Tissue distribution and elimination of estrogenic and anti-inflammatory catechol metabolites from sesaminol triglucoside in rats

Article abstract of DOI:10.1021/jf1009632

Sesaminol triglucoside (STG) is the main sesame (Sesamum indicum L.) lignan. Like many other plant lignans, STG can be converted to the mammalian lignans by intestinal microbiota. The objectives of the present study were to investigate the distribution of STG metabolite in rats, and the effects of STG and its metabolite on in vitro inflammation and estrogenic activities. STG was metabolized via intestinal microflora to a biologically active catechol moiety which would then be absorbed into the body in rats. After oral administration of STG to Sprague-Dawley rats, the concentrations of major STG metabolites in rectum, cecum, colon, and small intestines are higher than those in liver, lung, kidney, and heart. Its concentration in brain is low but detectable. The present study demonstrates that STG may be metabolized to form the catechol metabolites first by intestinal microflora and then incorporated via intestine absorption into the cardiovascular system and transported to other tissues. Results showed that the catechol metabolites were found to be able to penetrate the tail end of intestines (large intestine) and go through urinary excretion. STG metabolites significantly reduced the production of IL-6 and TNF-α in RAW264.7 murine macrophages stimulated with lipopolysaccharide. The estrogenic activities of STG metabolites were also established by ligand-dependent transcriptional activation through estrogen receptors. This study clearly shows that STG has anti-inflammatory and estrogenic activities via metabolism of intestinal microflora.

Full text of DOI:10.1021/jf1009632

J. Agric. Food Chem. 2010, 58, 7693–7700 7693
DOI:10.1021/jf1009632
Tissue Distribution and Elimination of Estrogenic and
Anti-Inflammatory Catechol Metabolites from Sesaminol
Triglucoside in Rats
†
K
UO-CHING
J
AN,† KUO-LUNG
WANG,*,§ AND
K
C
U
,‡ YAN-HWA
C
HU
,
,§,
L
UCY UN
S
H
HI-TANG
HO*
†Food Industry Research & Development Institute, Hsinchu, Taiwan, ‡Department of Applied Chemistry,
National Chiayi University, Chiayi, Taiwan, §Graduate Institute of Food Science and Technology,
National Taiwan University, Taipei, Taiwan, and Department of Food Science, Rutgers University,
65 Dudley Road, New Brunswick, New Jersey 08901
Sesaminol triglucoside (STG) is the main sesame (Sesamum indicum L.) lignan. Like many other
plant lignans, STG can be converted to the mammalian lignans by intestinal microbiota. The
objectives of the present study were to investigate the distribution of STG metabolite in rats, and the
effects of STG and its metabolite on in vitro inflammation and estrogenic activities. STG was
metabolized via intestinal microflora to a biologically active catechol moiety which would then be
absorbed into the body in rats. After oral administration of STG to Sprague-Dawley rats, the
concentrations of major STG metabolites in rectum, cecum, colon, and small intestines are higher
than those in liver, lung, kidney, and heart. Its concentration in brain is low but detectable. The
present study demonstrates that STG may be metabolized to form the catechol metabolites first by
intestinal microflora and then incorporated via intestine absorption into the cardiovascular system
and transported to other tissues. Results showed that the catechol metabolites were found to be
able to penetrate the tail end of intestines (large intestine) and go through urinary excretion. STG
metabolites significantly reduced the production of IL-6 and TNF-R in RAW264.7 murine macro-
phages stimulated with lipopolysaccharide. The estrogenic activities of STG metabolites were also
established by ligand-dependent transcriptional activation through estrogen receptors. This study
clearly shows that STG has anti-inflammatory and estrogenic activities via metabolism of intestinal
microflora.
KEYWORDS: Sesaminol triglucoside; catechol metabolites; tissue distribution; inflammation; estro-
genic activity
1. INTRODUCTION
dibenzylbutanediol to dibenzylbutyrolactone, and reductive clea-
vage of furofuran rings. STG has methylenedioxyphenyl moieties
in its structure which may require additional oxidative demethy-
lenation of the methylenedioxyphenyl ring for conversion to
mammalian lignans (ST1, ST2, ST3, enterolactone, and en-
terodiol) (Figure 1). However, STG was metabolized via intestinal
microflora to a biologically active catechol moiety and then
absorbed into the body in rats (11).
There is no comprehensive information on the distribution and
elimination of sesaminol triglucoside catechol metabolites (ST-2
and ST-3) in vivo. In addition, the potential bioactivities of STG
metabolites are poorly understood. This study aimed to investi-
gate the distribution of STG metabolites in vivo, and their in vitro
anti-inflammatory and estrogenic activities.
The metabolism of plant lignans involves gut microbial pro-
cesses (1). Despite the structural diversity of plant lignans from
different sources, most of them may undergo conversion to enter-
odiol (END) and enterolactone (ENL) by gut microbiota (2-5).
The main sources of lignans in Western diets include cereals and
cereal products (6, 7). Plant lignans, such as matairesinol, secoiso-
lariciresinol, pinoresinol, lariciresinol, and sesamin, are converted to
enterolignans END and ENL by intestinal microbiota (3). A diet
rich in sesame lignans has been shown to have many beneficial
physiological effects, which are mostly related to its lignan com-
pounds, such as sesaminol glucosides (STG). STG have less
bioactivity in vitro, but they have been reported to be converted
to bioactive phenolic compounds after oral administration (8-10).
Our previous studies reported that STG converted to mam-
malian lignans by intestinal microbiota involving the hydrolysis
of glucoside, demethylation of a methoxy group, oxidation of
2. MATERIALS AND METHODS
2.1. Chemicals. 2,20-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS), potassium persulfate, acetic acid, fluorescein
sodium salt, and sulfatase (type H-1, from Helix pomatia, containing
sulfatase and β-glucuronidase) were obtained from Sigma-Aldrich (Poole,
*To whom correspondence should be addressed. (C.-T.H.) E-mail:
ho@aesop.rutgers.edu; (L.S.H.) E-mail: lshwang@ntu.edu.tw.
© 2010 American Chemical Society
Published on Web 06/04/2010
pubs.acs.org/JAFC

7694 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Jan et al.
Figure 1. Chemical structures of sesaminol triglucoside and its metabolites (11).
Dorset, UK). The reagents lipopolysaccharide (LPS, Escherichia coli
serotype O55: B5), pyrrolidine dithiocarbamate (PDTC), and concanava-
lin (Con) A (Sigma Chemical Company; St. Louis, MO, USA) were all
analytical grade and dissolved in phosphate buffer saline as a stock. 2,20-
Azobis(2-amidinopropane) dihydrochloride (AAPH) and XAD-2 gel
were purchased from Aldrich (Milwaukee, WI, USA). Tetramethylbenzi-
dine (TMB) was provided by Clinical Science Products (Mansfield, MA,
USA). Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid)
was purchased from Fluka (St. Gallen, Switzerland). General anaerobic
medium (GAM) broth was provided by Nissui (Tokyo, Japan). Antibody
of IL-6 was provided by BD Pharmingen (San Diego, CA, USA). Anti-
body of TNF-R was provided by R&D (Minneapolis, MN, USA). All
other chemicals used were of analytical grade. Liquid chromatographic
grade solvents were obtained from Mallinckrodt Baker (Phillipsburg, NJ,
USA). Triply deionized water (Millipore, Bedford, MA, USA) was used
for all preparations.
2.2. Extraction and Isolation of Sesaminol Triglucoside (STG).
For the isolation of STG, sesame seeds were defatted with n-hexane and
extracted with 80% MeOH. The 80% MeOH extract was charged into an
Amberlite XAD-2 column and eluted with H2O, 20% MeOH, 40%
MeOH, and 60% MeOH. The 60% MeOH fraction was then purified
by preparative HPLC under the following conditions: column Cosmosil
ODS (250 ꢀ 20 mm i.d.), solvent MeOH, flow rate 4 mL/min to obtain
STG with 99% purity (12).
2.3. Preparation of Sesaminol Triglucoside (STG) Metbolites.
STG metabolites were prepared according to the method of Jan et al (11).
The incubationof STG with human intestinal bacterial mixture (300 mL of
GAM broth) and isolation of STG metabolites have been described
previously. Similarly, two major metabolites ST-2: [4-(((3R,4R)-5-(6-
hydroxybenzo[d][1,3]dioxol-5-yl)-4-(hydroxymethyl)tetrahydrofuran-3-yl)-
methyl)benzene-1,2-diol] and ST-3: [4-(((3S,4R,5S)-5-(6-hydroxybenzo[d]-
[1,3]dioxol-5-yl)-4- (hydroxymethyl)tetrahydrofuran-3-yl)methyl)benzene-
1,2-diol] were isolated and identified as described previously (11).
2.4. Animals and Diets. The experimental protocol was approved
by the National Laboratory Animal Center (Taipei, Taiwan) Inbred male
Sprague-Dawley rats [body wt 275 ( 25 g, mean ( SD] were housed in
pairs in cages in a room with controlled temperature (20-22 °C), relative
humidity (50-70%), and a 12-h light: dark cycle (lights on at 0700 h). The
rat diet was AIN 93M diet (Purina Mills, St. Louis, MO, USA). Rats
consumed their food ad libitum and had unlimited access to water; their
weight and food consumption were determined weekly.
2.5. Distribution Experiment. SD rats (n = 6) were administered
via gastric gavage, 500 mg/kg of body weight STG dissolved in normal
saline at three daily doses (1500 mg/kg/day) for four days. After consum-
ing the STG diet for 4 days, rats were anesthetized in the morning on the
fourth day (500 mg/kg), without overnight fasting, using CO2 as a carrier.
The liver, lung, brain, plasma, small intestines, cecum, colon and rectum
were collected after the administration on the fourth day. The intestines of
rats were rinsed free of its contents, which would normally be a standard
procedure for estimating intestine tissue concentrations. Rats were fully
bled via the abdominal aorta. Blood (8-12 mL) was collected in heparin
tubes and plasma was subsequently prepared in centrifuge tubes by
centrifuging for 20 min at 1000g and 4 °C. After blood collection, the
tissues were dissected, weighed, and immediately frozen in liquid nitrogen.
Preparation of Samples. All tissues were lyophilized before further
processing. Rat tissues were pooled per intake group and ground and
homogenized (Polytron). Liver, kidney, lung, brain, small intestines,
cecum, colon and rectum required additional homogenization in the mill.
Samples were stored in airtight containers at -20 °C.
Whole rat tissues were weighed in 50-mL tubes and homogenized
(Polytron) in 10 mL of 0.5 mol/L sodium acetate buffer (NaOAc, pH 5.0,
with 200 mg/mL ascorbic acid)/g tissue with a vortex. The samples were
deproteinized with 100% acetonitrile. The extract was transferred to a 25
mL tube and centrifuged for 10 min at 10000g and 4 °C. The supernatant
was then transferred to a clean tube and the residue was extracted two
more times. The organic solvent from the supernatant was evaporated by
nitrogen at 50 °C. Tubes were weighed before and after evaporation of
extraction solvent. Subsequently, the residues were dissolved with 1 mL 0.1
M NaOAc buffer (pH 5.0, with 200 mg/mL ascorbic acid). Each sample
(100 μL) was either hydrolyzed with 50 μL enzyme (1000 U sulfatase) in 0.1
M NaOAc buffer (pH 5.0, with 200 mg/mL ascorbic acid)/g of tissue for
incubation at 37 °C, or not hydrolyzed but processed immediately with the
addition of the same volume of NaOAc buffer without enzyme
mix (13-15). Subsequently, all samples were deproteinized with 250 μL
100% acetonitrile and centrifuged for 10 min at 10000g and 4 °C. After
centrifugation, 1 mL supernatant was injected into the LC-MS system.
Plasma and tissues samples were analyzed by a similar methodas described
previously (16-18).
2.6. Excretion Experiment. Rats (n = 6) were administered via
gastric gavage, 500 mg/kg body weight STG in normal saline for four days.
Urine and feces samples were collected at the following time periods: 0-24 h
after administration of STG. Urine and feces from rats housed in

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J. Agric. Food Chem., Vol. 58, No. 13, 2010 7695
metabolic cages were collected for the 24 h experiment. Ascorbic
acid (200 mg/mL) was added to urine samples, which were then stored
at -80 °C until analysis.
Preparation of Feces Samples. The feces were lyophilized before further
processing. The feces were pooled per intake group and ground and
homogenized (Polytron and mill). Samples were stored in airtight contain-
ers at -80 °C.
The extractions of feces in the rats were dealt with the procedures
outlined in section 2.5. Urinary STG metabolites analysis was performed
using a previously described method with slight modifications in rat
excreta (19). Briefly, 2 mL of 0.1 mol/L sodium acetate buffer (pH 4.5)
and 50 μL of 1000 U sulfatase activity in 0.1 mol/L sodium acetate
buffer were added to 1 mL urine sample, and the mixture was incubated in
a 37 °C water bath overnight to hydrolyze the conjugates of STG
metabolites.
Subsequently, all samples were deproteinized by centrifuging for 10 min
at 10000 ꢀ g and 4 °C. A 250 μL of 100% acetonitrile was added to each
sample before deproteinization. After centrifugation, 1 mL supernatant
was injected into the LC-MS system.
2.10. Transactivation Assay for Estrogenic Activity. In the
transactivation assay, vectors containing GAL4-hERR (or β) ligand
binding domain (LBD) chimeric receptors and the (UAS) 4-alkaline
phosphatase reporter genes were cotransfected into CHO-K1 cells. Treat-
ment of cotransfected cells with samples containing active compounds that
can bind to ER LBD will then trigger the binding of GAL4 to the UAS
sequence upstream of the reporter gene and activate the transcription of
the reporter gene, that is, the secreted form of the human placental alkaline
phosphatase (secreted alkaline phosphatase; SEAP). By measuring the
alkalinephosphatase activity, the estrogen activity can thenbe determined.
Vectors containing chimeric receptor constructs used were pBK-CMV-
Gal4-hERR-ligand-binding domain (Gal4-hERRLBD) and the pBK-
CMV-Gal4-hERβ- ligand-binding domain (Gal4-hERβLBD), respec-
tively. LBD (833-1785 bp) of hERR was cloned by RT-PCR. hER cDNA
was a kind gift from Dr. J. A. Gustaffson, Department of Medical
Nutrition, Karolinska Institute, Huddinge. The LBD (721-1593 bp)
was cloned by PCR. The LBDs of hERR and hERβ were excised and
ligated to pBKCMV containing GAL4. The correct in-frame fusions were
confirmed by sequencing. The reporter gene, pBK-CMV-(UAS) 4-tk-
alkaline phosphatase (AP), was kindly provided by Dr. J. A. Gustaffson.
The LBDs of hERR and hERβ were excised and ligated to pBKCMV
containing GAL4. The correct in-frame fusions were confirmed by
sequencing. These vectors were transformed into E. coli (XL1-Blue) and
extracted by QIAGEN Plasmid Midi Kits after cultivation. CHO-K1 cells
(American Type Culture Collection, Rockville, MD) were grown at 37 °C
in a 5% CO2 atmosphere in Ham’s F-12 medium supplemented with 10%
fetal bovine serum (Gibco BRL, Rockville, MD. USA). Near confluence,
cells were removed with trypsin-EDTA, seeded at a density of 2.0 ꢀ 104
cells/well into 96-well plates in the same medium supplemented with 10%
of charcoal-treated FBS, and incubated for 24 h at 37 °C, 5% CO2. Using
Fugene 6 (Roche) or Lipofectamine (GIBCO) as the transfection reagent
according to the manufacturer’s instruction with some modification, cells
were cotransfected with appropriate amounts of pBK-CMV-Gal4-hERR
(or β) and pBK-CMV-(UAS) 4-tk-alkaline phosphatase (AP) in 100 μL of
serum-free Ham’s F-12 medium containing the appropriate amount of the
transfection reagent. After 5 h, cells were treated with Ham’s F-12 medium
containing 10% serum replacement (TCM) (Celox, St. Paul, MN, USA)
and vehicle (DMSO), 17β-estradiol (E2), or test samples (END, ENL,
STG, ST1, and ST2) in appropriate concentrations. Test samples dissolved
in DMSO (100 μM/100 μL) were diluted to appropriate concentrations
with Ham’s F-12 medium containing 10% serum replacement immediately
before use. After 48 h, 50 μL of culture medium was transferred to a new
96-well plate for the reporter gene (activity of alkaline phosphatase, AP)
assay. As previously described, an equal volume of SEAP assay solution
containing 20 mM p-nitrophenyl phosphate (pNPP), 1 mM MgCl2, 10
2.7. Determination of Sesaminol Triglucoside (STG) Metabo-
lites in Tissues and Excreta. Determination of STG metabolites were
carried out by LC-MS/MS analysis. These analyses were performed on a
Thermo HPLC system equipped with electrospray-ionization ion trap
mass spectrometer (ThermoFinnigan LXQ Advantage, San Jose, CA,
USA). The separation was achieved using YMC Hydrosphere C18 column
(2.0 ꢀ 150 mm I.D.; 5 μm, YMC, Tokyo, Japan). For the operation in MS/
MS mode, a mass spectrometer with electrospray ionization (ESI) was
used. During the analyses, the ESI parameters were set as follows: capillary
voltage, 49 V for negative mode; source voltage, 4.5 kV; source current,
100 μA; sheath gas flow rate, 35 au; capillary temp, 350 °C; tube lens
voltage, -110 V. The collision energy of m/z 359 [M - H]- was adjusted to
maximize the intensity of the deprotonated molecular ion (precursor) as
30% and the collision energy was also adjusted to optimize the product ion
signals as 30% for sesaminol triglucoside metabolites analysis. The
selected reaction monitoring (SRM) was used to monitor the transition
of the molecule to the product ion for sesaminol triglucoside metabolites
analysis. All LC-MS/MS data were processed by the Xcalibur version 2.0
data acquisition software.
For the quantitation of ST2 and ST3 in the tissues and excreta, the
standard curves for these two metabolites were established. The linear
regression analysis showed that the correlation coefficients of all standard
curves were better than 0.995 over the range of 1-100 nmol. Limits of
quantification(LOQ) werecalculatedfrom the limits of detections with the
LOQ being determined as five times the limit of detection (LOD). The
quantification limits ranged from 1.2 nmol for ST3 to 2.1 nmol for ST2.
After STG administration, the STG metabolites could be detected in the
tissues and excreta.
2.8. Oxygen Radical Absorbance Capacity (ORAC). The auto-
mated ORAC assay was carried out on a Multiple-Detection Microplate
Reader (Atlanta, GA, USA) with fluorescent filters (excitation wave-
length, 485 nm; emission wavelength, 528 nm). In the final assay mixture
(0.25 mL total volume), fluorescein (38.4 nm/L) was used as a target of free
radical attack, with AAPH (3.2 mmol/L) as the ROO3 generator and
sample (50 μL). Trolox (6.25-50 μmol) was used as the control standard.
The analyzer was programmed to record the fluorescence of fluorescein
every 5 min after addition of AAPH. Final results were calculated on the
basis of the difference in the area under the fluorescein decay curve
between the blank and each sample. The antioxidative activity of a sample
was determined from its ability to protect the fluorescence of the indicator
in the presence of peroxyl radicals. Calculations of the final ORAC values
were followed by Prior et al. (20).
2.9. Trolox Equivalent Antioxidant Activity (TEAC). ABTS
was dissolved in water to a 7 mM concentration. ABTS radical cation
(ABTS•þ) was produced by reacting ABTS stock solution with 2.45 mM
potassium persulfate (final concentration) and allowing the mixture to
stand in the dark at room temperature for 12-16 h before use. For the
study of STG metabolites, the ABTS•þ solution was diluted with PBS, pH
7.4, to an absorbance of 0.70 ((0.02) at 734 nm and equilibrated at 30 °C.
The percentage inhibition of absorbance at 734 nm was calculated and
plotted as a function of concentration of antioxidants and of trolox for the
standard reference data (21).
mM L-homoarginine, and 1 M diethanolamine, pH 9.8, was added and
mixed (22, 23). Absorbance was read at 405 nm at 15 min. Fold of
activation was calculated by taking the AP activity of vehicle-treated cells
as 1. Cell viability was examined using the MTS assay. The MTS assay was
performed using the CellTiter 96 AQueous One Solution Cell Proliferation
Assay kit (Promega Inc., Madison, WI). In brief, cells growing in the log
phase were plated on 96-well plates the day before sample treatment and
end point determination was performed following the manufacturer’s
instruction. Absorbance of the color development in sample treated and
untreated cells were measured in a Bio-Rad Microplate reader. Data of
transactivation obtained were considered valid only in experiments in
which sample treatment did not alter cell growth and viability.
2.11. Macrophage Cell Culture Experiment. The macrophage
RAW264.7 cells (Bioresource Collection and Research Center, Hsinchu,
Taiwan) were cultured in DMEM containing 10% fetal bovine serum
(FBS), 2 mM glutamine, 1% nonessential amino acid, and 1 mM sodium
pyruvate, and maintained in a humidified incubator at 37 °C in 5% CO2.
In this experiment, the RAW264.7 cells were seeded on 96-well plates at
cell density of 2 ꢀ 104 cells/200 μL/well. The cells were then pretreated with
various concentrations of STG metabolites for 1 h before adding 100 ng/
mL LPS for stimulated incubation. STG metabolites were dissolved in
DMSO for cell treatment (final DMSO concentration e0.1% in medium)
and had no direct toxic effect on RAW264.7 cells.
2.12. Assay of Cytokine Production in RAW264.7 Cells. The
production of cytokines tumor necrosis factor (TNF)-R (R&D Systems,
Inc., Minneapolis, MN, USA) and interleukin (IL)-6 (PharMingen, San

7696 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Jan et al.
Figure 2. Massspectraof STG metabolites inthe plasma. (a) Mass fragmentation spectrum of MS/MS for ST-2(m/z 359 [M - H]-). (b) Massfragmentation
spectrum of MS/MS for ST-3 (m/z 359 [M - H]-).
Diego, CA, USA) in the cell supernatants was assayed by a commercial
ELISA kit. Briefly, anti-IL-6 and TNF-R antibodies were coated to 96-well
plates. After incubation, the wells in the plate were washed and blocked
with blocking solution (PBS buffer containing 1% bovine serum albumin;
Sigma), and then washed and properly diluted supernatant or serum was
added. After incubation, the wells were washed and biotin-conjugated
anti-IL-6 and TNF-R antibodies were added for 2 h. The wells were then
washed and horseradish peroxidase-conjugated streptavidin (Pierce Che-
mical Co.) was added for 30 min, washed, and incubated with ABTS or
TMB (24). Absorbance at 405 or 620 nm was measured using ELISA
reader (EL311, Bio-TEK Inc.). The data were calculated according to the
cytokine standard curves.
2.13. Statistical Analysis. All samples were extracted in triplicate.
Sesaminol triglucoside metabolite concentrations were expressed in
μmol/g of tissue or μmol/mL plasma or urine. Tissues of six rats were
pooled before analysis. Data were analyzed by t-test and ANOVA analysis
of variance, and differences were considered statistically significant at
p < 0.05 and p < 0.01.
3. RESULTS
3.1. Distribution of STG Metabolites in Rats. A full scan in
negative ion mode (scan range from m/z 50 to 500) was used to
identify the metabolites of STG. A tube lens offset voltage of
-110 eV was applied for the determination of ST-2 and ST-3,
(precursor ion is 359 [M - H]-). The collision energy for collision-
Figure 3. Mass spectrum and HPLC chromatograms of STG metabolites
in the urine of rats. (a) A280 and (b) SRM for STG metabolites (m/z 359,
ST-2 and ST-3).
induced dissociation (CID) was adjusted to 30% to produce the
main product ion at m/z 221, as shown in Figure 2. Panels a and b
of Figure 2 show the MS/MS spectra of collected rat plasma after
sesaminol triglucoside administration (500 mg/kg, p.o.), with
mass transitions of m/z 359 [M - H]- f 221, 203, and 191 for
ST-2 and ST-3 with sensitive selective reaction monitoring (SRM)
mass spectrometry.
ST-3 may be, at first, metabolized and absorbed from intestines
and then transported to the other tissues (liver, lung, kidney, and
brain) (Table 1).
It is interesting to note that, although ST-3 is the stereoisomer
of ST-2, the concentrations of ST-2 were significantly higher than
ST-3 in rat tissues. In the front of large intestine (cecum), the
concentration ST-2 was already significantly higher than ST-3.
The highest ST-2 concentrations were found in colon and cecum
(Table 2). It is also noted that after 24 h the concentrations of
ST-2 and ST-3 in the intestines (cecum, colon, and rectum) were
higher than intissues inrat (Tables 1 and 2). The exclusion of ST-2
and ST-3 were determined in urine and feces by LC-MS/MS
within 24 h after administration of STG to rats. In urinary
excretion (Table 2), the concentrations of ST-2 were significantly
higher than ST-3. Higher concentration of ST-2 was found in the
feces after administration.
To investigate the distribution of enterolignans in rats, an
analytical method was developed. The LC-MS/MS profile of ST-
2 and ST-3 in rat urine after sesaminol triglucoside administration
showed many peaks (Figure 3a); therefore, it is necessary to use
more sensitive SRM mass spectrometry to analyze the entero-
lignans. Figure 3b shows the SRM MS/MS spectrum of the same
rat urine sample as used for Figure 3a with mass transitions of
m/z 359 [M - H]- f 221 for ST-2 and ST-3. Two clearly sepa-
rated peaks corresponding to ST-2 and ST-3 were obtained.
Tissue concentrations of ST-2 and ST-3 were analyzed by LC-
MS/MS. They were widely distributed in rat tissues, with the
highest concentration in plasma and the lowest in brain. ST-2 and

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J. Agric. Food Chem., Vol. 58, No. 13, 2010 7697
Table 1. Tissue Concentration of ST-2 and ST-3 in Rata
concentration
(μmol/mL or μmol/g)
liver
lung
kidney
heart
plasma
brain
ST-2
ST-3
0.30 ( 0.01c
0.13 ( 0.01bc
0.47 ( 0.02b
0.14 ( 0.01bc
0.29 ( 0.01c
0.14 ( 0.01bc
0.42 ( 0.01b
0.15 ( 0.01bc
36.23 ( 1.37a
4.61 ( 0.39a
0.14 ( 0.01d
0.12 ( 0.01bcd
a Data are expressed as mean ( S.D. Data in the same row with different letters are significantly different at p < 0.05.
Table 2. Intestine Concentration of ST-2 and ST-3 in Rata
concentration
(μmol/mL or μmol/g)
rectum
cecum
colon
small intestines
feces
urine
ST-2
ST-3
11.90 ( 0.04c
3.28 ( 0.08b
20.77 ( 0.60a
2.62 ( 0.06bc
19.87 ( 0.16a
7.45 ( 0.87a
11.90 ( 0.04c
0.189 ( 0.01e
16.22 ( 0.09b
1.62 ( 0.15c
6.44 ( 0.08d
0.58 ( 0.01d
a Data are expressed as mean ( S.D. Data in the same row with different letters are significantly different at p < 0.05.
Table 3. Antioxidant Activities of Sesaminol Triglucoside and Its Metabolitesa
ABTS•þ scavenging effect
ORAC assay
sample
TEAC (μM)b
Trolox equivalent (μM)c
sesaminol triglucosdie
ST1 (sesaminol)
ST-2
0.26 ( 0.06c
2.32 ( 0.02b
6.13 ( 0.08a
0.77 ( 0.03c
7.51 ( 0.30b
10.69 ( 1.30a
a Each value represents mean ( SD from three different experiments (each
experiment was conducted in triplicate). Data in the same column with different
letters are significantly different at p < 0.05. b TEAC is the mM of a Trolox solution
having the antioxidant capacity equivalent to a 3 μmol solution of the sample under
investigation. c The concentration of all samples was 2 μmol.
3.2. Radical Scavenging Activity of STG and Its Metabolites in
Vitro. The methylenedioxyphenyl moiety of sesaminol was chan-
ged to the 3,4-dihydroxyphenyl (catechol) moiety and aliphatic
alcohol substituted tetrahydrofuran moiety in ST-2 both in vitro
and in vivo (11). The major metabolite ST-2 was found in tissues
of rats. We examined whether the metabolites had antioxidative
activities in vitro in comparison with trolox. As shown in Table 3,
the free radical scavenging activities (ABTS•þ) of STG (sesaminol
triglucoside), ST-1 (sesaminol), and ST-2 were 0.26( 0.06, 2.32(
0.02, and 6.13 ( 0.08%, respectively. STG showed weak free
radical scavenging activity, but ST-1 and ST-2 showed strong
activity at 3 μmol. In the ORAC assay, STG did not show
antioxidative activity, but ST-1 and ST-2 showed strong activity
at 2 μmol.
Figure 4. Transactivation of hERR and hERβ in CHO K1 cell stimulated
by 0.01 nmol of E2, sesaminol triglucoside and its metabolites. E2 (17β-
estradiol, 0.01 nmol) was the positive control. TAM (tamoxifen, 20 nmol)
and RAL (raloxifene, 2 nmol) were the negative controls. At a similar
concentration of 10 μmol, END (enterodiol), ENL (enterolactone), STG
(sesaminol trigluocisde), S (sesaminol), and ST2 (the major metabolite of
sesaminol triglucoside) were treated in the transactivation assay. Signifi-
cant difference from the E2 group was analyzed. Values not sharing the
same letter are significantly different from one another by one-way ANOVA
(*p < 0.05 and **p < 0.01).
3.3. Estrogenic Activity of Sesaminol Triglucoside and Its Me-
tabolites. Endogenous estrogen, 17β-estradiol (E2), and STG
metabolites all dose-dependently increased alkaline phosphatase
(AP) activity in the transactivation assay (data not shown). This
reporter was transfected into CHO-K1 cells, which were cultured
in the absence or presence of estradiol. The AP activity was
increased in the presence of estrogen. The presence and absence of
estradiol in cells treated with lignans at its concentration was
confirmed estrogenic activity from lignans. The potential estro-
genic activity of each lignan was explored by investigating the
ability of the lignans to compete with estradiol for binding to the
estrogen receptor of CHO-K1 cells. If the lignan was acting as a
strong estrogen, the absence of estradiol could be expected to
increase its transactivation of hERR and hERβ. Additionally, the
treatment of enterolignan increased the E2 cotreated activity,
indicating that the sesaminol metabolites-induced transcription
was ER-mediated. There were significant differences in the
expression indices of hERR and hERβ treated with STG and its
metabolites. The expression of hERR and hERβ were lower in
cells cultured without E2 than treated with E2. The expression of
hERβ was higher than hERR in the E2-maintained and -deprived
cell cultures. STG showed less expression of hERR and hERβ.
Accordingly, in cell cultures, the treatment with ST2 or its further
metabolite ENL increased the hERβ expression in the presence or
absence of E2. No significantly changes in hERR and hERβ
expression were measured by the antiestrogens, tamoxifen
(TAM) and raloxifene (RAL). However, the presence or absence
of ST1 significantly affected hERβ expression in cell cultures.
STG metabolites showed significant ER activity in the transacti-
vation assay (Figures 4 and 5). At a similar concentration of
10 μmol, STG metabolites (ENL and ST2) showed significant
activation of hERβ that was more than 1.5 fold that of 0.01 nmol
of E2 (Figure 4) in the E2-maintained cell cultures. The metabo-
lites (ENL, ST2) of STG activated hERβ, but the STG had no ER
activity. The modest results observed with STG metabolites in the
E2-maintained and -deprived cell cultures may be partially
explained by the fact that STG metabolites are a partial ER
agonist.
3.4. Inhibitory Activity of STG and Its Metabolites on Lipo-
polysaccharide-Induced Inflammation. The cells were treated with
LPS alone or with various concentrations of STG and its
metabolites. At concentrations used in this study, none of the
LPS or STG and its metabolites treatments caused toxicity to cells

7698 J. Agric. Food Chem., Vol. 58, No. 13, 2010
Jan et al.
Figure 5. Transactivation of hERR and hERβ in CHO K1 cell stimulated
by sesaminol triglucoside and its metabolites. E2 (17β-estradiol, 0.01
nmol) was the positive control. TAM (tamoxifen, 20 nmol) and RAL
(raloxifene, 2 nmol) were the negative control. At a similar concentration
of 10 μmol, END (enterodiol), ENL (enterolactone), STG (sesaminol
trigluocisde), S (sesaminol), and ST2 (the major metabolite of sesaminol
triglucoside) were treated in the transactivation assay. Significant differ-
ence from the No E2 group was analyzed. Values not sharing the same
letter are significantly different from one another by one-way ANOVA (*p <
0.05 and **p < 0.01).
as judged by the MTS assay. The pretreatment of RAW264.7 cells
with32, 63, and 125 μg/mLSTG and its metabolites, ST1 and ST2
for 1 h had no significant effect on cell viability (data not shown).
As shown in Figure 6, STG did not suppress TNF-R secretion at a
concentration of 125 μmol. On the other hand, ST1 and ST2 dose-
dependently decreased LPS-induced IL-6 and TNF-R secretion
from RAW 264.7 cells. ST2 (125 μmol) significantly inhibited
TNF-R more than IL-6 secretion in LPS-stimulated RAW264.7
cells (0.876 and 0.004 ng/mL, respectively).
Figure 6. Effects of sesaminol triglucoside and its metabolite on (a) TNF-
R, (b) IL-6 secretion from LPS-stimulated RAW264.7 cells. Values are
expressed as means ( SD of three independent experiments with
triplicates each and statistically analyzed by using Student’s t-test. A
significant difference from STG is indicated *p < 0.05 and **p < 0.01.
4. DISCUSSION
In a previous study, we found that STG was metabolized to
enterolignans via intestinal microflora, absorbed through tail end
of intestinal walls (large intestine), and then passed into the portal
vein or lymphatic absorption into the cardiovascular system
before it was transported to other tissues (25). Transit time of
material through the large intestine is an important factor
affecting the availability of dietary components to the host,
primarily because colonic bacterial fermentation can influence
circulating concentrations of compounds produced by colonic
bacteria (3, 26). Our previous study showed that a portion of
dietary STG was absorbed and metabolized, and major portion
of STG was removed in the feces after consumption (25). We
observed only limited conversion of STG to END and ENL by in
vitro fermentation with human and rat fecal microbiota; we also
detected ST-2 and ST-3 in the tissues of rats after administration
of STG. Therefore, it is assumed that STG is partially metabo-
lized in the intestines to ST-2 and ST-3; they will then be further
metabolized and absorbed by the intestines to hydroxylated
metabolites, which are then excreted in urine. The concentrations
of ST-2 in intestines were remarkably higher than those of ST-3.
The concentrations of ST-2 in tissues were in the order of plasma
. intestines . lung ≈ heart ≈ liver ≈ kidney ≈ brain. The
concentrations of ST2 in both plasma and intestines were
remarkably higher than END determined in a previous study .
STG was metabolized to stereoisomers (ST-2 and ST-3) by
intestinal microbiota. The concentration of ST-3 was extremely
low compared to ST-2 in rats. ST-2 (major metabolite) had higher
bioavailability compared to STG. The exact role of ST2 found in
this study has to be further examined in vivo.
It was previously reported that STG has weak bioactivity in
vivo, but the mechanism of this effect was unclear. Furthermore,
flavonoids such as catechin, epicatechin, quercetin, kaemferol,
and cyanidin generally show antioxidative effects, because they
have a catechol moiety in their structures (27, 28). In this study,
we found that two of the metabolites of STG, ST-1, and ST-2
having a catechol moiety were novel antioxidants with much
higher radical scavenging activities than STG. Previous studies
noted that lignans (sesamin and sesamolin) of sesame oil regu-
lated LPS-induced nitric oxide production in the murine micro-
glia cell lines BV-2. Lignans significantly inhibited LPS-
stimulated IL-6 mRNA and protein, and to a lesser degree
TNF-R (29). STG exhibited a range of pharmacological activities
on LPS-induced inflammatory reaction and its underlying me-
chanism in cultured astrocytes. STG inhibited LPS-induced
generation of nitric oxide (NO) and reactive oxygen species
(ROS), as well as inhibited LPS-induced cytosolic phospholipase
A2 (cPLA2), cyclooxygenase 2 (COX-2), and inducible nitric
oxide synthase (iNOS) expression (30). However, following STG
metabolism, we observed that ST2 had significantly higher
activity than STG in inhibiting LPS stimulated TNF-R and IL-6
production in RAW 264.7 cells. The previous study demonstrated

Article
J. Agric. Food Chem., Vol. 58, No. 13, 2010 7699
that dietary STG inhibited the development of colonic precancer-
ous lesions in vivo. The beneficial effect of STG might be
attributed to the antioxidative property (31). Our previous studies
reported that sesaminol triglucoside (STG) converted to ST-2 by
intestinal microbiota. The concentrations of ST-2 in the intestines
(cecum, colon, and plasma) were higher than other tissues in rat.
Furthermore, our results of the measurement of tissues distribu-
tion, anti-inflammatory and antioxidative activities clearly de-
monstrated that ST-2 played a significant role.
Phytoestrogens are plant-derived compounds that can interact
with estrogen receptors (ER) and exhibit estrogenic/antiestro-
genic activities. Phytoestrogens can bind to ER and have a higher
affinity for ERβ than for ERR (32). Penttinen-Damdimopou-
lou (33) reported that diet could modulate E2-induced ER-
mediated responses in vivo. The dietary sources of lignans and
isoflavones could modulate estrogen signaling in vivo. The
expression of ERβ was significantly increased in prostate and
uterus with a diet rich in sesame pericarp (30%) (34). According
to the present results, we detected that the metabolite of STG
(ST2) was endowed with estrogenic activity, which was likely to
be exerted through the contribution of ER-dependent pathways.
In comparison with ENL, ST2 had similar estrogenicity. This
study supports the notion that dietary supplementation with STG
be converted to ST2 which exhibits higher estrogenic activity than
STG.
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