SULT1A3-mediated regiospecific 7-O-sulfation of flavonoids in caco-2 cells can be explained by the relevant molecular docking studies

Article abstract of DOI:10.1021/mp200400s

Flavonoids are polyphenolic compounds with various claimed health benefits, but the extensive metabolism by uridine-5′-diphospho- glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) in liver and intestine led to poor oral bioavailabilities. The effects of structural changes on the sulfonation of flavonoids have not been systemically determined, although relevant effects of structural changes on the glucuronidation of flavonoids had. We performed the regiospecific sulfonation of sixteen flavonoids from five different subclasses of flavonoids, which are represented by apigenin (flavone), genistein (isoflavone), naringenin (flavanone), kaempherol (flavonol), and phloretin (chalcone). Additional studies were performed using 4 monohydroxyl flavonoids with a -OH group at the 3, 4′, 5 or 7 position, followed by 5 dihydroxyl flavonoids, and 2 trihydroxyl flavonoids by using expressed human SULT1A3 and Caco-2 cell lysates. We found that these compounds were exclusively sulfated at the 7-OH position by SULT1A3 and primarily sulfated at the 7-OH position in Caco-2 cell lysates with minor amounts of 4′-O-sulfates formed as well. Sulfonation rates measured using SULT1A3 and Caco-2 cell lysates were highly correlated at substrate concentrations of 2.5 and 10 μM. Molecular docking studies provided structural explanations as to why sulfonation only occurred at the 7-OH position of flavones, flavonols and flavanones. In conclusion, molecular docking studies explain why SULT1A3 exclusively mediates sulfonation at the 7-OH position of flavones/flavonols, and correlation studies indicate that SULT1A3 is the main isoform responsible for flavonoid sulfonation in the Caco-2 cells.

Full text of DOI:10.1021/mp200400s

Article
pubs.acs.org/molecularpharmaceutics
SULT1A3-Mediated Regiospecific 7-O-Sulfation of Flavonoids in
Caco-2 Cells Can Be Explained by the Relevant Molecular Docking
Studies
Shengnan Meng,†,‡ Baojian Wu,‡ Rashim Singh,‡ Taijun Yin, John Kenneth Morrow,§ Shuxing Zhang,§
and Ming Hu*,‡
†Department of Pharmaceutics, School of Pharmaceutical Sciences, China Medical University, Shenyang, Liaoning 110001, China
‡Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 1441 Moursund Street,
Houston, Texas 77030, United States
§The Integrated Molecular Discovery Laboratory, Department of Experimental Therapeutics, MD Anderson Cancer Center, 1515
Holcombe Boulevard, Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: Flavonoids are polyphenolic compounds with various
claimed health benefits, but the extensive metabolism by uridine-5′-
diphospho-glucuronosyltransferases (UGTs) and sulfotransferases
(SULTs) in liver and intestine led to poor oral bioavailabilities. The
effects of structural changes on the sulfonation of flavonoids have not
been systemically determined, although relevant effects of structural
changes on the glucuronidation of flavonoids had. We performed the
regiospecific sulfonation of sixteen flavonoids from five different
subclasses of flavonoids, which are represented by apigenin (flavone),
genistein (isoflavone), naringenin (flavanone), kaempherol (flavonol),
and phloretin (chalcone). Additional studies were performed using 4
monohydroxyl flavonoids with a −OH group at the 3, 4′, 5 or 7 position,
followed by 5 dihydroxyl flavonoids, and 2 trihydroxyl flavonoids by
using expressed human SULT1A3 and Caco-2 cell lysates. We found
that these compounds were exclusively sulfated at the 7-OH position by SULT1A3 and primarily sulfated at the 7-OH position in
Caco-2 cell lysates with minor amounts of 4′-O-sulfates formed as well. Sulfonation rates measured using SULT1A3 and Caco-2
cell lysates were highly correlated at substrate concentrations of 2.5 and 10 μM. Molecular docking studies provided structural
explanations as to why sulfonation only occurred at the 7-OH position of flavones, flavonols and flavanones. In conclusion,
molecular docking studies explain why SULT1A3 exclusively mediates sulfonation at the 7-OH position of flavones/flavonols,
and correlation studies indicate that SULT1A3 is the main isoform responsible for flavonoid sulfonation in the Caco-2 cells.
KEYWORDS: SULT1A3, flavonoids, Caco-2, molecular docking
after oral administration,8,9 significant amounts of sulfates were
also found.9 In the mouse intestine, a significant portion of the
INTRODUCTION
Flavonoids, a class of phenolic compounds widely distributed in
■
nature, have been postulated to possess significant biological
activities in prevention of diseases such as cancer, inflammation,
coronary heart diseases and other age-related illnesses.1,2
Currently, chemopreventive agents using this class of
compounds are yet to be approved, and their low bioavailability
is one of the top reasons why their development was
impeded.3,4 Many studies from this and other laboratories
have demonstrated that extensive first-pass metabolism by
phase II conjugating enzymes including uridine-5′-diphospho-
glucuronosyltransferases, or UGTs, and sulfotransferases, or
SULTs, causes the observed low bioavailabilities.5,6
absorbed aglycons, such as genistein and apigenin, were
conjugated and both sulfates and glucuronides were excreted
into the lumen.10 Thus, the published studies provided strong
evidence that rapid conjugation via sulfonation is one of the
main reasons for flavonoids’ low bioavailabilities in vivo.
Sulfonation reactions are catalyzed by several members of the
SULT superfamily, which play important roles in the regulation
of the levels and activities of flavonoids.
Received: August 11, 2011
Revised: December 20, 2011
Accepted: February 19, 2012
Published: February 21, 2012
Flavonoids, including flavones and flavonols, are known to
have low oral bioavailabilities.7 Although flavonols such as
kaempferol and quercetin were mainly present as glucuronides
© 2012 American Chemical Society
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Mammalian cytosolic SULTs have been divided into several
gene families and subfamilies based on their amino acid
sequence identity and catalytic properties.11 Human SULT1A3
is one of the most important SULT1 isoforms for the
metabolism of phenolic compounds and a good model isoform
for investigating structure−activity relationships.12 In addition,
preliminary data suggested that this isoform is highly active
against flavonoids compared to other SULT isoforms such as
SULT1A1 and SULT1E1 (not shown). Moreover, expression
studies in Caco-2 TC-7 cells showed that SULT1A3 was the
most abundant SULT isoform found in these cells.13 Caco-2
TC-7 cells are a model of the human intestinal epithelial cells.
Originally derived from human colon cancer, Caco-2 cells
possess many of the properties of the normal human small
intestinal cells including expression of various phase II enzymes
(such as UDP-glucuronosyltransferase, sulfotransferase, etc.)
known to be present in normal human enterocytes. Caco-2 cells
are a useful and well-accepted tool for studying the metabolic
characteristics of flavonoids in vitro.4,14−19
Table 1. Chemical Structures of Flavonoids Used in This
Study
Previously, many laboratories, including ourselves, have
determined the effects of structural changes on the
glucuronidation of flavonoids using the Caco-2 models, animal
intestinal models, animal and human microsomes, and
expressed human UGTs.20−23 However, there did not appear
to be any systematic studies on the effects of structural changes
on the sulfonation of flavonoids. In one recent study of 2
catechins and 2 flavanones, it was shown that they inhibited the
sulfonation by multiple SULT isoforms including SULT1A3 at
very high concentrations,24 not highly relevant to in vivo
conditions where the concentrations are much lower.
Therefore, the purpose of this study was to determine how
structural changes affect the sulfonation of the flavonoids using
SULT1A3 and Caco-2 cell lysates. There were a total of 16
flavonoids used in this study (Table 1). Five different subclasses
of flavonoids are represented by apigenin (flavone), genistein
(isoflavone), naringenin (flavanone), kaempherol (flavonol),
and phloretin (chalcone), and these five compounds are
analogues of apigenin, all with 3 hydroxyl groups at the 4′,5,7
positions, although kaempherol also has a 3-OH group. We
then determined the sulfonation of 4 monohydroxyl flavonoids
with a −OH group at the 3, 4′, 5 or 7 position, followed by 5
dihydroxyl flavonoids, and 2 additional trihydroxyl flavonoids.
Molecular docking techniques were then used to explain the
observed positional preference in sulfate formation by
examining the potential binding sites of these flavonoids inside
active pockets of SULT1A3 crystal structures.
triplicate. Briefly, expressed SULT1A3 (final protein concen-
tration of about 0.00248−0.00493 mg/mL) was mixed with
flavonoids (final concentration of 2.5 or 10 μM) in 50 mM
potassium phosphate buffer (pH 7.4). The cofactor PAPS (0.1
mM, final concentration) was added last to the reaction mixture
(total volume 200 μL), and the mixture was incubated in a 37
°C shaking water bath (speed = 200 rpm) for 5−30 min. The
shorter reaction time (5 min) was for compounds that were
rapidly metabolized to ensure that percentages metabolized
would not exceed 35%, which is the upper linear range in the
amount of sulfate formed vs time curve. The longer reaction
time was used to ensure that slowly metabolized compounds
will have produced a reasonable peak area for accurate
measurement (above the lowest quantifiable concentration in
the linear response range). A more detailed description of the
various reaction time and enzyme quantities is presented in the
Supporting Information.
MATERIALS AND METHODS
■
Materials. Naringenin, phloretin, genistein, apigenin,
kaempferol, 3-hydroxyflavone, 4′-hydroxyflavone, 5-hydroxy-
flavone, 7-hydroxyflavone, 5,4-dihydroxyflavone, 5,7-dihydroxy-
flavone, 7,4′-dihydroxyflavone, 3,4′-dihydroxyflavone, 3,7-dihy-
droxyflavone, 3,5,7-trihydroxyflavone and 3,7,4′-trihydroxyfla-
vone were purchased from Indofine Chemicals (Somerville,
NJ). Expressed human SULT isoforms were purchased from
XenoTech LLC (Lenexa, KS). 3′-Phosphoadenosine-5′-phos-
phosulfate (PAPS) and sulfatase from Aerobacter aerogenes were
purchased from Sigma-Aldrich (St. Louis, MO). All other
materials, analytical grade or better, were used as received.
Sulfonation Activities of Expressed Human SULT1A3.
Sulfonation activities of flavonoids in expressed human
SULT1A3 were measured using the published procedures
with minor modifications.20 All experiments were performed in
Two substrate concentrations were used for the character-
ization studies since characterization should be performed at
two or more concentrations. The low concentration (2.5 μM)
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was chosen here because it was close to the Km values (reported
here or elsewhere), and we did not use a lower concentration
much less than 2.5 μM (e.g., 1 μM) because molecular
extinction coefficients of some compounds precluded the
determination of their metabolite concentrations at a substrate
concentration much less than 2.5 μM. The high concentrations
were chosen because the rates of formation at this
concentration were usually close to the Vmax values. At any
rate, at a substrate concentration of 2.5 μM, the final enzyme
protein concentration (in the reaction mixture) was 0.00248
mg/mL for 3,7DHF and 3,5,7THF and was 0.00493 mg/mL
for naringenin, apigenin, kaempferol, 7HF, 5,4′DHF, 5,7DHF,
7,4′DHF, and 3,7,4′THF. The incubated time was 5 min for
naringenin, apigenin, kaempferol, 7HF, 5,4′DHF, 5,7DHF,
7,4′DHF, 3,7DHF, 3,5,7THF, and 3,7,4′THF, but 30 min for
phloretin, genistein, 4′HF, 5HF, 3HF, and 3,4′DHF. Different
incubation time was necessary to ensure that percentages of a
substrate metabolized did not exceed 35% but enough
metabolite was formed to facilitate detection by UV. At the
substrate concentration of 10 μM, the final enzyme protein
concentration was the same as the substrate concentration at
2.5 μM for every compounds, but the incubation time was 10
min for naringenin, apigenin, kaempferol, 7HF, 5,4′DHF,
5,7DHF, 7,4′DHF, 3,7DHF, 3,5,7THF, and 3,7,4′THF, but 30
min for phloretin, genistein, 4′HF, 5HF, 3HF, and 3,4′DHF.
The reaction was stopped by the addition of 50 μL of solution,
consisting of 94% acetonitrile and 6% formic acid containing
100 μM testosterone or 100 μM 5-hydroxyflavone as the
internal standard. Testosterone was used as internal standard
for naringenin, phloretin, genistein, 3HF,4′HF, 5HF, 7HF,
7,4′THF, 3,7,4′THF, 5,7,4′THF (apigenin), and 3,5,7,4′QHF
(kaempferol); whereas 5-hydroxyflavone was used for 3,7DHF.
5,7DHF, 5,4′DHF, and 3,5,7THF. The reaction mixture
containing the internal standard was centrifuged at 13,000
rpm for 20 min, and the supernatant was directly subjected to
UPLC for analysis.
For the determination of kinetic profile of sulfonation, four
flavonoids (i.e., 7HF, 7,4′DHF, 3,7DHF, 5,7DHF) in the
concentration range of 0.039−20 μM were used. This
concentration range was used because of our prior experience
with the study of sulfonation of apigenin by Caco-2 TC cell
lysates.15
Cell Culture. Cloned Caco-2 cells, TC-7, were a kind gift
from Dr. Monique Rousset of INSERM U178 (Villejuit,
France). The Caco-2 cells have been routinely used in this lab
for more than two decades, and in here the culture conditions
for growing Caco-2 cells were the same as those described
previously.20,21 The cells were used 14 days after seeding in the
current study.
Preparation of Caco-2 Cell Lysate for Sulfonation
Studies. Cell lysates were prepared using freshly collected
Caco-2 cells, and used for measuring rates of sulfate formation.
For cell lysate preparation, cells were first washed in ice-cold
PBS (pH7.4) and scraped off and put into in a centrifuge tube.
After it was centrifuged at 3000 rpm for 2 min, the supernatant
was removed, and cell pellets were mixed with ice-cold 50 mM
pH 7.4 potassium phosphate buffer. The cell suspension was
sonicated using Aquasonic 150D sonicator (VWR Scientific,
Bristol, CT) for 30 min in short pulses at the maximum power
(135 average watts) in an ice-cold water bath (the temperature
was ∼0 °C). The resulting cell lysate was then harvested and
pooled. It was used fresh or frozen at −80 °C until use, for
measuring the rates of conjugate formation (sulfonation
activities were maintained at −80 °C with a single defrosting
action). The protein concentration of the cell lysate was
determined using the BCA protein assay, using the bovine
serum albumin as the standard.
Measurement of Sulfonation Activities in Caco-2 Cell
Lysates. The incubation procedures for measuring sulfonation
activities using Caco-2 cell lysates were similar to those using
expressed human SULT1A3 except the concentration of Caco-
2 cell lysates in the final reaction mixture was about 0.465−1.86
mg/mL. All reactions were performed in triplicate. The reaction
time was adjusted to 5−60 min (except for genistein, where a
reaction time of 480 min was used) for the different flavonoids,
and the reason for using different reaction time here is the same
as stated previously when using SULT1A3. The substrate
concentrations were again 2.5 and 10 μM, the same as those
used for SULT1A3. Sulfonation activities are expressed in
nanomoles per minute per milligram of protein for Caco-2 cell
lysates.
UPLC Analysis of Flavonoids and Their Sulfate.
Flavonoids as well as their respective sulfates were analyzed
by a common chromatographic method: system, Waters
Acquity UPLC with photodiode array detector and Empower
software; column, BEH C18, 1.7 μm, 2.1 × 50 mm; mobile
phase A, 100% aqueous buffer (2.5 mM NH4Ac, pH 7,4);
mobile phase B, 100% acetonitrile,; flow rate 0.45 mL/min;
gradient, 0 to 2.0 min, 10−30% B (or mobile phase B), 2.0 to
3.0 min, 30−40% B, 3.0 to 3.5 min, 40−60% B, 3.5 to 4.0 min,
60−90%, 4.0 to 5.0 min, 90%-10% B, 5.0 to 5.5 min, 10% B and
injection volume, 10 μL. Naringenin, phloretin and their
respective sulfates were analyzed at 286 nm; genistein,
kaempferol, 3HF and their respective sulfates were analyzed
at 254 nm. Apigenin, 3,4′DHF, 3,7DHF, 5,4′DHF, 7,4′DHF,
3,7,4′THF and their respective sulfates were analyzed at 340
nm. 4′HF, 7HF and their sulfates were analyzed at 320 nm and
310 nm respectively. 5HF, 5,7DHF and their respective sulfates
were analyzed at 268 nm. 3,5,7THF and 3,5,7THF-sulfates
were analyzed at 263 nm. Linearity was established in the range
of 0.3−10 μM (a total of 6 concentrations were used) for 5HF
and 0.3−20 μM (a total 7 of concentrations were used) for
other compounds. Analytical methods for each compound were
validated for interday and intraday variation using six samples at
three concentrations (20, 5, and 0.625 μM). Precision and
accuracy for all compounds were in the acceptable range of 85%
to 115%.
Quantification of Flavonoid Sulfates. Since standards of
flavonoid sulfates of tested compounds could not be obtained
commercially, a previously published method25 was adapted for
quantification of sulfates although that method was originally
developed to quantify glucuronides. Briefly, the increase in peak
area of aglycon was compared with the decrease in the peak
area of sulfate after hydrolysis by sulfatase. Because 1 mol of
metabolite generates 1 mol of aglycon as the result of
hydrolysis, the change in concentration of aglycon as the result
of hydrolysis can be expressed as
ΔP
ΔP
FS
F
ΔC =
=
a
a
(1)
F
FS
where ΔPFS is the change in peak areas of flavonoid sulfate (or
FS), ΔPF is the change in the peak area of its corresponding
flavonoid aglycon (or F) obtained from the extracted samples
before and after hydrolysis, and aF and aFS are the slopes of the
corresponding calibration curves that goes through the origin.
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Equation 1 can be rearranged so that the term aFS was
expressed in terms of aF:
where Vmax is the maximum activation of enzyme activity, C is
the concentration of substrate and Km is the concentration of
substrate to achieve 50% of Vmax, and n is the Hill coefficient.
Eq 6 describes enzyme reactions with biphasic kinetics:
ΔP
ΔP
FS
a
=
a = Ka
F F
FS
(2)
S
V
K
C
C
V
K
C
C
m2
max1
max2
reaction rate =
+
where K represents the conversion factor of molar extinction
coefficients of sulfates to their corresponding aglycons. To
calculate the metabolite concentration (CFS), CFS should be
represented by ΔPFS and ΔPS through the conversion factor K,
which is provided as an average value determined at three
different substrate concentrations:
(6)
m1
where Vmax1 is the maximum enzyme velocity of the high
affinity phase, Vmax2 is the maximum velocity of the low affinity
phase, Km1 is concentration of substrate to achieve half of Vmax1
for the high-affinity phase, and Km2 is concentration of substrate
to achieve half of Vmax2 for the low-affinity phase.
When the enzymatic reactions showed substrate inhibition
kinetics (in which the substrate compound inhibits the
sulfonation, especially at higher concentrations), formation
rates (V) of flavonoid sulfates at various substrate concen-
trations (C) were fit to the following equation:
P
a
P
FS
FS
Ka
C
=
=
FS
(3)
FS
F
where PFS is the peak area of sulfates. Therefore, the
concentrations of sulfates could be estimated using the
corresponding calibration curve of the aglycons.
Confirmation of Flavonoid Sulfates Structure by LC−
MS/MS. An API 3200 QTrap triple quadrupole mass
spectrometer (Applied Biosystem/MDS SCIEX, Foster City,
CA), operated in negative ion mode, was used for identification
of the sulfates of flavonoids. The main working parameters for
the mass spectrometers were set as follows: ion spray voltage,
−4.0 kV; ion source temperature, 400 °C; the nebulizer gas
(gas 1), zero air, 40 psi; turbo gas (gas 2), zero air, 40 psi;
curtain gas, nitrogen, 20 psi. Flavonoid metabolites were
identified by MS full scan and MS2 full scan modes. The
conditions for separating flavonoids and their sulfates were
achieved by the same UPLC system and using the same
chromatographic conditions stated above. For preparation of
concentrated sulfate samples for identification purposes, the
sulfates were separated by solid phase extraction from the
sulfonation experimental samples, and reconstituted in a
smaller volume of 30% acetonitrile in water (i.e., concentrated).
Kinetics of Sulfonation. Rates of metabolism in expressed
human SULT1A3 were expressed as amounts of metabolites
formed per minute per milligram of protein, or nmol/min/mg.
Kinetic parameters were then obtained based on the fit to
various kinetic equations shown below based on profiles of
Eadie−Hofstee plots as described previously.23 If Eadie−
Hofstee plots were linear, formation rates (V) of flavonoid
sulfates at various substrate concentrations (C) were fit to the
standard Michaelis−Menten equation:
V
max1
reaction rate =
1 + (K /C) + (C/K )
(7)
m1 si
where Vmax1 is the maximum formation rate, C is the substrate
concentration, Km1 is the concentration of substrate to achieve
50% of (Vmax1), and Ksi is the substrate inhibition constant.
Statistical Analysis. One-way ANOVA with or without
Tukey−Kramer multiple comparison (post hoc) tests were
used to evaluate statistical differences. Differences were
considered significant when p values were less than 0.05 (or
p < 0.05).
Molecular Docking Analysis. Molecular docking was
employed to explain the apparent regiospecificity of SULT1A3.
To this end, we docked all flavonoids into the catalytic site of
the SULT1A3 crystal structure. These chemical structures were
minimized with root-mean-square gradient of 0.000001 in
MOE (Chemical Computing Group, Montreal, CA) based on
MMFF94x force field and partial charges. The docking program
GOLD (CCDC, Cambridge, UK) was used to perform docking
of these compounds against the crystal structure of SULT1A3
(PDB entry code: 2A3R).12 Hydrogen atoms were added in
GOLD and default parameters were used unless otherwise
stated. The cocrystallized substrate dopamine was removed
from the structure, but the PAPS cofactor was kept as part of
the protein. No structural water molecules were observed in the
active site, and hence all water molecules were deleted.
Based on the complex structure and literature reports,12,26
the active site was defined to include the following residues:
Tyr23, Pro47, Lys48, Thr51, Tyr76, Val84, Tyr139, Ala148,
Ser168, Tyr169, Leu247, Met248, Phe255, and A3P296
(PAPS). Ten additional residues were also considered as part
of the binding site, and they were treated as flexible during
docking (the maximum number allowed by GOLD: Ile21,
Phe24, Phe81, Asp86, Lys106, His108, Phe142, Glu146,
His149, and Tyr240. The maximum ligand flexibility and
maximum search efficiency was applied during docking runs.
To evaluate the regiospecificity of the sulfonation reactions, a
distance constraint (5.0−6.5 Å) from the 7-OH of each
substrate was applied to the sulfate group of PAPS. Addition-
ally, two hydrogen bonding constraints were applied from the
7-OH of each ligand to the imidazole of His108 and amine
group of Lys106 on SULT1A3, because these hydrogen
bonding interactions were observed in the crystal structure
and His108 is the catalytic residue for the sulfonation reaction.
V
C
max
+ C
V =
K
(4)
m
where Km is the Michaelis constant and Vmax is the maximum
rate of sulfonation.
When Eadie−Hofstee plots showed characteristic profiles of
atypical kinetics (sigmoidal autoactivation and biphasic
kinetics), the data from these atypical profiles were fit to eq
5 or 6, using the ADAPT II program. To determine the best-fit
model, the model candidates were discriminated using Akaike’s
information criterion (AIC), and the rule of parsimony was
applied. With regard to data showing sigmoidal kinetics, this
model was simply a rewriting of a Hill equation, and those
formation rates (V) of flavonoid sulfates at various substrate
concentrations (C) were fit to eq 5:
n
V
C
max
reaction rate =
n
n
K
+ C
(5)
m
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Figure 1. UPLC and LC−MS/MS profile of flavonoids and their mono-O-sulfates. Only 9 flavonoids with significant amounts of sulfates formed are
shown. Left panels (panels A1−I1) of each pair show the UPLC−UV trace with the retention time of each flavonoid, its respective metabolite(s)
(S), and internal standard (IS). Since the chromatograms of Nar, 7HF, and 3,7,4′-THF were collected at 286 nm, 320 nm, and 340 nm respectively,
their IS (100 μM testosterone) are not showed in the graph. The right panels (panels A2−I2) of each pair show MS2 spectra of the corresponding
flavonoid-7-O-monosulfates.
The top five docked solutions for each ligand were retained for
analysis.
(Figure 1), and no disulfates of any flavonoids were found. For
3HF, 4′HF, 5HF, 3,4′DHF and genistein, sulfates were not
detected when SULT1A3 was used under the conditions
described in Materials and Method.
Determination of the Conversion Factors for Quanti-
fication of Sulfates. The conversion factors for individual
sulfates of flavonoids were determined in order to quantify the
RESULTS
■
Confirmation of Flavonoid Sulfate Structures by LC−
MS/MS. The LC−MS/MS studies of the metabolites showed
that all sulfates generated in this study were monosulfates
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amounts of sulfates formed. The conditions under which these
conversion factors were generated including the wavelength,
types of enzymatic preparation used (Caco-2 cell lysates or
SULT1A3), and the conversion factor for each sulfate are listed
in Table 2. The conversion factors were in the range of 0.501
(for 3,5,7THF-7-O-sulfate) to 1.583 (for 3HF-3-O-sulfate).
mg protein (Figure 2B). It is interesting to note that
sulfonation rates of flavonols without the 7-OH group (4′HF
and 5HF) were too slow to be detected.
The sulfonation rates of another congeneric series of
flavonoids in the flavonol subclass (3HF, 3,4′DHF, 3,7DHF,
3,5,7THF, 3,7,4′THF, 3,5,7,4′QHF) were also used to
determine the effects of changes in number and position of
hydroxyl groups on the SULT1A3-mediated reaction rates. At a
concentration of 2.5 μM, the sulfonation activity followed the
rank order 3,7DHF (45.84 1.53) > 3,5,7THF (13.88 1.21)
Table 2. The Conversion Factors (K) of Various Flavonoid-
7-O-monosulfates
compd
K
wavelength (nm) used
> 3,7,4′THF (6.80
1.28) > 3,5,7,4′QHF (3.78
0.79) >
Nar
1.236
0.511
1.051
1.190
0.806
286
286
254
340
254
320
268
310
340
268
340
254
340
340
263
340
3,4′DHF (0) ∼ 3HF (0) nmol/h/mg of protein (Figure 2C).
At 10 μM, flavonol sulfonation followed the same rank order:
Phlor
Gen
3,7DHF (74.69
3,7,4′THF (11.79
1.05) > 3,5,7THF (24.98
0.33) > 3,5,7,4′QHF (5.77
0.49) >
0.22) >
Api (5,7,4′THF)
Kamp (3,5,7,4′QHF)
4′HF
3,4′DHF (0) ∼ 3HF (0) nmol/h/mg of protein (Figure 2C),
although the actual rates did change. Here, the sulfonation rate
of 3,7DHF was the fastest, which was highly significantly
different (p < 0.05) from the other compounds in this series.
Again, compounds without the 7-OH group were not sulfated.
Sulfonation of Flavonoids by SULTs in Caco-2 Cell
Lysates. Many investigators have shown that Caco-2 cells can
sulfonate flavonoids and secrete flavonoid sulfates.15,16,20,27,28
Here we performed a systematic study to determine how
changes in the structure of flavonoids affect their sulfonation in
Caco-2 cell lysates, which are known to express a high level of
SULT1A3.13
For the flavonoids from the five subclasses (naringenin,
phloretin, genistein, apigenin and kaempferol), the pattern of
metabolism as reflected by the rank order was almost the same
as those observed using SULT1A3 (Figure 2D), except that
kaempferol was metabolized a bit faster at lower concentration
than anticipated from SULT1A3 data (Figure 2D). In addition,
the rates were generally much slower than those using
expressed human SULT1A3, suggesting that the expression
level of the expressed SULT is quite high (>500-fold
enrichment), suggesting that the expression system is of high
quality.
0.824
a
5HF
ND
7HF
1.46
5,4′DHF
5,7DHF
0.960
1.198
1.041
1.583
0.873
1.46
7,4′DHF-
3HF
3,4′DHF
3,7DHF
3,5,7THF
3,7,4′THF
0.501
1.044
a
ND: not determined since no sulfate of 5HF was found.
Sulfonation of Flavonoids by Expressed Human
SULT1A3. Among the sixteen flavonoids, SULT1A3-mediated
sulfonation of 5 flavonoids (naringenin, phloretin, genistein,
apigenin and kaempferol), which were from 5 different
subclasses of flavonoids, was used to determine the effects of
backbone change. The sulfonation rates of flavonoids at the
substrate concentration of 2.5 μM by expressed SULT1A3
followed the following rank order: apigenin (19.00 1.58) >
kaempferol (3.78
0.79) ∼ naringenin (3.21
0.36) >
genistein ∼ phloretin (0 nmol/h/mg protein) (Figure 2A).
Apigenin, which belongs to flavone subclass, showed the fastest
sulfonation rates, whereas genistein (an isoflavone with phenol
at C-3 position) and phloretin (a chalcone with open ring)
showed extremely weak or undetectable sulfonation. Addition
of a free hydroxyl group at C-3 as in flavonols (kaempferol) or
saturation of the double bond at C2−C3 as in flavanones
(naringenin) appeared to similarly reduce the sulfonation as
compared to flavones (apigenin). The rank order of the
sulfonation rates at 10 μM showed slight variation compared to
those at 2.5 μM: apigenin (19.36 0.41) > naringenin (12.03
0.94) > kaempferol (5.77 0.22) > genistein ∼ phloretin (0)
nmol/h/mg protein (Figure 2A).
Correlation of Sulfonation Rates Obtained Using
SULT1A3 and Caco-2 Cell Lysates. To determine if the
SULT1A3 is the major isoform responsible for sulfonation of
flavonoids, correlations between the SULT1A3-mediated
sulfonation rates and sulfonation rates in Caco-2 cell lysates
were established at the substrate concentration of 2.5 μM and
10 μM (Figure 3). The results showed the correlation
coefficient at 2.5 μM was 0.736 when we set the intercept of
the regression line to zero, and was 0.764 when it was not (not
shown). At 10 μM substrate concentration, the correlation
coefficient was 0.963 when the regression line was forced
through the origin, and was 0.966 when the line was not forced
through the origin (not shown).
The sulfonation rates of a congeneric series of flavonoids in
the flavone subclass (5,4′DHF, 5,7DHF, 7,4′DHF and
5,7,4′THF, also included 4′HF, 5HF and 7HF) were used to
determine how sulfonation rates would change as a function of
the number and position of hydroxyl groups on the same
backbone. At 2.5 μM, sulfonation rates of 5,7DHF (62.30
2.54 nmol/h/mg of protein) were the fastest, followed by 7HF
(35.66 4.72), 5,7,4′THF (19.00 1.58), 7,4′DHF (7.54
0.34), 4′HF (0) and 5HF(0) nmol/h/mg protein (Figure 2B).
The sulfonation rates at 10 μM showed essentially the same
rank order: 5,7DHF(85.37 1.53) > 7HF(48.92 5.18) > 5,
7, 4′, THF(19.36 0.41) > 7,4′DHF(10.30 1.64) nmol/h/
There were strong correlations between formation rates of
sulfates derived from Caco-2 cell lysates and those derived from
SULT1A3 (Figure 3). The correlation was better at 10 μM than
at 2.5 μM, perhaps because reaction rates at higher
concentration approached that of the Vmax values, although
the exact reason for this is unclear.
Kinetics of Flavonoid Sulfonation by SULT1A3. We
determined the kinetics of sulfonation of four flavonoids.
Starting with 7-HF, we determined how addition of one
hydroxyl group at the 3, 5, and 4′ position affected the kinetics
of sulfonation. This study was conducted because the effects of
adding one hydroxyl group had inconsistent effects on
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Figure 2. Sulfonation of flavonoids by expressed human SULT1A3 (left three panels) and Caco-2 cell lysates (right three panels). Flavonoids were
from 5 apigenin analogues representing five subclasses of flavonoids (A, D), 6 flavones (B, E), and 6 flavonols (C, F). Experiments were conducted at
the concentration of 2.5 μM (open and dotted columns) and 10 μM (solid and filled columns). Amounts of monosulfate(s) formed were measured
using UPLC and quantified using the correction factors shown in Table 2. Rates of sulfonation were calculated as nmol/min/mg of protein. Each bar
is the average of three determinations, and the error bars are the standard deviations of the mean (n = 3).
sulfonation rates at the two concentrations (2.5 and 10 μM)
investigated, in that addition of these groups at 2.5 μM usually
decreased the rates (Figure 2), whereas addition of the same
groups at 10 μM usually increased the rates (Figure 2).
The results of the kinetic studies showed that sulfonation of
7HF followed substrate inhibition kinetics (Figure 4A1, 4A2),
with Km value of 2.80 μM and Vmax of 85.1 nmol/min/mg or an
intrinsic clearance (CLint) value of 30.4 mL/min/mg protein
(Table 3). Addition of a 4′-OH group did not change the
binding characteristics (Figure 4B1, 4B2) and resulted in a
similar Km value (3.42 μM). However, the Vmax values were
much smaller, suggesting that addition of 4′-OH really affected
the turnover rate of the reaction. In other words, addition of 4′-
OH decreased the capacity of SULT1A3 to form flavonoid-7-
sulfate. Indeed, addition of a 4′-OH group to 5,7DHF or
3,5,7THF all decreased the sulfonation rates at 10 μM.
Next, we determined how addition of a 3-OH group changed
the kinetics of sulfonation, and the results showed that both
kinetic profile (Figure 4C1, 4C2) and enzyme capacities
changed (Table 3). The kinetic profile is now autoactivation,
and the Km value is more than 2-fold smaller, and Vmax is more
than 4 times higher, resulting in a CLint value that is nearly 7-
fold higher. This would suggest that addition of 3-OH would
increase the sulfonation rates at both low and high
concentrations, at least for this set of compounds.
The effects of 5-OH substitution on kinetics of sulfonation at
the 7-OH group are similar to the effects of addition of 3-OH
position, in that kinetic profile (Figure 4D1, 4D2) and enzyme
capacities both changed. The effects of adding a 5-OH group
were more pronounced than the effects of adding a 3-OH
group in that we have larger overall Vmax values. Moreover,
sulfonation of these flavonoids did not display a substrate
inhibition pattern.
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basic residue Lys106 as well as with the deprotonated N-3 atom
on the His108 imidazole ring (N-1 tautomer). Additionally we
observed several other hydrogen bonds between His149 and
Glu146/Asp86 to the 5-OH and carbonyl groups (Figure 5),
respectively, which appears to stabilize the binding site as they
did for the binding of dopamine to the SULT1A3 protein.12
Also based on the comprehensive evaluation of these
parameters and interaction patterns (Table 4), all flavonoids
without 7-OH were not SULT1A3 substrates, in agreement
with our experimental findings. Similarly, we could explain why
isoflavones and chalcones were not substrates either. Although
they displayed high docking scores, these compounds did not
have correct docking poses as their 7-OH groups could not be
positioned appropriately toward the cofactor PAPS and the
catalytic residue His108,26 as demonstrated in Table 4. For
example, genistein showed a docking with its 7-OH group
oriented away from PAPS (the distance to PAPS is 14 Å). This
conformation also did not allow H-bonding with the critical
residue His108, which precludes sulfonation of genistein by
SULT1A3.
Furthermore, we examined how modification of chemical
structures could affect the reaction rates. In our docking study,
we found that the phenyl ring attached to the 2-position of the
chromone scaffold in flavones affects the binding as it is
surrounded by hydrophobic residues including Tyr76, Phe142,
Tyr240, Val241, and Leu247 (Figure 5). As a result, the
addition of a hydrophilic 4′-OH group reduces the docking
score drastically from 41.10 (as in 5,7DHF) to 24.63 (as in
apigenin or 5,7,4′THF in Table 4). This is consistent with our
observed sulfonation rate (at 2.5 μM) of 5,7,4′THF (19.00
1.58 nmol/h/mg) being 3 times slower than that of 5,7DHF
(62.30 2.54 nmol/h/mg). Similarly, the 3-OH substitution in
flavonols also decreases the sulfonation rate of 3,5,7THF (24.98
0.49 nmol/h/mg) as the hydroxyl group sterically limits the
flexibility of the 2-phenyl group (docking score 26.93) blocking
its appropriate interactions with those hydrophobic residues of
SULT1A3.
Figure 3. Correlation of sulfonation rates obtained from SULT1A3
with those obtained from Caco-2 cell lysates. Linear regression was
used to derive apparent correlations. The sulfonation rates of
flavonoids by expressed human SULT1A3 and Caco-2 cell lysates
were calculated the same as described in Figure 2. In panel A is shown
the correlation of sulfonation between SULT1A3 and Caco-2 cell
lysates at the substrate concentration of 2.5 μM (R2 = 0.7363), and
that at the substrate concentration of 10 μM (R2 = 0.963) is shown in
panel B. Each bar is the average of three determinations, and the error
bars are the standard deviations of the mean (n = 3).
DISCUSSION
■
We have shown, for the first time, through this systemic study
that SULT1A3-mediated sulfonation of flavonoids was highly
regiospecific for the 7-OH position of flavonoids excluding
isoflavones (represented by genistein) and chalcones (repre-
sented by phloretin) (Figure 2). This experimental observation
could be explained by using the molecular docking study, which
provided structural basis as to why 7-OH was the predominant
position for sulfonation and why isoflavone and chalcone were
not good substrates of SULT1A3. The strong correlation
between SULT1A3-mediated sulfonation rates and sulfonation
rates determined using the Caco-2 cell lysate lends support to
the hypothesis that SULT1A3 is the dominating isoform
responsible for sulfonation of 16 tested flavonoids in the Caco-
2 cells. Considering the fact that SULT1A3 is mainly expressed
by the human enterocytes but not by liver,29,30 this makes
SULT1A3 the likely major isoform responsible for flavonoid
sulfonation in human intestine in vivo.
Molecular Docking Analysis. We used molecular docking
to explain why a flavonoid is a substrate of SULT1A3 and shed
some light on the previously observed differences in sulfonation
rates. To this end, three criteria were evaluated: (1) the docking
score [we found substrates have higher scores than most
nonsubstrates (usually scores <20)]; (2) the distance between
7-OH and the phosphate of the cofactor PAPS [a range of 5.0−
6.5 Å was proposed based on the distance of 5.7 Å in crystal
structure 2A3R for D-dopamine, a prototypical substrate of
SULT1A3; if no 7-OH exists, we would evaluate other −OH
groups which are close to His108 as described in the next
criterion]; and (3) ability of the reactive OH group (e.g., 7-
OH) to form hydrogen bonding with the catalytic residue
His108 which acts as a catalytic base and deprotonates the 7-
OH during the sulfate transfer.26 The results are listed in Table
4.
Based on these three criteria, 5,7DHF was interpreted as the
most active substrate, in agreement with our experimental
observation. First, it has the highest docking score. Second, we
found that the 7-OH moiety was oriented correctly toward the
cofactor PAPS for the catalytic reaction (Figure 5), with an
appropriate distance of 5.8 Å from the sulfate group.12 Third,
the 7-OH group was able to form hydrogen bonds with the
We have shown convincingly for the first time that SULT1A3
is exclusively for (reasonably rapid) sulfonation at the 7-OH
position of a structurally diverse group of flavonoids.
Previously, SULT1A3 was shown to be highly regiospecific
and only sulfated 4-OH group of dopamine.31 This strong
regioselective sulfonation is very different from UGT-mediated
glucuronidation of flavonoids where one UGT isoform (e.g.,
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Figure 4. Kinetics of 7HF (A1), 7,4′DHF (B1), 3,7DHF (C1) and 5,7DHF (D1) sulfonation by SULT1A3. The sulfonation rates were determined
at the concentration range of 0.04 to 20 μM. The reaction time was controlled so that substrate concentration did not decrease substantially (usually
less than 30%) at the end of experiments, which lasted for of up to 30 min. Each point is the average of three determinations, and the error bars are
the standard deviations of the mean (n = 3). In the right four panels (panels A2−D2), the Eadie−Hofstee plots which are indicative of the
mechanism of reaction kinetics are shown. The apparent kinetic parameters and the fitting kinetic models are listed in Table 3.
UGT1A1) usually could metabolize compounds at multiple
−OH groups.22 This is consistent with the observation that
SULT1A3 has a small pocket size and highly restrictive binding
mode, especially for phenols.12,32 This restrictive binding
theory is supported by the fact that addition of a −OH
group at any of the three positions of the flavonoid backbone
(5, 3 or 4′) always substantially changes the kinetics of
sulfonation by affecting the kinetic profiles (5 and 3
substitution) or kinetic parameters (4′ and 5 substitution)
(Figure 4 and Table 3). It is also supported by the fact that no
disulfates of any flavonoid were found in the present study.
We have shown clearly that flavonoid-7-O-sulfonation in the
Caco-2 cells was the result of their metabolism by SULT1A3
expressed in these cells, because of the high degree of
correlation between rates of sulfonation in expressed
SULT1A3 and in Caco-2 cell lysate. This result is expected
since SULT1A3 is the predominant SULT isoform expressed in
Caco-2 TC-7 cells.13 For the congeneric series of flavonoids in
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Table 3. Apparent Kinetic Parameters of Metabolism of 7HF, 3,7DHF, 5,7DHF, and 7,4DHF by SULT1A3
a
kinetic param
Km1 (μM)
Vmax1 (nmol min−1 mg−1
max1/Km1 (mL min−1 mg−1
Km2 (mM)
Vmax2 (biphasic, nmol min−1 mg−1
max2/Km2 (mL min−1 mg−1
7HF
7,4′DHF
3,7DHF
5,7DHF
2.73
2.80
3.42
20.83
6.09
1.85
79.7
43.0
)
85.11
30.40
87.77
V
)
32.15
1 × 10−6
17.38
)
V
)
1.74 × 107
Ksi
23.12
0.996
32.50
13.81
0.994
3.44
R2
0.994
43.68
0.992
AIC
model
48.76
substrate inhibition
substrate inhibition
sigmoidal kinetics of Hill equation
biphasic kinetics
a
Kinetic parameters were obtained from using substrate inhibition, sigmoidal kinetics of Hill equation, and biphasic enzyme kinetics models as
described in Materials and Methods.
Table 4. Analysis of Parameters for Docked Flavonoids to
SUL1A3
the flavone subclass, the activity pattern in the Caco-2 cell
lysate was again very similar to those seen with SULT1A3, with
one notable exception in that a flavone with a 4′-OH but
without a 7-OH was also metabolized, albeit at a rate that is
almost minimal compared to those flavonoids with a 7-OH
group (Figure 2E). The latter is probably because Caco-2 cells
also express other SULT isoforms such as SULT1A1 and
SULT1E1.
For the congeneric series of flavonoids in the flavonol
subclass, the activity pattern was again similar between Caco-2
cell lysates and SULT1A3, and the notable exception is still that
a minor metabolite was also formed with flavonols that
contained a 4′-OH group but without a 7-OH group (Figure
2F).
Taken together, we determined the sulfonation of the same
16 flavonoids both in the expressed human SULT1A3 and in
the Caco-2 cell lysates, and found that these compounds were
mostly sulfated at the 7-OH group. However, there were some
minor differences in that 4′-O-sulfates were formed in the Caco-
2 cell lysates, albeit at much slower rates. Previously, sulfates of
genistein were identified in intact Caco-2 cells and Caco-2 cell
lysates that were grown for a longer period of time (19−21
days), but the formation rates were slow. However, other
isoflavones were metabolized at faster rates. Our own studies
have shown that genistein can be metabolized by SULT1A1
and SULT1E1,33 which were expressed at much lower levels in
Caco-2 TC-7 cells.13
docking
score
distance from 7-OH to
PAPS (Å)
7-OH H-bonding
a
compd
with His108
naringenin
phloretin
genistein
apigenin
kaempferol
4HF
20.11
27.01
23.66
24.63
27.93
15.69
15.95
33.34
17.92
41.10
24.14
14.61
16.64
26.01
26.93
26.68
6.8
3.1
14
yes
no
no
6.1
4.1
13.6
4
yes
yes
no
5HF
no
7HF
3.3
3.4
5.8
6.6
10.1
4
yes
no
5,4′DHF
5,7DHF
7,4′DHF
3HF
yes
yes
no
3,4′DHF
3,7DHF
3,5,7THF
3,7,4′THF
no
6.1
5.7
6.3
yes
yes
yes
a
If 7-OH exists, we evaluated 7-OH; otherwise, we evaluated all −OH
groups and recorded the one closest to His108 and PAPS.
Taken together, these three criteria allow us to explain our
experimental data as to why a compound is a substrate or not.
However, we must point out that these simplified parameters
are just some key features for substrates. In order to
demonstrate the complex substrate−SULT1A3 interactions,
expert knowledge is required to analyze the docking results of
each compound and interpret the modeling data (substrate or
nonsubstrate) case by case.
In conclusion, we have shown that SULT1A3 has a strong
and almost exclusive preference for regiospecific metabolism at
the 7-OH group of flavonoids (except isoflavones and
chalcones) and that SULT1A3-mediated metabolism of
flavonoids is mainly responsible for sulfonation of these
flavonoids in the Caco-2 cells. This strong preference coupled
with strong effects of −OH substitution (at any position) is
consistent with the notion that SULT1A3 has small binding
pocket for binding flavonoids in a limited number of restrictive
orientations, as shown by the molecular docking study.
Figure 5. The docked pose of 5,7DHF in SULT1A3. 5,7DHF (yellow
sticks) forms strong hydrogen bonding interactions (magenta dashed
lines) with residues Asp86, Lys106, His108, and Glu146. In addition,
hydrophobic interactions with Phe24, Phe81, Val84, Tyr139, Phe142,
Tyr240 and Phe255 also stabilize the binding. The 7-OH is close to
the catalytic residue His108 and orientated toward PAPS (light blue)
for sulfonation. The ribbon diagram represents the secondary structure
of SULT1A3.
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(10) Liu, Y.; Liu, Y.; Dai, Y.; Xun, L.; Hu, M. Enteric disposition and
recycling of flavonoids and ginkgo flavonoids. J. Altern. Complementary
Med. 2003, 9, 631−40.
(11) Glatt, H.; Boeing, H.; Engelke, C. E.; Ma, L.; Kuhlow, A.; Pabel,
U.; Pomplun, D.; Teubner, W.; Meinl, W. Human cytosolic
sulphotransferases: genetics, characteristics, toxicological aspects.
Mutat. Res. 2001, 482, 27−40.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental details and tables of reaction
conditions of sulfation activities of flavonoids. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) Lu, J. H.; Li, H. T.; Liu, M. C.; Zhang, J. P.; Li, M.; An, X. M.;
Chang, W. R. Crystal structure of human sulfotransferase SULT1A3 in
complex with dopamine and 3′-phosphoadenosine 5′-phosphate.
Biochem. Biophys. Res. Commun. 2005, 335, 417−23.
(13) Meinl, W.; Ebert, B.; Glatt, H.; Lampen, A. Sulfotransferase
forms expressed in human intestinal Caco-2 and TC7 cells at varying
stages of differentiation and role in benzo[a]pyrene metabolism. Drug
Metab. Dispos. 2008, 36, 276−83.
(14) Chen, J.; Lin, H.; Hu, M. Metabolism of flavonoids via enteric
recycling: role of intestinal disposition. J. Pharmacol. Exp. Ther. 2003,
304, 1228−35.
(15) Hu, M.; Chen, J.; Lin, H. Metabolism of flavonoids via enteric
recycling: mechanistic studies of disposition of apigenin in the Caco-2
cell culture model. J. Pharmacol. Exp. Ther. 2003, 307, 314−21.
(16) Murota, K.; Shimizu, S.; Miyamoto, S.; Izumi, T.; Obata, A.;
Kikuchi, M.; Terao, J. Unique uptake and transport of isoflavone
aglycones by human intestinal caco-2 cells: comparison of
isoflavonoids and flavonoids. J. Nutr. 2002, 132, 1956−61.
(17) Svehlikova, V.; Wang, S.; Jakubikova, J.; Williamson, G.; Mithen,
R.; Bao, Y. Interactions between sulforaphane and apigenin in the
induction of UGT1A1 and GSTA1 in CaCo-2 cells. Carcinogenesis
2004, 25, 1629−37.
(18) Walle, U. K.; Walle, T. Induction of human UDP-
glucuronosyltransferase UGT1A1 by flavonoids-structural require-
ments. Drug Metab. Dispos. 2002, 30, 564−9.
(19) Zhang, S.; Morris, M. E. Effect of the flavonoids biochanin A
and silymarin on the P-glycoprotein-mediated transport of digoxin and
vinblastine in human intestinal Caco-2 cells. Pharm. Res. 2003, 20,
1184−91.
AUTHOR INFORMATION
■
Corresponding Author
*1441 Moursund Street, Department of Pharmaceutical
Sciences, College of Pharmacy, University of Houston,
Houston, TX 77030. Tel: (713) 795-8320. Fax: (713) 795
8305. E-mail: mhu@uh.edu.
ACKNOWLEDGMENTS
■
This work is supported by National Institutes of Health Grant
GM-70737 to M.H. and the National Natural Science
Foundation of China (No. 81173123) to S.M.
ABBREVIATIONS USED
■
5,7,4′THF, apigenin, 5,7,4′-trihydroxyflavone; 3,5,7,4′QHF,
kaempferol, 3,5,7,4′-tetrahydroxyflavone; 4′HF, 4′-hydroxyfla-
vone; 5HF, 5-hydroxyflavone; 7HF, 7-hydroxyflavone;
5,4′DHF, 5,4′-dihydroxyflavone; 5,7DHF, 5,7-dihydroxyflavone;
7,4′DHF, 7,4′-dihydroxyflavone; 3HF, 3-hydroxyflavone;
3,4′DHF, 3,4′-dihydroxyflavone; 3,7DHF, 3,7-dihydroxyflavone;
3,5,7THF, 3,5,7-trihydroxyflavone; 3,7,4′THF, 3,7,4′-trihydrox-
yflavone; UPLC, ultraperformance liquid chromatography;
UGT, UDP-glucuronosyltransferases; SULT, sulfotransferase;
AIC, Akaike’s information criterion
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