Sesquiterpenes and neolignans from the roots of Illicium dunnianum

Article abstract of DOI:10.1080/10286020.2012.729507

Two new sesquiterpenes, dunnianoside I (1) and 8-oxo-6-hydroxy- dihydrophaseic acid methyl ester (2), a new glycoside, dunnianoside J (3), two new neolignans, dunnianeolignans A-B (4, 5), together with 10 known compounds (6-15), were isolated from the roots of Illicium dunnianum. The structures of these new compounds were elucidated by spectroscopic methods including 1D and 2D NMR, HR-ESI-MS, and chemical methods. Compounds 7, 8, and 13-15 exhibited potent antioxidant activities against Fe²⁺-cystine-induced rat liver microsomal lipid peroxidation, with IC50 values ranging from 4.2±0.4 to 38.4±2.6M.

Full text of DOI:10.1080/10286020.2012.729507

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Sesquiterpenes and neolignans from the
roots of Illicium dunnianum
Jian Bai a , Hui Chen a , Zhen-Feng Fang a , Shi-Shan Yu a , Shuang-
Gang Ma a , Yong Li a , Jing Qu a , Song Xu a , Jing-Hong Ren a , Hai-
Ning Lü a & Xia Chen a
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College , Beijing ,
100050 , China
Published online: 10 Oct 2012.
To cite this article: Jian Bai , Hui Chen , Zhen-Feng Fang , Shi-Shan Yu , Shuang-Gang Ma , Yong Li ,
Jing Qu , Song Xu , Jing-Hong Ren , Hai-Ning Lü & Xia Chen (2012) Sesquiterpenes and neolignans
from the roots of Illicium dunnianum , Journal of Asian Natural Products Research, 14:10, 940-949,
DOI: 10.1080/10286020.2012.729507
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Journal of Asian Natural Products Research
Vol. 14, No. 10, October 2012, 940–949
Sesquiterpenes and neolignans from the roots of Illicium dunnianum
Jian Bai, Hui Chen, Zhen-Feng Fang, Shi-Shan Yu*1, Shuang-Gang Ma, Yong Li, Jing Qu,
Song Xu, Jing-Hong Ren, Hai-Ning Lu¨ and Xia Chen
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China
(Received 6 July 2012; final version received 9 September 2012)
Two new sesquiterpenes, dunnianoside I (1) and 80-oxo-6-hydroxy-dihydrophaseic
acid methyl ester (2), a new glycoside, dunnianoside J (3), two new neolignans,
dunnianeolignans A–B (4, 5), together with 10 known compounds (6–15), were
isolated from the roots of Illicium dunnianum. The structures of these new compounds
were elucidated by spectroscopic methods including 1D and 2D NMR, HR-ESI-MS,
and chemical methods. Compounds 7, 8, and 13–15 exhibited potent antioxidant
activities against Fe2þ-cystine-induced rat liver microsomal lipid peroxidation, with
IC50 values ranging from 4.2 ^ 0.4 to 38.4 ^ 2.6 mM.
Keywords: Illicium dunnianum; Illiciaceae; sesquiterpene; neolignan; glycoside;
antioxidant activities
1. Introduction
ester (2), a new glycoside, dunnianoside J
(3), two new neolignans, dunnianeolignans
A–B (4, 5) (Figure 1), together with 10
known compounds including six neolignans
(6–11) and four lignans (12–15), were
isolated. Inthisstudy, we reportthe isolation
and structural elucidation of these new
compounds. The antioxidant activities of
compounds 1 and 4–15 are also reported.
The genus Illicium (Illiciaceae) is a rich
source of sesquiterpenes [1–3], neolignans
[4,5], and prenylated C6–C3 compounds
[6,7]. Illicium dunnianum is a toxic shrub
distributed in southern China and used as a
folk medicine for relieving pain and treating
rheumatism [8]. In our previous paper, 12
sesquiterpenes including 6 allo-cedrane
sesquiterpenes, 4 seco-prezizaane-type ses-
quiterpenes, and 2 monocyclofarnesane
sesquiterpenes with anti-inflammatory
activities were isolated from the roots of
this species [9]. As part of our continuing
search for novel bioactive compounds, we
investigated the chemical constituents of the
roots of I. dunnianum. As a result, two new
sesquiterpenes including a cuparane sesqui-
terpene glycoside, dunnianoside I (1), and a
monocyclofarnesane sesquiterpene, 80-oxo-
6-hydroxy-dihydrophaseic acid methyl
2. Results and discussion
The dry and powdered roots of
I. dunnianum were successively extracted
with EtOH–H2O (95:5, v/v) and EtOH–
H2O (70:30, v/v). The total EtOH extract
was absorbed by diatomite, and then
successively extracted with petroleum
ether (60–908C), CHCl3, EtOAc, and
MeOH. The CHCl3 and EtOAc extracts
were subjected to column chromatography
(CC) on silica gel, Sephadex LH-20,
*Corresponding author. Email: yushishan@imm.ac.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.729507
http://www.tandfonline.com

Journal of Asian Natural Products Research
941
Figure 1. The structures of compounds 1–5.
octadecyl silane (ODS), and preparative
reversed-phase HPLC, respectively, to
afford compounds 1–15.
129.9, and 127.9), 1 oxymethine (dC 77.3), 2
methylenes (dC 48.6 and 45.8), three
methyls (dC 26.9, 24.8, and 25.6), and two
quaternary carbons (dC 51.5 and 45.2). The
1H NMR spectroscopic data exhibited a set
of AA0BB0 couplings assignable to a
symmetrical 1, 4-disubstituted aromatic
ring at dH 7.96 (2H, d, J ¼ 8.5 Hz, H-8,
10) and 7.50 (2H, d, J ¼ 8.5 Hz, H-7, 11),
three methyl groups at dH 0.55 (3H, s,
H3-12), 1.17 (3H, s, H3-13), and 1.45 (3H, s,
H3-14), two methylene groups at dH 2.99
(1H, dd, J ¼ 14.5, 9.0 Hz, H-5a), 2.03 (1H,
dd, J ¼ 14.5, 2.5 Hz, H-5b), and dH
1.96–2.00 (2H, m, H-3). The 1H–1H
COSY spectrum showed the presence of a
CH2(3)–CH(4)–CH2(5) unit (Figure 2).
The NMR spectroscopic data of 1 were
found to be similar to those of b-cuparenol
[11,12], exceptforthepresenceofaglucosyl
moiety and a conjugated carbonyl group,
and the absence of a methyl group relative to
b-cuparenol. This indicated that the C-15
methyl group in b-cuparenol was replaced
by a carboxy group in 1 and the glucosyl
moiety was attached to C-4 in 1, which were
confirmed by HMBC correlations of H-8/C-
15, H-10/C-4, and H-4/C-10 (Figure 2).
Compound 1 was obtained as a white
amorphous powder, and its molecular
formula was deduced to be C21H30O8 by a
positive HR-ESI-MS ion at m/z 433.1843
[M þ Na]þ, which was supported by 13C
NMR and distortionless enhancement by
polarization transfer (DEPT) spectroscopic
data. The IR spectrum indicated the
presence of hydroxy (3333 cm21), carbonyl
(1693 cm21), and aromatic (1611, 1513, and
1454cm21) groups. The 1H and 13C NMR
spectroscopic data (Table 1) showed six
carbon resonances at dC 103.2, 77.9, 77.4,
74.8, 71.8, and 63.0, and an anomeric proton
signal at dH 4.35 (1H, d, J ¼ 8.0 Hz),
indicatingthepresenceofa glucosylmoiety.
The enzymatic hydrolysis of 1 afforded 1a
and a glucose, which was confirmed by TLC
with an authentic sample of glucose. The
large coupling constant (8.0Hz) of the
anomeric proton at dH 4.35 (H-10) suggested
that the glucose was in the b-configuration.
GC analysis established the D-configuration
of the glucose [10].
In addition, the 13C NMR and DEPT
spectra displayed 15 carbonresonances for a
conjugated carbonyl (dC 167.6), 6 aromatic
carbons (dC 153.4, 127.9, 129.9, 128.7,
In addition, the relative configuration
of 1 was elucidated by analysis of the
NOESY spectrum. NOE correlations

942
J. Bai et al.
Table 1. 13C and 1H NMR spectroscopic data for compounds 1, 2, and 3.
1a
2a
3b
Position
dC
d
H (J in Hz)
dC
d
H (J in Hz)
dC
dH (J in Hz)
1
2
3
4
51.5
45.2
48.6
77.3
167.1
115.9
153.7
129.6
170.2
116.4
155.7
71.5
6.15 s
6.06 br s
1.96–2.00 m
4.62 m
7.90 d (16.5)
6.56 d (16.5)
4.51 s
Ha 4.39 d (15.0)
Hb 4.16 d (15.0)
2.09 s
5
45.8 Ha 2.99 dd (14.5, 9.0) 131.3
Hb 2.03 dd (14.5, 2.5)
16
6
7
8
153.4
127.9
129.9
128.7
129.9
127.9
26.9
24.8
25.6
167.6
103.2
74.8
62.3
7.50 d (8.5)
7.96 d (8.5)
9
10
11
12
13
14
15
10
7.96 d (8.5)
7.50 d (8.5)
0.55 s
1.17 s
1.45 s
4.35 d (8.0)
3.18 dd (8.5, 8.0)
3.40 dd (8.5, 8.5)
88.2
82.5
103.7
75.1
4.30 d (7.8)
3.24 dd (9.0, 7.8)
3.36 t (9.0)
20
30
77.9
42.6 Hax 1.92 dd (14.0, 10.0) 78.1
Heq 2.25 dd (14.0, 7.0)
64.8
41.2 Hax 1.79 dd (13.5, 11.0) 78.1
Heq 1.89 dd (13.5, 7.0)
40
50
71.8
77.4
3.35 dd (8.5, 8.5)
3.32 m
Hax 3.87 m
71.6
3.28 overlap
3.27 overlap
60
63.0 Ha 3.84 dd (11.5, 2.5) 52.9
Hb 3.67 dd (11.5, 5.5)
62.8 Ha 3.87 dd (12.0, 1.8)
Hb 3.66 dd (12.0, 5.4)
70
80
18.5
178.5
14.6
1.36 s
90
1-OMe
1.09 s
3.68 s
51.3
a Measured in acetone-d6 at 500 MHz for proton and 125 MHz for carbon.
b Measured in methanol-d4 at 600 MHz for proton and 150 MHz for carbon.
(Figure 2) of H-4 with H-5a, H-7 and/or H-
11, and H3-12, and of H3-14 with H-5b and
H3-13 suggested that H-4 is trans to the
Me-14 in 1. The absolute configuration of
1 was determined by analysis of the
Rh2(OCOCF3)4-induced CD spectrum of
1a [13]. On the basis of the bulkiness rule
for secondary alcohols [13], a negative
Cotton effect at 350 nm indicated the 4R
configuration for 1. According to the
above relative configuration, the absolute
configuration for C-1 was determined as S.
Therefore, the structure of 1 was eluci-
dated as p-[(1S,4R)-1, 2, 2-trimethyl-4-O-
b-D-glucopyranosyl-cyclopentyl] benzoic
acid, named dunnianoside I (1).
1
Figure 2. Key HMBC ( ! ), H–1H COSY (—), and NOESY ( $ ) correlations of 1.

Journal of Asian Natural Products Research
943
Compound 2 was isolated as a color-
less amorphous solid with the molecular
formula of C16H22O7 determined by
positive HR-ESI-MS (m/z 349.1259
[M þ Na]þ). The characteristic absorption
bands of hydroxy (3437 cm21), carbonyl
spectroscopic data (Table 1) showed signals
for an olefinic proton at dH 6.06 (1H, br s, H-
2), a set of oxygen-bearing isolated methyl-
enes at dH 4.39 (1H, d, J ¼ 15.0 Hz, H-4a)
and 4.16 (1H, d, J ¼ 15.0 Hz, H-4b), a methyl
at dH 2.09 (3H, s, H3-5), and an anomeric
proton at dH 4.30 (1H, d, J ¼ 7.8 Hz, H-10).
Acid hydrolysis of 3afforded a glucose which
was confirmed by TLC with an authentic
sample of glucose, and the D-configuration
was determined by GC analysis [10]. The b-
(1763 cm21),
(1676 cm21), and olefinic (1638 cm21
groups were observed in the IR spectrum.
conjugated
carbonyl
)
1
The H NMR spectrum (Table 1) showed
signals for two tertiary methyls [dH 1.09
(3H, s, H3-90) and 1.36 (3H, s, H3-70)], a
methoxy [dH 3.68 (3H, s, 1-OCH3)], three
methylenes including an oxygen-bearing
isolated methylene [dH 1.92 (1H, dd,
J ¼ 14.0, 10.0 Hz, H-30ax) and 2.25 (1H,
dd, J ¼ 14.0, 7.0 Hz, H-30eq); dH 1.79 (1H,
dd, J ¼ 13.5, 11.0 Hz, H-50ax) and 1.89
(1H, dd, J ¼ 13.5, 7.0 Hz, H-50eq); dH 4.51
(2H, s, H2-6)], an oxymethine [dH 3.87
(1H, m, H-40)], and three olefinic protons
[dH 6.15 (1H, s, H-2), 6.56 (1H, d,
J ¼ 16.5 Hz, H-5), and 7.90 (1H, d,
J ¼ 16.5 Hz, H-4)]. The 13C NMR spec-
trum (Table 1) displayed 16 carbon signals
including four olefinic (dC 115.9, 153.7,
129.6, and 131.3) and two carbonyl (dC
167.1 and 178.5) carbons. According to
the above features, the NMR spectroscopic
data of 2 were very similar to those of 80-
oxo-6-hydroxy-dihydrophaseic acid [9],
except for the presence of an additional
methoxy group [dH 3.68 (3H, s, 1-OCH3)],
which indicated that the C-1 hydroxy in 80-
oxo-6-hydroxy-dihydrophaseic acid was
replaced by a methoxy group in 2. This
was confirmed by HMBC correlation of
the methoxy group proton at dH 3.68 with
the carbonyl carbon C-1 ((C 167.1). Thus,
2 was determined to be 80-oxo-6-hydroxy-
dihydrophaseic acid methyl ester (2).
anomeric configuration for the glucose was
3
0
0
deduced from its large J1 ;2 coupling
constant (J ¼ 7.8 Hz). Except for the glucosyl
moiety, the 13C NMR spectrum displayed five
carbon signals including a conjugated carbo-
nyl (dC 170.2) and two olefinic (dC 116.4 and
155.7) carbons. Furthermore, HMBC corre-
lations of H-2/C-1, C-3, and C-4; H3-5/C-2,
C-3, and C-4; H2-4/C-10, C-2, C-3, and C-5;
and H-10/C-4 indicated that the aglycone of 3
was 4-hydroxy-3-methylbut-2-enoic acid and
the glucosyl moiety was attached to C-4. In
the NOE spectrum, irradiation of H-2
enhanced H2-4 and H-10 resonances, whereas
irradiation of H3-5 enhanced H2-4 resonance,
revealing that the olefin moiety was in the E
configuration. Consequently, 3 was estab-
lished as (E)-4-O-b-D-glucopyranosyl-3-
methylbut-2-enoic acid and designated as
dunnianoside J (3).
Compound 4 was assigned the mol-
ecular formula C25H32O10 by a positive
HR-ESI-MS ion at m/z 515.1886
[M þ Na]þ. The IR spectrum indicated
the presence of hydroxy (3419 cm21),
carbonyl (1744 cm21), and aromatic
(1598, 1503, and 1460 cm21) groups. The
1H NMR spectroscopic data (Table 2)
showed the presence of a symmetrical 10,
30, 40, 50-tetrasubstituted aromatic ring [dH
6.81 (2H, s, H-20, 60)], an asymmetrical 1,
3, 4, 5-tetrasubstituted aromatic ring [dH
6.74 (2H, br s, H-4, 6)], four methoxys [dH
3.82 (6H, s, 30, 50-OCH3), 3.86 (3H, s,
7-OCH3), 3.73 (3H, s, 100-OCH3)], an oxy-
bearing methylene [dH 3.81 and 3.91 (each
1H, m, H-3a)], and a hydroxypropyl group
[dH 2.62 (2H, t, J ¼ 8.0 Hz, H-5a), 1.79
Compound 3 was isolated as a white
amorphous powder. Its molecular formula
was deduced to be C11H18O8 by a positive
HR-ESI-MS ion at m/z301.0899 [M þ Na]þ.
The IR spectrum displayed absorptions
ascribable to hydroxy (3348 cm21), conju-
gated carbonyl (1698 cm21), and olefinic
(1652 cm21
)
groups. The 1H NMR

944
J. Bai et al.
1
Table 2. 13C and H NMR spectroscopic data for compounds 4 and 5.
4a
5b
Position
dC
dH (J in Hz)
dC
dH (J in Hz)
2
3
3a
88.0
55.2
64.8
5.57 d (6.5)
3.53 m
3.91 m
3.81 overlap
6.74 br s
88.5
55.6
65.1
5.55 d (6.0)
3.45 m
3.85 overlap
3.76 overlap
6.72 br s
4
4a
5
5a
5b
5c
6
117.6
129.7
136.6
32.7
36.0
61.8
114.0
144.9
147.2
139.8
104.0
153.9
136.0
153.9
104.0
170.6
83.2
117.9
129.6
137.1
32.9
35.8
62.2
114.2
145.3
147.5
138.4
111.3
151.4
148.0
117.8
119.1
171.9
80.8
2.62 t (8.0)
1.79 m
3.56 t (7.0)
6.74 br s
2.63 t (8.0)
1.81 m
3.56 t (6.5)
6.73 br s
7
7a
10
20
6.81 s
6.81 s
7.04 s
30
40
500
6.90 overlap
6.90 overlap
600
100
2
4.45 t (4.0)
3.76 m
3.86 s
3.82 s
3.82 s
3.73 s
4.77 t (4.0)
3.96 m
3.86 s
300
63.1
56.5
56.6
56.6
63.7
56.7
56.5
7-OMe
30-OMe
50-OMe
100-OMe
3.83 s
52.0
52.6
3.74 s
a Measured in acetone-d6 at 500 MHz for proton and 125 MHz for carbon.
b Measured in methanol-d4 at 500 MHz for proton and 125 MHz for carbon.
(2H, m, H-5b), 3.56 (2H, t, J ¼ 7.0 Hz, H-
5c)]. The 13C NMR spectrum displayed 25
carbon resonances including a carbonyl
(dC 170.6), 12 aromatic (dC 117.6, 129.7,
136.6, 114.0, 144.9, 147.2, 139.8, 104.0,
153.9, 136.0, 153.9, and 104.0), and four
methoxy (dC 52.0, 56.6, 56.5, and 56.6)
carbon signals. All these findings indicated
that 4 was a dihydrobenzofuran neolignan
[14]. The NMR spectroscopic data of 4
were very similar to those of 200-[10-
[(2R,3S)-2,3-dihydro-3-hydroxymethyl-5-
(5c-hydroxyl propyl)-7-methoxy-2-benzo-
furanyl]-30,50-dimethoxy-40-phenoxy]-300-
hydroxy-propanoic acid butyl ester [14],
except that a methoxy group instead of an
n-butyl alcohol moiety was located at C-
100. This was confirmed by HMBC
correlation between the methoxy proton
(dH 3.73) and the carbonyl carbon C-100 (dC
170.6).
In addition, clear NOE correlation was
observed between H-2 and H2-3a in the
NOE experiment, indicating that H-2 and
H-3 were trans. The CD spectrum of 4
exhibited negative Cotton effects at 240
and 296 nm, which were very similar to
those of a synthetic compound 30, 4-di-O-
methylcedrusin [15], suggesting that 4
possesses a 2R, 3S configuration. The
absolute configuration for C-200 was
uncertain. According to the above anal-
ysis, 4 was elucidated as 200-{10-[(2R,3S)-2,
3-dihydro-3-hydroxymethyl-5-(3-hydro-
xypropyl)-7-methoxy-2-benzofuranyl]-30,
50-dimethoxy-40-phenoxy}-300-hydroxy-
propanoic acid methyl ester, named
dunnianeolignan A (4).

Journal of Asian Natural Products Research
945
The molecular formula of compound 5
was determined as C24H30O9 by positive
HR-ESI-MS (m/z 485.1790 [M þ Na]þ).
The NMR spectroscopic data of 5 were
similar to those of 4 except for the absence
of an aromatic methoxy group. Moreover,
Table 3. Antioxidant activities of compounds
7, 8, and 12–15a.
Compound
IC50 (mM)
7
8
12
13
14
4.6 ^ 0.5
8.5 ^ 0.8
38.4 ^ 2.6
14.7 ^ 1.2
10.2 ^ 1.1
4.2 ^ 0.4
23.4 ^ 2.5
1
the H NMR spectrum (Table 2) showed
signals for a 10, 30, 40-trisubstituted
aromatic ring [dH 6.90 (2H, overlap, H-
50, 60), 7.04 (1H, br s, H-20)] in 5 instead of
a symmetrical 10, 30, 40, 50-tetrasubstituted
aromatic ring in 4, indicating that 5 was
the 50-demethoxy derivative of 4. NOE
correlation of H-2 with H2-3a indicated
that H-2 and H-3 were trans. The CD
spectrum of 5 showed negative Cotton
effects at 241 and 290 nm, which were
similar to those of 4, suggesting that the
absolute configurations for C-2 and C-3 of
5 were identical to those of 4. Thus, 5 was
established as 200-{10-[(2R, 3S)-2,3-dihy-
dro-3-hydroxymethyl-5-(3-hydroxypro-
pyl)-7-methoxy-2-benzofuranyl]-30-meth-
oxy-40-phenoxy}-300-hydroxy-propanoic
acid methyl ester and named dunnianeo-
lignan B (5).
15
Vitamin Eb
a Data are mean ^ standard deviation (n ¼ 3). The
other compounds were inactive (IC50 . 50 mM).
b Positive control.
as the positive control. As shownin Table 3,
compounds 7, 8, and 13–15 exhibited more
significant antioxidant activities than the
positive control vitamin
E
(IC50
23.4 ^ 2.5 mM), with the IC50 values
ranging from 4.2 ^ 0.4 to 14.7 ^ 1.2 mM.
Compound 12 showed moderate antiox-
idant activity with the IC50 value of
38.5 ^ 2.6 mM, and the other tested com-
pounds were inactive (IC50 . 50 mM).
3. Experimental
The 10 known compounds including
six neolignans, dihydrodehydrodiconiferyl
alcohol (6) [16], (–)-dehydrodiconiferyl
alcohol (7) [17], simulanol (8) [18],
sakuraresinol (9) [19], 2, 3-dihydro-2-[30-
methoxy-40-(100, 300-dihydroxy-200-propy-
loxy)phenyl]-3-hydroxymethyl-7-meth-
oxy-5-benzofuranpropanol (10) [14], and
2,3-dihydro-2-(40-hydroxy-30-methoxy-
phenyl)-3-hydroxymethyl-7-methoxy-5-
(3-O-a-rhamnopyranosylpropan)benzo-
furan (11) [20], and four lignans, (–)-
secoisolariciresinol (12) [21], (–)-matair-
esinol (13) [22], aviculin (14) [23], and
(–)-secoisolariciresinol-O-a-L-rhamno-
pyranoside (15) [24] were identified by the
comparison of their NMR spectroscopic
data with those reported in the literature,
respectively.
3.1 General experimental procedures
Optical rotations were taken on a JASCO
P-2000 automatic digital polarimeter.
UV spectra were measured on a JASCO
V650 spectrophotometer. CD spectra were
measured on a JASCO J-815 spectrometer.
IR spectra were recorded on a Nicolet 5700
FT-IR spectrometer. NMR spectra were
recorded on an INOVA-500 spectrometer.
ESI-MS was measured on an Agilent 1100
Series LC/MSD ion trap mass spec-
trometer. HR-ESI-MS data were recorded
on an Agilent Technologies 6250 Accu-
rate-Mass Q-TOF LC/MS spectrometer.
Preparative HPLC was carried out on a
Shimadzu LC-6AD instrument with an
SPD-10A detector, using a YMC-Pack
ODS-A column (250 £ 20 mm, 5 mm). GC
data were recorded on an Agilent 7890A
instrument with an FID detector. Sephadex
LH-20 (Amersham Pharmacia Biotech
AB, Uppsala, Sweden), ODS (45–70 mm,
Compounds 1 and 4–15 were tested for
their antioxidant activities against rat liver
microsomal lipid peroxidation induced by
Fe2þ-cystine in vitro. Vitamin E was used

946
J. Bai et al.
Merck, Darmstadt, Germany), silica gel
(160–200, 200–300 mesh, Qingdao Mar-
ine Chemical Co., Ltd, Qingdao, China),
and diatomite (Sinopharm Chemical
Reagent Co., Ltd, Beijing, China) were
used for CC. TLC was carried out with
glass precoated with silica gel GF254 plates
(Qingdao Marine Chemical Co., Ltd,
Qingdao, China). Spots were visualized
under UV light or by spraying with 10%
H2SO4 in EtOH–H2O (95:5, v/v) followed
by heating.
MeOH–H2O (10:90 ! 100:0) to give
fractions 4-1 –C4-20 Fraction C4-7
C
.
(85 mg) was purified by preparative
HPLC using CH3CN–H2O (40:60) to
yield 13 (25.0 mg). Fraction C7 (2.8 g)
was subjected to an ODS CC, eluted with a
gradient of MeOH–H2O (10:90 ! 100:0)
to give fractions C7-1–C7-10. Fraction C7-4
(235 mg) was separated by preparative
HPLC using CH3CN–H2O (60:40) to
yield 12 (51.0 mg),
8 (9.0 mg), 6
(95.0 mg), and 7 (75.0 mg). Fraction C9
(6.0 g) was subjected to an ODS CC,
eluted with a gradient of MeOH–H2O
3.2 Plant material
(10:90 ! 100:0) to give fractions C9-1
–
9-15. Fraction C9-9 (85 mg) was purified
The roots of I. dunnianum were collected
from Guilin, Guangxi Province of China,
in November 2009, and identified by Prof.
Song-Ji Wei of Guang Xi College of
Traditional Chinese Medicine. A voucher
specimen (No. ID-S-2328) has been
deposited in the herbarium of the Depart-
ment of Medicinal plants, Institute of
Materia Medica, Chinese Academy of
Medical Sciences.
C
by preparative HPLC using CH3CN–H2O
(28:72) to yield 9 (17.0 mg).
The EtOAc extract (110 g) was sub-
jected to a silica gel CC (40 £ 8 cm, 160–
200 mesh) eluted with CHCl3 –MeOH
(50:1, 20:1, 10:1, 5:1, 1:1, 0:1, v/v) to
afford nine fractions E1–E9. Fraction E4
(4.5 g) was subjected to an ODS CC eluted
with
(10:90 ! 100:0) to yield fractions E4-1
4-9. Fraction E4-8 (170 mg) was subjected
a
gradient of MeOH–H2O
–
E
3.3 Extraction and isolation
to a Sephadex LH-20 CC eluted with
CH2Cl2–MeOH (50:50) to afford fractions
The roots of I. dunnianum (7.5 kg) were
air-dried, ground, and extracted three
times (2 h for each time) with EtOH–
H2O (3 £ 80 liters, 95:5, v/v) under con-
ditions of reflux (90–958C), and the
residue was refluxed with EtOH–H2O
(2 £ 64 liters, 70:30, v/v). The combined
EtOH extract was evaporated to near
dryness under reduced pressure to give
the crude extract (850 g), which was
absorbed by diatomite, and then succes-
sively extracted with petroleum ether (60–
908C) (10 liters), CHCl3 (10 liters), EtOAc
(10 liters), and MeOH (10 liters). The
CHCl3 extract (49 g) was subjected to a
silica gel CC (40 £ 7 cm, 160–200 mesh),
eluted with CH2Cl2–MeOH (100:1, 50:1,
20:1, 10:1, 5:1, 2:1, v/v) to yield nine
fractions C1–C9 on the basis of TLC
analysis. Fraction C4 (8.0 g) was subjected
to an ODS CC, eluted with a gradient of
E
4-8-1–E4-8-4. Fraction E4-8-1 (37 mg) was
purified by preparative HPLC using
CH3CN–H2O (21:79) to yield 10
(12.0 mg). Fraction E4-9 (156 mg) was
separated by preparative HPLC using
CH3CN–H2O (30:70) to yield 4 (7.5 mg).
Fraction E5 (8.4 g) was subjected to an
ODS CC eluted with a gradient of MeOH–
H2O (10:90 ! 100:0) to give 18 fractions
E
5-1–E5-18. Fraction E5-9 (140 mg) was
separated by preparative HPLC using
0.1% TFA in CH3CN–H2O (14:86) to
yield 2 (5.9 mg). Fraction E5-10 (3.1 g) was
subjected to a Sephadex LH-20 CC eluted
with
a
gradient of MeOH–H2O
(50:50 ! 100:0) to afford fractions E5-10-
1–E5-10-20. Fraction E5-10-6 (355 mg) was
separated by preparative HPLC using
0.1% TFA in CH3CN–H2O (18:82) to
yield 14 (6.2 mg), 11 (9.0 mg), 15

Journal of Asian Natural Products Research
947
(32.0 mg), 1 (14.0 mg), and 5 (4.7 mg).
Fraction E7 (20.5 g) was subjected to an
ODS CC eluted with a gradient of MeOH–
H2O (10:90 ! 100:0) to yield fractions
CD3OD) spectral data, see Table 1;
ESI-MS: m/z 279 [M þ H]þ, 301
[M þ Na]þ, 317 [M þ K]þ, 277
[M 2 H]2, 555 [2M 2 H]2; HR-ESI-
MS: m/z 301.0899 [M þ Na]þ (calcd for
C11H18O8Na, 301.0894).
E
7-1–E7-20. Fraction E7-7 (47 mg) was
purified by preparative HPLC using 0.1%
TFA in MeOH–H2O (10:90) to yield 3
(19.5 mg).
3.3.4 Compound 4
20
Syrup; ½aꢀD þ 31.8 (c 0.004, MeOH); UV
3.3.1 Compound 1
(MeOH) lmax (log 1) 204 (5.33), 237
(4.67), 281 (4.15) nm; CD (MeOH)
D1240 nm 27.20, D1296 nm 22.48; IR
(KBr) nmax 3419, 2939, 2881, 1744,
1598, 1503, 1460, 1423, 1330, 1215,
1126, 961, 834 cm21; for 1H NMR
(500 MHz, acetone-d6) and 13C NMR
(125 MHz, acetone-d6) spectral data, see
Table 2; ESI-MS: m/z 515 [M þ Na]þ,
531 [M þ K]þ, 527 [M þ Cl]2; HR-ESI-
MS: m/z 515.1886 [M þ Na]þ (calcd for
C25H32O10Na, 515.1888).
20
White amorphous powder; ½aꢀD 264.2 (c
0.083, MeOH); UV (MeOH) lmax (log 1)
203 (4.22), 240 (4.06), 279 (2.91) nm; IR
(KBr) nmax 3333, 2960, 1693, 1611, 1513,
1454, 1413, 1380, 1318, 1272, 1202, 1025,
926, 849, 780, 715 cm21; for 1H NMR
(500 MHz, acetone-d6) and 13C NMR
(125 MHz, acetone-d6) spectral data, see
Table 1; ESI-MS: m/z 433 [M þ Na]þ,
449 [M þ K]þ, 409 [M 2 H]2, 445
[M þ Cl]2, 819 [2M 2 H]2; HR-ESI-
MS: m/z 433.1843 [M þ Na]þ (calcd for
C21H30O8Na, 433.1833).
3.3.5 Compound 5
20
Syrup; ½aꢀD þ 6.6 (c 0.077, MeOH); UV
3.3.2 Compound 2
(MeOH) lmax (log 1) 207 (4.16), 229
(3.72), 280 (3.25) nm; CD (MeOH) lmax
(D1) 240 (20.88), 290 (20.28); IR (KBr)
Colorless amorphous solid; ½aꢀ2D0 234.2 (c
0.04, MeOH); UV (MeOH) lmax 206
(3.62), 261 (3.82) nm; IR (KBr) nmax 3437,
2943, 1763, 1701, 1676, 1638, 1618, 1508,
1459, 1438, 1379, 1237, 1201, 1052, 938,
n
max 3385, 2940, 1744, 1681, 1607, 1513,
1455, 1266, 1208, 1139, 803, 722 cm21
;
for H NMR (500 MHz, CD3OD) and 13C
NMR (125 MHz, CD3OD) spectral data,
see Table 2; ESI-MS: m/z 485 [M þ Na]þ,
501 [M þ K]þ, 497 [M þ Cl]2; HR-ESI-
MS: m/z 485.1790 [M þ Na]þ (calcd for
C24H30O9Na, 485.1782).
1
893 cm21
;
for 1H NMR (500 MHz,
acetone-d6) and 13C NMR (125 MHz,
acetone-d6) spectral data, see Table 1;
ESI-MS: m/z 349 [M þ Na]þ, 361
[M þ Cl]2, 439 [M þ CF3COO]2, 651
[2M 2 H]2; HR-ESI-MS: m/z 349.1259
[M þ Na]þ (calcd for C16H22O7Na,
349.1258).
3.4 Enzymatic hydrolysis of 1
Compound 1 (5 mg) and cellulose (15 mg)
were dissolved in H2O (1.5 ml) and were
stirred at 508C for 12 h. The reaction
mixture was extracted with EtOAc. The
EtOAc extract was evaporated to give 1a
(2.8 mg) and the aqueous layer was
3.3.3 Compound 3
20
White amorphous powder; ½aꢀD 242.9 (c
0.1, MeOH); UV (MeOH) lmax (log 1) 214
(3.91) nm; IR (KBr) nmax 3348, 2929,
1698, 1652, 1435, 1375, 1262, 1080, 954,
867 cm21
;
for 1H NMR (500 MHz,
concentrated in vacuo to afford a
monosaccharide.
CD3OD) and 13C NMR (125 MHz,

948
J. Bai et al.
3.4.1 Compound 1a
gradient: 1008C for 2 min, 1008C !
20
2808C (108C/min), 2808C for 5 min. The
same procedure was applied to the
authentic sample. By comparison with
retention time of the authentic sample
White amorphous solid; ½aꢀD 229.9 (c
0.23, MeOH); 1H NMR (500 MHz,
CDCl3): dH 8.01 (2H, d, J ¼ 8.4 Hz, H-8,
10), 7.41 (2H, d, J ¼ 8.4 Hz, H-7, 11), 4.61
(1H, m, H-4), 2.99 (1H, dd, J ¼ 14.5,
8.5 Hz, H-5a), 2.06 (1H, dd, J ¼ 14.5,
8.5 Hz, H-5b), 1.83 (2H, m, H-3), 1.49
(3H, s, H3-14), 1.21 (3H, s, H3-13), 0.55
(3H, s, H3-12); 13C NMR (125 MHz,
CDCl3): dC 170.6 (C-15), 153.4 (C-6),
129.6 (C-8, 10), 127.0 (C-7, 11), 126.6 (C-
9), 70.2 (C-4), 51.2 (C-1), 50.8 (C-3), 47.4
(C-5), 45.2 (C-2), 26.5 (C-12), 25.6 (C-
14), 24.5 (C-13); ESI-MS: m/z 271
[M þ Na]þ, 287 [M þ K]þ, 283
[M þ Cl]2; HR-ESI-MS: m/z 271.1307
[M þ Na]þ (calcd for C15H20O3Na,
271.1305).
(tR-
19.851 min), D-glucose
D
-glucose
(tR19.856 and 19.854 min) was identified
in the hydrolysate of 1 and 3.
3.6 Antioxidant activity assays
The antioxidant activities of compounds 1
and 4–15 were assessed by measuring the
inhibitory ratios of malondialdehyde in rat
liver microsomal lipid peroxidation
induced by Fe2þ-cystine as described
previously [25].
Acknowledgments
This project was financially supported by the
Natural Science Foundation of China (No.
201072234, No. 21132009), and the National
Science and Technology Project of China (No.
2012ZX09301002-002). We are grateful to the
Department of Instrumental Analysis, Institute
of Materia Medica, Chinese Academy of
Medical Sciences, and Peking Union Medical
College for measuring the IR, UV, NMR, and
MS spectra.
3.5 Acid hydrolysis and determination
of the absolute configuration of the sugar
moieties
According to the reported method [10],
compound 3 (2 mg) was hydrolyzed by
2 M HCl–H2O at 958C for 10 h. After
removal of HCl by evaporation and
extraction with EtOAc, the H2O extract
was evaporated and dried in vacuo to give
the monosaccharide residue. From the
residue, glucose was detected by TLC
[CH2Cl2:MeOH (5:2), Rf 0.43] with the
authentic sample. The residue was dis-
solved in pyridine (1 ml) containing L-
cysteine methyl ester hydrochloride (2 mg)
and heated at 608C for 2 h, then evaporated
under N2 stream and dried in vacuo. The
residue was dissolved in 0.2 ml of N-
trimethylsilylimidazole and heated at 608C
for 2 h. The reaction mixture was parti-
tioned between n-hexane and H2O (2 ml
each), and the n-hexane extract was
analyzed by GC (Agilent 7890A) under
the following conditions: capillary column
HP-5 (30 m £ 0.32 mm £ 0.25 mm); detec-
tor FID; carrier gas N2, flow rate 1 ml/min;
detector temperature 2808C; injection
temperature 2508C; oven temperature
Note
1. On the occasion of Professor De-Quan
Yu’s 80th birthday, this special issue is
dedicated to him for his huge contributions
to natural product chemistry. He and his
colleagues have achieved almost a hundred
of traditional Chinese medicines or Chi-
nese herbs study and found hundreds of
novel compounds, most of which had
prominent bioactivities.
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