Metabolism of Chuanxiong Rhizoma decoction: Identification of the metabolites in WZS-miniature pig urine

Article abstract of DOI:10.1016/j.fitote.2015.07.001

Abstract Chuanxiong Rhizoma (CR), a well-known traditional Chinese medicine originated from the rhizome of Ligusticum chuanxiong Hort., was effective for treating various vascular diseases. To identify the metabolites of CR in vivo, the drug-containing urine samples of WZS-miniature pigs after orally administrated CR decoction were collected, after sequential column chromatography 17 metabolites (M1-M17) were isolated from the methanol extract of the urine samples. Their structures, including nine phthalides (M1-M9) and eight phenolic acids (M10-M17), were identified by spectroscopic means. Among them, 8 were new ones (M1-M6, M11-M12). On the basis of the structures of identified metabolites, seven original constituents, including 2 phthalides (senkyunolideI/H) and 5 phenolic acids (ferulic acid, isoferulic acid, caffeic acid, 3-hydroxycinnamoyl acid and 4-hydroxybenzonic acid) were deduced to be the major absorbed original constituents of CR in vivo. This is the first study on the metabolites of CR decoction in non-rodent animal (WZS-miniature pig), the results will give an insight into the metabolism profiles of phthalides and phenolic acids in CR decoction in vivo.

Full text of DOI:10.1016/j.fitote.2015.07.001

Fitoterapia 105 (2015) 177–186
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Metabolism of Chuanxiong Rhizoma decoction: Identification of the
metabolites in WZS-miniature pig urine
Lei Zhang, Dong-Hui Yang ⁎, Xin Zhao, Feng Xu, Jing Liang, Qi-Le Zhou, Xiao-Yan Yang, Xiu-Wei Yang ⁎
Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 May 2015
Received in revised form 28 June 2015
Accepted 1 July 2015
Available online 7 July 2015
Chuanxiong Rhizoma (CR), a well-known traditional Chinese medicine originated from the rhizome of Ligusticum
chuanxiong Hort., was effective for treating various vascular diseases. To identify the metabolites of CR in vivo, the
drug-containing urine samples of WZS-miniature pigs after orally administrated CR decoction were collected,
after sequential column chromatography 17 metabolites (M1–M17) were isolated from the methanol extract
of the urine samples. Their structures, including nine phthalides (M1–M9) and eight phenolic acids (M10–
M17), were identified by spectroscopic means. Among them, 8 were new ones (M1–M6, M11–M12). On the
basis of the structures of identified metabolites, seven original constituents, including 2 phthalides
(senkyunolideI/H) and 5 phenolic acids (ferulic acid, isoferulic acid, caffeic acid, 3-hydroxycinnamoyl acid and
4-hydroxybenzonic acid) were deduced to be the major absorbed original constituents of CR in vivo. This is the
first study on the metabolites of CR decoction in non-rodent animal (WZS-miniature pig), the results will give
an insight into the metabolism profiles of phthalides and phenolic acids in CR decoction in vivo.
Keywords:
Ligusticum chuanxiong
Phthalides
Phenolic acids
Metabolites in vivo
WZS-miniature pig
©
2015 Elsevier B.V. All rights reserved.
1
. Introduction
acid and tetramethylpyrazine, have been identified mainly by analyzing
their MS information, respectively [12–26]. There was only one report
on the overall metabolism of CR extract in rat's plasma [27], which led
to the identification of 13 prototype constituents and 12 conjugated me-
tabolites basing on HPLC–MS information.
In the present study, the CR metabolites existed in the drug-
containing urine samples of WZS-miniature pigs after orally adminis-
trated CR decoction, were isolated using a series of column chromatog-
raphies, which led to 17 compounds, and their structures were
elucidated on the basis of MS and spectroscopic analyses.
Chuanxiong Rhizoma (CR), the rhizome of Ligusticum chuanxiong
Hort. (Chuanxiong), is a well-known traditional Chinese medicine that
is commonly prescribed for the treatment of migraine and various car-
diovascular diseases, such as angina pectoris and ischemic stroke
[1–3] and endocrine disorders [4] in clinical practice.
To date, a lot of chemical constituents have been isolated from CR,
mainly including two types of phthalides (ligustilide, senkyunolide A-
S, butylidenephthalide, and phthalide dimers, etc.) [5–9] and phenolic
acids (ferulic acid, isoferulic acid and caffeic acid, etc.) [10], and alka-
loids (tetramethylpyrazine) [11]. However, which were responsible
for CR activities are still uncertain.
2. Materials and methods
Generally, a traditional Chinese medicine (TCM) is administered in the
form of decoction, there usually have multiple components. As known,
only the absorbed constituents have the most possibilities for being its ef-
fective constituents, so, it is important to understand the absorptive con-
stituents of a TCM and their in vivo metabolite profiles. To the best of our
knowledge, there were much research on the metabolites of the pure
compounds in CR, and more than 30 in vivo metabolites, which originated
from Z-ligustilide, butylphthalide, butylidenephthalide, ferulic acid, caffeic
2.1. Materials
The CR materials were purchased from Yaoxing Medicinal Materials
Company (Anguo, Hebei Province, China) and were identified as the
dried rhizome of Ligusticum chuanxiong Hort. (Umbelliferae) by D.-H.
Yang. The voucher specimen (No. 6332) was deposited in Herbarium
of Pharmacognosy, School of Pharmaceutical Sciences, Peking Universi-
ty. The dry powders of CR crude drugs (15 kg) were immersed in 150 L
of water for 1 h, and then decocted for 1.5 h. After filtration, the residue
was decocted again with 90 L of water for 1 h. The two filtered extracts
were combined and concentrated to about 7 L (2.14 g crude drug/mL)
by rotatory evaporation in vacuum at 60 °C and stored at −20 °C before
use.
⁎
Corresponding authors at: No. 38, Xueyuan Road, Haidian District, Beijing 100191, PR
China.
E-mail addresses: ydhui@bjmu.edu.cn (D.-H. Yang), xwyang@bjmu.edu.cn
X.-W. Yang).
(
http://dx.doi.org/10.1016/j.fitote.2015.07.001
367-326X/© 2015 Elsevier B.V. All rights reserved.
0

178
L. Zhang et al. / Fitoterapia 105 (2015) 177–186
2
.2. Chemicals and reagents
containing urine extract (1.12 kg), which was stored at −20 °C be-
fore further isolation. Blank urine sample was pre-treated by the
same procedure.
HPLC-grade acetonitrile and methanol were purchased from Fisher
Scientific (Fairlawn, NJ, USA) and Tianjin Damao Chemical Company
Tianjin, China). Analytical-grade formic acid was purchased from
MREDA Technology (USA). Purified water was prepared by a Milli-Q
water purification system (Millipore, Bedford, MA, USA). Analytical-
grade methanol and ethanol were provided by Peking Chemical Factory
(
2.5. Extraction and isolation
The drug-containing urine extract (1.10 kg) was dissolved in 1 L of
distilled water, and then chromatographed on a XAD-2 macroporous
resin column (5 kg, 2 × 30 cm) using gradient elution of methanol–
water solutions, which gave 3 fractions of Fr.1 (eluted by water), Fr.2
(
Beijing, China). XAD-2 macroporous resin (Amberlite, Philadelphia,
USA) and Sephadex LH-20 (GE health care, Buckinghamshire, UK)
were used for isolation.
(
eluted by 20% methanol), and Fr.3 (eluted by 50% methanol). By
analyzing their HPLCs, the major constituents originating from CR
decoction were detected in Fr. 2 and Fr. 3, while Fr. 1 was mainly
composed of endogenous components (Fig. 1). Therefore, Fr. 2 and Fr.
2
.3. Animal protocol
Eight male WZS-miniature pigs (each weighing about 60 kg) were
3
were merged for the following chemical isolation, and 17 compounds
M1–M17) were isolated after consecutive column separations on
semi-preparative HPLC and Sephadex LH-20.
purchased from the Institute of Animal Science, Chinese Academy of Ag-
ricultural Sciences. The pigs were raised in metabolic cages in an envi-
ronmentally controlled room (25 °C and 60% relative humidity) for
consecutive 7 days before the experiment. Each pig was orally adminis-
tered with 125 mL of distilled water (twice per day, 8:00 a.m. and
(
2.6. Equipment and chromatographic conditions
5
:00 p.m.) in the first 2 days for collecting the blank urine sample.
Then an equal dose (125 mL, 5.4 g crude drug/kg) of the CR decoction
twice per day, 8:00 a.m. and 5:00 p.m.) was administered in the follow-
The HPLC chromatography system consisted of Shimadzu LC-
0A pumps, a SPD-M20A PDA detector (Shimadzu, Japan), and a
(
2
ed 4 days, and the drug-containing urine samples were collected since
the first administration.
All the procedures were in strict accordance with the guidelines for
Animal Experimentation of Peking University (Beijing, China), and the
protocols were approved by the Animal Biomedical Ethical Committee
of Peking University (Approval No. LA2011-058).
Phenomenex ODS column (250 × 4.6 mm, 5 μm; Phenomenex,
Inc.), developed with a linear gradient from 3% to 60% CH
CN/H
containing 0.3% trifluoroacetic acid) in 95 min followed by
3
2
O
(
3
additional 5 min elution with 100% CH CN at a flow rate of 1.0
mL/min and UV detection elution with 280 nm.
Semi-preparative HPLC chromatography system consisted of Agilent
1
100 pumps equipped with an DAD detector (Agilent Co.) and a
Phenomenex ODS column (250 × 21.2 mm, 10 μm; Phenomenex,
Inc.), using CH CN-H O (containing 0.3% trifluoroacetic acid) as eluent
2
.4. Urine samples
3
2
Prior to the drug administration, the blank urine sample within
8 h (40 L) was collected; then the drug-containing urine samples
were continuously collected until 12 h after the last administration,
5% ethanol as preservative was added into the urine samples
100 mL/L) during collection. The urine sample (approximately 20
L/day) was filtered through absorbent cotton, and equal amount of
methanol was added into the urine sample to precipitate endoge-
nous protein and carbamide. After vacuum suction filtration, the
filtrate was evaporated in vacuum at 50 °C to obtain the drug-
at a flow rate of 3 mL/min.
4
MS data of the isolated compounds were measured on Waters Xevo
G2-S QT of mass spectrometer. The IR absorption spectra were obtained
on a JASCO IR-810 spectrophotometer. Circular dichroism (CD) and in-
duced CD spectra were taken on a JASCO J-810 spectro polarimeter
(JASCO Co.).
NMR spectra were performed in an inverse probe of Bruker-AVIII-
500 and Bruker-AVIII-500 spectrometer with TMS as the internal stan-
dard, and coupling constants (J values) were given in Hertz.
9
(
Fig. 1. The HPLCs of blank urine sample and fractions of drug-containing urine samples from XAD-2 resin column chromatography eluted by 20% methanol (Fr.2) and 50% methanol (Fr.3),
respectively.

L. Zhang et al. / Fitoterapia 105 (2015) 177–186
179
−
2
.7. Data of the compounds (M1–M17)
M1, slight yellow solid, HR–ESI–MS m/z 263.0894 [M + Na] (calcd
M13, slight yellow solid, HR–ESI–MS m/z 250.0720 [M–H] (calcd
−
for C12
H
12NO
5
: m/z 250.0715). The NMR data were shown in Tables 4
+
and 5.
+
−
for C12
677.8, 1202.6 and 1039.7 (C_O) cm . The NMR data were shown in
Table 1.
M2, slight yellow solid, HR–ESI–MS m/z 263.0896 [M + Na] (calcd
H
16
O
5
Na : m/z 263.0895); IR (KBr) νmax 3391.24 (–OH), 1748.3,
M14, slight yellow solid, HR–ESI–MS m/z 264.0878 [M–H] (calcd
−
1
−
1
for C13
H
14NO
5
: m/z 264.0872). The NMR data were shown in Tables 4
and 5.
+
+
M15, slight yellow solid, ESI–MS m/z 242.13 [M + Na] (calcd for
+
+
for C12
1
H
16
O
5
Na : m/z 263.0895), IR (KBr) νmax 3401.6 (–OH), 1749.4,
C
12
H
13NO
3
Na : m/z 242.0788). The NMR data were shown in Tables
−
1
677.2 and 1203.5 (C_O) cm . The NMR data were shown in Table 1.
4 and 5.
+
1
M3, slight yellow solid, HR–ESI–MS m/z 263.0892 [M + Na] (calcd
M16, slight yellow solid, H NMR (400 MHz, DMSO) δ: 7.21 (1H, t,
J = 8.0, 7.6 Hz, H-3), 7.09 (1H, d, J = 7.6 Hz, H-5′), 7.03 (1H, s, H-6′),
6.83 (1H, d, J = 8 Hz, H-2′), 7.48 (1H, d, J = 15.6 Hz, H-4′), 6.42 (1H,
d, J = 16 Hz, H-2). 13C NMR (100 MHz, DMSO) δ: 167.6 (C-1), 157.6
(C-3′), 143.9 (C-3), 135.5 (C-1′), 129.8 (C-5′), 119.1 (C-6′), 119.1 (C-
2), 117.3 (C-2′), 114.4 (C-4′).
+
for C12
H
16
O
5
Na : 263.0895), IR (KBr) νmax 3420.0 (–OH), 1749.4,
−
1
1
654.9, 1026.3 and 1002.2 (C_O) cm . The NMR data were shown
in Table 1.
M4, slight yellow solid, HR–ESI–MS m/z 253.0715 [M–H] (calcd for
−
−
C
1
12 13 6
H O : m/z 253.0712), IR (KBr) νmax 3415.1 (–OH), 1750.2, 1680.0,
−
1
1
202.8 and 1032.6 (C_O) cm . The NMR data were shown in
M17, slight yellow solid, H NMR (400 MHz, DMSO) δ: 12.48 (1H, s, –
Table 2.
M5, slight yellow solid, HR–ESI–MS m/z 253.0721 [M–H] (calcd for
COOH), 10.32 (1H, s, −OH), 7.79 (2H, d, J = 8 Hz, H-2,6), 6.82 (2H, d,
J = 8.6 Hz, H-3,5). C NMR (100 MHz, DMSO) δ: 167.6 (–COOH),
162.1 (C-1), 132.0 (C-3,5), 121.8 (C-4), 115.6 (C-2,6).
3. Results and discussion
−
13
+
C
12
H
13
O
6
: m/z 253.0712), IR (KBr) νmax 3401.9 (–OH), 1677.2, 1201.9
−
1
and 1116.3 (C_O) cm . The NMR data were shown in Table 2.
+
M6, slight yellow solid, HR–ESI–MS m/z 291.0848 [M + Na] (calcd
+
for C13
H
16
O
6
Na : m/z 291.0845), IR (KBr) νmax 3401.9 (–OH), 1677.2,
−
1
1
201.9 and 1116.3 (C_O) cm . The NMR data were shown in
Table 2.
M7, slight yellow solid, HR–ESI–MS m/z 399.1294 [M–H] (calcd for
3.1. Structure elucidation
−
The identified 17 compounds (M1–M17), including nine phthalide-
related compounds (M1–M9) and eight phenolic acid-related
compounds (M10–M17), were determined on the basis of their spectro-
scopic data (Fig. 2). Their chemical structures were identified as: Z-
(6S,7R,10R)-3-butylidene-4,5,6,7-tetrahydro-6,7,10-trihydroxyphthalide
(M1), Z-(6S,7R,10S)-3-butylidene-4,5,6,7-tetrahydro-6,7,10-trihydro-
xyphthalide (M2), Z-(6R,7S)-3-butylidene-4,5,6,7-tetrahydro-6,7,10-
trihydroxyphthalide (M3), E-(6S,7R)-3-butylidene-4,5,6,7-tetrahydro-
11-carboxylic-6,7-dihydroxyphthalide (M4), Z-(6S,7R)-3-butylidene-
4,5,6,7-tetrahydro-11-carboxylic-6,7-dihydroxyphthalide (M5), (6S,7R)-
3-butylidene-4,5,6,7-tetrahydro-11-carboxylic-6,7-dihydroxyphthalide-
11-methyl ether (M6), Z-(6S,7R)-3-butylidene-4,5,6,7-tetrahydro-6,7-
dihydroxyphthalide-7-O-β-D-glucuronide (M7), E-(6S,7R)-3-butylidene-
4,5,6,7-tetrahydro-6,7-dihydroxyphthalide-6-O-β-D-glucuronide (M8),
E-(6S,7R)-3-butylidene-4,5,6,7-tetrahydro-6,7-dihydroxyphthalide-7-O-
β-D-glucuronide (M9), N-(4,5-dihydro-feruoyl)-glycine (M10), N-
(3′-hydroxy-4,5-dihydro-cinnamoyl)-glycine-methyl ether (M11), N-
(4,5-dihydro-5-carbonyl-feruoyl)-glycine (M12), N-(3′-hydroxy-4′-
methoxy-cinnamoyl)-glycine (M13), N-(3′,4′-dimethoxy-cinnamoyl)-
glycine (M14), N-cinnamoyl-glycine-methyl ether (M15), trans-3-
hydroxycinnamoyl acid (M16), and 4-hydroxybenzoic acid (M17).
Among them, eight compounds (M1–M6, M11–M12) are new ones,
and the other 9 known compounds were first isolated as absorptive
−
18 23
C H O10: m/z 399.1291), IR (KBr) νmax 3395.4 (–OH), 1742.8, 1676.3,
−
1
1
260.4, 1112.6 and 1041.0 (C_O) cm . The NMR data were shown in
Table 3.
M8, slight yellow solid, HR–ESI–MS m/z 399.1287 [M–H] (calcd for
−
−
C
1
18 23
H O10: m/z 399.1291), IR (KBr) νmax 3395.4 (–OH), 1742.8, 1676.3,
−
1
260.4, 1112.6 and 1041.0 (C_O) cm . The NMR data were shown in
Table 3.
M9, slight yellow solid, HR–ESI–MS m/z 399.1284 [M–H] (calcd for
−
−
C
1
18 23
H O10: m/z 399.1291), IR (KBr) νmax 3395.4 (–OH), 1742.8, 1676.3,
−
1
260.4, 1112.6 and 1041.0 (C_O) cm . The NMR data were shown in
Table 3.
M10, slight yellow solid, ESI–MS m/z 252.08 [M–H] (calcd for
−
−
C
12
H
14NO
5
: m/z 252.0877). The NMR data were shown in Tables 4
and 5.
M11, slight yellow solid, HR–ESI–MS m/z 260.0896 [M + Na]+
+
(
(
calcd for C12
H
15NO
4
Na : m/z 260.0899), IR (KBr) νmax 3403.9
−
1
–NH−), 1740.3, 1648.5, 1234.2 and 1024.9 (C_O) cm . The
NMR data were shown in Tables 4 and 5.
M12, slight yellow solid, HR–ESI–MS m/z 266.0664 [M–H] (calcd
−
−
for C12
1
6
H12NO : m/z 266.0670), IR (KBr) νmax 3395.4 (–OH), 1742.8,
−
1
676.3, 1260.4, 1112.6 and 1041.0 (C_O) cm . The NMR data were
shown in Tables 4 and 5.
Table 1
1
13
a
H NMR (δ) and C NMR data (δ) for M1, M2 and M3 in MeOD, δ in ppm, J in Hz .
No.
δ
H
δ
C
Senkyunolide R
M1
M2
M3
Senkyunolide R
M1
M2
M3
1
3
170.25
149.1
155.5
18.21
25.08
71.02
65.44
127.8
115.70
68.69
169.3
149.3
153.9
16.8
23.7
69.6
169.3
149.3
153.8
16.8
23.7
69.6
168.7
149.1
153.9
19.5
24.2
69.0
3
4
5
6
7
7
8
9
1
a
2.47–2.63 (2H, m)
1.89–2.03 (2H, m)
3.96 (1H, ddd, 5.4, 3.4, 2.4)
4.25 (1H, d, 3.4)
2.50–2.58 (2H, m)
1.92–2.00 (2H, m)
3.97 (1H, d, 2.4)
4.27 (1H, d, 3.6)
2.56–2.61 (2H, m)
1.95–2.05 (2H, m)
3.99 (1H, s)
2.55–2.73 (1H, m, 4a) 2.53–2.55 (1H, m, 4b)
1.98–2.03 (1H, m) 1.84–1.88 (1H, m)
3.81 (1H, dt, 11, 3.5)
4.29 (1H, d, 2.4)
4.45 (1H, d, 3.5)
64.0
64.0
61.8
a
125.4
109.4
35.2
125.4
109.4
35.1
126.6
109.6
35.1
5.35 (1H, d, 8.8)
4.61 (1H, dt, 8.8, 6.4)
1.52–1.63 (1H, m)
5.53 (1H, t, 8.0)
2.42–2.47 (2H, m)
5.55 (1H, t, 7.8)
2.53–2.55 (2H, m)
5.53 (1H, t, 8.0)
2.53–2.55 (2H, m)
0
1
1.64–1.75 (1H, m)
3.92 (1H, q, 6.4)
1.22 (3H, d, 6.5)
3.94 (1H, q, 6.0)
1.23 (3H, d, 6.6)
3.92 (1H, q, 6.0)
1.22 (3H, d, 6.0)
31.16
9.96
66.5
21.8
66.5
21.8
66.4
21.8
1
0.94 (3H, t, 7.3)
a
13
1
C NMR data (δ) were measured at 125 MHz for M1–M3. H NMR data (δ) were measured at 500 MHz for M1–M3.

180
L. Zhang et al. / Fitoterapia 105 (2015) 177–186
Table 2
1
13
a
H NMR (δ) and C NMR data (δ) for M4, M5 and M6 in MeOD, δ in ppm, J in Hz .
No.
δ
H
δ
C
Senkyunolide H
M4
M5
M6
Senkyunolide H
M4
M5
M6
1
3
3
4
169.2
148.5
153.0
19.1
169.1
148.7
153.9
16.8
168.5
148.6
153.9
19.5
169.3
149.2
154.0
16.7
a
2.36–2.42 (1H, m) 2.62–2.69 (1H, m)
1.79–1.85 (1H, m) 2.10–2.17 (1H, m)
2.50–2.56 (2H, m)
1.92–1.99 (2H, m)
2.58–2.69 (1H, m)
2.52–2.55 (2H, m)
1.91–2.02 (2H, m)
2
.46–2.54 (1H, m)
1.96–2.02 (1H, m)
.84–1.89 (1H, m)
5
26.7
23.7
24.2
23.6
1
6
7
7
8
9
1
4.06 (1H, ddd, 7.9, 3.9, 2.5)
4.61 (1H, d, 3.9)
3.96 (1H, d, 2.5)
4.27 (1H, d, 3.0)
3.79 (1H, dt, 3.5, 5.0)
4.44 (1H, d, 3.0)
3.79 (1H, q, 3.5)
4.27 (1H, d, 2.5)
71.9
68.0
126.3
114.1
28.2
69.7
64.0
125.5
111.0
21.2
68.9
61.7
126.7
111.2
21.2
69.6
64.0
128.3
111.6
21.1
a
5.31 (1H, t, 7.9)
2.36 (2H, dt, 7.9, 7.4)
1.50 (2H, tq, 7.4, 7.4)
5.50 (1H, t, 7.5)
2.68–2.63 (2H, m)
2.63–2.68 (1H, m)
5.48 (1H, t, 7.5)
2.58–2.69 (2H, m)
2.46–2.54 (2H, m)
5.48 (1H, t, 7.5)
2.65–2.69 (2H, q, 7)
2.57–2.59 (2H, m)
0
22.4
32.4
32.5
32.4
2
.50–2.56 (1H, m)
1
1
1
2
0.95 (3H, t, 7.4)
13.7
174.8
175.5
175.1
50.9
3.69 (3H, s)
a
13
1
C NMR data (δ) were measured at 125 MHz for M4–M6. H NMR data (δ) were measured at 500 MHz for M4–M6.
constituents or metabolites of the CR decoction in vivo, the NMR data of
known compounds (M7–M9, M10, M13–M15) are reported here for
the first time.
(Fig. 4). The NOE effect for H-4 (δ2.5, 2H, m) upon irradiation at H-8
(δ5.5, 1H, t, 8.0 Hz) in M1–M3 indicated the Δ3,8-configuration was in
trans. The absolute configurations at C-6 (δ69.6/69.6/69.0) and C-7
(δ64.0/64.0/61.8) were determined by induced CD spectra after adding
7
16 5
M1–M3 showed the molecular formula of C12H O in HRESI–MS,
1
13
and UV λmax at 280 nm. H NMR and C NMR spectra of them were
similar to senkyunolide R (C12 ) [7], except for the branched
2 4
Mo (AcO) (ligand-to-metal ratio: 0.6/0.8) in DMSO (Fig. 5), the 6S- and
H
16
O
5
7R-configurations in both M1 and M2 were determined due to the pos-
itive Cotton effect at 305–310 nm [29], whereas the 6R- and 7S-
configuration in M3 due to its negative Cotton effect at 305–310 nm.
The absolute configuration at C-10 in M1 and M2 were identified by
adding metal chelating agent Rh (OCOCF ) . To eliminate the interfer-
2 3 4
ence of 6,7-dihydroxy groups, the acetonide on C-6 and C-7 of M1 and
M2 were synthesized to obtain two compounds of M1a and M2a, and
chain signals at C-8, C-9, C-10 and C-11 (Table 1), which suggested
that M1–M3 should be similar to senkyunolide R. Basing on the 4 proton
signals in the branched chain of M1–M3 at δ 5.53/5.55/5.53 (H-8, 1H, t,
J = 8.0 Hz), 2.42/2.53/2.53 (H-9, 2H, m), 3.92/3.94/3.92 (H-10, 1H, q,
J = 6.4 Hz) and 1.22/1.23/1.22 (H-11, 3H, d, J = 6.5 Hz), it was
suggested that one hydroxyl group was attached to C-10 (δ 66.5/66.5/
6
6.4) [5,6], which could be proved by the correlations of H8/H9, H9/
then their induced CD spectra were detected after adding Rh
2 3 4
(OCOCF )
1
1
H10, H10/H11 in H– H COSY spectrum, as well as the correlations
between H-9 and C-3 (δ 149.3/149.3/149.1) in HMBC spectrum
(ligand-to-metal ratio: 0.6/0.8) in CHCl . Basing on the positive Cotton ef-
3
fect at 350 nm in the induced CD spectrum of M1a [30,31] and negative
Cotton effect at 350 nm in M2a, 10R-configuration in M1 and 10S-
configuration in M2 were determined. In conclusion, the structures of
M1–M3 were established as Z-(6S,7R,10R)-3-butylidene-4,5,6,7-
(
Fig. 3). Thus, the planar structure of M1–M3 was deduced as 3-
butylidene-4,5,6,7-tetrahydro-6,7,10-trihydroxyphthalide. There were
three chiral centers (C-6, C-7, and C-10) and one double bond (Δ3,8)
in M1–M3. The relative configuration of the 6,7-dihydroxy groups was
deduced to be in cis basing on the J6,7 value of 3.6 Hz which was similar
to that (J6,7 = 3.9 Hz) of senkyunolide H [5,7], as well as the NOESY cor-
relation between H-6 (δ3.97/3.99/3.81) and H-7 (δ4.27/4.29/4.45)
tetrahydro-6,7,10-trihydroxyphthalide
(M1),
Z-(6S,7R,10S)-3-
butylidene-4,5,6,7-tetrahydro-6,7,10-trihydroxyphthalide (M2), and Z-
(6R,7S)-3-butylidene-4,5,6,7-tetrahydro-6,7,10-trihydroxyphthalide
(M3), respectively.
Table 3
1
13
a
H NMR (δ) and C NMR data (δ) for M7, M8 and M9 in MeOD, δ in ppm, J in Hz .
No.
δ
H
δ
C
M7
M8
M9
M7
M8
M9
1
3
169.9
148.7
154.5
16.3
168.2
147.9
153.3
16.8
169.0
148.1
155.0
17.4
3
4
5
a
2.55–2.57 (2H, m)
1.94–2.0 (1H, m) 2.03–2.21 (1H, m)
2.40–2.44 (2H, m)
1.84–1.86 (1H, m)
2.49 (2H)
1.82 (2H, s)
23.1
22.0
22.8
1
.90–1.93 (1H, m)
6
7
7
8
9
1
1
1
2
3
4
5
6
4.27 (1H, s)
4.38 (1H, d, 2.0)
3.88 (1H, s)
4.25 (1H, s)
4.00 (1H, d, 4)
4.24 (1H, d, 4)
66.4
70.9
123.5
113.4
27.7
21.9
12.7
102.6
73.0
76.3
77.6
60.5
125.3
112.3
27.5
21.7
13.7
103.0
73.0
75.9
66.2
69.2
123.1
113.3
27.6
21.7
13.7
102.4
73.1
75.9
a
5.50 (1H, t, 8)
5.48 (1H, t, 8)
2.26 (2H, q, 8)
1.46 (2H, q, 8)
0.91 (3H, t, 8)
4.39 (1H, d, 8)
2.88 (1H, t, 8)
3.15 (1H, t, 8)
3.28 (1H, t, 8)
3.65 (1H, d, 8)
5.56 (1H, t, 8)
2.29 (2H, q, 8)
1.46 (2H, q, 8)
0.92 (3H, t, 8)
4.48 (1H, d, 8)
2.92 (1H, t, 8)
3.15 (1H, t, 8)
3.30 (1H, t, 8)
3.63 (1H, d, 8)
2.37 (2H, q, 7.5)
1.55 (2H, q, 7.5)
0.99 (3H, t, 7.5)
4.68 (1H, d, 7.5)
3.25 (1H, t, 7.5)
3.42 (1H, t, 9)
0
1
′
′
′
′
′
′
3.47 (1H, t, 10)
3.77 (1H, d, 9.5)
71.8
75.0
169.9
71.4
75.7
170.2
71.5
75.8
170.2
a
13
1
C NMR data (δ) were measured at 125 MHz for M7 and at 100 MHz for M8–M9. H NMR data (δ) were measured at 500 MHz for M7 and at 400 MHz for M8–M9.

L. Zhang et al. / Fitoterapia 105 (2015) 177–186
181
Table 4
1
a
H NMR (δ) for M10–M15 in DMSO (M11 and M14 were in MeOD), δ in ppm, J in Hz .
No.
M10
M11
M12
M13
M14
M15
2
4
5
3.99 (2H, s)
3.89 (2H, s)
3.79 (2H, d, 4)
3.87 (2H, s)
3.85 (2H, d, 4.5)
6.55 (1H, d, 15.5)
7.34 (1H, d, 15.5)
7.15 (1H, d, 2)
3.87 (2H, d, 5.6)
6.60 (1H, d, 16)
7.38 (1H, d, 15.6)
7.18 (1H, s)
3.98 (2H, d, 5.6)
2.53 (2H, t, 15.5)
2.87 (2H, t, 15.5)
6.86 (1H, s)
2.50 (2H, t, 6.5)
2.84 (2H, t, 7.5)
6.64 (1H, s)
6.72 (1H, d, 13.2)
7.46 (1H, d, 13.2)
7.60 (1H, s)
2
3
4
5
6
1
3
4
′
′
′
′
7.45 (1H, s)
7.42–7.38 (3H, m)
6.59 (1H, d, 8)
7.06 (1H, t, 7.5)
6.67 (1H, d, 7.5)
3.68 (3H, s)
6.71 (1H, d, 8)
6.66 (1H, d, 8)
6.85 (1H, d, 8)
7.52 (1H, d, 8)
6.80 (1H, d, 8)
7.01 (1H, d, 8.5)
6.99 (1H, d, 8.2)
7.13 (1H, d, 8.2)
′
7.58 (1H, s)
3.65 (3H, s)
-OCH
3
′-OCH
′-OCH
3
3.85 (3H, s)
8.18 (1H, s)
3.81 (3H, s)
3.78 (3H, s)
3.80 (3H, s)
8.27 (1H, s)
3
3.85 (3H, s)
8.07 (1H, s)
CO–NH–
8.45 (1H, t, 5.5)
8.55 (1H, s)
a
1
H NMR data (δ) were measured at 500 MHz for M10 and at 400 MHz for M11–M15.
M4 and M5 showed the same molecular formula of C12
H
14
O
6
and the
UVλmax at 280 nm. The H NMR and C NMR data (Table 2) were sim-
ilar to those of senkyunolide H (C12 ), except for the signals at C-9,
could not be observed due to the little amount of M6. Therefore, the
double bond configuration (Δ3,8) is still uncertain. Based on the above
spectral data, the structure of M6 was established as (6S,7R)-3-
butylidene-4,5,6,7-tetrahydro-11-carboxylic-6,7-dihydroxyphthalide-
11-methyl ether.
1
13
H O
16 4
C-10 and C-11 [5,6]. In place of the methyl signals at δ13.7 (C-11) in
senkyunolide H, there is one carboxyl signal at δ 174.8 (M4)/175.5
(
1
M5). The correlation between H-10 (δ2.63/2.46) and C-11 (δ 174.8/
75.5) in HMBC spectra of M4/M5, indicated that the carboxyl group
M7–M9 had the same molecular formula of C18
NMR spectra of them were similar to those of senkyunolide I/H
(C12 ), except for a group of glucuronosyl signals, including six car-
24
H O10. The UV and
was attached to at C-10 (Fig. 3). Thus, the planar structure of M4 and
M5 were deduced as 3-butylidene-4,5,6,7-tetrahydro-11-carboxylic-
6
16 4
H O
13
bon signals at δ 102.6, 73.01, 76.3, 71.8, 75.0, and 169.9 in C NMR, as
well as five aliphatic proton signals at δ 4.68 (1H, d, J = 7.5 Hz), 3.25
(1H, t, J = 7.5 Hz), 3.42 (1H, t, J = 9 Hz), 3.47 (1H, t, J = 10 Hz) and
,7-dihydroxyphthalide. There were also two chiral centers at C-6 and
C-7 and one double bond (Δ3,8) in the structures of M4 and M5. The J
values of the vicinal coupling between H-6 and H-7 were 3.0 Hz in M4
and M5, indicating that the relative configuration at C-6 and C-7 was
in cis [5,7]. Based on the positive Cotton effect at 305 nm in the induced
CD spectrum of M4 and M5, the absolute configuration of C-6,7 was
established as 6S,7R [29] (Fig. 5). The NOE effect for H-4 upon irradiation
at H-8 in M5 suggested the 3,8-trans configuration. Therefore, the struc-
ture of M4 was identified as E-(6S,7R)-3-butylidene-4,5,6,7-tetrahydro-
1
3.77 (1H, t, J = 9.5 Hz) in H NMR (Table 3). In HMBC spectrum, the pro-
ton signal at δ4.68 (in M7)/4.48 (in M9) of glucuronide moiety were cor-
relative with the carbon signal at δ 70.9/69.2 (C-7), indicating that the
glucuronosyl moiety of M7 and M9 were linked to C-7 (Fig. 3). The conju-
gating position of the glucuronosyl group of M8 was deduced to be at C-6
based on the correlation between C-6 (δ 77.6) and H-1′(4.76) in HMBC
spectrum. Thus, the planar structure of M7 and M9 were deduced as 3-
butylidene-4,5,6,7-tetrahydro-6,7-dihydroxyphthalide-7-O-β-D-
glucuronide; M8 were 3-butylidene-4,5,6,7-tetrahydro-6,7-dihydroxy
phthalide-6-O-β-D-glucuronide. Similarly, the J6,7 of M7–M9 were all
less than 3.9 Hz, indicating the 6,7-cis relative configuration. The NOE
for H-4 upon irradiation at H-8 were observed only in M7 indicating the
Δ3,8-trans configuration in M7 and Δ3,8-cis configuration in M8 and
M9 (Fig. 4). To establish the absolute configuration of C-6 and C-7, 2 mg
of M7–M9 were hydrolyzed by 50 μL of β-glucuronidase (in 5 mL acetic
acid-sodium acetate at 37 °C for 3 h) to obtain their aglycones of M7a,
M8a and M9a, respectively. Then M7a, M8a and M9a were solved in
1
(
1-carboxylic-6,7-dihydroxyphthalide; M5 was elucidated as Z-
6S,7R)-3-butylidene-4,5,6,7-tetrahydro-11-carboxylic-6,7-
dihydroxyphthalide.
1
16 6
M6 showed the molecular formula of C13H O . The UV, H NMR and
13
C NMR spectra were similar to M4/M5, except for one more methyl
signal (δ 4.26, s, 3H), which was correlated with carbonyl carbon signal
at δ 174.8 (C-11) in HMBC spectrum (Fig. 3). Thus, the planar structure
of M6 was deduced as 3-butylidene-4,5,6,7-tetrahydro-11-carboxylic-
6
,7-dihydroxyphthalide-11-methyl ether. Similarly, the relative
configuration of C-6 and C-7 in it was deduced to be in cis basing on
the J values 3.0 Hz of the vicinal signals H-6 and H-7 [5,6], and the abso-
lute configuration was further established as 6R and 7R on the basis of
the positive Cotton effect at 310 nm in the induced CD of M6 [29]
2 4
DMSO and mixed with Mo (AcO) to obtain their induced CD spectrum
[29] (Fig. 5). All of them showed the positive Cotton effect at
305 nm, indicating the 6S- and 7R-configuration for M7–M9. There-
fore, their structures were determined as Z-(6S,7R)-3-butylidene-
(
Fig. 5). It was a pity that the NOE correlation between H-4 and H-8
4
(
,5,6,7-tetrahydro-6,7-dihydroxyphthalide-7-O-β-D-glucuronide
M7), E-(6S,7R)-3-butylidene-4,5,6,7-tetrahydro-6,7-dihydroxy
phthalide-6-O-β-D-glucuronide (M8), and E-(6S,7R)-3-butylidene-
,5,6,7-tetrahydro-6,7-dihydroxyphthalide-7-O-β-D-glucuronide
Table 5
1
3
a
C NMR (δ) for M10–M15 in DMSO (M11 and M14 were in MeOD), δ in ppm, J in Hz .
4
No.
M10
M11
M12
M13
M14
M15
(M9). The possible planar structures of glucuronided senkyunolide I/
H were just deduced on the basis of MS data [28], yet the
glucuronided metabolites of senkyunolide I/H were first identified
here.
1
2
3
4
5
174.4
40.8
171.3
37.7
171.9
41.8
175.8
38.5
171.1
40.7
166.8
46.2
192.4
128.2
111.5
151.9
147.5
114.9
123.7
171.4
40.9
171.3
40.8
170.4
40.1
165.6
118.5
139.5
126.3
115.6
147.8
148.3
110.9
121.6
165.5
119.4
139.2
127.5
110.1
150.1
148.8
111.8
121.3
165.4
121.4
139.4
134.7
127.6
128.9
129.6
128.9
127.6
51.7
M11 showed the molecular formula of C12
max at 230 nm, 280 nm and a characteristic shoulder peak at
320 nm. There are four aromatic protons at δ 6.59 (1H, d, J = 8 Hz),
.64 (1H, s), 6.67 (1H, d, J = 7.5 Hz), and 7.06 (1H, t, J = 7.5 Hz)
which belong to an ABCD system, one methoxyl signal at δ 3.68
3H, s), one methylene signal at δ 3.89 (2H, s) and a pair of triplets
proton signals of a –CH – group at δ 2.50 (2H, t, J = 7.5 Hz)
–CH1
and 2.84 (2H, t, J = 7.5 Hz) in H NMR spectrum (Table 4). According
to the HMBC spectrum, the proton signals of –CH –CH – group
correlated with the aromatic carbon at δ 143.7 (C-1′) and a carbonyl
4
H15NO , as well as UV
31
32.6
λ
1
2
3
4
5
6
1
3
4
′
′
′
′
′
′
132.4
111.7
144.5
147.4
114.8
120.3
143.7
116.2
158.5
114.1
130.4
120.5
52.6
6
(
-OCH
′-OCH
′-OCH
3
2
2
3
55
55.6
55.5
55.4
3
55.5
2
2
a
13
C NMR data (δ) were measured for M10 at 125 MHz and M11–M15 at 100 MHz.

182
L. Zhang et al. / Fitoterapia 105 (2015) 177–186
Fig. 2. The structures of M1–M17. New compounds were marked with *.
carbon at δ 175.8 (C-3), indicated that the –CH
attached between C-1′ and C-3. The methylene proton signal at δ
.89 (H-2) that correlated to the carboxylic carbon signals at δ
2
–CH
2
– group was
175.8 (C-3) and 171.9 (C-1), suggested that the methylene was at-
tached to C-3 and C-1. Basing on the molecular formula of
C H15NO for M11, it was deduced to have one carboxylic group
12 4
3
Fig. 3. Key HMBC and 1H– H COSY correlations of M1–M9.
1

L. Zhang et al. / Fitoterapia 105 (2015) 177–186
183
Fig. 4. Key NOESY correlations of M1–M3, M5, M7 and M9.
Fig. 5. The CD spectra of in situ formed Mo-complexes of M1 (A), M2–M3 (B), M4–M6 (C), deglucuronidation products of M7a–M9a (D) and in situ formed Rh-complexes of M1a–M2a (E).

184
L. Zhang et al. / Fitoterapia 105 (2015) 177–186
and one acyl amino group. The correlation between methoxyl proton
at δ 3.68 (3H, S) and carboxylic carbon at δ 171.9 (C-1) in HMBC
spectrum, indicated the existence of a of carboxylic ester in M11
isolated from Salvia bicolor Desf. growing in Egypt [36] and the roots
of Rheum officinale, respectively [37]. Their structures were identified
by H and C NMR data, which were consistent with those in references
[38,39].
1
13
(
(
Fig. 6). Therefore, the structure of M11 was determined to be N-
3′-hydroxy-4,5-dihydro-cinnamoyl)-glycine-methyl ether.
1
M12 showed the molecular formula of C12
H
5
15NO . In the H NMR
3.2. Discussion
spectrum, three aromatic protons at δ 7.52 (1H, d, J = 8.0 Hz), δ 7.45
1H, s), and δ 6.85 (1H, d, J = 8 Hz) belonging to an ABX system, one
methoxyl signal at δ 3.81 (3H, s) and two methylene signals at δ 3.87
2H, s) and δ 3.79 (2H, d, J = 4 Hz) were observed (Table 4). The struc-
ture of M12 was deduced to possess one carboxylic group and one
acylamino group combining the molecular formula information of
(
As well known, it is usually difficult and incorrect to determine the
exact structures of TCM metabolites only by HPLC-DAD-MS techniques.
WZS-miniature pig is not only similar with human in anatomy and
physiology, but could also provide much more amounts of samples
which will facilitate metabolites isolation and structural elucidation.
Seventeen metabolites (M1–M17), including 8 new compounds
(M1–M6, M11–M12), were isolated and identified from the methanol
extract of drug-containing urine sample of WZS-miniature pigs after
orally administrated CR decoction in this study. M1–M6 were the me-
tabolites derived from the prototype compounds of senkyunolide I/H
under the reactions of hydroxylation, methylation, carboxylation, and
methylation (Fig. 7). M11 was deduced as a methylated and glycine
conjugated product of 3-hydroxy-cinnamoyl acid (M16) after hydroge-
nated in phase-I metabolic reaction. M12 was derived from ferulic acid
by enoltautomerizing after hydroxylation at 4,5 position of ferulic acid
(Fig. 8).
Nine known compounds were first identified as the metabolites
of CR, including three isomers of glucuronide conjugates derived from
senkyunolide I/H (M7, M8, M9) (Fig. 7), four glycine conjugates deriving
from hydrogenated isoferulic acid, isoferulic acid, ferulic acid/caffeic acid,
and cinnamic acid (M10, M13–M15), as well as two prototype com-
pounds of 3-hydroxy-cinnamoyl acid (M16) and 4-hydroxybenzonic
acid (M17) (Fig. 8).
(
12 5
C H15NO . According to the correlations of methylene proton signal at
δ 3.87 with carbonyl carbon signal at δ 192.4 (C-5) and carbonyl carbon
in acylamino group at δ 166.8 (C-3) in HMBC spectrum, it was suggested
that the methylene group was attached to the carbonyl carbon and
2
acylamino carbonyl carbon to form a –CO–CH –CO–NH– group. Similar
to M11, the methylene proton signal at δ 3.79 (H-2) correlated to both
carbonyl carbon signals at δ166.8 (C-3) and 171.1 (C-1) in HMBC spec-
trum, they thus were deduced to form a glycine moiety [32]. The carbon
signal at δ 192.4 (C-5) was also correlated to the aromatic proton signals
at δ 7.45 (H-2′) and δ 7.52 (H-6′), inferring that C-5 was bonded with the
aromatic ring (Fig. 6). Thus, the structure of M12 was determined to be
N-(4,5-dihydro-5-carbonyl-feruoyl)-glycine.
M10 and M13–M15 were identified according to their H and 13C
NMR data (Table 4 and Table 5) as well as their HMBC spectra (Fig. 6).
All of them were the glycine conjugate deriving from hydrogenated
isoferulic acid (M10), isoferulic acid, ferulic acid or caffeic acid (M13–
M14), and cinnamic acid (M15), respectively. M10 could be prepared by
N-(carboxyalkyl) dihydoferul amides and used as antioxidants and tyros-
inase inhibitors [33]. M14 existed in the form of metabolites of eugenol
methyl ethers in rat [34]. M15 was prepared for photo crosslinkable
cinnamic acid-glycosamino glycan derivative [35].
1
It was interesting that glycine conjugated metabolites for phenolic
acids in CR were found to be the main metabolic reaction in miniature
pigs. Although metabolites of glycine conjugation have been reported
in human urine and bile [40,41], this is the first time to report them as
the in vivo metabolites of constituents of CR.
M16 (trans-3-hydroxycinnamoyl acid) and M17 (4-hydroxybenzoic
acid) are two known prototype compounds in CR, which have been
Fig. 6. Key HMBC correlations of M10–M14.

L. Zhang et al. / Fitoterapia 105 (2015) 177–186
185
Fig. 7. The proposed metabolic pathways of senkynolide I/H-derived metabolites of CR decoction in vivo.
According to the structure skeletons of the identified compounds, 7
original compounds in CR, including two phthalides (senkyunolide I/H)
and 5 phenolic acids (ferulic acid, isoferulic acid, caffeic acid, 3-
hydroxy-cinnamoyl acid and 4-hydroxybenzonic acid), were deduced
to be the absorbed active constituents (Figs. 7 and 8). Ligustilides, com-
monly recognized as the major constituents in the essential oil of CR,
were not detected in the CR decoction, therefore, none ligustilide-
derived metabolites were separated from the drug-containing urine sam-
ple. Based on the previous studies [42,43], ligustilides are unstable at high
temperature or under light exposure and could be rapidly transformed
into senkyunolide I/H or their isomers when being decocted for a while.
Senkyunolide H/I were reported to possess the activities of anti-
migraine [44], reducing hydrogen peroxide-induced oxidative damage
in cultured PC12 cells [12] and human liver HepG2 cells [45], and reduc-
ing the morphological damage to red blood cells induced by concanavalin
A [46]. Ferulic acid and caffeic acid showed the antioxidative [47], anticar-
cinogenic [48] and antihypertensive [49] activities. Caffeic acid and
cinnamic acid could ameliorate glucose metabolism in insulin-resistant
mouse hepatocytes [50]. Senkyunolide I/H, and ferulic acid could pene-
trate blood brain barrier in vivo [51,52]. Thus, the phthalides and phenolic
acids in CR, and their metabolites in vivo should be responsible for the
bioactivities of CR decoction. The interactions between the pharmacolog-
ical and pharmacokinetic activity of phthalide-derived metabolites and
phenolic acid-derived metabolites in CR are worth to be further studied.
Fig. 8. The proposed metabolic pathways of ferulic acid, isoferulic acid and 3-hydroxycinnamonyl acid (M16)-derived metabolites of CR decoction in vivo.

186
L. Zhang et al. / Fitoterapia 105 (2015) 177–186
[
[
22] M.Y. Su, T.T. Zhou, M.J. Zhou, The characterization of the metabolites of caffeic acid
4
. Conclusions
n
in rats by LC/MS , Chin. J. Mod. Appl. Pharm. 26 (2009) 501–505.
23] L. Wei, M. Zhou, T. Zhang, W. Hou, L. Wang, J. Xu, Isolation and identification of two
The absorbed constituents and metabolites of CR decoction in vivo
glucuronide conjugates in rats metabolized by caffeic acid, Chin. Tradit. Herb. Drugs
41 (2010) 949–952.
were first studied. Seventeen metabolites, including 8 new ones and 9
known ones, were isolated by column chromatography from the drug-
containing urine samples of WZS-miniature pigs after oral administra-
tion of CR decoction. Their chemical structures were identified by MS,
NMR, and CD information. Deducing from the structures of the identi-
fied metabolites, 7 original constituents in CR, including 2 phthalides
[
[
[
[
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