Sesquiterpenes from the roots of Illicium dunnianum

Article abstract of DOI:10.1016/j.phytochem.2012.05.015

Six allo-cedrane sesquiterpenes, four seco-prezizaane-type sesquiterpenes, two monocyclofarnesane sesquiterpenes, together with four known sesquiterpenes, were isolated from the roots of Illicium dunnianum. The structures were elucidated by spectroscopic methods including 1D and 2D NMR. The absolute configuration of 10 was determined by a CD experiment. Compounds 11 and 13 showed potent activities against the release of β-glucuronidase in rat polymorphonuclear leukocytes induced by platelet-activating factor in vitro, with IC₅₀ values of 2.10 ± 0.40 and 1.93 ± 0.57 μM, respectively. All compounds were evaluated for cytotoxicities against five human cancer cell lines (A549, Bel-7402, BGC-823, HCT-8, and A2780) in the MTT assay, but none of them exhibited activity at concentrations tested (10 ^-5-10^-7 M).

Full text of DOI:10.1016/j.phytochem.2012.05.015

Phytochemistry 80 (2012) 137–147
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Sesquiterpenes from the roots of Illicium dunnianum
⇑
Jian Bai, Hui Chen, Zhen-Feng Fang, Shi-Shan Yu , Wen-Jie Wang, Yang Liu, Shuang-Gang Ma, Yong Li,
Jing Qu, Song Xu, Jun-Hong Liu, Fen Zhao, Nan Zhao
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, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 August 2011
Received in revised form 27 February 2012
Available online 11 June 2012
Six allo-cedrane sesquiterpenes, four seco-prezizaane-type sesquiterpenes, two monocyclofarnesane ses-
quiterpenes, together with four known sesquiterpenes, were isolated from the roots of Illicium dunnia-
num. The structures were elucidated by spectroscopic methods including 1D and 2D NMR. The
absolute configuration of 10 was determined by a CD experiment. Compounds 11 and 13 showed potent
activities against the release of b-glucuronidase in rat polymorphonuclear leukocytes induced by plate-
Keywords:
Illicium dunnianum
Illiciaceae
allo-Cedrane sesquiterpenes
seco-Prezizaane-type sesquiterpenes
Monocyclofarnesane sesquiterpenes
Polymorphonuclear leukocytes
Cytotoxicity
let-activating factor in vitro, with IC50 values of 2.10 0.40 and 1.93 0.57 lM, respectively. All com-
pounds were evaluated for cytotoxicities against five human cancer cell lines (A549, Bel-7402, BGC-
823, HCT-8, and A2780) in the MTT assay, but none of them exhibited activity at concentrations tested
(10ꢀ5–10ꢀ7 M).
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
6-hydroxy-dihydrophaseic acid (12), together with four known
sesquiterpenes, tashironin (13) (Fukuyama et al., 1995), tashironin
Species of the genus Illicium L. (Illiciaceae) are known to be
characterized by a variety of structurally unique sesquiterpenes
such as seco-prezizaane-type (Huang et al., 2002; Schmidt, 1999;
Yamada et al., 1968), anislactone (Huang et al., 2001b; Kouno
et al., 1989c), and allo-cedrane sesquiterpenes (Schmidt et al.,
2001), some of which were found to exhibit intriguing neurotro-
phic activities (Huang et al., 2001a; Kubo et al., 2009; Yokoyama
et al., 2002). Illicium dunnianum is a toxic shrub distributed in
Southern China and used as a folk medicine for relieving pain
and treating rheumatism (Zhang, 1989). In previous papers, seco-
prezizaane-type sesquiterpenes with anti-inflammatory activities
(Tang et al., 2009), prenylated C6–C3 compounds with cytotoxici-
ties (Ma et al., 2011), and neolignan glycosides (Tang et al., 2007)
were isolated from I. oligandrum. As part of our ongoing search
for structurally unique and biologically interesting Illicium sesqui-
terpenes, the chemical constituents of the roots of I. dunnianum
were investigated, which resulted in the isolation of six new
allo-cedrane sesquiterpenes possessing a tetracyclic ketal struc-
A (14) (Song et al., 2007), 14-O-benzoylfloridanolide (15) (Huang
et al., 2000b), and neoanisatin (16) (Yamada et al., 1968) (Fig. 1).
In this paper, the isolation and structure elucidation of these
new compounds are described. The activities against the release
of b-glucuronidase in rat polymorphonuclear leukocytes (PMNs)
induced by platelet-activating factor (PAF) and cytotoxicities of
compounds 1–16 are also assayed.
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 suc-
cessively extracted with petroleum ether (60–90 °C), CHCl3, EtOAc,
and MeOH. The CHCl3 and EtOAc extracts were subjected to
column chromatography on silica gel, Sephadex LH-20, ODS, and
preparative reversed-phase HPLC, respectively, to afford com-
pounds 1–16.
Compound 1 was isolated as a colorless amorphous solid, and its
molecular formula was deduced to be C23H28O6 by ESIMS ions at m/z
401 [M+H]+, 423 [M+Na]+, 439 [M+K]+, 399 [M–H]ꢀ, 435 [M+Cl]ꢀ and
by a positive HRESIMS ion at m/z 423.1784 [M+Na]+ (calcd. for
423.1778), which was supported by 13C NMR and DEPT spectro-
scopic data. The IR spectrum indicated the presence of hydroxy
(3485 cmꢀ1), carbonyl (1713 cmꢀ1), and phenyl (1612 and
ture, tashironin B (1), tashironin C (2), 2a-hydroxytashironin (3),
2a
-acetoxytashironin (4), 2 -hydroxytashironin A (5), and 2a-
a
acetoxytashironin A (6), four new seco-prezizaane-type sesquiter-
penes, dunnianolide A–D (7–10), two new monocyclofarnesane
sesquiterpenes, 80-oxo-6-hydroxy-dihydrophaseic acid (11) and
⇑
Corresponding author. Tel.: +86 10 63165326; fax: +86 10 63017757.
E-mail address: yushishan@imm.ac.cn (S.-S. Yu).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.05.015

138
J. Bai et al. / Phytochemistry 80 (2012) 137–147
7'
6'
5'
1'
4'
1' 2'
10
3
3'
15
7'
12
8
1
5
13
13
1
7'
9'
5'
1'
7'
3'
8'
1'
1
1''
2''
3'
5'
5'
15
1
3'
15
1
O
O
10
10
5'
15
1
7'
1'
7
O
10
1
7
O
5
7'
12
OH
1'
5
3
3
O
O
7
3'
1
14
1''
12
14
3''
3
5
1
12
13
14
2
5''
13
D
6
1
2
2
7''
1
9'
8'
5
3
1
5'
1'
7'
3'
Fig. 1. Chemical structures of compounds 1–16.
1458 cmꢀ1) groups. The 1H and 13C NMR spectroscopic data (Tables
1 and 2) showed the presence of a p-methyl benzoyl group [dH 2.41
(3H, s, H3-80), 7.86 (2H, d, J = 8.0 Hz, H-30, 70), 7.22 (2H, d, J = 8.0 Hz,
H-40, 60); dC 165.6 (C-10)]. In addition to the p-methyl benzoyl group,
the 1H NMR spectrum of 1 showed signals due to two tertiary meth-
yls [dH 1.05 (3H, s, H3-13) and 1.23 (3H, s, H3-12)], a secondary
methyl [dH 1.20 (3H, d, J = 7.0 Hz, H3-15)], an isolated methylene
[dH 2.14 and 2.60 (each d, J = 18.5 Hz, H-8)], and an oxygen-bearing
isolated methylene [dH 3.85 and 4.21 (each d, J = 9.0 Hz, H-14)].
Moreover, the 1H–1H COSY spectrum verified the presence of a
CH3(15)–CH(1)–CH2(2)–CH2(3) moiety (Fig. 2). The 13C NMR and
DEPT spectra of 1 displayed an oxygenated methine carbon at dC
76.3 (C-10) and six quaternary carbons including a carbonyl carbon
at dC 210.9 (C-7) and a ketal-type carbon at dC 110.0 (C-11). The
above features suggested that 1 exhibited a molecular framework
similar to 11-O-debenzoyltashironin, which was previously isolated
form I. merrillianum (Huang et al., 2001a). Furthermore, by compar-
ing the 13C NMR spectroscopic data of 1 with those of 11-O-deben-
zoyltashironin, it was observed that signal for C-11 in 1 was
down-shifted to dC 110.0 from dC 106.4 in 11-O-debenzoyltashiro-
nin. This indicated that the p-methyl benzoxy group was connected
to C-11, which was further confirmed by NOESY correlation (Fig. 3)
between H3-12 (dH 1.23) and the aromatic H-30 and/or H-70 (dH 7.86).
Extensive HMBC and COSY correlations as shown in Fig. 2 confirmed
this structure. The relative configuration of 1 was determined based
on a NOESY experiment. NOESY correlations of H-10 to both of H3-15
and H-8b, of H-14a to H-3b, and of H3-13 to H3-12 were observed to
establish the relative configuration as shown in Fig. 3. Thus, the
structure of 1 was determined as shown in Fig. 1 and designated
as tashironin B (1).
Compound 2 was obtained as a colorless amorphous solid. Its
molecular formula was established as C24H28O6 by positive HRE-
SIMS (m/z 435.1788 [M+Na]+). The ESI mass spectrum exhibited a
peak (m/z 131, [C8H7CO]+) attributable to the presence of a cinnam-
oyl group. This was confirmed by analysis of the 1H and 13C NMR
spectroscopic data (Tables 1 and 2), which included signals of five
aromatic protons at dH 7.51 (2H, dd, J = 7.5, 2.0 Hz, H-50, 90) and
7.38–7.41 (3H, H-60, 70, 80), two olefinic protons at dH 6.38 (1H, d,
J = 16.0 Hz, H-20) and 7.69 (1H, d, J = 16.0 Hz, H-30), six aromatic
carbons at dC 133.8 (C-40), 128.3 (C-50, 90), 129.0 (C-60, 80), and
130.9 (C-70), two olefinic carbons at dC 116.8 (C-20) and 147.1 (C-
30), and a carbonyl carbon at dC 165.9 (C-10). The geometry of the
disubstituted double bond of the cinnamoyl moiety was deter-
mined as E by the large vicinal coupling constant value (16.0 Hz)
of the olefinic proton between H-20 (dH 6.38) and H-30 (dH 7.69).
The NMR spectroscopic data of 2 closely resembled those of 11-
O-debenzoyltashironin (Huang et al., 2001a), except for the pres-
ence of an additional cinnamoyl group. The resonance for C-11
was shifted downfield from dC 106.4 in 11-O-debenzoyltashironin
to dC 110.0 in 2, which suggested that the cinnamoyl group was
esterified at C-11. The relative configuration of 2 was the same
as that of 1 according to NOESY correlations. Therefore, the struc-
ture of 2 was assigned as shown in Fig. 1 and named tashironin C
(2).
Compound 3 was assigned the molecular formula C22H26O7 by
positive HRESIMS (m/z 425.1587 [M+Na]+). Comparison of the

J. Bai et al. / Phytochemistry 80 (2012) 137–147
139
Table 1
13C NMR spectroscopic data for compounds 1–10 (125 MHz for 1–10).a.
Position
1
2
3
4
5
6
7
8
9
10
49.3
76.4
44.5
96.6
48.6
77.2
209.5
46.9
51.8
41.1
174.4
21.8
16.1
65.9
9.8
166.2
129.3
129.6
128.6
133.2
128.6
129.6
1
2
3
4
5
6
7
8
9
38.7
31.0
34.1
85.6
54.4
60.8
210.9
44.0
52.3
76.3
110.0
9.8
14.6
74.3
38.7
31.0
34.0
85.2
54.5
60.6
210.6
44.0
52.4
76.1
110.0
9.6
14.4
74.1
49.7
79.9
44.2
84.3
54.0
61.0
45.1
82.1
43.6
83.6
54.3
60.9
209.7
42.1
53.1
75.4
109.5
9.8
14.5
74.3
49.7
79.9
44.0
84.3
53.8
60.9
210.2
44.1
53.5
75.6
109.1
9.8
14.5
74.4
11.5
165.1
126.3
160.5
33.3
21.2
41.0
16.7
45.1
82.2
43.6
83.6
54.1
60.8
209.9
42.0
52.9
75.5
108.8
9.7
14.6
74.3
11.3
165.0
126.2
160.6
33.3
21.2
41.1
16.6
46.3
40.1
81.6
49.2
53.2
76.8
40.9
99.6
50.7
43.5
106.1
52.3
48.9
132.0
146.2
39.9
45.5
81.1
37.7
51.8
72.7
175.8
12.9
27.0
73.5
13.7
103.8
73.9
77.8
71.3
77.8
62.7
133.5
137.3
50.5
75.8
86.1
25.5
56.8
73.3
176.8
22.7
62.1
65.4
22.2
210.0
44.0
53.7
75.5
10
11
12
13
14
15
10
39.7
176.1
7.9
109.9
9.8
14.4
74.4
11.5
14.1
70.8
10.0
13.5
13.5
11.3
165.6
126.3
130.1
129.2
144.8
129.2
130.1
21.7
165.9
116.8
147.1
133.8
128.3
129.0
130.9
129.0
128.3
165.7
129.0
130.0
128.6
133.9
128.6
130.0
165.7
129.0
130.0
128.6
134.0
128.6
130.0
166.2
129.8
129.5
128.4
133.2
128.4
129.5
20
30
40
50
60
70
80
90
100
200
300, 700
400, 600
500
171.2
21.4
171.2
21.4
166.0
133.4
129.4
128.7
129.9
a
Measured in CDCl3 for compounds 1–6, 9, and 10, in acetone-d6 for compound 7, and in methanol-d4 for compound 8.
Table 2
1H NMR spectroscopic data for compounds 1–6 (500 MHz in CDCl3, J in Hz).
Position
1
2
3
4
5
6
1
2
2.27, m
2.27, m
2.14, m
4.30, dd (8.0, 5.5)
2.42, m
5.15, dd (7.5, 5.5)
2.12, m
4.27, dd (7.5, 5.5)
2.39, m
5.13, dd (7.5, 5.5)
H
a
2.09, m
Ha
2.08, m
Hb 1.75, m
1.50, m
2.55, m
2.60, d (18.5)
2.14, d (18.5)
4.07, s
1.23, s
1.05, s
4.21, d (9.0)
3.85, d (9.0)
1.20, d (7.0)
Hb 1.74, m
1.50, m
2.50, m
2.59, d (18.5)
2.16, d (18.5)
4.05, s
3
3b
a
1.58, d (14.5)
2.94, dd (14.5, 8.5)
2.77, d (18.5)
2.15, d (18.5)
4.00, br s
1.25, s
1.06, s
4.11, d (9.0)
3.85, d (9.0)
1.30, d (7.0)
1.58, d (15.5)
3.07, dd (15.5, 9.5)
2.74, d (18.5)
2.14, d (18.5)
4.01, s
1.25, s
1.05, s
4.14, d (9.0)
3.86, d (9.0)
1.26, d (7.0)
1.53, d (14.5)
2.93, dd (14.5, 8.5)
2.72, d (18.5)
2.09, d (18.5)
3.88, br s
1.14, s
1.02, s
4.06, d (9.0)
3.80, d (9.0)
1.29, d (7.0)
1.55, d (15.0)
3.06, dd (15.0, 9.5)
2.69, d (18.5)
2.08, d (18.5)
3.89, s
1.14, s
1.01, s
4.10, d (9.0)
3.80, d (9.0)
1.25, d (7.0)
8a
8b
10
12
13
14a
14b
15
20
1.17, s
1.03, s
4.17, d (9.0)
3.84, d (9.0)
1.21, d (7.0)
6.38, d (16.0)
7.69, d (16.0)
30
7.86, d (8.0)
7.22, d (8.0)
7.97, d (7.5)
7.44, t (7.5)
7.59, t (7.5)
7.44, t (7.5)
7.97, d (7.5)
7.97, d (7.5)
7.44, t (7.5)
7.59, t (7.5)
7.44, t (7.5)
7.97, d (7.5)
40
2.54, m
1.79, m
2.48, m
2.07, s
2.55, m
1.79, m
2.48, m
2.07, s
50
7.51, dd (7.5, 2.0)
7.38–7.41, (overlap)
7.38–7.41, (overlap)
7.38–7.41, (overlap)
7.51, dd (7.5, 2.0)
60
7.22, d (8.0)
7.86, d (8.0)
2.41, s
70
80
90
200
2.06, s
2.05, s
10-OH
3.92, br s
4.02 br s
4.07, br s
NMR spectroscopic data (Tables 1 and 2) of 3 with those of tashi-
ronin (13) (Fukuyama et al., 1995) indicated that the structure of
3 was very close to that of 13, with the exception of the signal
assigned to C-2, where the resonance at dC 31.1 (C-2) in 13 was
shifted downfield to dC 79.9 (C-2) in 3. This indicated that a proton
at C-2 in 13 was replaced by a hydroxy group in 3, which was sup-
ported by HMBC correlations between H-2 (dH 4.30) and both of
C-4 (dC 84.3) and C-15 (dC 11.5). In addition, the relative configura-
tion of 3 was deduced from NOE correlations. H-2, OH-10, and the
C-15 methyl group were all b-oriented, which were confirmed by
NOE enhancements of H-2 and H-10 by irradiation of H3-15.
According to the above analysis, the structure of 3 was elucidated
as 2a-hydroxytashironin (3).
The molecular formula of compound 4 was established as
C
24H28O8 by positive HRESIMS (m/z 467.1687 [M+Na]+). Further-
more, the 1H and 13C NMR spectra (Tables 1 and 2) of 4 showed
close resemblance to those of 3, except for the presence of an addi-
tional O-acetyl group [dC 171.2 (C-100), dH 2.06 (3H, s, H3-200)]
relative to 3. It was observed that the signal for H-2 was down-
field-shifted from dH 4.30 in 3 to dH 5.15 in 4, and H-2 showed

140
J. Bai et al. / Phytochemistry 80 (2012) 137–147
O
HO
O
O
O
O
O
HO
OH
O
O
O
OH
O
O
HO
O
O
HO
OH
OH
1
7
9
O
OH
O
O
O
O
O
OH
OH
O
HO
COOH
O
O
H
10
11
1H -1H COSY
HMBC
Fig. 2. Key HMBC and 1H–1H COSY correlations of 1, 7, 9, 10, and 11.
an HMBC correlation with the carbonyl carbon at dC 171.2 (C-100),
which suggested that 4 was the 2-O-acetyl derivative of 3. More-
over, the relative configuration of the C-2 acetoxy group was
unambiguously proven by a NOE experiment, which showed inter-
actions from H-2 to both H3-15 and H-3b, and from H3-15 to H-10,
GC analysis established the d-configuration of the glucose (Hara
et al., 1987).
Additionally, the 1H and 13C NMR spectroscopic data (Tables 1
and 3) indicated the presence of a trisubstituted olefinic bond [dH
5.94 (1H, dd, J = 3.0, 1.5 Hz, H-3); dC 132.0 (C-3) and 146.2 (C-4)],
a tertiary methyl [dH 1.25 (3H, s, H3-13)], two secondary methyls
[dH 1.13 (6H, d, J = 7.0 Hz, H3-12, 15)], an oxygen-bearing isolated
methylene [dH 3.70 and 3.33 (each d, J = 9.5 Hz, H-14)], two meth-
thereby confirming the
Thus, 4 was established as 2
a
-orientation of the C-2 acetoxy group.
-acetoxytashironin (4).
a
Compound 5 was assigned the molecular formula C22H30O7 by
positive HRESIMS (m/z 429.1861 [M+Na]+). The 13C NMR spectro-
scopic data (Table 1) of 5 were very similar to tashironin A (14)
(Song et al., 2007) except that the signal for C-2 at dC 31.0 in 14
was shifted downfield to dC 79.9 in 5, which suggested that 5
was the 2-hydroxy derivative of 14. The location of the hydroxy
group at C-2 was further confirmed by the HMBC correlations of
H-2 (dH 4.27) to C-4 (dC 84.3) and C-15 (dC 11.5). Additionally,
the relative configuration of the C-2 hydroxy group was deter-
mined on the basis of NOESY correlations. H3-15 showed NOESY
ylenes [dH 2.28 (1H, ddd, J = 14.0, 7.0, 3.0 Hz, H-2a), 2.08 (1H, ddd,
J = 14.0, 9.5, 1.5 Hz, H-2b); dH 1.62 (1H, dd, J = 13.5, 1.5 Hz, H-8
a),
2.34 (1H, dd, J = 13.5, 4.5 Hz, H-8b)], and a carbonyl carbon at dC
175.8 (C-11). The 1H–1H COSY spectrum showed the presence of
a CH3(15)–CH(1)–CH2(2)–CH(3) = partial unit (Fig. 2). Except for
the glucosyl moiety, the NMR spectroscopic data of 7 were found
to be similar to those of 3,4-dehydrofloridanolide (Huang et al.,
2000a). However, the signal for H3-12 at dH 1.13 split into a doublet
coupled to quartet of a doublet for H-6 at dH 1.77. The resonance
for C-6 was up-shifted from dC 74.0 in 3,4-dehydrofloridanolide
to dC 45.5 in 7. These findings indicated that 7 was the 6-deoxy
derivative of 3,4-dehydrofloridanolide, which was further con-
firmed by the presence of a CH3(12)–CH(6)–CH(7)–CH2(8) moiety
based on a COSY experiment (Fig. 2). Moreover, the AB doublet
assignable to H2-14 were shifted downfield from dH 3.09 and
3.34 in 3,4-dehydrofloridanolide to dH 3.33 and 3.70 in 7, suggest-
ing that the glucosyl moiety was linked to C-14. This was sup-
ported by an HMBC correlation (Fig. 2) between H-14a (dH 3.70)
and the anomeric carbon at dC 103.8 (C-10). The NOESY spectrum
showed cross-peaks between H3-15 and H-10, between H3-12
interactions with H-2 and H-10, indicating the
a-orientation of
the C-2 hydroxy group. Therefore, 5 was deduced to be 2
a-
hydroxytashironin A (5).
The molecular formula of compound 6 was deduced to be
24H32O8 by positive HRESIMS (m/z 471.1971 [M+Na]+). The NMR
C
spectroscopic data (Tables 1 and 2) of 6 were closely related to
those of 5 except for the presence of an additional O-acetyl group
and the shift of signal corresponding to H-2 from dH 4.27 in 5 to dH
5.13 in 6. In HMBC spectrum, the signal at dH 5.13 (H-2) showed a
cross-peak with the O-acetyl carbonyl carbon resonance at dC 171.2
(C-100), which confirmed that the acetoxy group was connected to
C-2. The NOE correlations of 6 were identical to those observed
for 5, indicating that these two compounds had the same relative
configuration. Accordingly, the structure of 6 was elucidated as
and H-14a, and between H-6 and both H3-13 and H-8
the -orientation of Me-13 and the b-orientation of Me-15, Me-12,
and OH-10 (Fig. 3). Accordingly, the structure of 7 was elucidated
as 14-O-b- -glucopyranosyl-6-deoxy-3,4-dehydrofloridanolide,
a, indicating
a
2a-acetoxytashironin A (6).
D
Compound 7 was obtained as a colorless amorphous solid. Its
and designated as dunnianolide A (7).
molecular formula was deduced to be C21H32O9 by positive HRE-
SIMS (m/z 451.1939 [M+Na]+). The IR absorption bands at
3437 cmꢀ1 and 1718 cmꢀ1 indicated the presence of hydroxy and
carbonyl groups, respectively. In the 13C NMR spectrum, six carbon
resonances at dC 103.8 (C-10), 73.9 (C-20), 77.8 (C-30), 71.3 (C-40),
77.8 (C-50), and 62.7 (C-60) were readily assigned to be a glycosyl
moiety. The hydrolysis of 7 with 2 M HCl–H2O afforded a glucose,
which was confirmed by TLC with an authentic sample of glucose.
The large coupling constant (8.0 Hz) of the anomeric proton at dH
4.21 (H-10) indicated that the glucose was in the b-configuration.
The molecular formula of compound 8 was deduced to be
C
15H22O7 by analysis of positive HRESIMS (m/z 337.1261 [M+Na]+).
The 1H and 13C NMR spectroscopic data of 8 (Tables 1 and 3) were
similar to those of 13,14-dihydroxy-3,4-anhydrofloridanolide
(Schmidt et al., 2001). The major differences were that signal for
H3-15 (dH 1.28) was shifted downfield and changed into a singlet,
and resonances for C-1 and C-15 were down-shifted by 37.1 and
8.4 ppm, respectively, in comparison with 13,14-dihydroxy-3,4-
anhydrofloridanolide. This suggested that 8 was the 1-hydroxy
derivative of 13,14-dihydroxy-3,4-anhydro-floridanolide. The

J. Bai et al. / Phytochemistry 80 (2012) 137–147
141
3'
10
8
14
2
3
12
15
15
13
1
5
6
14
1
9
8
5
3
13
12
10
10
12
8
15
1
8
15
1
5
14
6
5
14
2
2
3
3
13
12
13
6
3
1
5
5'
9'
1'
3'
7'
Fig. 3. Key NOESY correlations of 1, 7, 9, 10, and 11.
relative configuration of 8 was identical with that of 7 and 13,14-
dihydroxy-3,4-anhydrofloridanolide according to NOESY correla-
tions. Consequently, 8 was established as 1,13,14-trihydroxy-3,
4-anhydrofloridanolide, and named dunnianolide B (8).
to pseudomajucin (Kouno et al., 1989b), except for the presence of
an additional benzoyl group. By comparing the 1H and 13C NMR
spectroscopic data of 9 with those of pseudomajucin, it was found
that signals at dH 4.42 (H-2) and dC 73.6 (C-2) in pseudomajucin
were shifted downfield to dH 5.52 (H-2) and dC 76.8 (C-2) in 9,
respectively, which indicated that the hydroxy group at C-2 in
pseudomajucin was substituted by a benzoyloxy group in 9. This
was further confirmed by an HMBC correlation (Fig. 2) between
H-2 (dH 5.52) and the carbonyl carbon at dC 166.2 (C-10). The relative
configuration of 9 was deduced from a NOESY experiment (Fig. 3).
The NOESY spectrum showed cross-peaks between H-2 and both
Compound 9 was isolated as colorless needles, and its molecular
formula C22H26O6 was determined by positive HRESIMS (m/z
387.1804 [M+H]+). Its IR spectrum displayed absorptions ascribable
to hydroxy (3423 cmꢀ1), carbonyl (1747 and 1716 cmꢀ1), and
phenyl (1601, 1585, and 1451 cmꢀ1) groups. The 1H and 13C NMR
spectroscopic data (Tables 1 and 3) showed the presence of a
benzoyl group [dH 8.00 (2H, d, J = 7.5 Hz, H-30, 70), 7.44 (2H, t,
J = 7.5 Hz, H-40, 60), and 7.55 (1H, t, J = 7.5 Hz, H-50); dC 166.2 (C-
10)]. Except for the benzoyl group, the 13C NMR spectrum showed
another 15 carbon signals, which were categorized by DEPT exper-
iment as three methyls (CH3-12, CH3-13, CH3-15), four methylenes
including an oxygenated methylene (CH2-3, CH2-8, CH2-10, and O-
CH2-14), three methines including an oxygenated methine (CH-1,
O-CH-2, and CH-6), and five quaternary carbons including a car-
bonyl carbon at dC 176.1 (C-11) and a hemiketal-type carbon at dC
106.1 (C-7). The above features indicated that 9 was closely similar
of H-1 and H-3
that the benzoyloxy group and Me-15 were b-oriented. The correla-
tions of H3-12 to H-14b, of H-6 to H-3 and H-8 , and of H-14a to
H-10b confirmed the b-orientation of Me-12 and the -orientation
a, and between H3-15 and H-10a, which indicated
a
a
a
of Me-13. Accordingly, 9 was elucidated as 2b-benzoyloxy-pseudo-
majucin, and designated as dunnianolide C (9).
Compound 10 was obtained as a colorless amorphous solid.
Its molecular formula was established as C29H30O8 by positive
HRESIMS (m/z 529.1820 [M+Na]+). Its IR spectrum showed absorptions

142
J. Bai et al. / Phytochemistry 80 (2012) 137–147
Table 3
1H NMR spectroscopic data for compounds 7–10 (500 MHz, J in Hz).a.
Position
7
8
9
10
1
2
2.04, m
2.29, m
5.52, br d (4.0)
2.36, m
5.57, br d (5.0)
H
a
2.28, ddd (14.0, 7.0, 3.0)
H
a
2.25, dd (16.5, 1.5)
Hb 2.08, ddd (14.0, 9.5, 1.5)
Hb 2.56, d (16.5)
3
5.94, dd (3.0, 1.5)
5.81, br s
H
a
2.31, dd (15.5, 4.5)
Ha 3.13, dd (16.0, 6.0)
Hb 2.47, d (15.5)
Hb 2.46, d (16.0)
6
7
1.77, qd (7.0, 2.5)
4.47, m
1.84, q (7.0)
4.17, br s
8
8b
10
a
1.62, dd (13.5, 1.5)
2.34, dd (13.5, 4.5)
4.06, d (8.0)
2.66, d (14.0)
1.70, dd (14.0, 3.5)
4.09, s
1.90, d (14.0)
2.36, d (14.0)
Ha 3.00, d (18.5)
Hb 2.90, d (18.5)
1.08, d (7.0)
2.78, d (15.5)
2.89, d (15.5)
Ha 3.24, d (19.0)
Hb 2.40, d (19.0)
1.46, s
12
13
14
1.13, d (7.0)
1.25, s
Ha 3.70, d (9.5)
Hb 3.33, d (9.5)
1.13, d (7.0)
1.32, s
3.25, s
3.91, s
1.06, s
1.24, s
Ha 3.92, d (9.5)
Hb 3.69, d (9.5)
1.08, d (7.0)
Ha 4.68, d (12.0)
Hb 4.58, d (12.0)
1.09, d (7.0)
15
10
20
30
40
50
60
1.28, s
4.21, d (8.0)
3.22, (overlap)
3.31, (overlap)
3.30, (overlap)
3.24, (overlap)
Ha 3.79, ddd (11.5, 6.0, 2.0)
Hb 3.63, dd (11.5, 6.0)
8.00, d (7.5)
7.44, t (7.5)
7.55, t (7.5)
7.44, t (7.5)
7.96, d (7.5)
7.45, t (7.5)
7.56, t (7.5)
7.45, t (7.5)
70
8.00, d (7.5)
7.96, d (7.5)
7.96, d (7.5)
7.47, t (7.5)
7.58, t (7.5)
300, 700
400, 600
500
10-OH
20-OH
30-OH
40-OH
60-OH
3.97, d (8.0)
3.89, d (3.5)
4.11, (overlap)
4.11, (overlap)
3.58, dd (11.5, 6.0)
a
Measured in acetone-d6 for compound 7, in methanol-d4 for compound 8, and in CDCl3 for compounds 9 and 10.
ascribable to hydroxy (3361 cmꢀ1), -lactone (1773 cmꢀ1), carbonyl
c
(1719 cmꢀ1), and phenyl (1601, 1584, and 1451 cmꢀ1) groups. Two
benzoyl groups were readily observed in the 1H and 13C NMR
spectroscopic data (Tables 1 and 3). In addition, the 1H and 13C NMR
spectra showed the presence of two tertiary methyls [dH 1.24 (3H, s,
H3-13); 1.46 (3H, s, H3-12)], a secondary methyl [dH 1.09 (3H, d,
J = 7.0 Hz, H3-15)], two isolated methylenes [dH 2.78 and 2.89 (each
d, J = 15.5 Hz, H-8); dH 3.24 and 2.40 (each d, J = 19.0 Hz, H-10)], an
oxygen-bearing isolated methylene [dH 4.68 and 4.58 (each d,
J = 12.0 Hz, H-14)], and two carbonyl carbons at dC 174.4 (C-11) and
dC 209.5 (C-7). The NMR spectroscopic data of 10 closely resembled
those of (6R)-pseudomajucinone (Huang et al., 2002). However, it
was observed that signals for H-2, H-14a, and H-14b were shifted
downfield from dH 4.12, 3.62, and 3.53 in (6R)-pseudomajucinone to
dH 5.57, 4.68, and 4.58 in 10, respectively. This indicated that two
benzoyl groups were esterified at C-2 and C-14, respectively, which
were further confirmed by HMBC correlations (Fig. 2) between H-2
(dH 5.57) and the carbonyl carbon at dC 166.2 (C-10) and between H-
14a (dH 4.68) and C-100 (dC 166.0). Moreover, the signal for H3-12 (dH
1.46) changed to a singlet and resonance for C-6 in 10 was shifted
downfield by 30.9 ppm, in comparison with (6R)-pseudomajucinone,
which suggested that the proton at C-6 in (6R)-pseudomajucinone
was replaced by a hydroxy group in 10.
Fig. 4. Octant projection of 10.
effect at 302 nm for the n ? p⁄ transition indicated that the abso-
lute configuration of 10 was (1S, 2R, 4S, 5R, 6R, 9R) (Fig. 4), consis-
tent with the core configuration of (6R)-pseudomajucinone (Huang
et al., 2002), whose absolute configuration have been established by
X-ray crystallographic analysis. Therefore, 10 was determined to be
(1S, 2R, 4S, 5R, 6R, 9R)-2,14-dibenzoyloxy-6-hydroxy-pseud-
omajucinone, and named dunnianolide D (10).
In addition, the relative configuration of 10 was elucidated by
analysis of NOESY spectrum. NOESY correlations (Fig. 3) between
H-2 and both of H-1 and H-3a, and between H3-15 and H-10a sug-
gested that 2-benzoyloxy group and Me-15 were b-oriented. The
interactions between H3-12 and H-14b, and between H3-13 and
both of H-3
a and H-8a reflected the b-orientation of Me-12 and
Compound 11 was isolated as colorless granules with the molec-
ular formula of C15H20O7 determined by positive HRESIMS (m/z
335.1099 [M+Na]+). The characteristic absorption bands of hydroxy
(3313 cmꢀ1), carbonyl (1763 cmꢀ1), conjugated carbonyl (1687
the -orientation of Me-13. The absolute configuration of 10 was
a
determined by analysis of its CD spectrum. On the basis of the
octant rule for cyclohexanone (Kirk, 1986), the negative Cotton

J. Bai et al. / Phytochemistry 80 (2012) 137–147
143
Table 4
13C and 1H NMR spectroscopic data for compounds 11 and 12a.
Position
11
12
b
b
c
c
c
c
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
2
3
4
5
6
10
20
30
169.7
117.9
153.0
130.6
130.9
63.0
82.8
89.8
42.2
167.5
116.5
153.3
129.7
130.9
62.4
82.5
88.2
42.6
167.6
115.2
153.9
128.3
133.6
62.4
83.0
86.7
46.5
6.10, s
6.15, s
6.11, s
7.83, d (16.5)
6.42, d (16.5)
4.43, s
7.89, d (16.5)
6.54, d (16.5)
4.50, s
7.89, d (16.5)
6.55, d (16.5)
4.48, s
Hax 1.84, dd (14.0, 10.0)
Heq 2.26, dd (14.0, 7.0)
Hax 3.84, m
Hax 1.71, dd (13.5, 11.0)
Heq 1.90, dd (13.5, 7.0)
Hax 1.91, dd (14.0, 10.0)
Heq 2.25, dd (14.0, 6.5)
Hax 3.87, m
Hax 1.79, dd (13.5, 11.0)
Heq 1.88, dd (13.5, 7.0)
Hax 1.76, dd (13.5, 10.0)
Heq 1.99, dd (13.5, 7.0)
Hax 4.10, m
Hax 1.69, dd (13.5, 10.0)
Heq 1.83, dd (13.5, 7.0)
40
50
65.2
40.9
64.8
41.1
65.4
45.0
60
70
80
53.5
18.4
181.0
52.8
18.5
178.6
49.2
19.9
76.8
1.34, s
1.35, s
1.08, s
1.09, s
Ha 3.77, dd (7.0, 1.5)
Hb 3.66, d (7.0)
0.91, s
90
14.5
1.08, s
14.6
16.5
a
b
c
Measured at 500 MHz for proton and 125 MHz for carbon.
Measured in methanol-d4.
Measured in acetone-d6.
cmꢀ1), and olefinic (1641 and 1603 cmꢀ1) groups were observed in
the IR spectrum. The 1H NMR spectrum of 11 (Table 4) showed
signals for two tertiary methyls [dH 1.08 (3H, s, H3-90), dH 1.35 (3H,
s, H3-70)], three methylenes including an oxygen-bearing isolated
methylene [dH 1.91 (1H, dd, J = 14.0, 10.0 Hz, H-30ax) and 2.25 (1H,
dd, J = 14.0, 6.5 Hz, H-30eq); dH 1.79 (1H, dd, J = 13.5, 11.0 Hz,
H-50ax) and 1.88 (1H, dd, J = 13.5, 7.0 Hz, H-50eq); dH 4.50 (2H, s,
H2-6)], an oxymethine [dH 3.87 (1H, m, H-40)], and three olefinic
protons [dH 6.15 (1H, s, H-2), 6.54 (1H, d, J = 16.5 Hz, H-5), and
7.89 (1H, d, J = 16.5 Hz, H-4)]. The 13C NMR and DEPT spectra dis-
played six nonprotonated carbons including an olefinic (dC 153.3,
C-3), two carbonyl (dC 167.5, C-1 and 178.6, C-80), a quaternary (dC
52.8, C-60), and two oxygenated quaternary (dC 82.5, C-10 and 88.2,
C-20) carbons together with nine protonated carbons. The 1H–1H
COSY spectrum established the sequence of CH2(30)–CH(O)(40)–
CH2(50) and an olefin moiety (Fig. 2). The large vicinal coupling
constant (16.5 Hz) between H-4 (dH 7.89) and H-5 (dH 6.54) sug-
gested that the olefin moiety was in the E configuration. According
to the above features, the NMR spectroscopic data (CD3OD) of 11
were very similar to those of rel-5-(3S,8S-dihydroxy-1R,5S-di-
methyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pent-
adienoic acid (Kikuzaki et al., 2004), a derivative of dihydrophaseic
acid (Milborrow, 1975), except for the absence of a signal for a
tertiary methyl group and the presence of resonance for an oxy-
gen-bearing isolated methylene group [dH 4.50 (2H, s, H2-6)], which
indicated that a proton of the methyl group at C-3 in rel-5-(3S,8S-
dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-
methyl-2Z,4E-pentadienoic acid was replaced by a hydroxy group in
11. This was further confirmed by HMBC correlations between the
hydroxymethyl protons at dH 4.50 (H2-6) and the olefinic carbons
at dC 116.5 (C-2), 153.3 (C-3), and 129.7 (C-4) (Fig. 2).
NMR spectroscopic data (Table 4) very resembled those of 11.
Comparing the 1H and 13C NMR spectra of 11, the signals due to
an isolated oxymethylene group were observed at dH 3.77 (1H,
dd, J = 7.0, 1.5 Hz, H-80a) and 3.66 (1H, d, J = 7.0 Hz, H-80b) in the
1H NMR and at dC 76.8 in the 13C NMR spectra of 12, with the dis-
appearance of the carbonyl carbon at dC 178.6 assignable to C-80 in
11. In the HMBC spectrum of 12, the oxymethylene proton at dH
3.66 (H-80b) was correlated with C-10 (dC 83.0), C-20 (dC 86.7),
C-50 (dC 45.0), and C-60 (dC 49.2), indicating that C-80 was an oxy-
methylene carbon. The NOESY correlation between H-80b (dH
3.66) and H-40ax (dH 4.10) confirmed that the orientation of the
oxymethylene was axial. Thus, 12 was determined to be 6-hydro-
xy-dihydrophaseic acid (12).
Compounds 1–16 were assessed for their inhibitory activities
against the release of b-glucuronidase in rat PMNs induced by
PAF in vitro. Ginkgolide B was used as a positive control (IC50
2.92 0.49 lM). Meanwhile, the tested compounds were not cyto-
toxic against rat PMNs in the MTT assay at a concentration of 10ꢀ5
M (See Supplementary data). As shown in Table 5, compounds 11
and 13 exhibited significant inhibitory activities, with IC50 values
of 2.10 0.40 and 1.93 0.57 lM, respectively. Compounds 1–3, 8,
9, 15, and 16 showed moderate activities with the inhibitory rates
ranging from 32.4 1.4% to 43.4 3.3% at a concentration of 10ꢀ5
M, while compounds 4–7 exhibited weak activities with the
inhibitory rates ranging from 19.4 2.7% to 28.6 3.5% at the
same concentration, and the other compounds were inactive. Pri-
mary discussion of structure–activity relationship revealed that
for allo-cedrane sesquiterpenes, the benzoyloxy group at C-11 in
13 could significantly increase the activity in relative to the p-
methyl benzoyloxy group in 1, the O-cinnamoyl group in 2, and
the 11-O-2-methylcyclopent-1-enecarboxyl moiety in 14, while
the C-2 hydroxy group in 3 and the C-2 acetoxy group in 4 cause
a drastic decrease in activity comparing with 13. Thus, the occur-
rence of a benzoyloxy group at C-11 in addition to the lack of a
hydroxy or acetoxy group at C-2 may be structural prerequisites
for the activities against the release of b-glucuronidase in rat
PMNs induced by PAF. In addition, the 80-carbonyl group in 11
maybe involved in the activity.
The relative configuration of 11 was deduced from a NOESY
experiment, in which the correlations between H2-6 and both H-
2 and H-5 verified the conjugated diene was 2E,4E-configuration.
The cross-peaks between H-30ax and both H3-70 and H-5, between
H-50ax and both H3-90 and H-5, and between H-40 and both H-30eq
and H-50eq were observed to establish the relative configuration as
shown in Fig. 3. Thus, 11 was elucidated as 80-oxo-6-hydroxy-dihy-
drophaseic acid (11).
Cytotoxic activities of compounds 1–16 were evaluated against
five human cancer cell lines (A549, Bel-7402, BGC-823, HCT-8, and
A2780) in the MTT assay, with adriamycin as a positive control
Compound 12 was assigned the molecular formula C15H22O6
obtained from positive HRESIMS (m/z 321.1307 [M+Na]+). Its

144
J. Bai et al. / Phytochemistry 80 (2012) 137–147
Table 5
HRESIMS data were recorded on an Agilent Technologies 6250
Accurate-Mass Q-TOF LC/MS spectrometer. Analytical HPLC was
performed on an Agilent 1100 Series instrument with a DAD detec-
Potency of compounds 1–16 in inhibiting the release of b-glucuronidase in rat PMNs
induced by PAFa.
Compound
IC50
(
l
M)
Inhibition rate (%)b
tor, using a YMC-Pack ODS (250 ꢁ 4.6 mm, 5
was performed on a Shimadzu LC-6AD instrument with an SPD-10A
m).
lm). Preparative HPLC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.10 0.40
>10
1.93 0.57
>10
>10
>10
40.4 3.3
35.8 2.6
32.4 1.4
20.3 2.0
26.6 3.3
19.4 2.7
28.6 3.5
40.9 1.6
33.4 2.0
ꢀ5.3 5.2
65.8 3.0
ꢀ34.5 4.6
65.0 4.1
ꢀ6.5 3.2
43.4 3.3
34.5 2.8
58.7 1.6
detector, using a YMC-Pack ODS-A column (250 ꢁ 20 mm, 5
l
GC data were recorded on an Agilent 7890A instrument with an
FID detector. Sephadex LH-20 (Amersham Pharmacia Biotech AB,
Sweden), ODS (45–70 lm, Merck), silica gel (160–200, 200–300
mesh, Qingdao Marine Chemical Co. Ltd., China), and diatomite
(Sinopharm Chemical Reagent Co. Ltd., China) were used for column
chromatography (CC). TLC was carried out with glass precoated
with silica gel GF254 plates (Qingdao Marine Chemical Co. Ltd., Chi-
na). Spots were visualized under UV light or by spraying with 10%
H2SO4 in EtOH–H2O (95:5, v/v) followed by heating. Solvents
[petroleum ether (60–90 °C), CHCl3, EtOAc, MeOH, CH2Cl2, and
EtOH] were of analytical grade and purchased from Beijing Chemi-
cal Company, Beijing, China.
Ginkgolide Bc
2.92 0.49
a
b
c
Data are mean standard deviation (n = 3).
4.2. Plant material
Inhibition rate (%) at 10
Positive control.
lM.
Roots ofI. dunnianum were collected in Guangxi Province, 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) was deposited in the herbarium of the Department of
Medicinal plants, Institute of Materia Medica, Chinese Academy
of Medical Sciences.
(IC50 0.23 0.16, 0.53 0.04, 0.97 0.92, 0.61 0.25, 0.63 0.27
l
M, respectively). None of them exhibited significant activities in
the concentration range of 10ꢀ5–10ꢀ7 M.
Some of the sesquiterpene lactones isolated from the genus Illic-
ium were considered as responsible for the neurotoxicity of these
plants (Kouno et al., 1989a; Yang et al., 1990, 1991). Such as anisat-
in with a b-lactone structure acts as a picrotoxin-like, non-compet-
itive GABA antagonist (Kudo et al., 1981; Matsumoto and Fukuda,
1982). However, some sesquiterpenes have been found not to be
neurotoxic, but to show neurotrophic activities (Fukuyama and
Huang, 2005; Fukuyama et al., 2001, 1993; Huang et al., 2001a,c;
Kubo et al., 2009; Yokoyama et al., 2002). These properties influ-
ence the application of the active sesquiterpene lactones as poten-
tial anti-inflammatory agents. Further studies are necessary to
clarify the toxicological activities of these compounds.
4.3. Extraction and isolation
Roots of I. dunnianum (7.5 kg) were air-dried, ground, and ex-
tracted three times (2 h for each time) with EtOH–H2O (3 ꢁ 80 l,
95:5, v/v) under conditions of reflux (90–95 °C), and the residue
was refluxed with EtOH–H2O (2 ꢁ 64 l, 70:30, v/v). The combined
EtOH extract was evaporated to near dryness under reduced pres-
sure to give the crude extract (850 g), which was absorbed by diat-
omite, and then successively extracted with petroleum ether (60–
90 °C) (10 l), CHCl3 (10 l), EtOAc (10 l), and MeOH (10 l). 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. Frac-
tion C4 (8.0 g) was subjected to a ODS CC, eluted with a gradient of
MeOH–H2O (10:90 ? 100:0) to give fractions C4-1–C4-20. Fraction
C4-5 (102 mg) was purified by preparative HPLC using CH3CN–H2O
(20:80, 8 ml/min) to yield compound 16 (54.0 mg, tR 36 min, purity
97.1%). Fraction C4-6 (122 mg) was separated by preparative HPLC
3. Concluding remarks
Twelve new sesquiterpenes including six allo-cedrane sesquiter-
penes (1–6), four seco-prezizaane-type sesquiterpenes (7–10), and
two monocyclofarnesane sesquiterpenes (11 and 12), were isolated
from the roots of I. dunnianum along with four known sesquiter-
penes (13–16). allo-cedrane sesquiterpenes 1–6 possesses a rare
oxatetracyclic system with a cagelike ketal structure, correspond-
ing to a key precursor in the biogenesis of seco-prezizaane-type ses-
quiterpenes (Fukuyama et al., 1995; Schmidt et al., 2001). The
discovery of compounds 1–6 is a further addition to the diversity
of allo-cedrane sesquiterpenes. In addition, the activities against
the release of b-glucuronidase in rat PMNs induced by PAF and
cytotoxicities of the isolated compounds were also investigated.
using CH3CN–H2O (40:60, 8 ml/min) to afford compounds
9
(42.0 mg, tR 33 min, purity 97.4%) and 15 (25.2 mg, tR 40 min, pur-
ity 97.7%). Fraction C4-8 (65 mg) was separated by preparative
HPLC using CH3CN–H2O (50:50, 8 ml/min) to yield compound 4
(9.3 mg, tR 29 min, purity 92.4%). Fraction C4-10 (250 mg) was ap-
plied to preparative HPLC using CH3CN–H2O (50:50, 7 ml/min) to
yield compound 14 (112.0 mg, tR 41 min, purity 96.3%). Fraction
C4-11 (60 mg) was separated by preparative HPLC using CH3CN–
H2O (48:52, 8 ml/min) to yield compound 6 (21.0 mg, tR 39 min,
purity 96.9%). Fraction C4-12 (0.5 g) was subjected to Sephadex
LH-20 CC eluted with CH2Cl2–MeOH (1:2) and then further purified
by preparative HPLC using CH3CN–H2O (60:40, 7 ml/min) to yield
compounds 1 (3.0 mg, tR 36 min, purity 91.8%), 2 (6.1 mg, tR
34 min, purity 93.8%), 10 (3.1 mg, tR 30 min, purity 96.3%), and
13 (156.0 mg, tR 38 min, purity 99.2%). Fraction C6 (3.8 g) was ap-
4. Experimental
4.1. General experimental procedures
Melting points were measured on a XT-5B micromelting point
apparatus and were uncorrected. 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 at 25 °C. ESIMS was measured on
an Agilent 1100 Series LC/MSD ion trap mass spectrometer.
plied to an ODS CC eluted with
a gradient of MeOH–H2O
(10:90 ? 90:10) to give nine fractions C6-1–C6-9. Fraction C6-2
(215 mg) and C6-3 (280 mg) were subjected to Sephadex LH-20
CC eluted with MeOH and then further purified by preparative
HPLC using CH3CN–H2O (41:59, 7 ml/min) and (45:55, 7 ml/min),

J. Bai et al. / Phytochemistry 80 (2012) 137–147
145
to yield compounds 3 (27.1 mg, tR 24 min, purity 98.0%) and 5
(46.0 mg, tR 22 min, purity 99.0%), respectively.
4.3.6. 2a-Acetoxytashironin A (6)
Colorless needles (EtOAc); mp 154–155 °C; ½a D20
ꢂ
+6.3 (c 0.09,
The EtOAc extract (110 g) was subjected to a silica gel CC
(50 ꢁ 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 applied to an ODS CC eluted with a gradient of MeOH–
H2O (10:90 ? 100:0) to yield nine fractions E4-1–E4-9. Fraction
E4-3 (160 mg) was submitted to a Sephadex LH-20 CC eluted with
MeOH, then purified by preparative HPLC using 0.1% TFA in
CH3CN–H2O (12:88, 7 ml/min) to afford compound 11 (27.1 mg, tR
20 min, purity 96.2%). Fraction E4-5 (150 mg) was separated by pre-
parative HPLC using CH3CN–H2O (28:72, 7 ml/min) to yield com-
pound 7 (20.0 mg, tR 22 min, purity 96.7%). 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 E5-1–E5-18. Fraction E5-4
(70 mg) was separated by preparative HPLC using 0.1% TFA in
CH3CN–H2O (5:95, 7 ml/min) to yield compound 8 (9.0 mg, tR
30 min, purity 92.1%). Fraction E5-6 (125 mg) was subjected to a
Sephadex LH-20 CC eluted with MeOH, and further purified by pre-
parative HPLC using 0.1% TFA in CH3CN/H2O (11:89, 7 ml/min) to
yield compound 12 (5.1 mg, tR 17 min, purity 95.2%). (The purity
of isolated compounds was determined by HPLC).
MeOH); UV (MeOH) kmax (loge) 200 (3.69), 236 (3.84) nm; IR
(KBr) mmax 3516, 2973, 1728, 1712, 1641, 1266, 1060, 1028,
879 cmꢀ1; For 13C and 1H NMR spectroscopic data, see Tables 1
and 2; ESIMS m/z 471 [M+Na]+, 487 [M+K]+, 447 [M–H]ꢀ, 483
[M+Cl]ꢀ; HRESIMS m/z 471.1971 [M+Na]+ (calcd. for C24H32O8Na,
471.1989).
4.3.7. Dunnianolide A (7)
Colorless amorphous solid;
½
a 2D0
ꢂ
+9.3 (c 0.06, MeOH); UV
(MeOH) kmax (log
e
) 202 (3.94), 233 (2.99), 280 (2.56) nm; IR
(KBr) mmax 3437, 2929, 1719, 1102, 1078, 1040, 756 cmꢀ1; For 13C
and 1H NMR spectroscopic data, see Tables 1 and 3; ESIMS m/z
429 [M+H]+, 451 [M+Na]+, 467 [M+K]+, 463 [M+Cl]ꢀ; HRESIMS m/
z 451.1939 [M+Na]+ (calcd. for C21H32O9Na, 451.1939).
4.3.8. Dunnianolide B (8)
Colorless amorphous solid; ½a D20
ꢂ
+70.2 (c 0.08, MeOH); UV
(MeOH) kmax (loge) 205 (3.50) nm; IR (KBr) mmax 3331, 2934,
1725, 1677, 1205, 1086, 923, 763 cmꢀ1; For 13C and 1H NMR spec-
troscopic data, see Tables 1 and 3; ESIMS m/z 337 [M+Na]+, 651
[2M+Na]+, 427 [M+CF3COO]ꢀ; HRESIMS m/z 337.1261 [M+Na]+
(calcd. for C15H22O7Na, 337.1258).
4.3.1. Tashironin B (1)
Colorless amorphous solid;
½
a 2D0
–15.7 (c 0.1, MeOH); UV
ꢂ
(MeOH) kmax (log ) 204 (3.81), 241 (3.79), 274 (2.80) nm; IR
e
(KBr) mmax 3485, 2951, 2911, 1713, 1612, 1458, 1416, 1386, 1284,
1030, 751 cmꢀ1; For 13C and 1H NMR spectroscopic data, see Tables
1 and 2; ESIMS m/z 401 [M+H]+, 423 [M+Na]+, 439 [M+K]+, 399 [M–
H]ꢀ, 435 [M+Cl]ꢀ; HRESIMS m/z 423.1784 [M+Na]+ (calcd. for
4.3.9. Dunnianolide C (9)
Colorless needles (n-Hexane–EtOAc); mp 162–164 °C; ½a D20
ꢂ
–
39.8 (c 0.06, MeOH); UV (MeOH) kmax (loge) 201 (4.06), 230
C
23H28O6Na, 423.1778).
(4.02), 274 (2.88) nm; IR (KBr) mmax 3423, 2972, 2938, 2880,
1748, 1716, 1601, 1585, 1493, 1452, 1317, 1281, 1110, 1015,
714 cmꢀ1; For 13C and 1H NMR spectroscopic data, see Tables 1
and 3; ESIMS m/z 387 [M+H]+, 409 [M+Na]+, 425 [M+K]+, 385
[M–H]ꢀ, 421 [M+Cl]ꢀ; HRESIMS m/z 387.1804 [M+H]+ (calcd. for
4.3.2. Tashironin C (2)
Colorless amorphous solid; ½a D20
ꢂ
–43.3 (c 0.03, MeOH); UV
(MeOH) kmax (loge) 206 (4.11), 218 (4.16), 283 (4.33) nm; IR
(KBr) mmax 3445, 2944, 1712, 1635, 1450, 1386, 1334, 1284, 1173,
1027, 769 cmꢀ1; For 13C and 1H NMR spectroscopic data, see Tables
1 and 2; ESIMS m/z 435 [M+Na]+, 451 [M+K]+, 131 [C8H7CO]+, 411
[M–H]ꢀ, 447 [M+Cl]ꢀ; HRESIMS m/z 435.1788 [M+Na]+ (calcd. for
C22H27O6, 387.1802).
4.3.10. Dunnianolide D (10)
Colorless amorphous solid;
½
a 2D0
–37.8 (c 0.1, MeOH); CD
ꢂ
C24H28O6Na, 435.1778).
(MeOH) kmax (d
e
) 280 (+0.19), 302 (ꢀ0.17); UV (MeOH) kmax (log
e)
203 (3.80), 229 (4.01), 274 (3.02) nm; IR (KBr) mmax 3361, 2964,
2930, 1774, 1719, 1601, 1584, 1468, 1451, 1315, 1272, 1025,
713 cmꢀ1; For 13C and 1H NMR spectroscopic data, see Tables 1
and 3; ESIMS m/z 529 [M+Na]+, 545 [M+K]+, 505 [M–H]ꢀ, 541
[M+Cl]ꢀ; HRESIMS m/z 529.1820 [M+Na]+ (calcd. for C29H30O8Na,
529.1833).
4.3.3. 2
Colorless amorphous solid;
(MeOH) kmax (log ) 201 (4.30), 233 (4.17), 276 (3.14) nm; IR
a-Hydroxytashironin (3)
½
a 2D0
–19.1 (c 0.1, MeOH); UV
ꢂ
e
(KBr) mmax 3516, 2973, 2934, 1720, 1600, 1493, 1452, 1391, 1278,
1024, 712 cmꢀ1; For 13C and 1H NMR spectroscopic data, see Tables
1 and 2; ESIMS m/z 403 [M+H]+, 425 [M+Na]+, 441 [M+K]+, 437
[M+Cl]ꢀ; HRESIMS m/z 425.1587 [M+Na]+ (calcd. for C22H26O7Na,
425.1571).
4.3.11. 80-Oxo-6-hydroxy-dihydrophaseic acid (11)
Colorless granules (n-hexane–EtOAc); mp 164–165 °C; [½a D20
ꢂ
–
4.3.4. 2
Colorless amorphous solid;
(MeOH) kmax (log ) 203 (3.21), 232 (3.41), 275 (2.38) nm; IR
a-Acetoxytashironin (4)
65.3 (c 0.1, MeOH); UV (MeOH) kmax (loge) 200 (3.81), 259 (4.18)
½
a 2D0
+4.4 (c 0.09, MeOH); UV
ꢂ
nm; IR (KBr) mmax 3313, 2974, 2937, 2895, 1763, 1688, 1641,
1603, 1382, 1243, 1050, 884 cmꢀ1; For 13C and 1H NMR spectro-
scopic data, see Table 4; ESIMS m/z 335 [M+Na]+, 647 [2M+Na]+,
623 [2M–H]ꢀ; HRESIMS m/z 335.1099 [M+Na]+ (calcd. for
e
(KBr) mmax 3438, 2963, 2927, 1720, 1638, 1275, 1024, 713 cmꢀ1
;
For 13C and 1H NMR spectroscopic data, see Tables 1 and 2; ESIMS
m/z 467 [M+Na]+, 483 [M+K]+, 443 [M–H]ꢀ, 479[M+Cl]ꢀ; HRESIMS
m/z 467.1687 [M+Na]+ (calcd. for C24H28O8Na, 467.1676).
C15H20O7Na, 335.1101).
4.3.5. 2
a
-Hydroxytashironin A (5)
4.3.12. 6-Hydroxy-dihydrophaseic acid (12)
Colorless granules (EtOAc); mp 144–145 °C; ½a D20
ꢂ
–13.6 (c 0.1,
) 198 (3.81), 238 (4.14) nm; IR
Colorless needles (EtOAc); mp 104–106 °C; ½a D20
ꢂ
–10.9 (c 0.07,
MeOH); UV (MeOH) kmax (log
e
MeOH); UV (MeOH) kmax (loge) 197 (3.86), 256 (4.18) nm; IR
(KBr) mmax 3510, 2950, 2908, 1708, 1641, 1260, 1111, 1067, 1029,
767 cmꢀ1; For 13C and 1H NMR spectroscopic data, see Tables 1
and 2; ESIMS m/z 407 [M+H]+, 429 [M+Na]+, 445 [M+K]+, 405
[M–H]ꢀ, 441 [M+Cl]ꢀ; HRESIMS m/z 429.1861 [M+Na]+ (calcd. for
(KBr) mmax 3465, 3368, 2937, 2884, 1686, 1642, 1610, 1421, 1379,
1248, 1234, 1037, 885 cmꢀ1; For 13C and 1H NMR spectroscopic
data, see Table 4; ESIMS m/z 321 [M+Na]+, 619 [2M+Na]+, 297
[M–H]ꢀ, 595 [2M–H]ꢀ; HRESIMS m/z 321.1307 [M+Na]+ (calcd.
for C15H22O6Na, 321.1309).
C22H30O7Na, 429.1884).

146
J. Bai et al. / Phytochemistry 80 (2012) 137–147
4.4. Acid hydrolysis and determination of the absolute configuration of
the monosaccharide (Hara et al., 1987)
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 Pek-
ing Union Medical College for measuring the IR, UV, NMR, and MS
spectra.
Compound 7 (8 mg) was hydrolyzed by 2 M HCl–H2O (15 ml) at
95 °C for 10 h. The reaction mixture was diluted with H2O and
extracted with EtOAc (3 ꢁ 15 ml). The aqueous layer was evapo-
rated to dryness under vacuum, diluted repeatedly with H2O, and
evaporated in vacuo to give a neutral monosaccharide residue. From
the residue, glucose was detected by TLC [CH2Cl2:MeOH (5:1), Rf
0.43] with authentic sample. The residue (2 mg) was dissolved in
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
05.015.
pyridine (1 ml) containing L-cysteine methyl ester hydrochloride
(2 mg) and heated at 60 °C for 2 h, then evaporated under N2 stream
and dried in vacuo. The residue was dissolved in N-trimethylsilylim-
idazole(0.2 ml) and heated at 60 °C for 2 h. The reaction mixture was
partitioned between n-hexane and H2O (2 ml each), and the n-hex-
ane extract was analyzed by GC (Agilent 7890A) under the following
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Acknowledgments
This project was supported by the Natural Science Foundation
of China (No. 201072234, No. 21132009), and the National Science

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