Identification and characterization of sesaminol metabolites in the liver

Article abstract of DOI:10.1021/jf901939m

Sesame seeds contain a number of antioxidants, such as sesamin, sesamolin, sesaminol, and sesaminol glucosides. Sesaminol triglucoside was reported to suppress oxidative stress In vivo, but little is known about the metabolism of this potentially Importan

Full text of DOI:10.1021/jf901939m

J. Agric. Food Chem. 2009, 57, 10429–10434 10429
DOI:10.1021/jf901939m
Identification and Characterization of Sesaminol Metabolites in
the Liver
M
IKA
MOCHIZUKI
,
†,‡,§ YOSHIKAZU
T
SUCHIE
,
†,§ YOSHIMASA
,§
NAKAMURA
,
AND
T
OSHIHIKO
O
SAWA
*
‡Department of Health and Nutrition Faculty of Psychological and Physical Science, Aichi Gakuin
University, Nisshin, Aichi 470-0195, Japan, §Laboratory of Food and Biodynamics, Graduate School of
Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, and Department of Biofunctional
Chemistry, Division of Bioscience, Graduate School of Natural Science and Technology,
Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. † These authors contributed
equally to this work.
Sesame seeds contain a number of antioxidants, such as sesamin, sesamolin, sesaminol, and
sesaminol glucosides. Sesaminol triglucoside was reported to suppress oxidative stress in vivo, but
little is known about the metabolism of this potentially important compound. Therefore, we have
studied the metabolites of sesaminol formed in the rat liver S9 mix and excreted in the liver of rats
ingesting sesaminol triglucoside for 24 h. Nuclear magnetic resonance (NMR) and mass spectro-
metry (MS) analyses revealed that rat liver S9 mix transformed the sesaminol into a catechol-type
metabolite. On the basis of a previous study with sesame lignans by culturing the genus Aspergillus,
sesaminol-6-catechol was identified as the major metabolite. Sesaminol was further converted into
500-methylated sesaminol-6-catechol by catechol-O-methyltransferase. Moreover, we successfully
detected these metabolites in the liver of rats ingesting the sesaminol triglucoside.
KEYWORDS: Sesame lignans; sesaminol; metabolism; rat liver microsome; antioxidant
INTRODUCTION
antioxidant activities stronger than the parent compounds (11).
Moreover, Kang et al. (12) have reported that sesaminol gluco-
sides, which are also very weak antioxidant compounds present in
defatted sesame flour, can decrease the susceptibility to oxidan-
tive stress in hypercholesterolemia rabbits. Therefore, the bioa-
vailability and metabolism of sesaminol glucosides need to be
determined to clarify the function of the absorbed sesame lignans
in vivo.
In this study, we investigated the in vitro metabolism of
sesaminol, using the S9 fraction of the rat liver and identified
sesaminol-6-catechol as the major metabolite. We also demon-
strated that catechol-O-methyltransferase (COMT) can catalyze
the conversion of sesaminol-6-catechol into the 500-methylated
derivative in vitro. This is the first report showing the successful
detection of these metabolites in the liver of rats ingesting
sesaminol triglucoside.
For a long time, sesame has been categorized as one of the
traditional healthy foods in East Asia. It contains linoleic-acid-
rich oil and small quantities of lignans consisting of sesamin,
sesaminol, sesamolin, and sesaminol triglucoside (Figure 1) (1).
These lignans have been reported to have their own unique
biological effects, including an antihypertensive effect (2), hypo-
cholesterolemic activity (3), and antioxidative activities (4). In
contrast, the information about the bioavailability of sesame
lignans is limited mainly because of their extensive metabolism.
The metabolism of sesame lignans appears to include a reduction
reaction (5), but there is little systematic studies concerning the
metabolites in humans. Nakai et al. (6) and Liu et al. (7) indicated
that sesamin undergoes cleavage of methylenedioxyphenyl
(MDP) groups to catechol or the methoxy catechol in vitro and
in rats, respectively. Sesamin, having no phenolic hydroxyl group
and little radical scavenging activity, prevents liver damage
caused by alcohol or carbon tetrachloride (8), 7,12-dimethylbenz-
[R]anthracene-induced rat mammary carcinogenesis (9), and
hypertension (2, 10), suggesting that sesamin metabolites might
be at least in part involved in these effects. Recently, it has been
reported that the incubation of sesaminol and sesamin with
Aspergillus species resulted in the production of sesaminol-6-
catechol and sesamin-6-catechol, respectively, both of which have
MATERIALS AND METHODS
Chemicals. Sesaminol (a mixture of sesaminol and 6-episesaminol)
and sesaminol triglucoside (sesaminol-20-O-β-
D-glucopyranosyl-(1f2)-[β-
D-glucopyranosyl-(1f6)]-β-D-glucopyranoside) were kindly supplied by
Takemoto Oil and Fat Co., Ltd. (Aichi, Japan). The rat S9 mix was
purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Cofactor I
consisted of MgCl2 (8 μmol), KCl (33 μmol),
D-glucose-6-phosphate
(5 μmol), NADPH (4 μmol), NADH (4 μmol), Na2HPO4 (100 μmol),
and NaH2PO4 (100 μmol), all of which were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of
the highest purity commercially available.
*To whom correspondence should be addressed. Telephone: þ81-
52-789-4126. Fax: þ81-52-789-5296. E-mail: osawat@agr.nagoya-u.
ac.jp.
© 2009 American Chemical Society
Published on Web 10/13/2009
pubs.acs.org/JAFC

10430 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Mochizuki et al.
A portion of a liver (0.5 g) was homogenized with ethyl acetate (2 mL)
containing 50 μM butylated hydroxytoluene (BHT) and 50 mM ethylene-
diaminetetraacetic acid (EDTA). After the centrifugation (3500 rpm) for
10 min at room temperature, the supernatants were evaporated to dryness
and reconstituted in 100 μL of methanol. After centrifugation (15000 rpm)
for 5 min at 4 °C, the supernatants were used for the HPLC-
electrochemical detection (ECD) analysis. The rats tissue extracts were
analyzed by HPLC-ECD. The extracts were injected into a HPLC
column (4.6 ꢀ 250 mm inner diameter, Develosil C30-UG-5 column,
Nomura). The mobile phase was composed of water containing 50
mmol/L citric acid and 50 mmol/L lithium acetate (solvent A) and
methanol containing 50 mmol/L citric acid and 50 mmol/L lithium acetate
(solvent B). The gradient program was as follows: initial (60% A), 20 min
(50% A), 40 min (43% A), 55 min (20% A), and 60 min (0% A) at the flow
rate of 1.0 mL/min. The elution was monitored by a coulometric electro-
chemical detector (Coularray system, MC medical, Tokyo, Japan), with
the working potentials set at þ0, 100, 200, 450, and 500 mV.
HPLC-MS Analysis. The HPLC-MS analysis was carried out
using a PLATFORM II (Micromass, Manchester, U.K.). Separation was
performed using a Develosil ODS-HG column (250 ꢀ 4.6 mm inner
diameter, Develosil ODS-HG-5 column). Solvent A was 5% acetic acid in
40% methanol at the flow rate of 1 mL/min. Operation was in MS mode,
and electrospray ionization (ESI) was used. During the analysis, the ESI
parameters were set as follows: the cone voltage was set at -35 V; the
capillary voltage was set at -2.8 kV; and the source temperature was set at
70 °C. The select ion mode (SIM) was used for the detection of sesaminol
(370) and sesaminol metabolites (358 and 372).
1H NMR and 13C NMR Analysis. Analysis of the sesaminol
metabolites isolatedfromthe rat S9 microsomal incubation was performed
in a DMSO-d6 solution using a Bruker APX 400.
The structure of the sesaminol metabolite collected by HPLC was
assigned by NMR spectroscopy, incorporating 1H NMR and 13C NMR,
heteronuclear multiple-bond correlation (HMBC), and heteronuclear
multiple-quantum correlation (HSQC). The NMR experiments were
performed by a Bruker ARX 400. The measurements were taken at 400
MHz (1H) and 100 MHz (13C) frequencies.
Figure 1. Chemical structures of (A) sesaminol triglucoside, (B) sesami-
nol, (C) sesaminol-6-catechol, (D) sesamin, and (E) sesamolin.
Enzymatic Reaction in Vitro. The incubation mixture contained the
pooled rat S9 fraction (approximately 20 mg of protein) and 100 mM
sesaminol [dissolved in dimethyl sulfoxide (DMSO), final concentration of
0.1%] in 9 mL of cofactor I, as desctibed above. After incubation at 37 °C
for 24 h, the reaction was terminated by adding 10 mL of ethyl acetate (2
times). The control was incubated in the absence of sesaminol, analyzed by
high-performance liquid chromatography (HPLC), and then characterized
by liquid chromatography-mass spectrometry (LC-MS) (see below). The
combined organic phase was evaporated to dryness and stored at -80 °C.
Compound A for the HPLC analysis was fractionated from the sesaminol
metabolites by preparative HPLC using a Develosil C30-UG-5 column
(20 ꢀ 250 mm inner diameter, Nomura, Chemical Co., Ltd., Aichi, Japan)
and UV detection at 300 nm. The column was eluted with a linear gradient
from solvent A (water) to solvent B (methanol) at the flow rate of 7.0
mL/min in 45 min. The gradient program was as follows: initial (60% A),
40 min (20% A), and 45 min (0% A) (retention time of 25 min).
Assay for Radical Scavenging Activity. The sample was measured
for antioxidative activity using the DPPH radical scavenging system by
HPLC analysis according to the reported method (13). Sesamolin,
sesamin, sesaminol, sesaminol triglucoside, and the isolated lignans were
dissolved in DMSO and assayed at a final concentration of 150 μM. A 100
μL portion of the sample solution was mixed with 2 mL of 20 mg/mL
DPPH in ethanol and 1 mL of a 100 mM Tris-HCl buffer (pH7.4). The
mixture was vigorously shaken, left to stand for 40 min at room
temperature in the dark, and then subjected to HPLC analysis. The
analyses were performed using a TSKgel Octyl-80Ts column (4.6 ꢀ 150
mm, Tosoh, Ltd., Tokyo) at ambient temperature and a spectrophoto-
metric detector (517 nm) with a mobile phase of methanol/water (70:30,
v/v) at the flow rate of 1 mL/min. The activity was evaluated from the
difference in the decreasing peak area of the DPPH radical detected
between the blank and the sample. The values were reported as the mean of
the three measurements.
Methylation by the Rat Liver S9 Fraction. The methylation of
sesaminol was examined using the rat S9 mix. The incubation mixture
(final volume of 50 mL) consisted of cofactor I, 2000 units of COMT,
1 mM dithiothreitol(DTT), 1 mM MgCl2, 2 mM S-adenosyl-L-methionine
(SAM), approximately 20 mg/mL rat S9 fraction protein, and 100 mM
sesaminol (dissolved in DMSO, final concentration of 0.1%). The
incubation was conducted at 37 °C for 24 h. The reaction was quenched
by extracting 2 times with 100 mL of ethyl acetate. The organic extract was
combined, dried in vacuo, and stored at -80 °C. The sample was
reconstituted with methanol assisted by centrifugation, and the super-
natant was injected into the HPLC detection system. Compounds B1 and
B2 were then fractionated by HPLC using a Develosil C30-UG-5 column
and UV detection at 300 nm with a mobile phase of water/methanol
(40:60, v/v) at the flow rate of 7.0 mL/min (retention times of 22 and 26
min). The analysis was carried out using LC-MS (see below) and nuclear
magnetic resonance (NMR). The 1H and 13C NMR spectra were obtained
using a Bruker ARX 400 instrument (400 MHz for 1H and 100 MHz for
13C) in dimethyl sulfoxide-d6.
RESULTS AND DISCUSSION
Identification of Sesaminol Metabolites Using Rat Liver S9 Mix.
To identify the metabolites of sesaminol in vivo, we examined the
metabolic conversion of sesaminol in vitro using the rat liver S9
mix. The HPLC chromatogram of the sample, obtained from a
3 h incubation of sesaminol with the rat liver S9 mix, revealed the
formation of a major metabolite (peak A) with the retention time
of 18 min (Figure 2). In the control group incubated without the
NADPH generating system, we detected no significant peak,
except for the parent compound (data not shown). The major
metabolite was then isolated and purified using preparative
HPLC. The 1H NMR data for the metabolite is shown in Table 1.
Assignment of the 1H NMR has been achieved on the basis of the
chemical-shift value and the splitting pattern. The data for the
metabolite was quite similar to those of sesaminol, except for the
methylene signal intensity. Theproton signalof the methylendioxy
Animals and Diets. The female Wistar rats, obtained from Chubu
Kagaku Shizai Co., Ltd. (Nagoya, Japan), were 6 weeks old (weighing
120-160 g). The animals were housed in an air-conditioned room under 12
h dark/12 h light cycles. After prefeeding with the control diet (CLEA
Rodent Diet CE-2) for a week, the rats were randomly divided into two
groups and fed the diet of each group [group 1, water; group 2, sesaminol
triglucoside (sesame seed lees-derived) 0.9 mmol/kg with water] for 24 h.
All of the animal protocols were approved by the Animal Experiment
Committee of the Graduate School of Bioagricultural Sciences, Nagoya
University. The samples were stored at -80 °C until used for the
measurements of the sesaminol metabolites.

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10431
Figure 2. HPLC analysis of the product of the 3 h incubation of sesaminol
with rat S9 mix. A chromatogram is shown using a photodiode array (PDA)
detector at 300 nm. For identification of peak A, see Table 1.
Table 1. 1H and 13C NMR Spectroscopic Data of Peak A Formed in the
Incubation Mixture of S9 Mix with Sesaminol and Its Chemical Structure
Figure 3. Mass spectra of peak A at m/z 356 identified in rat S9 mix.
further metabolized into the methylated ones by COMT.
Figure 4A shows the HPLC chromatogram of the incubation of
the rat liver microsome with sesaminol-6-catechol and the cofac-
tor [phosphate buffer (pH 7.4), COMT, DTT, and SAM, as
described in the Materials and Methods]. The HPLC chromato-
gram showed the formation of two major metabolites (peaks B1
and B2) with retention times of 22 and 26 min, respectively. The
LC-MS analysis gave [M - H]- ions at m/z 371 for both peaks,
clearly suggesting that the metabolites are the methylated deri-
vatives of sesaminol-6-catechol. The NMR spectral data of these
metabolites (peaks B1 and B2) are presented in Table 2 and
Figure 5A. Peak B1 of the 1H NMR spectrum (Figure 5A) from
these metabolites indicated that the methoxyl group (MeO) was
assigned to 3.7 ppm (500-MeO), and the other proton signals were
almost identical to those of sesaminol-6-catechol. The signals in
the H and C NMR spectra were assigned from the HMBC
data (Figure 5B). The methoxyl proton signal at δH 3.76(s) was
found to be correlated with C-500 (δC, 147.4), indicating that the
methoxyl group was substituted at C-500. Taken together with the
LC-MS data (Figure 4B), it is concluded that its molecular
formula was determined to be 6-(400-hydroxy-500- methoxy-
phenyl)-2-[200-hydroxy-40,50-(methylenedioxy)phenyl-3,7-dioxabi-
cyclo[3,3,0]-octane (500-methylated sesaminol-6-catechol) . As for
the 1H and 13C NMR analyses of peak B2, the chemical shifts of
the proton and carbon signals were quite similar to those of the
500-methylated metabolite, 500-methylated sesaminol-6-catechol.
Nevertheless, in the HMBC analysis, the correlation of the
methoxyl proton signal in peak B2 was quite different from that
of peak B1. We observed the HMBC correlations among the
methoxyl group (δH, 3.74), 200 proton (dd, 6.72), and 400 carbon
(δC, 147.4), suggesting that the methoxyl group was located at C-
400. The LC-MS analysis further suggested that those two
compounds have similar features in their mass spectra (data not
shown). We finally identified peak B2 as 400-methylated sesami-
nol-6-catechol. Computer modeling showed that the energetically
most favorable binding orientation of a catecholamine in the
moiety in the metabolite was 8.8 ppm with a signal intensity of
two protons, whereas that in sesaminol was 5.5 ppm with a signal
intensity of four protons. As shown in Figure 3, the LC-MS
analysis of the metabolite gave [M - H]- ions at m/z 357,
suggesting that the metabolite has the same molecular weight as
sesaminol-6-catechol, which have been isolated from the fermen-
tation products of the sesaminol glucosides (11). Kang et al.
reported that sesaminol aglycone was detected in abundant
quantities in the serum and liver of rabbits fed the sesaminol
glucosides. This indicates that sesaminol is the principal metabo-
lite of sesaminol glucosides, because the dietary sesaminol gluco-
sides are gradually hydrolyzed by β-glucosidase in the bacterial
flora (14). In this study, a metabolic process that converts gluco-
sides into an aglycone sesaminol may be necessary for further
metabolism by the liver microsome enzymes. In vitro, sesaminol-
6-catechol has been reported to show a strong antioxidative
activity (11). Flavonoids, such as catechin and quercetin, show
general antioxidative effects, because they have a catechol moiety
in their structure (15). Therefore, the newly formed catechol
structure of sesaminol-6-catechol is essential for the high anti-
oxidative activity, such as a radical scavenging activity (11).
Methylation by the Rat Liver S9 Fraction. COMT is well-
known to catalyze the O-methylation of a wide array of
catechol-containing substrates, including exogeous flavonoids,
1
13
using S-adenosyl-L-methionine as the methyl donor. We next
examined the possibility that sesaminol-6-catechol could be

10432 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Mochizuki et al.
Table 2. 1H and 13C NMR Spectroscopic Data of Peak B1 Formed in the
Incubation Mixture of S9 Mix, COMT, and SAM with Sesaminol and Its
Chemical Structure
On the other hand, sesamol, a phenolic compound having the
partial structure of sesaminol, was partially metabolized into the
catechol and 5-methylated catechol, possibly through the CYP-
depended metabolism, while the glucuronide is the predominate
form in the urine (18). Taken together, the ingested sesaminol
might, at least in part, be demethylenated and then converted into
500-methylated sesaminol-6-catechol by COMT and/or to the
further conjugated form by the phase II metabolism in the liver.
Radical Scavenging Activity of the Sesame Lignans. It has been
reported that sesame lignans, such as sesamin, sesamolin, and
sesaminol glucosides, exhibited in vivo antioxidant activities when
orally administered to animals (12, 19-22). On the other hand,
Suja et al. reported that sesamin and sesaminol glucoside showed
no radical scavenging activity in vitro in contrast to that of
sesaminol and sesamolin (23). We next examined the antioxidant
potentials of the sesaminol triglucoside and the three major in vivo
metabolites using the assay for the DPPH radical scavenging
activity (Figure 7). In the assay, sesaminol, sesaminol-6-catechol,
and 500-methylated sesaminol-6-catechol showed significant radi-
cal scavenging activities, whereas the sesaminol triglucoside did
not. The presence of adjacent hydroxyl groups on the flavonoids
has been reported to play an important role in the potent
antioxidative activity, because of their capacity for radical-
scavenging or chelating metal ions (24). Compounds sesaminol,
sesaminol-6-catechol, and 500-methylated sesaminol-6-catechol,
having hydroxyl groups, might be necessary for the structure to
contain more than its group moieties for the scavenging effect
against the hydroxyl radical or free radicals. In particular,
sesaminol-6-catechol showed the most potent activity because
of the unique ortho-dihydroxyl group.
Figure 4. HPLC chromatogram of the incubation of the rat S9 mix, COMT,
and SAM with sesaminol (mixture). (A) UV absorption at 300 nm. (B)
LC-MS analysis of peak B1. The trace is an extracted ion profile [m/z 371 for
methylated sesaminol-6-catechol] of a single linear scan. (C) Expanded
section of the chromatogram showing the production of sesaminol metabolites
from sesaminol (mixture). (D) Negative-ion ESI mass spectra of peak B1.
active site of COMT led to methylation of the hydroxyl group at
the meta position. In the case of quercetin, the 30-hydroxyl group
is situated at the meta position with respect to the main part of the
molecule (16). In this study, we suggested that the methylation of
the 500 position is more favorable than that of the 400 position for
the methylation of sesaminol-6-catechol.
Identification of Sesaminol Metabolites in Rat Liver. To demon-
strate the physiological significance of sesaminol metabolites
identified from the in vitro study, the metabolism of sesaminol
was examined using the liver of rats fed the sesaminol glucosides.
Representative HPLC-ECD chromatograms before and after
the intake of the sesaminol triglucoside are shown in Figure 6.
Besides sesaminol, significant amounts of sesaminol-6-catechol
and 500-methylated sesaminol-6-catechol (the three major rat liver
S9 mix metabolites) were detected on the basis of their retention
time and UV spectra of the standard samples. Kang et al. showed
that dietary sesaminol glucosides could not be detected in the
serum or liver, because sesaminol triglucoside is highly water-
soluble and gradually hydrolyzed by β-glucosidase in the bacter-
ial flora (12). The present study is consistent with the previous
report showing that sesaminol is the principal metabolite of the
sesaminol triglucoside (12). We concluded that the rat liver can
metabolize sesaminol into not only sesaminol-6-catechol but
also 500-methylated sesaminol-6-catechol. However, we detected
500-methylated sesaminol-6-catechol, whereas 400-methylated
sesaminol-6-catechol could not be in Figure 6. Recently, Jan et
al. demonstrated that sesaminol triglucoside could be converted
to enterolactone and enterodiol by rat intestinal microflora (17).
In conclusion, this study identified the three related com-
pounds, sesaminol, sesaminol-6-catechol, and 500-methylated se-
saminol-6-catechol, as the major metabolites in vitro. These meta-
bolites were also identified, for the first time, as in vivo meta-
bolites, which might be generated by the action of hepatic
cytochrome P450 enzymes or COMT. Furthermore, these meta-
bolites have significant antioxidant activities, strongly suggest-
ing their physiological role in the in vivo antioxidant activity of
the orally administered sesaminol glucosides. A recent report
demonstrated that sesaminol triglucoside could be converted to

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10433
Figure 5. (A) 1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz). (B) HMBC spectra of peak B1.
Figure 7. Antioxidantive activity of six sesame lignan compounds. The
values are represented as the means ( standard deviation (SD) (N = 3).
ACKNOWLEDGMENT
We thank Masanori Inayoshi (Takemoto Oil and Fat Co.,
Ltd.) for providing the sesame lignans.
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Received June 13, 2009. Revised manuscript received September 12,
2009. Accepted September 15, 2009.