
galloyl moieties within the structure are required for
enhancing lipase inhibition (Nakai et al., 2005).
Given this, it appears that increasing the hydroxyl moieties
in the C3-position of flavonoids contribute to their lipase
inhibitory potential. In addition to the effect of galloylation in
flavan-3-ols at the C3 position, there is also a dramatic
increase in the lipase inhibitory activity when a hydroxyl
moiety in the C3 position is present like in the case of
quercetin and luteolin, wherein quercetin contains a hydroxyl
moiety at the C3 position, whereas luteolin does not.
Furthermore, replacing the C3 hydroxyl moiety with a
benzene ring in the case of genistein, an isoflavone, also
dramatically decreases its inhibitory potency. In a study of
quercetin esterified with various acyl chains of different
length at the C3-position, it was found that there is a direct
relationship between lipase inhibitory activity and the length
of the acyl chain attached to the C3-position (R2 ¼ 0.91)
(Gatto et al., 2002), which indicates that changes in this
position in the flavonoid skeleton may indeed affect lipase
inhibitory activity.
Although Sergent et al. (2012) indicated that no direct
SAR of flavonoids toward lipase inhibitory activity exists, it
seems that planarity, hydroxylation and galloylation at the C3-
position may affect lipase inhibitory activity. However, this
indeed needs to be validated by screening a wider range of
flavonoids for lipase inhibitory activity. Furthermore, 2D and
3D QSAR analysis, especially pharmacophore or COMFA/
COMSIA, will definitely be helpful in analyzing precisely
which structural features contribute to activity and in mapping
the precise location of functional groups in the molecule that
enhance lipase inhibitory activity.
Unlike studies on pancreatic lipase interaction, the direct
interaction of flavonoids with fats has not been well studied
(Bordenave et al., 2014; Zhang et al., 2014). In a review by
Bordenave et al. (2014), a list of calculated octanol–water
partition coefficients for different flavonoids was reported,
which could be used to explain the ability of flavonoids to
stabilize emulsions and partition to the fat region. Therefore,
it can be said that flavonoids interact with fats by means of
hydrophobic interactions, which are rather weak. Using a
Caco-2/HT29-MTX co-culture model, Jailani & Williamson
(2014) found that co-administration of flavonoids with
commonly used dietary oils altered the absorption behavior
of flavonoids depending on their hydrophobicity. For
instance, addition of oil significantly increased the production
of quercetin and kaempferol conjugates (especially sulfates) at
the basolateral compartment by up to three and four times,
respectively. The transport of galangin through the cells was,
however, inhibited by the presence of oils on the apical side,
which is surprising since galangin is more hydrophobic and
should partition with the fat more than quercetin and
kaempferol. Although, it is unclear from this article if the
fat was emulsified in the cell culture medium. Emulsification
is necessary to ensure that the compounds of interest do not
partition to the fat phase and float away from the cells, and
 DOI: 10.3109/03602532.2014.1003649 QSAR of flavonoids during digestion, absorption, distribution and metabolism 5
Table 1. In vitro inhibitory activity of flavonoids against human and pancreatic lipase.

Flavonoid IC50 Lipase References

(+)-catechin 3,5-di-O-gallate 0.42 ± 0.03 mg/mL HL (Ivanov et al., 2011)
(+)-catechin 3-O-gallate 2.02 ± 0.11 mg/mL HL (Ivanov et al., 2011)
()-catechin 3-O-gallate 2.45 ± 0.23 mg/mL HL (Ivanov et al., 2011)
()-epicatechin 3-O-gallate 3.22 ± 0.25 mg/mL HL (Ivanov et al., 2011)
Hesperidin 32 mg/mL PL (Kawaguchi et al., 1997)
Neohesperidin 46 mg/mL PL (Kawaguchi et al., 1997)
Narirutin 4400 mg/mL PL (Kawaguchi et al., 1997)
Naringin 4400 mg/mL PL (Kawaguchi et al., 1997)
Epigallocatechin gallate 0.8 ± 0.1 mM PL (Sergent et al., 2012)
Kaempferol 13.4 ± 4.1 mM PL (Sergent et al., 2012)
Quercetin 21.5 ± 9.4 mM PL (Sergent et al., 2012)
Luteolin 100 mM PL (Sergent et al., 2012)
Genistein 4100 mM PL (Sergent et al., 2012)
Naringenin 4100 mM PL (Sergent et al., 2012)
Catechin 4100 mM PL (Sergent et al., 2012)
Epicatechin 4100 mM PL (Sergent et al., 2012)
Epigallocatechin 88 mg/mL PL (Yoshikawa et al., 2002)
()-Epiafzelechin-(4b -4 8)-()-40 -O-methylepigallocatechin 270 mg/mL PL (Yoshikawa et al., 2002)
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()-Epicatechin-(4b -4 8)-()-40 -O-methylepigallocatechin 68 mg/mL PL (Yoshikawa et al., 2002)

()-epiafzelechin 3-O-gallate 2.58 mM PL (Nakai et al., 2005)
()-epicatechin 420 mM PL (Nakai et al., 2005)
()-epicatechin 3-O-gallate 0.45 mM PL (Nakai et al., 2005)
()-epicatechin 3-O-(30 -O-methyl)gallate 0.68 mM PL (Nakai et al., 2005)
()-epigallocatechin 420 mM PL (Nakai et al., 2005)
()-epigallocatechin 3-O-gallate 0.35 mM PL (Nakai et al., 2005)
()-epigallocatechin 3,5-di-O-gallate 0.10 mM PL (Nakai et al., 2005)
()-epigallocatechin 3-O-p-coumaroate 0.89 mM PL (Nakai et al., 2005)
(+)-catechin 420 mM PL (Nakai et al., 2005)
()-catechin 3-O-gallate 0.54 mM PL (Nakai et al., 2005)
(+)-gallocatechin 420 mM PL (Nakai et al., 2005)
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()-gallocatechin 3-O-gallate 0.44 mM PL (Nakai et al., 2005)

()-gallocatechin 3,5-di-O-gallate 0.21 mM PL (Nakai et al., 2005)
8-C-ascorbyl ()-epigallocatechin 0.65 mM PL (Nakai et al., 2005)
8-C-ascorbyl ()-epigallocatechin 3-O-gallate 0.79 mM PL (Nakai et al., 2005)

HL, human pancreatic lipase; PL, porcine pancreatic lipase.

also to allow penetration to the mucus layer, as will be
discussed later in this article.
In parallel, an in vivo test using pig models revealed that
higher levels of dietary fat content increased flavonoid
absorption, which was attributed to the increased secretion
of bile salts forming micellar structures during administration
of a high fat diet (Lesser et al., 2004). This in vivo study
confirms the importance of micellar incorporation of flavon-
oids for intestinal absorption. The increased absorption of
flavonoids upon fat ingestion also implies that the interaction
of flavonoids with pancreatic lipases is indeed minimal.
Interaction of flavonoids with food proteins and
proteolytic enzymes
Perhaps the most popular interaction in the study of flavonoid
bioavailability is the interaction of the flavonoids with food
proteins. This effect does not only reduce the bioavailability
of the flavonoid but also of the food protein itself.
Unfortunately, studies on the structure–affinity relationship
of flavonoids to food proteins have been limited to mainly
milk proteins.
In a study on bovine milk proteins (BMP) (Xiao et al.,
2011; Xu & Chen, 2011), the affinity of flavonoids toward
such proteins was highly affected by their molecular proper-
ties and structure, which may be summarized as follows:
(1) Hydroxylation of the A and B rings significantly
improves binding to BMP.
(2) Methylation and methoxylation of such rings therefore
diminishes their affinity.
(3) Glycosylation of the flavonoids strongly reduces the
affinity of flavonoids toward BMP by 1–2 orders of
magnitude.
(4) Hydrogenation of the C2¼C3 double bond of the C ring
strongly reduces binding affinity by up to 75-fold.
Planarity is therefore a key structural requirement for
affinity.
(5) Galloylation of catechins significantly improved binding
affinity by at least 100-fold.
On a quantitative perspective, Xu & Chen (2011) argued that
the binding affinity of flavonoids to BMP is increasing with
increasing partition coefficient (XlogP3) and has a negative
correlation with the number of hydrogen bond acceptors and
donors. This suggests that hydrophobicity of the flavonoids
plays a crucial role in the binding of flavonoids with food
proteins. The same conclusion was made when analyzing
the potential of food proteins as carriers for flavonoids (Bohin
et al., 2012). Furthermore, it was suggested that the
topological polar surface area (TPSA) of flavonoids decreases
with increasing binding affinity, which explains the low
affinity of flavonoid glycosides to BMP compared to their
aglycone counterparts (Xu & Chen, 2011). However, the data
presented in this article did not give a reliable quantitative
relationship because of the low correlation values (R2 ¼ 0.42
for XlogP3 versus binding affinity; R2 ¼ 0.27 for TPSA versus
 6 G. B. Gonzales et al.
binding affinity). However, a higher correlation between the
Mulliken electronegativity and the binding affinity was found
(R2 ¼ 0.65). Despite a higher correlation, this molecular
property may not fulfill the requirements for a predictive
QSAR model. Nonetheless, these structural information
provide a qualitative view on the important molecular features
that affect binding of flavonoids with food proteins.
According to Xiao et al. (2011), flavonoids lose their anti-
oxidative properties when bound to food proteins.
Furthermore, it is apparent that the molecular features
important for food protein binding are the same features
that enhance a-amylase and a-glucosidase inhibitory activ-
ities. This is not surprising since the enzymes are after all
proteins themselves. However, one must therefore be careful
in the use of flavonoids as anti-obesity and/or anti-diabetic
agents as co-administration of a high protein diet may reduce
their functionality. Furthermore, when the dietary interven-
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tion is aimed at increasing the oral bioavailability of
flavonoids, high protein diets may not be co-administered as
these diminish the bioavailability of the flavonoid in question.
Proteolytic enzymes have also been investigated for their
interaction with flavonoids. However, these studies have
focused on the inhibitory activity of flavonoids with relevance
to intracellular proteases and not the gastric and intestinal ones.
Intestinal barrier
Intestinal mucus layer: a missing link in the study of flavonoid
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bioavailability
Numerous studies have attempted to understand the mechan-
isms of absorption and increase the intestinal absorption of
flavonoids. Cellular models, such as Caco-2 cells, have been
extensively used to simulate the intestinal permeability of
flavonoids (Barrington et al., 2009; Tammela et al., 2004;
Tian et al., 2009). These in vitro experiments revealed that
hydrophobic flavonoids are better absorbed (Barrington et al.,
2009; Liu & Hu, 2002; Tammela et al., 2004; Tian et al.,
2009), which may be explained by their enhanced permeation
through the phospholipid bilayer of the cell membrane. In
parallel, flavonoid glycosides are poorly absorbed through the
intestinal cell models due to the presence of sugar moieties,
which increase hydrophilicity, thus reducing membrane
permeability (Dai et al., 2008).
However, in contrast with these in vitro findings, animal
and human studies showed that certain flavonoid glucosides
are absorbed more efficiently than their aglycone forms. For
instance, ingested quercetin glucosides are absorbed twice as
much as the corresponding aglycones in humans (Hollman
et al., 1995). The same observation was obtained from feeding
studies using pigs (Cermak et al., 2003), rats (Morand et al.,
2000) and dogs (Reinboth et al., 2010). Although several
hypotheses have been formulated, such as effective deglyco-
sylation by bacteria or enzymes of the epithelial cells
(Cermak et al., 2003), current literature has failed to provide
a concrete explanation for this behavior.
In general, successful strategies to increase flavonoid
bioavailability rely on increasing their hydrophilic nature.
A review of these techniques can be found in Thilakarathna &
Rupasinghe (2013). Briefly, incorporation of flavonoids to
borneol/methanol eutectic mixtures, micro-emulsions,
Drug Metab Rev, Early Online: 1–16
polyvinylpyrrolidone dispersion, lecithin complexation and
cyclodextrin complexation have been effective (Thilakarathna
& Rupasinghe, 2013), as well as the incorporation of
flavonoid aglycones into nanocrystals (Li et al., 2013).
Furthermore, as earlier stated, fat content in the diet of pigs
increased the oral bioavailability of quercetin, probably due to
the formation of flavonoid-mixed micelles (Lesser et al.,
2004). All of these methods increase aqueous solubility of the
flavonoid aglycone although the mechanisms of improved
absorption remain unclear (Shen et al., 2011).
From these studies, we may conclude that two driving
forces are triggering intestinal absorption of flavonoids, i.e.
on one hand, deglycosylation of the flavonoid glycosides is an
important step prior to intestinal absorption (Day et al., 1998;
Németh et al., 2003), but on the other hand, aqueous forms of
flavonoids or flavonoid glucosides render higher amounts of
flavonoid metabolites in the blood. This may be explained by
the existence of an aqueous barrier between the gut and the
epithelial cells that only allows the penetration of hydrophilic
flavonoids and/or flavonoid glycosides and that deglycosyla-
tion only occurs after the compound has penetrated through
such barrier. Cermak et al. (2003) also hypothesized that
hydrophilic quercetin monoglucoside concentrates at the
brush border where they are deglycosilated by the epithelium,
thus releasing the aglycone that passively diffuses through the
cells.
The gastrointestinal tract is protected by a mucus layer
(90–98% water content), which permits nutrient uptake whilst
excluding bacteria and other toxic compounds. Studies on
penetration of bacteria and model food particles have been
done, but extensive research on barrier function and health
remains scarce (Macierzanka et al., 2011; Mackie et al.,
2012). In order for flavonoids to be absorbed, they must
therefore be able to travel through adherent the mucus layer of
the intestines (Georgiades et al., 2014). As far as we know, the
effect of this mucus barrier on the intestinal absorption of
flavonoids and its glycosides is currently unknown. We
hypothesize that the hydrophobic nature of flavonoid
aglycones limits penetration, thus making it inaccessible to
the cells. Flavonoid glycosides, however, may be able to
penetrate through the mucus layer. They are then deglycosy-
lated on the cell surface, thus leading to absorption. As also
mentioned earlier, aglycones interact more with digestive
enzymes and food matrix components than their aglycone
counterparts. This may explain why there seems to be a higher
absorption of flavonoid glucosides in vivo than in vitro,
considering that current cell-based models (usually Caco-2
cells) do not produce a mucus layer. Currently, cellular
models involving co-culture of enterocytes with mucus-
producing cells are increasingly being used to overcome this
limitation, although data are scarce (Béduneau et al., 2014).
Recently, research on the interaction of tea-derived
flavonoids with isolated intestinal mucins has shown that
flavan-3-ols, especially epigallocatechin gallate (EGCG),
bind to mucins leading to cross-linking and aggregation
(Georgiades et al., 2014). Mucins are 10–40 mDa proteins
glycosylated via proline, threonine and/or serine residues that
comprise 2–5% of the mucus layer. Other components are
DNA (0.02%), lipids (1–2%), salts (1%), proteins, cells and
cellular debris and water (90–98%) (Lai et al., 2009). This
 DOI: 10.3109/03602532.2014.1003649 QSAR of flavonoids during digestion, absorption, distribution and metabolism
binding therefore automatically limits the bioavailability of
flavan-3-ols. The interaction of gastrointestinal mucus to
other types of flavonoids has unfortunately not been reported
in literature to date.
Furthermore, a study on the penetration of particles
through intestinal mucus revealed that a net negative charge
(caused by adsorption of bile salts) is essential to allow
transport of particles through the mucus layer due to its
innately negative surface charge (z-potential ¼ 10–11 mV)
(Macierzanka et al., 2011). Positively charged particles, such
as anthocyanins, may therefore bind to the mucus and thus not
reach the cells, unless they are covered with bile salts or
incorporated in mixed micelles. This, however, demands
further investigation.
Overall intestinal permeability
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Flavonoids, upon reaching the small intestines, undergo
hydrolysis of their glycosidic moieties and severe phase 1
and 2 metabolism, which include glucuronidation, sulfation
and methylation (Crozier et al., 2010; Hollman, 2004; Manach
et al., 2005; Scalbert & Williamson, 2000). Therefore, the
bioavailability of the parent flavonoid structures remains a big
determinant for its bioactivity (Thilakarathna & Rupasinghe,
2013). The structure of flavonoids has long been regarded as a
critical determining criterium for bioavailability (Hollman,
2004; Scalbert & Williamson, 2000). Figure 2 summarizes the
For personal use only.
findings of a much quoted review involving 97 bioavailability
studies (Manach et al., 2005) with regards to bioavailability of
flavonoids.
Figure 2 provides a qualitative view on the absorbability of
flavonoids in the intestines. However, due to the extremely
wide range and variability in terms of structure within
each group, it is difficult to generalize the absorbability
of flavonoids only based on which group they belong to.
For instance, methylation of the hydroxyl groups of the
Figure 2. Bioavailability of flavonoids
(Manach et al., 2005).
7
flavonols has been found to significantly increase its passage
through intestinal cell models (Walle, 2007; Wen & Walle,
2006). Furthermore, the degree of glycosylation and the type of
sugar has been shown to affect the bioavailability of the
flavonoids compared to their aglycone counterpart (Crozier
et al., 2010; Karakaya, 2004; Morand et al., 2000). For
instance, administration of quercetin-3-O-glucoside to rats and
pigs resulted in higher levels of quercetin metabolites in
plasma compared to rutin (quercetin-3-O-glucorhamnoside),
which was almost undetected in plasma until three hours after
intake. This delayed peak in plasma concentration was
attributed to the microbial deglycosylation of rutin in the
duodenum and the lack of rhamnosidases in the epithelium
(Cermak et al., 2003; Morand et al., 2000). Since the study of a
wider range of flavonoids in human trials presents financial,
technical and ethical hurdles, researchers have focused on the
use of intestinal cell models to further study the intestinal
uptake of flavonoids.
Predictive models have also been increasingly important in
drug design and discovery over the past decade (van de
Waterbeemd & Gifford, 2003; Wessel et al., 1998). Several
predictive models for intestinal absorption have been proposed,
however, not focusing on flavonoids as such. An influential
example is the ‘‘Lipinski rule-of-5’’, which resulted from the
analysis of the World Drug Index (Lipinski et al., 2001). In this
rule-of-5, compounds that are likely absorbable through the
intestines contain at most 5 H-bond donors, 10 H-bond
acceptors, have a molecular weight of4500 Da and the Log P
(lipophilicity index)45. Applying this rule to flavonoids, it can
be said that structures containing many hydroxyl, glycosidic
and galloyl moieties are less likely to be absorbed through the
intestines. In contrast, methylated compounds lose their H-
bond acceptor/donor properties and have higher Log P values,
which make them highly absorbable.
Another approach used for the prediction of intestinal
absorption of potential drugs was proposed by Egan et al.
 8 G. B. Gonzales et al.
(2000) who analyzed the drug-likeness of thousands of known
orally delivered drugs, which have been assayed for Caco-2 cell
permeability. Using pattern recognition, it was found that two
molecular descriptors, polar surface area (PSA) and AlogP98
(lipophilicity index), play a crucial role in determining
intestinal absorption. Using this principle, Gonzales et al.
(2014b) showed the potential of 36 flavonoids from different
classes (Tian et al., 2009; Wang et al., 2011) to be absorbed
through the intestines, using a commercially available ADMET
prediction software (Accelrys, San Diego, CA).
Using the model proposed by Egan et al. (2000), it was
shown that glycosylated flavonoids fall outside the intestinal
absorption region (99% confidence) along with flavonoids
with more than six hydroxylation points within the aglycone
structure. All the other compounds, such as those with 56
hydroxyl groups, isoflavones and methylated flavonoids, are
projected to have over 90% absorbability. Increasing the PSA
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seems to have a negative effect on absorbability, while
hydrophobicity of the flavonoids favor intestinal absorption.
This principle, in fact, has been the foundation of many other
intestinal permeability models (Egan et al., 2000). However,
these rules must be viewed as mere guidelines and not
absolute cut-offs (van de Waterbeemd & Gifford, 2003).
QSAR devoted to flavonoids remains scarce in literature.
This is due to the limited amount of in vitro studies
comprising a wide range of flavonoids, and the maximum
number of flavonoids analyzed in a single study so far is 36,
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albeit from different chemical classes (Tian et al., 2009). The
development of such models, however, are useful in high-
throughput screening of bioavailable flavonoids or when
synthesizing flavonoid-based drugs. In the study of Tian et al.
(2009), it was found that the octanol/water partition coeffi-
cient correlated well with the apparent permeability of
isoflavones, flavones and dehydroflavones. However, there
was a poor correlation with flavonols, which they considered
as due to high cell accumulation, with some exceptions due to
efflux mechanisms and compounds instability. This therefore
means that lipophilicity is a good indicator for many other
types of flavonoids, but this alone cannot explain intestinal
permeability characteristics of flavonoids.
In our previous study (Gonzales et al., 2014b), we have
developed both 2D and 3D QSAR models to explain and
predict intestinal absorption of flavonoids using their apparent
permeability in Caco-2 cells. For the 2D model, SMLR
and PLSR models were generated. The 2D models yielded
high internal and external predictabilities (SMLR:
R2training ¼ 0.93, R2test ¼ 0.82, Q2 ¼ 0.77; PLSR: R2training ¼ 0.93,
R2test ¼ 0.90, Q2 ¼ 0.67). The descriptors used in the model,
however, contained indices (electrotopological state descrip-
tors, weighted holistic invariant molecular (WHIM) descrip-
tors, etc.), which are not directly interpretable, especially
when ascertaining which molecular properties influence
permeability. To improve the interpretability of the perme-
ation model, we employed a pharmacophore-based COMSIA,
which resulted in a highly predictive (R2training ¼ 0.96,
R2test ¼ 0.95) and robust (Q2 ¼ 0.63) model. The model
included only hydrogen bond acceptors and donors as
molecular descriptors since adding hydrophobicity to the
model did not improve its predictive performance, whereas
steric and electrostatic parameters did not contribute to the
Drug Metab Rev, Early Online: 1–16
intestinal permeability of flavonoids. According to the model,
the number and location of the hydrogen bond donors and
acceptors contribute to the intestinal uptake of flavonoids.
Considering that hydrogen bond acceptor and donor groups
influence permeability of flavonoids, it is therefore evident
that flavonoid glycosides are poorly transported through
Caco-2 cells, especially when the glucose moiety is attached
to the C3-position. This model also agrees with the previously
discussed ‘‘rule-of-5’’, however, more quantitative and pre-
dictive. Due to its high predictability, this model can therefore
be used as a method for screening a database of flavonoids
and flavonoid-like compounds in the search for highly
bioavailable compounds.
Glucuronidation of flavonoids
Poor bioavailability of flavonoids has been related to
extensive phase 2 metabolism, which is primarily caused by
conjugating enzymes in the intestines. Glucuronidation of
endogenous and exogenous compounds is mediated by several
isoforms of UDP-glucuronosyltransferase (UGT) which is
found both in the intestines and the liver. It was reported that
80% of the metabolic pathway of flavonoids is caused by this
enzyme (Wu et al., 2011b,c). In fact, glucuronidation has been
the most studied metabolic fate of flavonoids, wherein it was
demonstrated that glucuronidation is regiospecific and is
strongly affected by the structure of the flavonoid (Tang et al.,
2012; Wong et al., 2009; Wu et al., 2011a,b,c; Zhang et al.,
2006). In this section, both intestinal and hepatic glucur-
onidation are discussed since they share the same mechanism
although with different kinetics.
In general, SAR of flavonoids to in vitro glucuronidation
activity of UGT isoforms can be summarized as follows:
(1) Flavonols are the least glucuronidated flavonoids.
(2) When the C3–OH group is removed (in the case of
flavones), glucuronidation activity increases.
(3) Glucuronidation is even more intensified when the
C2¼C3 bond is removed (in the case of flavanones),
which relates to the importance of structural planarity
(Lewinsky et al., 2005).
(4) Addition of hydroxyl groups in the A ring was also
found to increase glucuronidation while glycosylation
decreases it.
(5) In a study of monohydroxylflavones exposed to human
jejunum S9 fraction, it was found that glucuronidation is
favored in the following order: C30 and C644C40 , C3,
C20 , C744C5 hydroxyflavone (Zhang et al., 2005).
However, using mouse liver S9 fraction, it seemed that
the C7 position is more preferred than C3, C30 or C6
(Tang et al., 2012).
(6) In dihydroxyflavones (dHF), 30 ,7-dHF had the highest
intrinsic clearance compared to a 20 ,7dHF and 40 ,7dHF.
This, together with the result from monohydroxylflavones,
indicates the importance of the C30 -hydroxyl moiety for
the effective glucuronidation of flavonoids in general
(Wong et al., 2009). However, it was observed that
glucuronidation was limited to the C7 and C3-hydroxyl
groups using mouse liver S9 fraction (Tang et al., 2012).
(7) Increasing the number of hydroxyl groups on both A and
B rings (except for 40 -OH moiety) enhances the
 DOI: 10.3109/03602532.2014.1003649 QSAR of flavonoids during digestion, absorption, distribution and metabolism
glucuronidation activity of flavones, while adding a
3-OH hydroxyl group does not seem to have an effect
(Lewinsky et al., 2005; Wong et al., 2009).
Using UGT1A9, one of the 9 UGT1A isoforms that has
been reported to have a higher glucuronidation activity to
flavonoids at the C3–OH position, Wu et al. (2011c)
developed a predictive 3D QSAR model with good internal
and external predictabilities using the Vmax and CLint as
dependent variables (Q2 ¼ 0.74, R2training ¼ 0.98, R2test ¼ 0.74;
Q2 ¼ 0.56, R2training ¼ 0.94, R2test ¼ 0.63, respectively). In this
model, training molecules were first aligned using a pharma-
cophore model that consisted of the glucuronidation site (the
C3–OH moiety) and two aromatic rings (A and B rings). It
was found that hydroxyl groups in both A and B rings
contribute largely to the glucuronidation susceptibility of the
flavonoid at the C3–OH position. Bulk substituents, perhaps
due to a methyl group, near the C5 and C6 positions also
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contribute to glucuronidation activity. Although UGT1A9 is
primarily expressed in the liver, whereas other isoforms (1A 8
and 1A10) are found in the intestines, it was found
nonetheless that UGT1A9 share the same regioselectivity as
UGT1A8 and 1A10 (Wu et al., 2011d). However, a report that
specifically modeled UGT1A8 or 1A10 could not be found in
literature.
Wu et al. (2011c) also argued that it is necessary to model
regiospecific glucuronidation individually to obtain a global
glucuronidation rate, considering the many potential glucur-
For personal use only.
onidation sites in the flavonoid structure. However, the
existence of an ‘‘accurate prediction’’ model for glucuronida-
tion, which do not model regiospecific glucuronidation (Wu
et al., 2012), may indicate that modeling regiospecific
glucuronidation has not been a successful strategy and may
require further investigation and validation. In this model (Wu
et al., 2012), however, 146 very structurally different
molecules (flavonoids, phenolic acids, etc.) were aligned,
which makes the results of their COMFA/COMSIA model
less reliable, considering that molecular alignment is a crucial
step in COMFA and COMSIA.
Sulfation of flavonoids
Another phase 2 metabolism pathway is mediated by
sulfotransferases (SULTs), which are primarily responsible
for catalyzing the sulfation of flavonoids and other drug-like
compounds. This pathway is important in the understanding
of flavonoid bioavailability since sulfated flavonoids have
been reported to be effluxed back into the intestinal lumen
and thus are not well absorbed (Barrington et al., 2009).
Among its many isoforms, the mammalian SULT1A3 has
been reported to be the most important isoforms for the
metabolism of phenolic compounds, such as flavonoids
(Meng et al., 2012; Wu et al., 2011a). Literature regarding
the structure–sulfation relationship on a wide range of
flavonoids remains scarce. However, according to several
studies, it was found that there is a preference for sulfation at
the C7–OH moiety (Meng et al., 2012; Wu et al., 2011a; Yang
et al., 2011). In a study of 16 different flavonoids from
different flavonoid classes, it was clearly shown that sulfation
occurred mostly at the C7–OH position, and removing this
hydroxyl moiety reduces or inhibits sulfation. Furthermore,
9
any attachment to the C3-position (whether a hydroxyl
group or the B ring) reduces the sulfation activity (Meng
et al., 2012).
To explain the sulfation behavior of the SULT1A3 isoform,
docking studies using the crystal structure of SULT1A3 have
been done. It was found that the C7–OH group can easily be
docked to the active site of SULT1A3 and yields the highest
fit and docking score. Furthermore, the 7-OH group is able to
form hydrogen bonds with the basic residue Lys106 and the
deprotonated N-3 atom on His108. It was also found that the
phenyl ring at the C2-position (C ring except for isoflavones)
interacts with the hydrophobic residues Tyr76, Phe142,
Tyr240, Val241 and Leu 247, such that hydroxylation of
this ring (for instance C4,–OH) drastically reduces its binding
affinity. Similarly, hydroxylation at the C3-position intro-
duces unwanted bulk or steric hindrance, which affects the
flexibility of the C2-phenyl group, thus again reducing
binding affinity (Meng et al., 2012; Wu et al., 2011a).
Interaction of flavonoids with ATP-binding cassette
Adding to the long list of gate-keepers that guard our body
from unwanted chemicals entering the systemic circulation
are certain membrane pumps or efflux transporters called
ATP-binding cassette (ABC) transporters. These membrane-
bound transporters have lately received much attention not
only due to their effect on flavonoid bioavailability but also
on the bioavailability of other pharmacologically active
compounds, i.e. anti-cancer drugs (Morris & Zhang, 2006).
ABC transporters act as ATP-dependent efflux pumps
found in both apical and basal membranes of epithelial cells
(Figure 3). The most pharmacologically relevant ones are
found in the apical membrane and include P-glycoprotein,
multidrug resistance protein (MRP) 2 and breast cancer-
resistant protein (BCRP) (Alvarez et al., 2010). The function
and interaction of flavonoids with ABC transporters espe-
cially its consequence to the absorption of other drugs have
been reviewed elsewhere (Alvarez et al., 2010; Morris &
Zhang, 2006). In this study, we focus on the SAR of
flavonoids to P-glycoprotein, MRP and BCRP, including
inhibition and other interactions. Since flavonoids are both
substrates and inhibitors of these transporters, their
Figure 3. ABC transporters on the apical and basal compartments of
Caco-2 cells. P-glycoprotein (P-gp), breast cancer resistance protein
(BCRP), multidrug resistance protein (MRP) (Alvarez et al., 2010).
 10 G. B. Gonzales et al.
interaction with ABC transporters therefore has a direct link
to their intestinal uptake and ultimately their bioavailability.
P-glycoprotein
P-glycoprotein is a 170-kDa transmembrane protein, which is
known for actively transporting drugs out of the cells in an
ATP-dependent manner (Boumendjel et al., 2002; Kothandan
et al., 2011). Structural analysis of the protein sequence
showed 1276 amino acids in mice, comprising of two
homologous halves containing a transmembrane domain
(TMD) composed of six membrane-spanning a-helices
involved in efflux and drug binding, and a cytosolic
nucleotide-binding domain with characteristic Walker motifs
A and B and an ABC transport S signature, which have been
shown to possess ATPase activity (Boumendjel et al., 2002;
Kothandan et al., 2011; Morris & Zhang, 2006). This
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transporter has a rather broad spectrum of substrates including
many therapeutic drugs. Such substrates have been observed
to share similar properties, such as being hydrophobic,
positively charged or neutral and having a planar structure
(Morris & Zhang, 2006).
It is known that flavonoids exert P-glycoprotein inhibition,
based on the physical properties earlier described. In general,
the following observations regarding flavonoid-P-glycopro-
tein interaction have been reported in literature (Boumendjel
et al., 2002; Conseil et al., 1998, 2000; Morris & Zhang,
For personal use only.
2006):
(1) Flavonols, chalcones and flavones are generally active,
while flavanones and isoflavones have lower P-glycopro-
tein-inhibitory activities.
(2) The hydroxyl groups at the 5- and 3 positions are
required for inhibitory activity against P-glycoproteins.
(3) Highly hydrophilic compounds, such as rutin and
other flavonoid glycosides, do not interact with
P-glycoproteins.
(4) Increasing the number of hydroxyl groups in the A and
C rings increases the affinity of flavonoids to
P-glycoprotein.
(5) Ring B attached to the C2 position entails higher affinity
than when it is attached to the C3 position, in the case of
isoflavones.
(6) The presence of the C2¼C3 double bond increases the
affinity of flavonoids to P-glycoprotein.
(7) Hydroxylation at the C40 and C30 position reduces
flavonoid-P-glycoprotein affinity, whereas methoxylation
at the C40 position increases it.
Wang et al. (2005) built a QSAR model of 57 structurally
diverse flavonoids using 248 molecular descriptors comprising
of ClogP, electrotopological state descriptors, molecular
connectivity indices calculated by Molconn-Z (SYBYL soft-
ware package, St Louis, MO). Initially, the molecular descrip-
tors were subjected to principal components analysis (PCA) to
reduce the number of molecular descriptors to four principal
components, which represented 85% total variance. Three
data-mining techniques were then used, back-propagation
neural network, PLSR and Bayesian-regularized neural net-
work, wherein the latter yielded the most predictive and robust
model. Although predictive, direct interpretation of which
molecular descriptors were responsible for P-glycoprotein
Drug Metab Rev, Early Online: 1–16
inhibition is rather not obvious due to PCA being a diminution
technique. Another approach was made by Sheu et al. (2010),
wherein SMLR was used to create a QSAR model from an
in vitro analysis of 22 flavonoids using human colorectal
carcinoma (HCT15) cells. The model with the highest adjusted
R2 was reported as (Sheu et al., 2010):
log activity ðat 50 mM flavonoidÞ
¼ 81:34 ð7OHÞ  50:37 ð30 OHÞ  65:93 ð40 OHÞ
þ 58:81 ð50 OHÞ þ 40:83 ðflavanolÞ  101:9 ðisoflavonesÞ
þ 12:13 ðC log PÞ þ 115:81


Adjusted R2 ¼ 0:7126, F ¼ 4:53, p 5 0:02
In this model, it was observed that hydroxylation at positions
C30 and C40 and the position of the B ring (isoflavones)
negatively affected the modulation activity of flavonoids to P-
glycoproteins. Conversely, hydroxylation at C7, C50 and
ClogP positively influences the activity of flavonoids against
P-glycoproteins. Although the adjusted R2 value is relatively
high, validation steps were not performed in this study, which
is extremely important when making QSAR models.
Therefore, the robustness and actual predictability of this
model cannot be assured. Nonetheless, the results of the
model complemented well with the generally observed
structural trends, as enumerated above.
Kothandan et al. (2011) on the other hand used docking
and 3D QSAR approaches (COMFA and COMSIA) to explain
the structural features of flavonoids relevant for P-glycopro-
tein inhibition (Kothandan et al., 2011). For both COMFA
and COMSIA analysis, both ligand-based alignment (atom-
atom alignment) and receptor-guided alignment (based on
docking pose) were employed. The technique yielded pre-
dictive and robust models, as indicated by their internal and
external predictability (R2internal and R2external values), cross
validated R2 values (Q2) and internal prediction parameter by
progressive scrambling (Q*2) as listed in Table 2.
As listed in Table 2, receptor-guided alignment of flavon-
oids prior to COMFA and COMSIA yielded better predictive
performance. This implies that the interactions of the
flavonoids with the amino acid residues at the binding site
of the protein are of prime importance to determine efficacy.
Based on their docking results, it seems that the potential sites
for conjugation include Asn1043, Asp1049, Pro1051 and
Asn1248. The success of the COMFA and COMSIA methods
to produce a highly predictive model also indicates that the
location of certain functional groups indeed play a role in the
interaction of the flavonoids with P-glycoprotein. According
to their COMFA and COMSIA receptor-guided model,
hydrophobic and bulky, possibly methylated moieties
Table 2. COMFA and COMSIA of flavonoids against P-glycoprotein
activity (Kothandan et al., 2011).
Model Parameter/s Q2 R2int R2ext Q*2
COMFAligand-based Steric 0.75 0.95 0.80 0.64
COMFAreceptor-guided Steric 0.71 0.98 0.84 0.50
COMSIAligand-based Steric 0.81 0.94 0.79 0.68
COMSIAreceptor-guided Hydrophobic 0.81 0.99 0.94 0.59
 DOI: 10.3109/03602532.2014.1003649 QSAR of flavonoids during digestion, absorption, distribution and metabolism
around the A ring increase the affinity of the flavonoid toward
the P-glycoprotein-binding site.
Multidrug resistance proteins
MRP is another type of ABC transporter consisting of nine
members, which vary in terms of substrate specificity, tissue
distribution and intracellular location. The most commonly
studied are MRP1 and MRP2. Although both transporters
share many substrates and approximately 49% sequence
similarity (van Zanden et al., 2005), MRP1 is usually found
on the basal membrane, whereas MRP2 is found on the apical
membrane of intestinal cells (shown in Figure 3) (Alvarez
et al., 2010). MRP1 is also less substrate specific than MRP2.
Overexpression of both MRP1 and MRP2 has been found to
confer multidrug resistance to a variety of anti-cancer drugs
(van Zanden et al., 2005). Being on the apical region, MRP2
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is, however, more physiologically relevant (Alvarez et al.,
2010) than MRP1 and is thus expected to limit oral
bioavailability and facilitate the biliary and renal secretion
of its metabolites (Morris & Zhang, 2006). However,
considering the stricter substrate specificity of MRP2, only
a few flavonoids have been found to inhibit MRP2, which
implies that a QSAR is difficult to establish (Morris & Zhang,
2006; van Zanden et al., 2005).
Using stepwise linear regression, with MRP1 inhibition as
the dependent variable and five structural descriptors (number
For personal use only.
of OCH3 groups, number of OH groups, log dihedral angle of
the C3–C2–C10 –C20 bonds, lipophilicity and number of
pyrogallol and catechol moieties), van Zanden et al. (2005)
reported that MRP1 inhibition by flavonoids may be
explained by the following equation:
%inhibition¼45:46þ18:94ð#OCH3 Þþ12:47ð#OHÞ


48:25 ðlog dihedral angleÞ R2 ¼0:77, p50:001
The results of this model suggest that the dihedral angle of the
B and C rings negatively affects inhibition, which implies that
a rather planar structure should be a prime requirement for
MRP1 inhibition. Furthermore, increasing the number of
methoxyl groups and increasing the number of hydroxyl
groups both increase the flavonoid-induced inhibition of
MRP1 to a relatively similar degree. This result is, however,
somewhat odd since increasing the number of OCH3 moieties
in the flavonoid structure should cause a decrease in the
amount of hydroxyl groups in the structure. Furthermore, the
lack of essential validation steps in the modeling procedure
makes this model a rough estimation rather than an absolute
reliable model.
In the case of MRP2, the amount of tested flavonoids is
rather limited. However, based on the report of Pedersen et al.
(2008) on the MRP inhibition of 191 different compounds,
some of which are flavonoids, molecular features necessary
for MRP2 inhibition include higher molecular weights,
increased lipophilicity and aromaticity. Most of the flavonoids
in the dataset however fall under the ‘‘non-inhibitors’’
category after employing OPLS-DA. This goes to show that
while flavonoids may interact with MRP1, their interaction
with MRP2 is not supported by current literature.
11
Breast cancer-resistance proteins
A recent addition to the ABC transporter family is the BCRP,
which is a 655-amino acid protein with a molecular weight of
72.1 kDa. Unlike other ABC transporters that contain 12
TMDs and two ATP-binding sites, BCRP only consists of six
TMDs and one ATP-binding domain; hence, it is called a half
ABC transporter and may require the formation of a
homodimer to function (Morris & Zhang, 2006; Zhang
et al., 2005). BCRP is ubiquitously located in the body with
high mRNA expression in the placenta and lower level in the
brain, prostate, intestines, testis, ovary and liver. Substrate
specificity is somewhat similar yet much more restrictive than
P-glycoproteins (Gandhi & Morris, 2009; Nicolle et al.,
2009). However, it has been previously reported that BCRP
expression in the intestines is higher than P-glycoproteins, and
thus may restrict more the oral bioavailability of its substrates
(Morris & Zhang, 2006). BRCP is located at the luminal side
of the intestines and thus at the apical side of polarized cells
(Gandhi & Morris, 2009; Morris & Zhang, 2006; Nicolle
et al., 2009).
A number of researchers has extensively studied the SAR
of flavonoids with BCRP, and their findings can be
summarized as follows (Ahmed-Belkacem et al., 2005;
Gandhi & Morris, 2009; Katayama et al., 2007; Zhang
et al., 2005):
(1) The presence of the C2¼C3 double bond increases BCRP
inhibition, thus implying the necessity for structure
planarity.
(2) Hydroxylation at positions C5 and C7 (moderate effect)
increases BCRP inhibition, while methylation at such
positions reduce activity.
(3) Placing the B ring to position C3 (in the case of
isoflavones) eliminated BCRP inhibitory activity.
(4) Hydroxylation at positions C3, C6 and C8 reduces its
activity, while methoxylation of these hydroxyl moieties
reverses the effect. In the case of the C40 position,
OCH34OH4H.
(5) Addition of a prenyl moiety to positions C6 and C8
increases the BCRP-inhibitory activity.
(6) Glycosylation reduces the inhibitory activity.
Zhang et al. (2005) developed a predictive QSAR model
for the flavonoid-mediated BCRP inhibition, which involved a
screening of 25 flavonoids from various classes. Using
genetic algorithm as a feature selection technique, an initial
number of 115 molecular descriptors was reduced to 3, which
were then used as independent variables in a MLR model with
pEC50 as the dependent variable (Zhang et al., 2005).
The resulting model is as follows:
pEC50 ¼ 1:156 logP þ 0:891 SdssC acnt  0:176 Dy þ 0:480

R2training ¼ 0:852, Q2 ¼ 0:784, R2test ¼ 0:922,
p50:0001, n ¼ 25Þ
where logP is a calculated octanol–water partition coefficient,
SdssC_acnt is the count of all ¼C5 groups in the molecule
and Dy is the moment of displacement between the center-of-
mass and the center-of-dipole along the inertial Y-axis (Zhang
et al., 2005). The produced model resulted in high internal
and external predictability, as well as good robustness as
 12 G. B. Gonzales et al.
depicted by the high Q2 value after leave-one-out cross
validation. This model is therefore a valid and predictable
QSAR model for the BCRP inhibition by flavonoids. This
model also suggests that lipophilicity and the presence of the
C2¼C3 bond and aromaticity play a positive role in BCRP
inhibition.
Another approach was to use 3D linear solvation energy
descriptors obtained from VolSurf and molecular interaction
fields. Employing PLS regression, a model (R2training ¼ 0.77,
R2test ¼ 0.67, Q2 ¼ 0.70) was developed to predict BCRP
inhibitory activity of flavonoid and flavonoid-like compounds
(n ¼ 34) (Nicolle et al., 2009). In the model by Nicolle et al.
(2009), it was found that BCRP inhibition is mainly driven by
large and polarizable hydrophobic volumes with minimal H-
bond donor capacity. Unlike the model presented by Zhang
et al. (2005), as discussed above, Nicolle et al. (2009) argued
that their approach considers more 3D molecular properties,
Drug Metabolism Reviews Downloaded from informahealthcare.com by Gazi Univ. on 02/03/15
which implies that the model describes the overall intermo-
lecular interactions of the flavonoids. In the case of Zhang
et al. (2005), where a genetic algorithm was used to select the
independent variables, much of the molecular information is
not described in the model. However, it must be considered
that although Nicolle et al. (2009) argued that their model
represented more molecular interactions, the model proposed
by Zhang et al. (2005) seemed to perform better in terms of
predictability. Furthermore, since MLR was used, model
interpretation is simpler.
For personal use only.
To outline the molecular fields that describe the differences
in inhibitory potency of flavonoids, as well as to provide a
visual presentation of the important molecular features
contributing to BCRP inhibitory activity, Pick et al. (2011)
performed a 3D QSAR analysis using COMFA and COMSIA.
Based on the Q2 values, the effect of the individual and
combination of fields were analyzed, and the field/s that yield a
higher Q2 value were used for further analysis. They found that
hydrogen bonding field alone yielded the highest Q2 (0.62) for
COMFA analysis, while the combination of electrostatic and
hydrogen bond acceptor fields provided higher predictability
for COMSIA analysis. According to the results of their
COMSIA analysis (R2 ¼ 0.88, Q2LOO ¼ 0.62) using electrostatic
and hydrogen bond acceptor fields, the presence of hydrogen
bond acceptors around C5–C7 are favored for BCRP inhib-
ition, which indicates that hydroxyl and methoxyl groups in
these regions increase the activity. A C3–OH moiety on the
contrary leads to reduced activity. A negative charge at the C-3
position is favored, whereas a positive charge at the C2 and C4
regions is favored, which implies the importance of polariz-
ability. This also suggests the higher BCRP inhibitory activity
of flavones compared to flavanones. In general, the results of
this model corresponded well with the earlier described 2D
models, except for the importance of the C2¼C3 double bond
that is not highlighted in this model. Nonetheless, this approach
provides a simple and interpretable visual representation of the
QSAR of flavonoids to BCRP inhibition.
Flavonoids in systemic circulation and their
distribution to target tissues
Once compounds or their conjugated metabolites manage to
pass through the intestinal barrier, they must then be
Drug Metab Rev, Early Online: 1–16
transported through the circulatory system to their target
tissues. However, the bioactivity of these compounds may be
influenced by their affinity to proteins inherent in the blood. As
with food proteins earlier discussed, flavonoids have also been
shown to interact with some blood proteins (Bolli et al., 2010;
Boulton et al., 1998; Xiao & Kai, 2011; Zsila et al., 2003).
Human serum albumin (HSA) is the most abundant protein
in plasma and serves as a depot and carrier for many different
types of compounds in blood, which then affects their
pharmacokinetics and metabolic fates (Bolli et al., 2010). In
fact, it was found that the distribution and metabolism of
many bioactive compounds are correlated with their affinities
toward HSA (Zsila et al., 2003). As the ‘‘free drug’’
hypothesis suggests, only compounds unbound to plasma
protein exert biological activity, which therefore suggests that
flavonoids with high-binding affinity toward HSA may lose
their biological activity once they reach the blood (Xiao &
Kai, 2011). As summarized by Xiao & Kai (2011) in their
review, the structure–affinity relationship of flavonoids to
plasma proteins is as follows:
(1) Increase in the hydroxyl groups of the A and B ring
increases the affinity of flavonoids to plasma protein,
whereas addition of a hydroxyl group at the C ring will
weaken it.
(2) Methylation of these hydroxyl groups further enhances
their binding to plasma proteins.
(3) The presence of the unsaturated bond at C2¼C3, typical
for flavonols, strengthens protein binding.
(4) Glycosylation greatly reduces plasma protein-binding
affinity.
(5) Galloylation increases the binding of catechins to plasma
proteins.
Given these characteristics, it can be implied that hydropho-
bicity plays a crucial role in protein binding; methylation,
C2¼C3 bond makes the flavonoid more lipophilic, whereas
glycosylation makes it hydrophilic. Indeed, it has been
previously reported that compounds with reduced lipophili-
city also exhibit reduced protein binding (Smith et al., 2010).
It is interesting to note that characteristics of flavonoids with
higher overall intestinal permeability are similar to the
structural requirements for protein binding. For instance,
while it has been reported that methylated flavonoids pass
through the intestinal cells intact (Wen & Walle, 2006), such
compounds have high plasma protein-binding affinities, as
discussed above (Xiao & Kai, 2011).
However, Smith et al. (2010) argued that the ‘‘free drug’’
hypothesis may have been misunderstood and that most
in vitro studies dealing with aglycones and purified plasma
proteins may not represent the in vivo situation. Furthermore,
as discussed in the earlier chapters, flavonoids experience
extensive first-pass metabolism, especially glucuronidation,
which therefore restricts the passage of aglycones through the
intestinal cells intact, although there have been very few
exemptions reported (Williamson, 2002). The use of agly-
cones on the study of plasma protein interaction therefore
raises the same issues as Williamson (2002) raised for the use
of aglycones for testing in vitro bioactivity; is this a valid
approach?
Unfortunately, to the best of our knowledge, there are no
studies regarding the comparative protein binding of
 DOI: 10.3109/03602532.2014.1003649 QSAR of flavonoids during digestion, absorption, distribution and metabolism 13

"hydrophobicity

"hydrophobicity
flavonoid metabolites, especially glucuronides. However, the
results of the in vitro assays mentioned above clearly show

Others

#C3–OH
that flavonoid glucuronides may have low plasma protein-
binding affinity because glucuronidation increases the aque-
ous solubility of flavonoids. This implies that glucuronides
may freely diffuse to the target tissues, where they are
deglucuronidated. Therefore, while glucuronidation has been
perceived as something that limits the oral bioavailability of

Position of B ring
the flavonoid aglycone (Wu et al., 2011b), it may well be that
this mechanism is the perfect carrier of flavonoids to target

on SAR with flavonoids but found to generally increase flavonoid bioavailability

on SAR with flavonoids but found to generally increase flavonoid bioavailability

tissues because of their potentially high free drug concentra-
tion in the blood. If in case any aglycone would be able to
pass through the intestines intact, they would face extensive

C2

C2

C3
C2
C2
C2
C2
binding to plasma proteins anyway, which reduces their
biological activity (Boulton et al., 1998; Xiao & Kai, 2011;
Zsila et al., 2003). In this regard, current strategies to improve
bioavailability by by-passing the glucuronidation stage may
Drug Metabolism Reviews Downloaded from informahealthcare.com by Gazi Univ. on 02/03/15

–Glycosyl
need to be re-evaluated.
One may argue that flavonoid glucuronides lose much of
their biological activity compared to their aglycone counter-

#
#

#
#
parts, which has been demonstrated by many in vitro studies
(Naomi et al., 2008; Thilakarathna & Rupasinghe, 2013).

Table 3. Summary of the effect of certain flavonoid structural features in various stages of digestion, absorption and distribution.
However, flavonoid aglycones are not found in the blood after
oral administration and yet, flavonoid-rich diets still resulted

C2¼C3
in profound physiological changes in the body (Perez-
Vizcaino et al., 2012; Terao et al., 2011). Flavonoid

"

"

"
"

"
"
on SAR with flavonoids

on SAR with flavonoids

metabolites must be converted back to its active aglycone
For personal use only.

form upon reaching target tissues, which has been confirmed

"denotes an increase in the interaction or occurrence and #denotes a decrease in the interaction or occurrence.
by a few studies.
Shimoi et al. (2001) first observed that luteolin mono- –OCH3
glucuronide was converted to its free aglycone form during #

"
#

#
"
inflammation using human neutrophils stimulated with
ionomycin/cytochalasin B and rats treated with lipopolysac-
charide. They concluded that b-glucuronidases are released
No studies

No studies

No studies

No studies
from stimulated neutrophils or certain injured cells, which
–OH

releases the active luteolin aglycone. Naomi et al. (2008) also

"C3

"C7
"

"

#
"

"
"
found that livers of hepatocarcinogenic rats have higher
b-glucuronidase activity than healthy rats, which indicated
Interaction with a-amylase and a-glucosidase

that b-glucuronidases are specifically activated in inflamma-

tory tissues, such as cancerous tissues (Naomi et al., 2008).
Recently, it has been reported that increased b-glucuronidase
Interaction with food carbohydrates

Interaction with ABC transporters

activity of injured cells is associated with mitochondrial

Overall intestinal permeability
Interaction with food proteins

dysfunction (Ishisaka et al., 2013).

Given these, it can be said that keeping a constant supply
Interaction with food fats

Plasma protein binding

of flavonoid glucuronides in the blood stream may be

Interaction with lipase
Interaction/occurrence

beneficial as tissues can secrete b-glucuronidases to release

Mucus penetration

Deglucuronidation
Glucuronidation

the bioactive aglycone when they are needed. Due to a high

clearance rate, it is therefore advised to have a constant dose
Sulfation

of flavonoids in the diet in order to keep a high level of

glucuronides in the blood. Going back to the comment of
Williamson (2002) on whether using aglycones to test in vitro
bioactivity as a valid approach, we believe that it is
Upper gastro-intestinal tract

considering that aglycones are indeed released locally to the

Systemic circulation and

damaged tissues. It was previously proposed by Actis-Goretta

et al. (2006) that a local enrichment is indeed possible, which
increases the concentration of the phenolic compound in situ.
target tissues
Brush border

Considering that production of b-glucuronidase may be

localized in damaged tissues, the concentration of aglycones
in that particular region may increase to high levels. This,
Site

however, needs to be validated. The next challenge would

 14 G. B. Gonzales et al.
then be to know the local/in situ concentration of flavonoids
and use this as the basis for designing in vitro bioactivity
assays to determine bioactive concentration of flavonoids,
instead of depending on the concentration of flavonoids found
in the blood as the maximum cut-off.
Conclusion and perspectives
In this review, we have shown that flavonoid structure greatly
influences its bioavailability, and to some extent, bioactivity.
Moreover, apart from processes occurring in the intestinal
epithelium previously reported, it was shown that other stages
in the digestion, absorption and distribution processes also
affect the final bioavailability of flavonoids. Table 3 lists a
summary of the key structural features of flavonoids and their
effect on the different stages described above.
As shown in Table 3, flavonoids interact with digestive
enzymes and food components in the diet. Interaction with
Drug Metabolism Reviews Downloaded from informahealthcare.com by Gazi Univ. on 02/03/15
proteins has been reported to reduce flavonoid bioavailability,
while the contrary was observed when flavonoids are co-
administered with starches and fats.
Studies have shown that flavonoids or flavonoid matrices
with high aqueous solubility promote intestinal absorption
in vivo, although in vitro studies show the contrary. This may
be due to the presence of a mucus layer, which may prevent
hydrophobic compounds to diffuse to the cells. It is
hypothesized therefore that deglucosylation happens after
passage through the mucus layer. Since current cell models
For personal use only.
(Caco-2) do not produce a mucus layer, this effect has usually
been overlooked. Therefore, the use of co-cultures with
mucus-producing cells may be a good strategy to fill this gap.
It is also of interest to see that the structural features that
improve overall intestinal permeability are not similar to those
required for glucuronidation, sulfation and ABC transporter
interaction. Therefore, certain flavonoids are able to pass
through intact because they are neither metabolized nor
effluxed. However, once these flavonoids reach the blood,
they may bind to plasma proteins, which ultimately reduce their
availability and bioactivity in the tissues. Glucuronidation,
which was previously considered as a barrier to flavonoid
bioavailability, may therefore be good to increase the ‘‘free
drug concentration’’, which is necessary for bioactivity.
Because certain cells secrete glucuronidases under stressed
conditions such as inflammation, we hypothesize that stressed
cells create much localized micro-environment of high
aglycone concentrations that may assist in the in situ protection
of the cells against the stress. For future research, we therefore
propose to investigate local flavonoid concentrations of
stressed cells or tissues to verify whether the use of aglycones
in in vitro bioassays is indeed a(n) (in)valid approach.
Declaration of interest
The authors declare that there is no conflict of interest.
The authors would like to thank the Special Research Fund
(BOF) of the Ghent University for their financial support.
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