Stereoselective conjugation, transport and bioactivity of S- and R-hesperetin enantiomers in vitro

Article abstract of DOI:10.1021/jf1008617

The flavanone hesperetin ((±)-4′-methoxy-3′,5,7- trihydroxyflavanone) is the aglycone of hesperidin, which is the major flavonoid present in sweet oranges. Hesperetin contains a chiral C-atom and so can exist as an S- and R-enantiomer, however, in nature 2S-hesperidin and its S-hesperetin aglycone are predominant. The present study reports a chiral HPLC method to separate S- and R-hesperetin on an analytical and semipreparative scale. This allowed characterization of the stereoselective differences in metabolism and transport in the intestine and activity in a selected bioassay of the separated hesperetin enantiomers in in vitro model systems: (1) with human small intestinal fractions containing UDP-glucuronosyl transferases (UGTs) or sulfotransferases (SULTs); (2) with Caco-2 cell monolayers as a model for the intestinal transport barrier; (3) with mouse Hepa-1c1c7 cells transfected with human EpRE-controlled luciferase to test induction of EpRE-mediated gene expression. The results obtained indicate some significant differences in the metabolism and transport characteristics and bioactivity between S- and R-hesperetin, however, these differences are relatively small. This indicates that for these end points, including intestinal metabolism and transport and EpRE-mediated gene induction, experiments performed with racemic hesperetin may adequately reflect what can be expected for the naturally occurring S-enantiomer. This is an important finding since at present hesperetin is only commercially available as a racemic mixture, while it exists in nature mainly as an S-enantiomer.

Full text of DOI:10.1021/jf1008617

J. Agric. Food Chem. 2010, 58, 6119–6125 6119
DOI:10.1021/jf1008617
Stereoselective Conjugation, Transport and Bioactivity
of S- and R-Hesperetin Enantiomers in Vitro
†
W
K
ALTER
ATHELIJN N. VAN
AARIT J. REIN,‡ FABIOLA
B
RAND,*,†,‡
J
IA
S
HAO,† ELISABETH F. HOEK
-
VAN DEN
H ,
IL
†
E
LK,† BERT
S
PENKELINK,† LAURA H. J. DE
H
AAN
,
†,‡
M
D
IONISI,‡ GARY
WILLIAMSON
,
‡,§ PETER J. VAN
†
B
LADEREN
,
AND
IVONNE M. C. M. RIETJENS
†Division of Toxicology, Wageningen University, Tuinlaan 5, PO Box 8000, 6703 HE Wageningen,
The Netherlands, ‡Nestle
´
Research Center, Nestec Ltd., Vers-chez-les-Blanc, PO Box 44,
1000 Lausanne 26, Switzerland, and §School of Food Science and Nutrition,
University of Leeds, Leeds LS2 9JT, U.K.
The flavanone hesperetin ((()-40-methoxy-30,5,7-trihydroxyflavanone) is the aglycone of hesperidin,
which is the major flavonoid present in sweet oranges. Hesperetin contains a chiral C-atom and so
can exist as an S- and R-enantiomer, however, in nature 2S-hesperidin and its S-hesperetin
aglycone are predominant. The present study reports a chiral HPLC method to separate S- and
R-hesperetin on an analytical and semipreparative scale. This allowed characterization of the
stereoselective differences in metabolism and transport in the intestine and activity in a selected
bioassay of the separated hesperetin enantiomers in in vitro model systems: (1) with human small
intestinal fractions containing UDP-glucuronosyl transferases (UGTs) or sulfotransferases (SULTs);
(2) with Caco-2 cell monolayers as a model for the intestinal transport barrier; (3) with mouse
Hepa-1c1c7 cells transfected with human EpRE-controlled luciferase to test induction of EpRE-
mediated gene expression. The results obtained indicate some significant differences in the
metabolism and transport characteristics and bioactivity between S- and R-hesperetin, however,
these differences are relatively small. This indicates that for these end points, including intestinal
metabolism and transport and EpRE-mediated gene induction, experiments performed with racemic
hesperetin may adequately reflect what can be expected for the naturally occurring S-enantiomer.
This is an important finding since at present hesperetin is only commercially available as a racemic
mixture, while it exists in nature mainly as an S-enantiomer.
KEYWORDS: Flavonoid; chirality; HPLC; stereoselectivity; conjugation
INTRODUCTION
cells, hesperetin aglycone can be conjugated by UDP-glucurono-
syltransferases (UGTs) and sulfotransferases (SULTs) into glu-
curonidated and sulfonated metabolites, respectively, which have
been found in vivo in both rat and human plasma (6, 7). The
intestinal barrier is believed to play a dominant role in the
conjugation of hesperetin (8) and in its limited bioavailability
because of efflux of the metabolites back to the intestinal lumen by
ABC transport proteins (9, 10).
Unlike many other classes of flavonoids, flavanones as well as
flavanols share a chiral carbon atom in position 2 and therefore
exist in an S- and R-configuration (Figure 1). 2S-Hesperidin is
naturally predominant in citrus fruits (11-13), and hesperidin
is present in fresh, sweet orange juice in an S:R ratio of at least
92:8 in favor of the 2S-epimer (14-16). Although in nature the
2S-epimer of hesperidin, and subsequently the S-hesperetin
enantiomer, is dominant, hesperetin and hesperidin are currently
only commercially available as a mixture of both stereoisomers.
As a result, studies on hesperetin and hesperidin generally do not
take the chirality into account whereas in theory the two enantio-
mers may display distinct kinetic and dynamic properties (17).
The flavanone hesperetin ((()-40-methoxy-30,5,7-trihydroxy-
flavanone) (Figure 1) is the aglycone of hesperidin (hesperetin
7-O-rutinoside), which is the major flavonoid present in sweet
oranges (Citrus sinensis) and orange juice but which can also be
found in other citrus fruits including lemon, lime, and mandarin
and someherbs (1). Hesperetin and hesperidinhave been reported
to provide health beneficial effects, including anticarcinogenic
properties and a reduced risk of osteoporosis (2-4).
Upon ingestion, hesperidin has to be hydrolyzed into hesperetin
aglycone by colonic microbiota prior to its absorption. Conversion
of the disaccharide hesperidin into the monosaccharide hesperetin
7-O-glucoside prior to consumption has been demonstrated to lead
to absorption already in the small intestine after deglucosylation
by phloridzin hydrolase and/or by facilitated transport into the
intestinal cells by a sodium dependent glucose transporter (e.g.,
SGLT1) followed by intracellular deglucosylation (5). In the intestinal
*To whom correspondence should be addressed. Phone: þ31-317-
482294. Fax: þ31-317-484931. E-mail: Walter.Brand@WUR.nl.
© 2010 American Chemical Society
Published on Web 05/04/2010
pubs.acs.org/JAFC

6120 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Brand et al.
Cell Lines. Caco-2 human colon carcinoma cells were obtained from
the American Type Culture Collection (Manassas, VA) and were cultured
as described earlier (9). Passage numbers 39 to 47 were used for the
experiments.
Hepa-1c1c7 mouse hepatoma cells stably transfected with the reporter
vector pTI(hNQO1-EpRE)Lucþ from Promega (Leiden, The Nether-
lands) carrying the EpRE from the human NQO1 gene regulatory region
between -470 and -448 (50-AGT CAC AGT GAC TCA GCA GAA
TC-30), coupled to a luciferase reporter gene, were obtained as described
previously (24). These transfected Hepa-1c1c7 cells will further be referred
to as EpRE-LUX cells.
Figure 1. Chemical structure of (-)-S- and (þ)-R-hesperetin (40-methoxy-
30,5,7-trihydroxyflavanone).
Identification of S-Hesperetin. Hesperidin naturally occurs predo-
minantly as the 2S-epimer (14-16). To acquire S-hesperetin, 2S-hesper-
idin from an orange (Citrus sinensis) was deglycosylated. To this end,
freshly prepared orange juice (0.5 mL) was added to 1 mL of nano-
pure water, 110 μL of 0.78 M sodium acetate (pH 4.8), 100 μL of 0.1 M
ascorbic acid, and 200 μL of crude preparation of β-glucuronidase from
Helix pomatia type HP-2, which also deglycosylates flavanone rutino-
sides (16, 25), and incubated overnight at 37 °C (16, 25). Then, 1 mL of
acetonitrile was added to precipitate the proteins, and the mixture was
vortexed for 1 min and centrifuged at 16000g for 5 min. The supernatant
was collected, the solvent was evaporated under nitrogen gas, and the
residue was dissolved in the mobile phase for chiral HPLC analysis.
Chiral HPLC-DAD Analysis. Chiral analyses of hesperetin were
performed on an HPLC system consisting of a Waters (Milford, MA)
Alliance 2695 separation module connected to a Waters 2996 photodiode
array detector (DAD) equipped with a ChromTech (Cheshire, UK)
analytical 150 mm ꢀ 4 mm Chiral-AGP column connected to a 10 mm ꢀ
4 mm guard column. Samples were centrifuged at 16000g for 4 min, and
20 μL was injected and isocratically eluted at a flow rate of 0.9 mL/min in
10 mM ammonium acetate (pH 5.0) containing 2% (v/v) isopropyl
alcohol, filtered through a membrane filter with a pore size of 0.45 μm
from Schleicher and Schuell (Dassel, Germany). The column was equili-
brated for 10 min before injection and washed with 15% (v/v) isopropyl
alcohol in nanopure water. DAD-UV spectra were detected between
200 and 420 nm, and HPLC chromatograms acquired at 280 nm were
used for quantification and presentation.
Semipreparative Separation of S- and R-Hesperetin. Semiprepara-
tive HPLC separation of the S- and R-enantiomers of hesperetin was
performed on an HPLC system consisting of an Uniflows Degasys DG-
2410 degasser (Tokyo, Japan), a Waters 600 fluid unit, and a controller
connected to a Waters 996 DAD (Milford, MA), equipped with a
ChromTech semipreparative 100 mm ꢀ 10.0 mm Chiral-AGP column
(Cheshire, UK). An injection volume of 100 μL of 500 μM of racemic
hesperetin in 25% (v/v) isopropyl alcohol in nanopure water was injected
and eluted at a flow rate of 5.6 mL/min in 10 mM ammonium acetate
(pH 5.0) containing 2% (v/v) isopropyl alcohol and filtered through a
membrane filter with a pore size of 0.45 μm from Schleicher and Schuell
(Dassel, Germany). Elution of both hesperetin enantiomers was followed
at 280 nm. Fractions containing the separate enantiomers were collected
and divided into Eppendorf tubes and freeze-dried. The resulting products
were dissolved in a small amount of methanol, pooled, and dried under a
flow of nitrogen and redissolved in a small amount of DMSO. To check
the enantiomeric purity (>95%), a sample of each preparation, 100-fold
diluted with 25% (v/v) isopropyl alcohol in nanopure water, was analyzed
by chiral HPLC-DAD. To precisely determine the hesperetin concentra-
tion, a 100- or 1000-fold diluted sample of each preparation in 20%
acetonitrile (v/v) in 0.1% trifluoroacetic acid in nanopure water was
analyzed by a-Chiral HPLC-DAD based on detection at 280 nm using a
10-point linear (R2 > 0.99) calibration line of relevant concentrations of
racemic hesperetin. On the basis of the outcome of this quantification, the
separated S- and R-hesperetin solutions in DMSO were further diluted
with DMSO to create 10 mM stock solutions. A sample dissolved in cell
culture medium did not demonstrate racemization after incubation at
37 °C for 2 h (data not shown). No other peaks were detected in the
chromatograms of the a-Chiral analysis of S- or R-hesperetin, when
chromatograms were analyzed at different wavelengths (data not shown).
Microsomal and Cytosolic Incubations. To study intestinal glucuro-
nidation of S- and R-hesperetin, incubations with human intestinal micro-
somes were performed as described before for racemic hesperetin (26).
The incubation mixtures (total volume 200 μL) contained 10 mM MgCl2,
For flavonoids, stereochemical properties have been reported to
influence, for example, the bioavailability of the flavanol catechin (18),
the estrogenic activity of the isoflavone metabolite equol (19,20), and
the plasma and urinary kinetics of hesperetin (16, 21) and may thus
very well affect both the intestinal metabolism and transport of
hesperetin as well as its biological effects.
Although several studies reported analytical methods to ana-
´
lyze S- and R-enantiomers of hesperetin, as reviewed by Yanez
et al. (11), the kinetic differences of S- and R-hesperetin were only
studied indirectly. After intravenous administration of racemic
hesperetin to rats, R-hesperetin had a significant 3.3-fold higher
area under the serum concentration-time curve (AUC) and
a 1.9-fold longer half-life, compared to S-hesperetin (after enzy-
matic hydrolysis of the metabolites in plasma samples) (16).
The aim of the present study was to develop a method for
separation of S- and R-hesperetin on an analytical and semi-
preparative scale using chiral HPLC with R1-acid glycoprotein
(AGP) as chiral selector and to characterize differences in the
intestinal conjugation and transport, and the activity in a selected
bioassay, of the two hesperetin enantiomers in in vitro models.
To that end, we performed incubations with microsomal and
cytosolic fractions of human small intestine with the separated
enantiomers in order to determine the apparent kinetics for
glucuronidation and sulfonation of S- and R-hesperetin. Further-
more, the stereoselective differences in intestinal metabolism
and transport were assessed using Caco-2 cell monolayers in a
two-compartment transwell system as a model for the intestinal
barrier. To test differences in a selected bioassay, S- and R-
hesperetin were tested in a reporter gene based bioassay quantify-
ing EpRE-(electrophile responsive element) mediated activation
of gene expression. EpRE-mediated activation of gene expression
is considered to contribute to the cancer preventive action of
chemoprotectivedietarycompoundsincludingflavonoids(22,23).
MATERIALS AND METHODS
Materials. Alamethicin (from Trichoderma viride), crude preparation
of β-glucuronidase (from Helix pomatia) type HP-2, hesperetin (purity
g95%, batch 015K1099), L
-ascorbic acid, and uridine 50-diphospho-
glucuronic acid (UDPGA) were obtained from Sigma (St. Louis, MO),
30-phosphoadenosine 50-phosphosulfate (PAPS) from Fluka (Buchs,
Switzerland), dimethyl sulfoxide (DMSO), dipotassium hydrogen phos-
phate trihydrate, EDTA disodium salt dehydrate, glacial acetic acid,
hydrochloric acid, potassium dihydrogen phosphate, and sodium acetate
trihydrate from Merck (Darmstadt, Germany), acetonitrile, isopropyl
alcohol, and methanol from Sigma-Aldrich (Steinheim, Germany), Tris
from Invitrogen (Carlsbad, CA), and ammonium acetate and trifluoro-
acteic acid from J. T. Baker (Philipsburg, NJ). Authentic standards of
hesperetin 7-O-glucuronide (purity >90%), hesperetin 30-O-glucuronide
(purity >90%), and hesperetin 7-O-sulfate (purity <50%) were provided
by Nestle Research Center (Lausanne, Switzerland). An orange (Citrus
´
sinensis) from South Africa was bought at a local store. All cell culture
reagents were purchased from Invitrogen (Paisley, UK). Pooled human
small intestinal microsomes (batch MIC318012) and pooled human small
intestinal cytosol (batch CYT318004) were purchased from Biopredic
(Rennes, France).

Article
J. Agric. Food Chem., Vol. 58, No. 10, 2010 6121
25 μg/mL alamethicin added from a 200 times concentrated stock solution
in methanol (final concentration 0.5% methanol), 0.1 mg/mL microsomal
protein, and 1 mM uridine 50-diphosphoglucuronic acid (UDPGA) in
50 mM Tris-HCl (pH 7.5). The reaction was started by addition of S- or
R-hesperetin from a 200 times concentrated stock solution in DMSO (final
concentration 0.5% DMSO) and incubated for 5 min at 37 °C. The
reaction was terminated by addition of 50 μL of acetonitrile. Under these
conditions, formation of hesperetin glucuronides was linear in time and
with the amount of protein (data not shown).
To study intestinal sulfonation of S- and R-hesperetin, incubations with
human intestinal cytosol were performed as described before for racemic
hesperetin (26). The incubation mixtures (total volume 100 μL) contained
5 mM MgCl2, 0.5 mg/mL cytosolic protein, and 100 μM 30-phospho-
adenosine 50-phosphosulfate (PAPS) in 50 mM potassium phosphate
(pH 7.4). The reaction was started by addition of S- or R-hesperetin from
a 100 times concentrated stock solution in DMSO (final concentration 1%
DMSO) and incubated for 5 min at 37 °C. The reaction was terminated by
addition of 25 μL of acetonitrile. Under these conditions, formation of
hesperetin sulfates was linear in time and with the amount of protein (data
not shown).
Metabolism and Transport by Caco-2 Cell Monolayers. Caco-2
cells were cultured in a humidified atmosphere of 5% CO2 and 95% air
at 37 °C and seeded at a density of 1 ꢀ 105 cells/cm2 in Costar 12-well
transwell plate inserts from Corning (Corning, NY) with an insert
membrane pore size of 0.4 μm and growth area of 1.12 cm2. The culture
medium consisted of Dulbecco’s modified Eagle’s medium (DMEM)
containing 25 mM HEPES buffer (pH 7.4), 4500 mg/mL glucose,
L-glutamine, and phenol red, supplemented with 10% (v/v) heat inacti-
vated (30 min at 56 °C) fetal bovine serum, 1% (v/v) minimal essential
medium nonessential amino acids (NEAA), and 0.1 mg/mL gentamicin.
The medium was changed three times a week. The transport experiment
was performed 18-19 days postseeding, for which the monolayers were
washed and further incubated with DMEM without phenol red. The
integrity of the monolayers was checked by measuring the trans-epithelial
electrical resistance (TEER) values with a Millicell ERS volt/ohmmeter
from Millipore (Bedford, MA). Only monolayers that demonstrated a
TEER value between 500 and 1000 Ω/cm2 were used. For the experiment,
exposure medium was prepared consisting of DMEM (without phenol
Figure 2. Chiral HPLC chromatograms of (A) racemic hesperetin, (B)
hesperetin resulting from deglycosylation of the 2S-hesperidin epimer from
citrus.
red) supplemented with 1% (v/v) NEAA and 1 mM L-ascorbic acid, which
was filtered through a sterile 0.2 μm filter unit from VWR (West Chester,
PA). The monolayers were exposed at the apical side to exposure medium
containing 10 μM S-hesperetin or R-hesperetin (final concentration 0.5%
DMSO). Samples of 150 μL were taken from the apical and basolateral
side 120 min upon addition, whereafter integrity of the monolayer was
reconfirmed. All samples were stored at -80 °C until further HPLC-DAD
analysis carried out as described earlier (9).
a-Chiral HPLC Analysis. a-Chiral gradient HPLC on a C18 column
was used to detect and quantify the amounts of hesperetin, hesperetin 7-O-
glucuronide, hesperetin 30-O-glucuronide, hesperetin 7-O-sulfate, and
hesperetin 30-O-sulfate using methods previously described (9, 26).
Enzyme Kinetics. To determine the kinetics for glucuronidation and
sulfonation, microsomal and cytosolic incubations were performed as
described above by varying the concentration of S- or R-hesperetin from
1 to 50 μM. Under the applied conditions, the formation of hesperetin 7-O-
glucuronide, hesperetin 30-O-glucuronide, hesperetin 7-O-sulfate, and
hesperetin 30-O-sulfate conjugates was linear in time and with the amount
of microsomal protein added (data not shown). The apparent maximum
incubated for 24 h, whereafter the cells had attached to the bottom and
formed a confluent monolayer. The plates were exposed to different
concentrations of S- or R-hesperetin. To this end, the culture medium
was removed and replaced by 100 μL of medium containing hesperetin at
the required concentration (0.5% DMSO). The plates were incubated for
another 24 h, whereafter the cells were washed with 0.5 ꢀ PBS and lysed by
addition of low salt buffer (10 mM Tris, 2 mM DTT, and 2 mM trans-1,2-
diaminocyclohexane-N,N,N0,N0-tetra-acetic acid monohydrate, pH 7.8).
After lysis, luciferase reagent (20 mM tricine, 1.07 mM (MgCO3)4-
Mg(OH)2, 2.67 mM MgSO4, 0.1 mM EDTA, 2 mM DTT, 0.47 mM
D-luciferin, 5 mM ATP; pH 7.8) was added and the luciferase activity
was measured using a Luminoskan Ascent luminometer from Thermo
Electron Corporation (Helsinki, Finland).
RESULTS
Analytical and Semipreparative Chiral HPLC-DAD Analyses.
Figure 2A depicts a chromatogram of the analytical chiral HPLC
analysis of racemic hesperetin, demonstrating the two enantio-
mers at retention times (tR) 25.5 and 29.0 min in a 41:59 ratio.
Both peaks demonstrated equivalentUV spectra, with a UVmax at
285.9 nm. The limit of detection, defined as the lowest concentra-
tion which can be detected (signal-to-noise ratio 3) of this method
was 0.6 μM, the limit of quantification, defined as the lowest
concentration which can be quantitatively determined (signal-
to-noise ratio 10) was 2 μM. To identify the S- and R-hesperetin
enantiomers, hesperidin from fresh orange juice, mainly pre-
sent as 2S-epimer, was deglycosylated to yield predominantly
S-hesperetin and analyzed (Figure 2B). The peak at tR 29.0 min
velocity (Vmax(app)) and apparent Michaelis-Menten constant (Km(app)
)
for the formation of the different phase II metabolites of S- and
R-hesperetin were determined by fitting the data to the Michaelis-Menten
steady-state model v = Vmax/(1 þ (Km/[S])), with [S] being the hesperetin
concentration, using the LSW data analysis toolbox (version 1.1.1) from
MDL Information Systems (San Ramon, CA).
EpRE-Lux Assay. EpRE-mediated induction of gene expression by
S- and R-hesperetin was tested using the EpRE-LUX luciferase reporter
gene assay as described earlier (24, 27). The EpRE-LUX cells were
cultured in alpha modified Eagle’s medium supplemented with 10% fetal
bovine serum, 50 μg/mL gentamicin, and 0.5 mg/mL G418 from Duchefa
(Haarlem, The Netherlands), in a humidified atmosphere of 5% CO2 and
95% air at 37 °C. Cell suspension (100 μL) with a density of 3 ꢀ 105 cells/
ml was seeded per well in a 96 well view-plate (Corning, NY) and

6122 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Brand et al.
(UVmax = 285.9 nm), was dominant in the hydrolyzed citrus
sample identifying the corresponding metabolite as S-hesperetin.
S- and R-hesperetin were successfully separated on a semipre-
parative scale and concentrated, yielding sufficient amounts to
perform small scale in vitro experiments. The racemic purity after
vacuum concentration and pooling of the collected samples was
>95% for both S- and R-hesperetin.
Microsomal Glucuronidation. Hesperetin is glucuronidated by
small intestinal microsomes into hesperetin 7-O-glucuronide and
30-O-glucuronide metabolites (26). The apparent Vmax and Km
values derived from the concentration dependent formation of
S-hesperetin 7-O-glucuronide and S-hesperetin 3-O-glucuronide
(Figure 3A), and R-hesperetin 7-O-glucuronide and R-hesperetin
30-O-glucuronide (Figure 3B) by human small intestinal micro-
somes, are presented in Table 1, as well as the apparent catalytic
efficiencies (Vmax/Km). For comparison, Table 1 also presents the
kinetic data obtained previously for racemic hesperetin (26). For
the glucuronidation of both hesperetin enantiomers, the rela-
tive contribution of the conjugation at position 7 and 30 is com-
parable. However, the affinity was higher (Km lower) for the
glucuronidation of S-hesperetin as compared to the glucuronida-
tion of R-hesperetin. Together with a slightly higher capacity
(Vmax), this results in a 5.2-fold higher catalytic efficiency for the
glucuronidation of S-hesperetin compared to R-hesperetin
(Table 1). The catalytic efficiencies obtained for 7-O-glucuronide
formation from S- and R-hesperetin were respectively 2.7-fold
higher and 2.5-fold lower than the catalytic efficiencies observed
with the racemic mixture, and the catalytic efficiencies obtained
for 30-O-glucuronide formation from S- and R-hesperetin were
respectively 2.5-fold higher and 1.6-fold lower compared to the
catalytic efficiencies observed with the racemic mixture.
Cytsosolic Sulfonation. Hesperetin is sulfonated by human
small intestinal cytosol into hesperetin 30-O-sulfate and 7-O-
sulfate metabolites (26). The apparent Vmax and Km values derived
from the concentration dependent formation of S-hesperetin
30-O-sulfate and S-hesperetin 7-O-sulfate (Figure 4A), and
R-hesperetin 30-O-sulfate and R-hesperetin 7-O-sulfate (Figure 4B)
by human small intestinal microsomes are shown in Table 2, as well
as the apparent catalytic efficiencies (Vmax/Km). For comparison,
Table 2 also presents the kinetic data obtained previously for racemic
hesperetin (26). Both hesperetin enantiomers are predominantly
sulfonated at position 30, although the capacity and the relative
contribution of the formation of 7-O-sulfonation to the total
sulfonation of R-hesperetin is higher as compared with the sulfona-
tion of S-hesperetin (Table 2). The catalytic efficiency obtained for
30-O-sulfate formation from S- and R-hesperetin were respectively
1.2-fold lower and 1.5-fold higher than the catalytic efficiencies
observed with the racemic mixture, and the catalytic efficiency
obtained for 70-O-sulfate formation from S- and R-hesperetin were
respectively equal and 2.8-fold higher compared to the catalytic
efficiencies observed with the racemic mixture (26).
Metabolism and Transport by Caco-2 Cell Monolayers. Caco-2
cell monolayers apically exposed to hesperetin metabolize it into
hesperetin 7-O-glucuronide and hesperetin 7-O-sulfate, which are
predominantly transportedtotheapicalsideofthe monolayer (9).
Figure 5 shows the percentages of the applied amount of
S-hesperetin or R-hesperetin that is metabolized into 7-O-glucuro-
nide or 7-O-sulfate metabolites as well as the percentage which
remained unmetabolized and the disposition of the metabolites
to the apical or basolateral side of the Caco-2 cell monolayer.
Exposure to R-hesperetin resulted in a significantly (p < 0.001)
higher amount of hesperetin 7-O-sulfate formed, amounting to
Figure 3. Concentration dependent formation of hesperetin 7-O-glucuro-
nide (b), hesperetin 30-O-glucuronide (9), and total hesperetin glucuro-
nides (2) from S-hesperetin (A) and R-hesperetin (B) enantiomers by
human small intestinal microsomes. Data points represent average activi-
ties of two measurements ( SD.
Table 1. Apparent Vmax ((SEM), Km ((SEM), and Catalytic Efficiency for the Glucuronidation of S- and R-Hesperetin by Human Small Intestinal Microsomes (n = 2)a
S-hesperetin
R-hesperetin
racemic hesperetinb
V
max(app)
catalytic efficiency
V
max(app)
catalytic efficiency
V
max(app)
catalytic efficiency
(μL min-1 mg
conjugation
reaction
Km(app) (nmol min-1 mg
(μL min-1 mg Km(app) (nmol min-1 mg
(μL min-1 mg Km(app) (nmol min-1 mg
(μM)
protein-1
)
protein-1
)
(μM)
protein-1
)
protein-1
)
(μM)
protein-1
)
protein-1
)
glucuronidation
(total)
7-O-
5.7 ( 1.7
6.1 ( 1.6
5.2 ( 1.7
12.5 ( 0.99
7.43 ( 0.56
5.08 ( 0.43
2189
21.3 ( 6.5
24.0 ( 8.0
18.9 ( 6.1
8.95 ( 1.24
4.40 ( 0.69
4.55 ( 0.63
420
7.3 ( 1.4
8.3 ( 1.9
6.4 ( 1.2
6.26 ( 1.67
3.80 ( 0.50
2.49 ( 0.49
858
1224
183
458
glucuronidation
30-O-
glucuronidation
971
240
391
a Catalytic efficiency was calculated as Vmax(app)/Km(app) b The kinetic data for racemic hesperetin are taken from our previous work (26).
.

Article
J. Agric. Food Chem., Vol. 58, No. 10, 2010 6123
129% (at the apical plus basolateral side) of the amount of
hesperetin 7-O-sulfate formed upon exposure to S-hesperetin
(Figure 5). The total amount of R-hesperetin 7-O-glucuronide
formed was 8% lower compared to the amount of S-hesperetin
7-O-glucuronide formed, and this difference was not significant
(Figure 5). The total amount of hesperetin metabolites and/or
aglycone transported over the Caco-2 monolayer did not differ
significantly for S- and R-hesperetin.
((0.2) at 25 μM and higher (Figure 6). Although at some of the
concentrations significant differences are found between the
induction factors by S- and R-hesperetin, the overall induction
however, especially at lower, and thus physiologically relevant,
concentrations does not demonstrate notable differences in
activation of EpRE mediated gene expression between both
hesperetin enantiomers.
Activation of EpRE-Controlled Gene Expression by Hesperetin.
Figure 6 shows the concentration dependent induction of EpRE-
mediated luciferase expression by S-hesperetin and R-hesperetin.
Exposure to both hesperetin enantiomers resulted in a dose
dependent induction of EpRE mediated gene expression: expo-
sure to S-hesperetin led to an 8.2-fold ((0.6) induction at 50 μM,
and exposure to R-hesperetin to a maximum induction of 6.0-fold
Figure 5. Amount of hesperetin 7-O-glucuronide (glucuronide), hesper-
etin 7-O-sulfate (sulfate), and hesperetin aglycone detected at the apical
and basolateral side of Caco-2 cell monolayers incubated for 120 min with
10 μM S-hesperetin or R-hesperetin added to the apical side. Mean ( SD
values shown (n = 8). ***, p < 0.001 significantly different.
Figure 4. Concentration dependent formation of hesperetin 7-O-sulfate
(9), hesperetin 30-O-sulfate (b), and total hesperetin sulfates (2) from
S-hesperetin (A) and R-hesperetin (B) enantiomers by human small
intestinal cytosol. Data points represent average activities of two measure-
ments ( SD.
Figure 6. Induction of EpRE-mediated gene transcription by R-hesperetin
(O) and S-hesperetin (b). Data are presented as mean ((SD) (n = 4). *,
p < 0.05; **, p < 0.01 significantly different compared with the induction by
corresponding the corresponding concentration of the other hesperetin
enantiomer.
Table 2. Apparent Vmax ((SEM), Km ((SEM), and Catalytic Efficiency for the Sulfonation of S- and R-Hesperetin by Human Small Intestinal Cytosol (n = 2)a
S-hesperetin
R-hesperetin
racemic hesperetinb
V
max(app)
catalytic efficiency
V
max(app)
catalytic efficiency
(μL min-1 mg Km(app) (nmol min-1 mg
Vmax(app) catalytic efficiency
(μL min-1 mg
conjugation
reaction
Km(app) (nmol min-1 mg
(μL min-1 mg Km(app) (nmol min-1 mg
(μM)
protein-1
)
protein-1
)
(μM)
protein-1
)
protein-1
)
(μM)
protein-1
)
protein-1
)
sulfonation
(total)
0.9 ( 0.2
0.96 ( 0.03
1067
1.6 ( 0.2
1.27 ( 0.04
803
0.8 ( 0.1
0.90 ( 0.29
1082
7-O-sulfonation 15.6 ( 2.5
30-O-sulfonation 0.6 ( 0.2
0.25 ( 0.03
0.80 ( 0.03
16
1455
11.5 ( 2.1
0.9 ( 0.3
0.51 ( 0.04
0.89 ( 0.04
44
1000
9.1 ( 1.5
0.6 ( 0.2
0.14 ( 0.04
0.79 ( 0.14
16
1240
a Catalytic efficiency was calculated as Vmax(app)/Km(app) b The kinetic data for racemic hesperetin are taken from our previous work (26).
.

6124 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Brand et al.
DISCUSSION
to note that interindividual variation in kinetics may be in the
same order of magnitude as or even larger than the differences
now observed between S- and R-hesperetin.
Although hesperidin naturally exists mainly as the 2S-epimer,
which upon intake is subsequently transformed into S-hesperetin,
practically all research on hesperidin and hesperetin using “pure”
compounds is actually on racemic mixtures, which are currently
the only forms of hesperidin and hesperetin commercially available.
Stereochemical differences have been proven to affect the bioavail-
ability of flavonoids, as was shown for example for catechin.
(þ)-Catechin (2R,3S-catechin) has been demonstrated to be up
to 2.1-fold more bioavailable compared to (-)-catechin (2S,3R-
catechin), partly due to increased intestinal absorption (18).
To study the differences between the “natural” S-hesperetin
and R-hesperetin, the present paper reports a method to separate
both enantiomers on an analytical and semipreparative scale,
allowing small-scale in vitro experiments with the separated
enantiomers. This allowed characterization of possible differ-
ences in intestinal metabolism and transport and the activity in
a selected bioassay of S- and R-hesperetin, by incubating S- or
R-hesperetin with human small intestinal microsomes and cytosol
and cofactors for phase II conjugation, with Caco-2 cell mono-
layers in a transwell transport model and with a transfected cell
line to test induction of EpRE-mediated gene expression.
Although the results obtained indicate some significant differ-
ences in metabolism, the differences in the metabolism and
transport of the two hesperetin enantiomers are relatively small.
The higher affinity and capacity toward S-hesperetin resulted in
an overall 5.2-fold higher catalytic efficiency for the formation of
S-hesperetin glucuronides as compared to R-hesperetin glucuro-
nides by human small intestinal microsomes (Table 1). The
differences between the sulfonation kinetics of S-hesperetin and
R-hesperetin are marginal, although the capacity for the forma-
tion of 7-O-sulfate from R-hesperetin is 2.8-fold higher compared
with the formation of 7-O-sulfate from S-hesperetin (Table 2,
Figure 4). Although relatively small, together these kinetic differ-
ences might explain the increased formation of hesperetin 7-O-
sulfate and reduced formation of hesperetin 7-O-glucuronide by
Caco-2 cell monolayers exposed to R-hesperetin as compared
to monolayers exposed to S-hesperetin (Figure 5).
In addition to possible differential kinetics for S- and
R-hesperetin, it is of interest to study whether different hesperetin
enantiomers would lead to different biological effects. It has been
reported for instance that stereochemical differences greatly
affect the estrogenic activity of the isoflavone metabolite equol:
S(-)-equol demonstrated high affinity for estrogen receptor
ERβ (Ki = 0.73 nM), whereas R(þ)-equol had a Ki of only
15.4 nM (19).
It has been reported that flavonols and catechins can activate
EpRE mediated gene expression because of their pro-oxidant
properties under certain conditions (27, 28) by which these
flavonoids could exert their chemopreventive action (23, 27).
The present study revealed that, at physiologically relevant
concentrations, S- and R-hesperetin were both able to induce
EpRE mediated gene expression to a similar extent (Figure 6).
However, this assay represents only one of the possible mechan-
isms by which hesperetin could exert biological effects.
In conclusion, in the case of hesperetin, whenever differences
were observed in the kinetics of S- and R-hesperetin, they turned
out to be moderate. The most explicit difference found was the
5.2-fold difference in the catalytic efficiency for the glucuronida-
tion of S-hesperetin compared to R-hesperetin. However, the
differences never resulted in the complete absence of a biochem-
ical pathway or kinetic route for one of the hesperetin enantio-
mers. Also, in a selected bioassay, the two enantiomers were
equally active. Taken together, it is concluded that for the end
points tested, including intestinal metabolism and transport and
EpRE-mediated gene induction, experiments performed with
racemic hesperetin may adequately reflect what can be expected
for the naturally occurring S-enantiomer. This is an important
finding since at present hesperetin is only commercially avail-
able as a racemic mixture, while it exists in nature mainly as an
S-enantiomer.
ABBREVIATIONS USED
ABC, ATP binding cassette; AGP, R1-acid glycoprotein; bw,
body weight; DAD, diode array detector; DMEM, Dulbecco’s
modified Eagle’s medium; DMSO, dimethyl sulfoxide; EpRE,
electrophile-responsive element; NEAA, nonessential amino
acids; PAPS, 30-phosphoadenosine 50-phosphosulfate; SD, stan-
dard deviation; SEM, standard error of the mean; SGLT1,
sodium-glucose cotransporter 1; SULT, sulfotransferase; TEER,
trans-epithelial electrical resistance; tR, retention time; UDPGA,
uridine 50-diphosphoglucuronic acid; UGT, UDP-glucuronosyl-
transferase.
Differences in the kinetics between S-hesperetin and R-hesper-
etin have been observed indirectly in vivo after intravenous
administration of racemic hesperetin (20 mg/kg bodyweight
(bw)) to male Sprague-Dawley rats, demonstrating a significant
(p < 0.05) 3.2-fold higher area under the serum concentra-
tion-time curve (AUC) and 1.9-fold (p < 0.05) longer serum
half-life for R-hesperetin compared to S-hesperetin, which had a
3.3-fold (p < 0.05) higher total clearance from serum (16). The
cumulative urinary excretion of R-hesperetin was 2.3-fold (p <
0.05) higher compared to S-hesperetin (16). In an earlier study,
however, oral administration of racemic hesperidin (200 mg/kg
bw) to a single rat revealed only slightly (∼15%) increased
cumulative 24 h urinary excretion of R-hesperetin compared to
S-hesperetin, while the cumulative urinary excretion of R-hesper-
etin compared to S-hesperetin was only ∼20% lower following
the administration of orange juice (containing 7.3 mg/kg bw
hesperidin in an S:R ratio of 6.8:1) to a healthy volunteer (21).
Although several of these effects appeared significant, they
were moderate in size, showing, similar to the observations in the
present study, differences between the kinetic parameters for
the S- and R-enantiomer of less than 5-fold. In addition, the
differences never resulted in the complete absence of a biochem-
ical pathway or kinetic route for one of the hesperetin enantio-
mers. From this it is concluded that kinetic data obtained with
the racemic mixture do give insight into what may happen to the
naturally predominant S-enantiomer of hesperetin. It is of interest
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Received for review March 3, 2010. Revised manuscript received April
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´
research grant from Nestle Research Center (Lausanne, Switzerland).