Isolation and identification of urinary metabolites of kakkalide in rats

Article abstract of DOI:10.1124/dmd.109.028555

Kakkalide is a major isoflavonoid from the flowers of Pueraria lobata (Willd.) Ohwi, possessing the protective effect against ethanol-induced intoxication and hepatic injury. The metabolism of kakkalide was investigated in rats. Thirteen metabolites were isolated by using solvent extraction and repeated chromatographic methods and identified by using spectroscopic methods including UV, IR, mass spectrometry, NMR, and circular dichroism experiments. Four new compounds were identified as irisolidone-7-Oglucuronide (M-1), tectorigenin-7-O-sulfate (M-2), tectorigenin-4′-O-sulfate (M-3), and biochanin A-6-O-sulfate (M-4) together with nine known compounds identified as irisolidone (M-5), tectorigenin (M-6), tectoridin (M-7), 5,7-dihydroxy-8, 4′-dimethoxyisoflavone (M-8), isotectorigenin (M-9), biochanin A (M-10), genistein (M-11), daidzein (M-12), and equol (M-13). The metabolic pathway of kakkalide was proposed, which is important to understand its metabolic fate and disposition in humans. Copyright

Full text of DOI:10.1124/dmd.109.028555

0090-9556/10/3802-281–286$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:281–286, 2010
Vol. 38, No. 2
28555/3552762
Printed in U.S.A.
Isolation and Identification of Urinary Metabolites of Kakkalide
in Rats
Xue Bai, Yuanyuan Xie, Jia Liu, Jialin Qu, Yoshihiro Kano, and Dan Yuan
Department of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang, China
Received June 2, 2009; accepted November 5, 2009
ABSTRACT:
Kakkalide is a major isoflavonoid from the flowers of Pueraria glucuronide (M-1), tectorigenin-7-O-sulfate (M-2), tectorigenin-4؅-
lobata (Willd.) Ohwi, possessing the protective effect against eth- O-sulfate (M-3), and biochanin A-6-O-sulfate (M-4) together with
anol-induced intoxication and hepatic injury. The metabolism of nine known compounds identified as irisolidone (M-5), tectorigenin
kakkalide was investigated in rats. Thirteen metabolites were iso- (M-6), tectoridin (M-7), 5,7-dihydroxy-8,4؅-dimethoxyisoflavone
lated by using solvent extraction and repeated chromatographic (M-8), isotectorigenin (M-9), biochanin A (M-10), genistein (M-11),
methods and identified by using spectroscopic methods including daidzein (M-12), and equol (M-13). The metabolic pathway of kak-
UV, IR, mass spectrometry, NMR, and circular dichroism experi- kalide was proposed, which is important to understand its meta-
ments. Four new compounds were identified as irisolidone-7-O- bolic fate and disposition in humans.
In China, Japan, and other Asian countries, Puerariae Flos has been
More recently, in China and Japan, phytochemicals containing
used to relieve some symptoms such as drunkenness, headache, and Puerariae Flos have become one of the better selling herbal medicines
red face and ameliorate liver injury caused by excessive drinking of for treatment of diseases such as alcohol intoxication and liver injury.
alcohol (Niiho et al., 1989; Song et al., 2001). Puerariae Flos is According to our previous study (Zhang et al., 2009), the contents of
botanically from the flowers of Pueraria lobata (Willd.) Ohwi, where kakkalide in the P. lobata flower and in its water extracts were more
kakkalide is a predominant isoflavone, and Pueraria thomsonii Benth, than 2 and 10%, respectively. Thus, peoples consuming phytochemi-
where tectoridin is a major isoflavone (Zhang et al., 2009). Pharma- cals containing Puerariae Flos may be exposed to high levels of
cologically, kakkalide, irisolidone-7-O-␤-D-xylopyranosyl-(136)-␤- kakkalide. As a phytoestrogen, the metabolic forms of an isoflavone
D-glucopyranoside, shows a wide spectrum of bioactivities such as would be responsible for its activity and side effects (Shin et al.,
hepatoprotection (Yamazaki et al., 1997; Han et al., 2003; Lee et al., 2006). Thus, it is necessary to understand the metabolic fate of
2005a), estrogenic effect (Shin et al., 2006), anti-inflammation (Park kakkalide.
et al., 2007), antihyperlipidemia (Min and Kim, 2007), antioxidation
The liquid chromatography-tandem mass spectrometry (LC-MSn)
(Kang et al., 2008), and others. The major metabolite in the blood of technique was applied to characterize the urinary and biliary metab-
rats orally given kakkalide was its aglycone irisolidone, but not olites of tectoridin and tectorigenin, the major isoflavones of P.
kakkalide itself. Han et al. (2003) isolated irisolidone and kakkalidone thomsonii (Chen et al., 2008; Zhang, 2008; Zhang et al., 2009). In
(irisolidone-7-O-␤-D-glucopyranoside) from an anaerobic medium these studies, the glucuronide or sulfate-conjugated position of several
containing kakkalide and human fecal bacteria, and they found that phase II metabolites cannot be definitively determined means of the
kakkalide exerts the protective activity against ethanol-induced mor- LC-MSn method alone. Isolation of metabolites and their further
tality and hepatic injury in mice only by oral route. On the other hand, structural confirmation on the basis of UV, IR, NMR, and mass
the activity of irisolidone was stronger than that of kakkalide whether spectrometry (MS) data are valuable as well.
it was given orally or intraperitoneally (Yamaki et al., 2002; Min and
In the present study, we conducted the systematic isolation of
Kim, 2007; Shin et al., 2006). In addition, some isoflavone aglycone urinary metabolites in rats given kakkalide orally and determined the
metabolites such as tectorigenin, glycitein, and genistein showed more structure by using chemical and spectroscopic experiments. On the
potent activity than their glycoside precursors (Yamaki et al., 2002). basis of the metabolite profile, the possible metabolic pathway of
These results indicate that kakkalide is in essence a prodrug.
kakkalide was proposed.
Materials and Methods
This work was supported by the Research Fund for the Doctoral Program of
Higher Education from the Ministry of Education of the People’s Republic of
China.
Materials and Chemicals. Kakkalide and irisolidone were isolated from
the flowers of P. lobata, and tectorigenin and tectoridin were isolated from the
flowers of P. thomsonii, following the previously reported methods of Chang
et al. (2009) and Yuan et al. (2009). The identity of these compounds was
confirmed by melting point, UV, IR, 1H and 13C NMR, and MS. The purity
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.028555.
ABBREVIATIONS: LC-MSn, liquid chromatography-tandem mass spectrometry; MS, mass spectrometry; HPLC, high-performance liquid chro-
matography; CC, column chromatography; CD, circular dichroism; ESI, electrospray ionization; TOF-MS, time-of-flight tandem mass
spectrometry.
281

282
BAI ET AL.
of kakkalide evaluated with high-performance liquid chromatography
quadrupole mass spectrometer. Parameters for analysis were set by using
(HPLC)-UV was 98.6%, and those of others were more than 95%. ␤-Glucu- full-scan negative ion mode with spectra acquired over a mass range from m/z
ronidase and sulfatase were purchased from Sigma-Aldrich (St. Louis, MO);
macroporous resin D101 was obtained from Fushun Xintai Fine Chemical
50 to 1000. The ESI source was set to the following conditions: drying gas (N2)
flow rate, 4.0 l/min; drying gas temperature, 190°C; nebulizer, 0.4 bar; capil-
Factory (Fushun, China); Sephadex LH-20 was from GE Healthcare (Uppsala, lary voltage, 3.2 kV.
NMR spectra were measured on a Bruker ARX-600 spectrometer, and
Sweden); ODS was obtained from YMC Co., Ltd. (Kyoto, Japan); and Silica
gel GF254 for thin-layer chromatography and silica gel column chromatogra-
phy (CC) were from Qindao Ocean Chemical Co. (Qingdao, China). Other
chemical reagents were of analytical or HPLC grade. Double-distilled water
was used in this study.
chemical shifts are given in ppm downfield relative to tetramethylsilane. All
compounds were dissolved in dimethyl sulfoxide-d6. UV spectra were obtained
using a Shimadzu UV-2201 spectrophotometer. Circular dichroism (CD) spec-
tra were recorded on a JASCO CD-2095 plus spectrophotometer (Jasco,
Tokyo, Japan). IR spectra were obtained on a Bruker IFS-55 IR spectrometer.
HPLC Condition. Waters HPLC system (Waters, Milford, MA) 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 35°C on a Kromasil C18 column (4.6 ϫ 250 mm, 5 ␮m;
Tianjin Scientific Instruments Co. Ltd., Tianjin, China). The on-line UV
spectra were recorded in the range of 200 ϳ 400 nm. The injection volume was
20 ␮l. The mobile phase consisted of a gradient system of solution A, water
containing 0.05% trifluoroacetic acid, and solution B, acetonitrile containing
0.05% trifluoroacetic acid 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.
Animals. Male Wistar rats (200 Ϯ 20 g b.wt.) were purchased from the
Animal Center of Shenyang Pharmaceutical University (Shenyang, China).
The experimental protocol was approved by the Ethics Review Committee for
Animal Experimentation of Shenyang Pharmaceutical University. They were
kept in a breeding room to be acclimated for 4 days before use. Normal foods
were available before experiments, and normal water was available at all times.
Urine Collection. Male Wistar rats were fasted 12 h before experiments.
Kakkalide (9 g) was given orally to 48 rats at a dose of 200 mg/kg and
administered reiteratively in an interval of 7 days (2 days for administration
and 5 days for recovery) for the collection of urine 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 urine samples to adjust pH to
5.0, and samples were subsequently placed in the refrigerator at Ϫ10°C.
Isolation of Metabolites. The cumulative urine samples (approximately 5
liters 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 (35:65) was further subjected to a Sephadex LH-20 column
eluting with MeOH-H2O (50:50) to give M-1, so were the fractions eluted with
MeOH-H2O (40:60) to yield M-2 and M-3 and the fractions eluted with
MeOH-H2O (50:50) to yield M-4 and M-7.
The 70% EtOH fraction was further separated by silica gel CC eluting with
a CHCl3-MeOH gradient solvent system. M-6 was given from the fraction
eluted with CHCl3-MeOH (120:1). The fraction eluted with CHCl3-MeOH
(100:1) was further subjected to a Sephadex LH-20 column eluting with
CHCl3-MeOH (1:1) to give M-9, so were the fractions eluted with CHCl3-
MeOH (70:1) to a Sephadex LH-20 column eluting with MeOH to yield M-11,
and the fractions eluted with CHCl3-MeOH (50:1) to a Sephadex LH-20
column eluting with MeOH to yield M-12.
The 95% EtOH fraction was further separated by silica gel CC eluting with
a CHCl3-MeOH gradient solvent system. M-5 was obtained from the fraction
eluted with CHCl3-MeOH (500:1). The fraction eluted with CHCl3-MeOH
(300:1) was further subjected to a Sephadex LH-20 column to give M-8,
eluting with CHCl3-MeOH (3:1), so were the fractions eluted with CHCl3-
MeOH (200:1) to a Sephadex LH-20 column eluting with CHCl3-MeOH
(1:1) to yield M-10, and the fraction eluted with CHCl3-MeOH (150:1) to
a Sephadex LH-20 column eluting with CHCl3-MeOH (1:1) to yield M-13.
Enzymatic Hydrolysis of Metabolites M-1, M-2, and M-3. M-1 (0.1 mg)
was incubated with ␤-glucuronidase (20 ␮l, 2000 units, type B-1) in 0.05 M
ammonium dihydrogen phosphate buffer (1.0 ml, pH 5.0) for 2 h at 37°C. The
hydrolysis of M-2 and M-3 (each 0.1 mg) with sulfatase (20 ␮l, 1000 units,
type H-1) was conducted following the hydrolytic method of M-1 as described
above. Each reaction mixture was extracted with ethyl acetate, and the organic
layer was evaporated to dryness in vacuo to give the aglycone powder, which
dissolved in 0.2 ml of methanol for HPLC-UV analysis.
Results
Isolation and Structural Elucidation of Kakkalide Metabolites.
Representative HPLC profiles showing the rat urinary metabolites are
given in Fig. 1. Kakkalide and its metabolites were selectively de-
tected at 265 nm due to their characteristic benzoyl group. Five major
metabolites, M-1, M-2/-3, M-5, and M-6, were clearly observed in rat
urine (Fig. 1). By means of repeated chromatographic methods on the
columns of silica gel, Sephadex LH-20, or reverse-phase ODS, 13
metabolites were isolated from the rat urine sample, including four
new compounds, a glucuronide conjugate, M-1 (2.5 mg), and three
sulfate ones, M-2 (3.5 mg), M-3 (3.0 mg), and M-4 (2.5 mg), together
with nine known isoflavones, M-5 (15 mg), M-6 (10 mg), M-8 (1.4
mg), M-9 (1.1 mg), M-10 (1.1 mg), M-11 (1.4 mg), M-12 (1.2 mg),
M-13 (2.4 mg), and M-7 (2.0 mg).
The maximal absorption at 263 to 265 nm in UV spectra and the
absorption bands at 1649 to 1659 cmϪ1 due to conjugated carbonyl
and at 1457 to 1616 cmϪ1 due to aromatic functions in IR spectra
indicate that M-1 ϳ M-12 have an isoflavone skeleton. The structures
of M-1 ϳ M-13, as shown in Fig. 2, were elucidated using UV, IR, 1H
and 13C NMR, MS, or CD techniques.
Metabolite M-1 (irisolidone-7-O-␤-D-glucuronide) was isolated as
a white amorphous powder. The molecular formula was determined to
be C23H21O12 from the [M Ϫ H]Ϫ quasimolecular ion peak at m/z
489.1024 (calculated 489.1027) in the ESI-TOF-MS. The [M Ϫ H]Ϫ
ion at m/z 489 and an important fragment ion at m/z 313 originating
from the eliminating 176 mass units (glucuronic acid) from [M Ϫ H]Ϫ
ion indicated that M-1 should be a glucuronide conjugate (Fig. 3A).
M-1 was hydrolyzed with ␤-glucuronidase to give the aglycone iri-
solidone, the identity of which was confirmed through cochromatog-
raphy with irisolidone standard by HPLC coupled to UV photodiode
array detection based on the same retention time and UV spectral. In
the 1H NMR spectrum, the characteristic isoflavone signal for H-2
was observed at ␦ 8.49 (1H, s). A singlet at ␦ 6.91 due to an aromatic
proton suggested that three substituents were linked to A-ring. The
protons due to an AAЈBBЈ aromatic system appeared at ␦ 7.53 (d, J ϭ
8.4 Hz, H-2Ј, 6Ј) and 7.01 (d, J ϭ 8.4 Hz, H-3Ј, 5Ј), indicating that
B-ring was substituted at C-4Ј. In addition, two singlets at ␦ 3.80 and
3.78 due to two methoxyl groups were observed. The resonances for
carbons and protons of the aglycone moiety had a close resemblance
Spectroscopic Methods. Electrospray ionization (ESI)-single quadrupole
MS (Shimadzu QP8000␣; Shimadzu Co. Ltd., Kyoto, 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 to 800, a heating capillary temperature at 250°C, and dry air
at a flow rate of 4.5 l ⅐ minϪ1
.
Time-of-flight tandem mass spectrometry (TOF-MS) (Bruker MicroTOF-Q
125; Bruker, Newark, DE) was used to acquire both the exact molecular weight
and the product ion spectra of any novel compounds detected by the single- to those of the known irisolidone, and they are assigned according to

METABOLITES OF KAKKALIDE IN RAT URINE
283
FIG. 1. A, HPLC-UV chromatograms of blank urine; B, a rat urine sample during 0 to 24 h after oral administration of kakkalide at a dose of 200 mg/kg; C, metabolite
standards.
FIG. 2. Proposed metabolic pathways of kakkalide. [ ] refers to possible intermediates that were not obtained in the present study.
the literature values of the 1H and 13C NMR data for irisolidone (Yin of irisolidone (Yasuda et al., 1994; Yin et al., 2006). The full assign-
et al., 2006). The signals of an anomeric proton (␦ 5.08, 1H, d, J ϭ 6.6 ments of carbon and proton signals are summarized in Table 1. Thus,
Hz) and a carboxylic group at C-6Ј at ␦c 171.0 indicated the presence M-1 was determined to be irisolidone 7-O-␤-D-glucuronide.
of a ␤-D-glucuronic acid moiety (Yasuda et al., 1994). The ␤-D-
Metabolite M-2 (tectorigenin-7-O-sulfate) was isolated as a yellow-
glucuronic acid moiety could be attached to C-7 position according to ish amorphous powder. IR spectrum showed an absorption band at
the upfield shift of C-7 (Ϫ0.8 ppm) and the downfield shifts of C-6 1038 cmϪ1 due to a sulfate group, suggesting the sulfate-conjugated
(ϩ1.1 ppm) and C-8 (ϩ0.2 ppm) relative to the corresponding signals structure of M-2. The molecular formula was determined to be

284
BAI ET AL.
FIG. 3. ESI-TOF-MS spectra of metabolite M-1, irisolidone-7-O-glucuronide (A); metabolite M-2, tectorigenin-7-O-sulfate (B); metabolite M-3, tectorigenin-4Ј-O-sulfate
(C); and metabolite M-4, biochanin A-6-O-sulfate (D).
TABLE 1
NMR of kakkalide metabolites M-1ϳM-4
M-1
M-2
M-3
M-4
Position
a
a
a
a
␦
␦
a (J in Hz)
␦
␦
a (J in Hz)
␦
␦
a (J in Hz)
␦
␦
a (J in Hz)
H
C
H
C
H
C
H
C
2
3
4
5
6
7
8
155.1
122.9
180.8
153.0
132.6
156.8
94.2
8.49, s
154.9
122.0
181.0
153.2
133.7
153.0
98.0
8.44, s
154.6
125.4
180.4
153.2
131.5
157.5
93.9
8.39, s
154.7
120.3
180.2
154.1
125.3
157.6
95.0
8.4, s
6.91, s
7.26, s
6.50, s
6.55, s
9
10
1Ј
2Ј,6Ј
3Ј,5Ј
4Ј
1Љ
2Љ
152.5
106.5
121.8
130.3
113.8
159.3
100.0
73.1
151.7
107.0
121.2
130.3
115.2
157.5
152.7
104.8
121.6
129.5
120.1
153.5
154.1
105.0
121.6
130.3
113.8
159.2
7.53, d (8.4)
7.01, d (8.4)
7.40, d (8.4)
6.83, d (8.4)
7.46, d (8.4)
7.22, d (8.4)
7.51, d (9.0)
7.00, d (9.0)
5.08, d (6.6)
3Љ
76.7
4Љ
71.9
5Љ
73.9
6Љ
6-OMe
4Ј-Ome
171.0
60.4
55.3
3.78, s
3.80, s
60.3
3.77, s
59.9
3.75, s
55.3
3.79, s
a All spectra were recorded on a Bruker ARX-600 spectrometer, in DMSO-d6.
1Љ ϳ 6Љ refer to the numbering of glucuronic acid moiety.
C16H12O9S from the [M Ϫ H]Ϫ quasimolecular ion peak at m/z in the 1H and 13C NMR spectrum closely resembled those of the
379.0117 (calculated 379.0118) in the ESI-TOF-MS. The negative known tectorigenin, and they are assigned according to the literature
ESI-MS displayed a [M Ϫ H]Ϫ ion peak at m/z 379 and a [M-SO3H]Ϫ values of the 1H and 13C NMR data for tectorigenin (Kang et al.,
ion peak at m/z 299, further indicating the presence of a sulfate group 2008). The sulfate moiety could be attached to C-7 position ac-
(Fig. 3B). M-2 was hydrolyzed by sulfatase to give the aglycone cording to a upfield shift of C-7 (Ϫ4.5 ppm) and the downfield
tectorigenin, the identity of which was also confirmed through co- shifts of C-6 (ϩ2.2 ppm), C-8 (ϩ4.0 ppm) together with H-8
chromatography with tectorigenin standard using HPLC-UV. More- (ϩ0.76 ppm), and C-10 (ϩ2.1 ppm) relative to the corresponding
over, the resonances for protons and carbons of the aglycone moiety signals of tectorigenin (Yasuda et al., 1994; Kang et al., 2008). The

METABOLITES OF KAKKALIDE IN RAT URINE
285
full assignments of carbon and proton signals are summarized in by comparing their UV, NMR, MS, or CD data (Tables 2) with the
Table 1. Thus, M-2 was determined to be tectorigenin-7-O-sulfate. reported values (Bashir et al., 1991; Yasuda et al., 1994; Yasuda and
Metabolite M-3 (tectorigenin-4Ј-O-sulfate) was isolated as a yellow- Ohsawa, 1998; Talukdar et al., 2000; Yin et al., 2006; Moriyasu et al.,
ish amorphous powder. IR spectrum also showed an absorption band 2007; Kang et al., 2008). M-6 ϳ M-13 were first isolated as the
at 1038 cmϪ1 due to a sulfate group. The molecular formula was metabolites of kakkalide.
determined to be C16H12O9S from the [M Ϫ H]Ϫ quasimolecular ion
Discussion
peak at m/z 379.0117 (calculated 379.0118) in the ESI-TOF-MS. The
[M Ϫ H]Ϫ ion peak at m/z 379 and a [M Ϫ SO3H]Ϫ ion peak at m/z
The metabolic pathways of soy isoflavones in vivo are related to
299 were clearly shown in ESI-MS (Fig. 3C). The enzymatic hydro- many reactions such as hydrolysis, O-methylation, glucuronida-
lysis of M-3 with sulfatase gave tectorigenin, the identity of which tion, sulfation, hydroxylation, decyclization, and reduction (Yasuda and
was also confirmed through cochromatography with tectorigenin stan- Ohsawa, 1998; Yasuda et al., 2001; Bursztyka et al., 2008). In the
dard using HPLC-UV. The resonances for protons and carbons of the present study, 13 metabolites were isolated from the urine of rats
aglycone moiety in the 1H and 13C NMR spectrum also closely given kakkalide orally and structurally confirmed on the basis of
resembled those of tectorigenin (Kang et al., 2008). The sulfate UV, IR, NMR, MS, and CD data. According to the metabolite
moiety could be attached to C-4Ј position according to an upfield shift profile, the possible metabolic pathways of kakkalide in rats are
of C-4Ј (Ϫ4.2 ppm) and the downfield shifts of C-3Ј and C-5Ј (ϩ4.9 proposed as shown in Fig. 2.
ppm) relative to the corresponding signals of tectorigenin (Yasuda et
Two phase I metabolites (M-5 and M-6) and three phase II metabolites
al., 1994; Kang et al., 2008). The full assignments of carbon and (M-1, M-2, and M-3) were clearly identified in the HPLC-UV profile of
proton signals are summarized in Table 1. Thus, M-3 was determined rat urine (Fig. 1). Irisolidone (M-5) is a major metabolite that is believed
to be tectorigenin-7-O-sulfate.
to be formed through the microbial hydrolysis of kakkalide in the gas-
Metabolite M-4 (biochanin A-6-O-sulfate) was isolated as a yellow- trointestinal tract, which is consistent with the previous literature (Han et
ish amorphous powder. The IR spectrum showed an absorption band al., 2003). Moreover, M-5 is also a reactive metabolite that can be
at 1045 cmϪ1 due to a sulfate group. The molecular formula was successively converted to a glucuronide-conjugate (M1) via further bio-
determined to be C16H12O9S from the [M Ϫ H]Ϫ quasimolecular ion transformation at C-7 position catalyzed by UDP-glucuronosyltrans-
peak at m/z 379.0117 (calculated 379.0118) in the ESI-TOF-MS. The ferases. Most importantly, we found that M-6 is another key metabolite
[M Ϫ H]Ϫ ion peak at m/z 379 and [M Ϫ SO3H]Ϫ ion peak at m/z 299 derived from the O-demethylation of M-5 at C-4Ј position catalyzed by
were also clearly shown in ESI-MS (Fig. 3D). The resonances for cytochrome P450 (Meyer et al., 2009), which has not been reported in the
protons and carbons of the aglycone moiety of M-4 in the 1H and 13
C
previous studies on the metabolism of kakkalide. It also subsequently
NMR spectrum closely resembled those of the known 6-hydroxybio- undergoes sulfate-conjugation at the C-4Ј and C-7 positions (Fig. 3)
chanin A, and they are assigned according to the literature values of catalyzed by sulfotransferases, forming M-2 and M-3. The total amount
1
the H and 13C NMR data for 6-hydroxybiochanin A (Horie et al., of M-5 and M-6 in urine samples is more than that of three conjugates
1996). The sulfate moiety could be attached to C-6 according to the (M-1 ϳ M-3). In addition, we also isolated some minor metabolites,
upfield shift of C-6 (Ϫ4.1 ppm) and downfield shifts of C-7 (ϩ4.1 including seven phase I metabolites (M-7 ϳ M-13) and a phase II
ppm) and C-5 (ϩ6.8 ppm) relative to the corresponding signals of metabolites (M-4). It is worth noting that the purity of the dosed kak-
6-hydroxybiochanin A (Yasuda et al., 1994; Horie et al., 1996). The kalide was found to be 98.6%, and that these minor metabolites were not
full assignments of carbon and proton signals are summarized in impurities in the dosed kakkalide according to HPLC analysis. In general,
Table 1. Thus, M-4 was determined to be biochanin A-6-O-sulfate.
the role of phase II metabolism in vivo is drug detoxification by means
Other Metabolites. M-5 ϳ M-13 were identified as irisolidone, of conjugation of phase I metabolites with endogenous substances to
tectorigenin, tectoridin, 5,7-dihydroxy-8,4Ј-dimethoxyisoflavone, iso- increase their water solubility and decrease or eliminate their biological
tectorigenin, biochanin A, genistein, daidzein, and equol, respectively, activity (Liska, 1998). The wide existence of phase I metabolites of
TABLE 2
1H NMR date of kakkalide metabolites M-5ϳM-13
␦
a (J in Hz)
H
Position
M5
M6
M7
M8
M9
M10
M11
M12
M13
2
8.38, s
8.33, s
8.44, s
8.45, s
8.37, s
8.36, s
8.31, s
8.28, s
3.89, t ␣ (10.5)
4.15, dd ␤ (10.5, 1.8)
2.96–3.04, m
2.78–2.88, m
6.86, d (8.4)
6.29, dd (8.4, 2.1)
6.19, d (2.1)
7.10, d (8.4)
3
4
5
6
7.95, d (9.0)
6.92, dd (9.0, 2.4)
6.84, d (2.4)
7.37, d (8.4)
6.79, d (8.4)
6.31, s
6.26, s
6.22, d (2.4)
6.39, d (2.4)
7.50, d (8.4)
7.00, d (8.4)
6.22, d (2.0)
6.42, d (2.0)
7.40, d (8.5)
6.83, d (8.5)
8
2Ј,6Ј
3Ј,5Ј
Glc-1
2
3
4
5
6
6.51, s
7.50, d (8.6)
7.00, d (8.6)
6.50, s
7.38, d (8.4)
6.83, d (8.4)
6.88, s
7.39, d (8.7)
6.82, d (8.7)
5.08, d (7.4)
3.30, m
3.30, m
3.16, m
7.50, d (9.0)
7.01, d (9.0)
7.36, d (8.4)
6.80, d (8.4)
6.72, d (8.4)
3.44, m
3.46, m ␣
3.70, m ␤
3.76, s
6-OMe
8-OMe
4Ј-OMe
3.79, s
3.76, s
3.75, s
3.77, s
3.77, s
3.74, s
3.79, s
a All spectra were recorded on a Bruker ARX-600 spectrometer, in DMSO-d6.

286
BAI ET AL.
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Address correspondence to: Dan Yuan, Department of Traditional Chinese
Medicines, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang,
110016, People’s Republic of China. E-mail: yuandan_kampo@163.com