Isolation and identification of urinary metabolites of tectoridin in rats

Article abstract of DOI:10.1080/10286020.2011.578069

Tectoridin is an active isoflavone from the rhizome of Belamcanda chinensis and flower of Pueraria thomsonii Benth, possessing an anti-inflammation action and the protective effect against ethanol-induced intoxication and hepatic injury. The metabolism of tectoridin was investigated in rats. Nine metabolites were isolated by using repeated chromatographic methods and identified by spectroscopic methods including UV, IR, mass spectrometry, and NMR experiments. A new compound was determined as tectorigenin-7-O-β-D-glucuronide (M-1), together with eight known compounds identified as irisolidone-7-O-β-D- glucuronide (M-2), tectorigenin-7-O-sulfate (M-3), tectorigenin-4′-O- sulfate (M-4), tectorigenin (M-5), irisolidone (M-6), isotectorigenin (M-7), genistein (M-8), and daidzein (M-9), respectively. The metabolic pathway of tectoridin was proposed, which is important to understand its metabolic fate and disposition in humans.

Full text of DOI:10.1080/10286020.2011.578069

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Isolation and identification of urinary
metabolites of tectoridin in rats
Xue Bai a , Jia-Lin Qu a , Jia Liu a , Jia-Hong Sun b & Dan Yuan a
a Department of Traditional Chinese Medicine , Shenyang
Pharmaceutical University , Shenyang, 110016, China
b Department of Pharmacy , Shenyang Pharmaceutical University ,
Shenyang, 110016, China
Published online: 22 Jun 2011.
To cite this article: Xue Bai , Jia-Lin Qu , Jia Liu , Jia-Hong Sun & Dan Yuan (2011) Isolation
and identification of urinary metabolites of tectoridin in rats, Journal of Asian Natural Products
Research, 13:7, 604-610, DOI: 10.1080/10286020.2011.578069
To link to this article: http://dx.doi.org/10.1080/10286020.2011.578069
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Journal of Asian Natural Products Research
Vol. 13, No. 7, July 2011, 604–610
Isolation and identification of urinary metabolites of tectoridin in rats
Xue Baia, Jia-Lin Qua, Jia Liua, Jia-Hong Sunb and Dan Yuana*
aDepartment of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang
110016, China; bDepartment of Pharmacy, Shenyang Pharmaceutical University, Shenyang
110016, China
(Received 7 December 2010; final version received 1 April 2011)
Tectoridin is an active isoflavone from the rhizome of Belamcanda chinensis and
flower of Pueraria thomsonii Benth, possessing an anti-inflammation action and the
protective effect against ethanol-induced intoxication and hepatic injury. The
metabolism of tectoridin was investigated in rats. Nine metabolites were isolated by
using repeated chromatographic methods and identified by spectroscopic methods
including UV, IR, mass spectrometry, and NMR experiments. A new compound was
determined as tectorigenin-7-O-b-D-glucuronide (M-1), together with eight known
compounds identified as irisolidone-7-O-b-D-glucuronide (M-2), tectorigenin-7-O-
sulfate (M-3), tectorigenin-40-O-sulfate (M-4), tectorigenin (M-5), irisolidone (M-6),
isotectorigenin (M-7), genistein (M-8), and daidzein (M-9), respectively. The
metabolic pathway of tectoridin was proposed, which is important to understand its
metabolic fate and disposition in humans.
Keywords: tectoridin; metabolism; glucuronidation; rat; urine
1. Introduction
tectoridin to rats by liquid chromatog-
raphy-tandem mass spectrometry, which
included tectorigenin, hydrogenated tec-
torigenin, mono-hydroxylated tectori-
genin, di-hydroxylated tectorigenin,
glucuronide-conjugated tectorigenin, and
sulfate-conjugated tectorigenin in urine
and tectorigenin, di-hydroxylated tector-
igenin, and sulfate-conjugated tectorigenin
in feces. Among these metabolites, how-
ever, the conjugated tectorigenin could not
be structurally confirmed due to the failure
in determination of the conjugated position
by LC-MSn technology. It has been
reported that glucuronidation of the 30- or
40-hydroxyl group in the B-ring of
flavonoids decreased the antioxidant
activity, whereas glucuronidation in the
A-ring affected antioxidant activity to a
lesser extent [14]. The above results
Tectoridin is the main active isoflavone of
Belamcanda chinensis, a Chinese tra-
ditional medicine for the treatment of
asthma and tonsillitis. The flower of
Pueraria thomsonii Benth, which has
been used to relieve some symptoms such
as drunkenness, headache, and red, also
contains high content (1–3%) of tectoridin
[1]. Pharmacologically, tectoridin shows
a wide spectrum of bioactivities such as
anti-inflammatory, antioxidative, hepato-
protective and cytotoxic activities against
tumor cells, etc [2–10].
Tectoridin was reported to be trans-
formed into tectorigenin by human intes-
tinal bacteria [11,12]. Chen et al. [13]
reported the identification of urinary and
fecal metabolites of tectoridin in vivo and
in vitro after oral administration of
*Corresponding author. Email: yuandan_kampo@163.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.578069
http://www.informaworld.com

Journal of Asian Natural Products Research
indicated that identification of the struc- 2. Results
605
tures of metabolites is important for
further pharmacological research. Thus,
the isolation of metabolites and their
structural confirmation on the basis of
UV, IR, NMR, and MS data are valuable
as well.
Representative HPLC profiles showing
the rat urinary metabolites are shown in
Figure 1. Tectoridin and its metabolites
were selectively detected at 265 nm due to
their characteristic benzoyl group. Six
major metabolites M-1–M-5 and M-7
were clearly observed in the urine of rats.
By means of repeated chromatographic
methods on the columns of silica gel,
Sephadex LH-20, or reverse phase ODS,
nine metabolites were isolated from the rat
urinary sample including one new com-
pound, a glucuronide conjugate M-1
In this study, we conducted the
systematic isolation of urinary metabolites
in rat given tectoridin orally, and deter-
mined their structures by means of
chemical and spectroscopic experiments.
On the basis of the metabolite profile, the
possible metabolic pathway of tectoridin
was proposed.
(A) 1.00
0.80
0.60
0.40
0.20
0
M5
(B) 1.00
0.80
×10
0.60
M7
M2
0.40
M1
0.20
0
M3
M4
(C) 1.00
0.80
0.60
0.40
0.20
0
M3
M4
M8
M2
M5
M1
M6
M7
M9
0
10
20
30
40
50
60
70
80
Figure 1. (A) HPLC-UV chromatograms of blank urine, (B) a rat urine sample during 0 to 24 h
after oral administration of tectoridin at a dose of 100 mg/kg, and (C) metabolite standards.

606
X. Bai et al.
(7.5 mg), together with eight known iso-
flavones, M-2 (1.9 mg), M-3 (6.5 mg), M-4
(2.8 mg), M-5 (13 mg), M-6 (7.4 mg), M-7
(1.1 mg), M-8 (1.1 mg), and M-9 (1.1 mg).
The maximal absorption at 263–
265 nm in UV spectrum and the absorption
bands at 1648–1659 cm21 due to con-
jugated carbonyl and at 1456–1622 cm21
due to aromatic functions in IR spectrum
indicated that M-1–M-9 had an isoflavone
skeleton. The structures of M-1–M-9, as
shown in Figure 2, were elucidated using
UV, IR, 1H and 13C NMR, and MS
techniques.
NMR spectrum, the characteristic isofla-
vone signal for H-2 was observed at d 8.43
(1H, s). A singlet at d 6.89 due to an
aromatic proton suggested that three
substituents existed in A-ring. The protons
due to an AA0BB0 aromatic system
appeared at d 7.39 (d, J ¼ 8.4 Hz, H-20,
60) and 6.81 (d, J ¼ 8.4 Hz, H-30, 50),
indicating that B-ring was hydroxylated at
C-40. Also a singlet at d 3.76 due to a
methoxyl group was observed. The reson-
ances for carbons and protons of the
aglycone moiety had a close resemblance
to those of the known tectorigenin, and
they are assigned according to the
Metabolite M-1 (tectoridin-7-O-b-D-
glucuronide) was isolated as a yellowish
amorphous powder. The molecular for-
mula was determined to be C22H19O12
from the quasi-molecular ion peak at m/z
475.0877 [M 2 H]2 in the ESI-TOF-MS.
The [M 2 H]2 ion at m/z 475 and an
important fragment ion at m/z 299
originating from the eliminating 176
mass units (glucuronic acid) from
[M 2 H]2 ion indicated that M-1 should
be a glucuronide conjugate. M-1 was
hydrolyzed with b-glucuronidase to give
the aglycone tectorigenin, the identity of
which was confirmed through co-chroma-
tography with tectorigenin standard by
HPLC coupled with ultraviolet photodiode
array detection based on the same reten-
tion time and UV spectral values. In the 1H
1
literature values of the H and 13C NMR
spectra for tectorigenin [1,15]. The signals
of an anomeric proton (d 5.07, 1H, d,
J ¼ 7.8 Hz) and a carboxylic group at C-600
at dc 171.2 indicated the presence of a b-D-
glucuronic acid moiety [16]. The b-D-
glucuronic acid moiety could be attached
to C-7 position according to the upfield
shift of C-7 (21.0 ppm) and the downfield
shifts of C-6 (þ1.0 ppm), C-8 (þ0.2 ppm),
and C-10 (þ1.6 ppm) relative to the
corresponding signals of tectorigenin
[1,16,17]. The full assignments of carbon
and proton signals are summarized in
Table 1. Thus, M-1 was determined to be
tectorigenin-7-O-b-D-glucuronide.
Other metabolites were identified as
irisolidone-7-O-b-D-glucuronide (M-2),
OCH
3
COOH
O
O
HO
O
O
O
O
HO
O
OH
OH
Glucuronidation
OH
H CO
H CO
3
3
OH
O
OH
OH
OH
OCH
OCH
4
3
M-7
M-6
M-2
Methylation
O
Methylation
O
Rearrangement
CH OH
COOH
2
1
O
8
O O
O
O
HO
9
7
OH
OH
2
3
OH
OH
Glucuronidation
Hydrolysis
2'
5'
6
OH
H CO
1'
OH
3'
4'
10
H CO
3
4
5
H CO
3
3
6'
O
OH
O
OH
O
OH
OH
OH
OH
Tectoridin
M-5
M-1
Sulfation
Demethoxylation
HO
HO
HO SO
3
HO
O
O
O
O
O
O
Dehydroxylation
H CO
3
H CO
3
OH
O
OH
OH
O
OH
OH
OSO H
3
OH
M-3
M-4
M-8
M-9
Figure 2. Proposed metabolic pathways of tectoridin.

Journal of Asian Natural Products Research
Table 1. 1H and 13C NMR of tectoridin metabolite M-1.
607
a
a
a
a
dH (J in Hz)
Position
dC
dH
Position
dC
2
3
4
5
6
7
8
9
154.8
122.1
180.9
152.9
132.6
156.8
94.2
152.2
106.5
121.1
8.43, s
20,60
300,50
400
130.2
115.2
157.5
100.1
73.1
76.8
71.9
73.9
171.2
60.3
7.39, d (8.4)
6.81, d (8.4)
100
5.07, d (7.8)
200
3
6.89, s
400
500
10
10
600
6-OMe
3.76, s
a
Notes: All spectra were recorded on a Bruker ARX-600 spectrometer, in DMSO-d6. 100 –600 refer to the
numbering of glucuronic acid moiety.
tectorigenin-7-O-sulfate (M-3), tectori-
genin-40-O-sulfate (M-4), tectorigenin (M-
5), irisolidone (M-6), isotectorigenin (M-7),
genistein (M-8), and daidzein (M-9),
respectively, by comparing their UV,
NMR, and MS data with the reported values
[15–22]. M-2, M-3, and M-6–M-9 were
first isolated as the metabolites of tectoridin.
sulfotransferase, forming M-3 and M-4.
Structural identification of tectorigenin-40-
O-b-D-glucuronide and M-4 was studied on
the basis ofLC-MSn databyChenetal. [13].
However, LC-MSn experiments could not
provide full evidence for the determination
of the glucuronidation or sulfation positions
because the 40-OH, 5-OH, and 7-OH
positions are all possible conjugated sites
for tectorigenin. In this study, tectorigenin-
7-O-b-D-glucuronide was isolated and
structurally confirmed using UV, NMR,
and MS, rather than tectorigenin-40-O-b-D-
glucuronide. Thus, the isolation of metab-
olite and the structural confirmation are
valuable for some polyhydroxy compounds.
M-7, the isomeric metabolite of M-5,
was also clearly detected in the HPLC
profiles of the rat urinary metabolites. A
similar pathway related to methoxyl group
migration was found in the urinary
metabolites of kakkalide in our previous
study, which produced two unusual
metabolites 5,7-dihydroxy-8,4-dimethox-
yisoflavone and isotectorigenin formed by
the rearrangement of irisolidone and
tectorigenin [18]. Furthermore, we found
that M-6 is another minor metabolite
derived from the O-methylation of M-5
at C-40 position catalyzed by methyltrans-
ferase [23], which has not been reported in
the previous study on the fecal and urinary
metabolites of tectoridin in rat [13].
3. Discussion
In this study, nine metabolites were isolated
from the urine of rats given tectoridin
orally, and structurally confirmed on the
basis of UV, IR, NMR, and MS data.
According to the metabolite profile, the
possible metabolic pathways of tectoridin
in rats are proposed as shown in Figure 2.
Two phase I metabolites (M-5 and M-7)
and four phase II metabolites (M-1, M-2, M-
3/M-4) were clearly identified in the HPLC-
UV profile of rat urine (Figure 1).
Tectorigenin (M-5), a major metabolite, is
believed to be formed through the microbial
hydrolysis of tectoridin in the gastrointesti-
nal tract, which was consistent with the
previous literature [13]. Moreover, M-5 is
also a reactive metabolite that can be
successively converted to a glucuronide
conjugate (M-1) via further biotransform-
ation at C-7 position catalyzed by UDP-
glucuronosyltransferases. And the sulfate
conjugation at the C-7 and C-40 positions
of M-5 (Figure 2) was catalyzed by

608
X. Bai et al.
It might subsequently undergo glucuro-
nide-conjugation at C-7 position, forming
M-2 (Figure 2). M-2 might also be formed
by M-1 via the methylation of 40-hydroxyl
group. In addition, we also isolated two
minor phase I metabolites (M-8 and M-9).
Kakkalide, the predominant isoflavone
of P. lobata, has a similar structure to
tectoridin. We isolated 13 metabolites
from the urine of rats after orally given
kakkalide [18]. This study showed that
most of the metabolites of tectoridin were
similar to those of kakkalide, which should
be ascribed to their common key metab-
olites, tectorigenin and irisolidone, in vivo.
Ethics Review Committee for Animal
Experimentation of Shenyang Pharmaceu-
tical University. They were kept in a
breeding room to be acclimated for 4 days
before use. Normal foods were provided to
the rats before experiments, so was normal
water at all times.
4.3 Urine collection
Male Wistar rats were fasted 12 h before
experiments. Tectoridin (2.5 g) was given
orally to 28 rats at a dose of 100 mg/kg and
reiteratively in an interval of 7 d (2 d for
administration and 5 d for recovery) for the
collection of urinary samples. The urine
samples were collected from 0 to 36 h.
During the collection, water and sugar
were available freely. Phosphoric acid was
added to urinary samples to adjust pH to
5.0, and they were placed in the refriger-
ator at 2108C subsequently.
4. Materials and methods
4.1 Materials and chemicals
Tectoridin and tectorigenin were isolated
from the flowers of P. thomsonii, and
irisolidone from the flowers of P. lobata,
following the previously reported methods
[24,25]. The identity of these compounds
was confirmed by melting point, UV, IR, 1H
and 13C NMR, and MS. The purity of
tectoridin evaluated with HPLC-UV was
above 98%, and that ofotherswas more than
95%. b-glucuronidase was purchased from
Sigma (St Louis, MO, USA), macroporous
resin D101 from Fushun Xintai Fine
Chemical Factory (Fushun, China), Sepha-
dex LH-20 from GE Healthcare (Uppsala,
Sweden), ODS from YMC Co., Ltd (Kyoto,
Japan), silica gel for column chromatog-
raphy (CC) from Qingdao Ocean Chemical
Co. (Qingdao, China), and MDS-5 RP (200-
300 mesh) from Beijing Medicine Technol-
ogy Center(Beijing, China). Other chemical
reagents were of analysis or HPLC grades.
Doubledistilledwaterwasusedinthisstudy.
4.4 Isolation of metabolites
The cumulative urinary samples (approxi-
mately 1.8 l in total) were thawed at room
temperature, and successively passed
through a macroporous absorption resin
D101 column eluting with a gradient of
EtOH–H2O (H2O, 30% EtOH, 70%
EtOH, and 95% EtOH elutions) to yield
four major fractions.
The 30% EtOH fraction was further
separated through an ODS open column
eluting with a gradient of MeOH–H2O
(10:90–100:0). The fraction eluted with
MeOH–H2O (30:70) was further subjected
to a Sephadex LH-20 column eluting with
MeOH–H2O (50:50) to yield M-1 and M-
2, so was the fraction eluted with MeOH–
H2O (40:60) to a Sephadex LH-20 column
eluting with MeOH–H2O (50:50) to yield
M-3 and M-4.
4.2 Animals
Male Wistar rats (220 ^ 20 g body
weight) were purchased from the Animal
Center of Shenyang Pharmaceutical Uni-
versity (Shenyang, China). The exper-
imental protocol was approved by the
The 70% EtOH fraction was further
separated through an MDS column
eluting with a gradient of MeOH–H2O
(10:90–100:0). The fraction eluted
with MeOH–H2O (60:40) was further

Journal of Asian Natural Products Research
609
separated by silica gel CC eluting with a
CHCl3–MeOH gradient solvent system.
The fraction eluted with CHCl3–MeOH
(50:1) was further subjected to a Sephadex
LH-20 column eluting with CHCl3 –
MeOH (1:1) to give M-8, so was the
fraction eluted with CHCl3–MeOH (30:1)
to a Sephadex LH-20 column eluting with
CHCl3–MeOH (1:1) to give M-9. The
fraction eluted with MeOH–H2O (70:30)
was further separated by silica gel CC
eluting with a CHCl3–MeOH gradient
solvent system. M-5 was given from the
fraction eluted with CHCl3 –MeOH
(100:1). The fraction eluted with CHCl3–
MeOH (80:1) was further subjected to a
Sephadex LH-20 column eluting with
CHCl3–MeOH (1:1) to give M-7.
Japan) was used in the beginning to scan
each fraction for novel metabolites. The
mass spectrometer was operated under the
following conditions: direct-infusion of
sample, ESI in positive and negative
mode, an electrospray voltage of 4.0 KV,
a mass scan range at m/z 50–800, a heating
capillary temperature at 2508C, and dry air
at a flow rate of 4.5 l/min.
TOF-MS (Bruker MicroTOF-Q 125,
Bruker, Newark, DE, USA) was used to
acquire both the exact molecular weight
and the product ion spectra of any novel
compound detected by the single quadru-
pole mass spectrometer. Parameters for
analysis were set using full scan negative
ion mode with spectra acquired over a
mass range from m/z 50 to 1000. The ESI
source was set to the following conditions:
drying gas (N2); flow rate, 4.0 l/min;
drying gas temperature, 1908C; nebulizer,
0.4 bar; capillary voltage, 3.2 KV.
The 95% EtOH fraction was subjected
to a Sephadex LH-20 column eluting with
CHCl3–MeOH (3:1) to give M-6.
NMR spectra were measured on a
Bruker ARX-600 spectrometer, and
chemical shifts are given in ppm downfield
relative to TMS. All compounds were
dissolved in DMSO-d6. UV spectra were
obtained using a Shimadzu UV-2201
spectrophotometer. IR spectra were
obtained on a Bruker IFS-55 infrared
spectrometer.
4.4.1 Tectoridin-7-O-b-D-glucuronide
(M-1)
A yellow amorphous powder; UV (MeOH)
max: 266 nm; IR (KBr) nmax (cm21):
l
3387, 1650, 1613, 1515, 1459; 1H and 13
C
NMR spectral data, see Table 1; HR-ESI-
TOF-MS: m/z 475.0877 [M 2 H]2 (calcd
for C22H19O12, 475.0871).
4.5 Enzymatic hydrolysis of metabolite
M-1
4.7 HPLC condition
Waters HPLC system (Waters Co., Mil-
ford, MA, USA) consisting of model 510
pump, automated gradient controller,
model 2996 photodiode array detector,
and Millennium32 PDA software was
used. HPLC analyses were carried out at
M-1 (0.1 mg) was incubated with b-
glucuronidase (20 ml, 2000 units, type B-1)
in0.05Mammoniumdihydrogenphosphate
buffer (1.0ml, pH 5.0) for 2 h at 378C. The
reaction mixture was extracted with ethyl
acetate, and the organic layer was evapor-
ated to dryness in vacuo to give the aglycone
powder, which was dissolved in 0.2 ml
methanol for HPLC-UV analysis.
358C on
a Kromasil C18 column
(4.6 £ 250 mm, 5 mm, Tianjin Scientific
Instruments Co. Ltd, Tianjin, China). The
on-line UV spectra were recorded in the
range of 200–400 nm. The injection
volume was 20 ml. The mobile phase
consisted of a gradient system of solution
A, water containing 0.05% TFA, and
solution B, acetonitrile containing 0.05%
4.6 Spectroscopic methods
ESI-single quadrupole MS (Shimadzu
QP8000a, Shimadzu Co. Ltd, Kyoto,

610
X. Bai et al.
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TFA at a flow rate of 0.8 ml/min. The
gradient program was as follows: linear
gradient from solution A-B (90:10, v/v) to
solution A-B (60: 40, v/v) in 48 min,
followed by linear gradient to solution A-
B (30:70, v/v) in 32 min.
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Acknowledgements
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This study was financially supported by the
Research Fund for the Doctoral Program of
Higher Education, China (No. 200801630007),
and the 2008 Scientific Technology Plan
Project from Science and Technology Depart-
ment of Liaoning Province, China (No.
20082260233).
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