Sesquiterpenes from the roots of illicium jiadifengpi

Article abstract of DOI:10.1055/s-0032-1328768

Two new sesquiterpenes (1, 2) and two new sesquiterpene glycosides (3, 4) with a seco-prezizaane skeleton, three new allo-cedrane sesquiterpene glycosides (5-7), and a new acorane sesquiterpene (8) as well as 15 known analogues were isolated from the roots of Illicium jiadifengpi used for the treatment of rheumatoid arthritis. The structures of these compounds were elucidated by extensive spectroscopic analysis and chemical methods. The absolute configurations of compounds 5-7 were confirmed by CD experiments. The configuration of compound 8 was assigned by single-crystal X-ray crystallographic analysis and CD experiments. Compounds 4, 6, 7, 14, and 23 showed moderate antiviral activities against Coxsackie virus B3, and all compounds were inactive when evaluated for their cytotoxic activities against five human tumor cell lines and neuroprotection. Georg Thieme Verlag KG Stuttgart New York.

Full text of DOI:10.1055/s-0032-1328768

1
056 Original Papers
Sesquiterpenes from the Roots of Illicium jiadifengpi
Authors
Guijie Zhang, Pengyu Zhuang, Xiaojing Wang, Shishan Yu, Shuanggang Ma, Jing Qu, Yong Li, Yunbao Liu, Yan Zhang,
Dequan Yu
Affiliation
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, Peopleʼs Republic of China
Key words
Abstract
The configuration of compound 8 was assigned
by single-crystal X‑ray crystallographic analysis
and CD experiments. Compounds 4, 6, 7, 14, and
23 showed moderate antiviral activities against
Coxsackie virus B3, and all compounds were inac-
tive when evaluated for their cytotoxic activities
against five human tumor cell lines and neuro-
protection.
"
l Illicium jiadifengpi
!
"
l Illiciaceae
Two new sesquiterpenes (1, 2) and two new ses-
quiterpene glycosides (3, 4) with a seco-prezi-
zaane skeleton, three new allo-cedrane sesquiter-
pene glycosides (5–7), and a new acorane sesqui-
terpene (8) as well as 15 known analogues were
isolated from the roots of Illicium jiadifengpi used
for the treatment of rheumatoid arthritis. The
structures of these compounds were elucidated
by extensive spectroscopic analysis and chemical
methods. The absolute configurations of com-
pounds 5–7 were confirmed by CD experiments.
"
l seco‑prezizaane sesquiter-
pene
"
l allo‑cedrane sesquiterpene
glycoside
l acorane sesquiterpene
"
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
mane (21) [9], cadinane-4β,5α,10β-triol (22) [10],
and bullatantriol (23) [9]. The following describes
the isolation and structure elucidation of the
!
Illicium jiadifengpi B.N. Chang (Illiciaceae) is dis-
tributed mainly in South China. Its cortex and root
bark have been used in Chinese folk medicine for
the treatment of rheumatoid arthritis. However,
many people have been seriously poisoned by its
use [1]. This threat of poisoning by I. jiadifengpi
prompted us to investigate the bioactive sub-
stances from the plant so as to evaluate its efficacy
and toxicity. In our previous research on the roots
of I. jiadifengpi, we isolated and determined the
structure of 19 diterpenes from the EtOAc extract;
some of them displayed significant activities
against Coxsackie virus B [2]. Our continuing ex-
amination of the root extract has resulted in the
characterization of eight new sesquiterpenes (1–
8). In addition, 15 known sesquiterpenes were
characterized: majucin (9) [3], neomajucin (10)
"
eight new sesquiterpenes (1–8) (l Fig. 1). The
antiviral activities against Coxsackie virus B3, cy-
totoxic activities against five human tumor cell
lines, and neuroprotective activities of the 23
compounds are also stated.
received
revised
accepted
Dec. 16, 2012
June 12, 2013
June 17, 2013
Bibliography
DOI http://dx.doi.org/
0.1055/s-0032-1328768
1
Results and Discussion
!
Published online July 22, 2013
Planta Med 2013; 79:
Compound 1 was isolated as colorless crystals. Its
molecular formula was determined to be
1
056–1062 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
C22H26O from the HR-ESIMS and NMR data. A de-
7
tailed NMR analysis indicated that 1 was a benzo-
yl-substituted pseudomajucin sesquiterpene and
that it was structurally similar to the known com-
pound 2β-benzoyloxypseudomajucin [11], except
for the presence of an additional hydroxyl group
at C-6. The HMBC correlations from H -12 (δ
Correspondence
Prof. Dr. Shishan Yu
State Key Laboratory of Bio-
active Substance and Function
of Natural Medicines
[4], (2R)-2-hydroxyneomajucin (11) [4], (2S*)-hy-
Institute of Materia Medica
Chinese Academy of Medical
Sciences and Peking Union
Medical College
No. 1 Xian Nong Tan
Beijing 100050
Peopleʼs Republic of China
Phone: + 861063165326
Fax: + 861063017757
yushishan@imm.ac.cn
droxyneomajucin (12) [5], (2R*)-hydroxy-3,4-de-
hydroxyneomajucin (13) [5], 2-oxoneomajucin
3
H
1.24)/H -13 (δ 1.00) to C-6 (δC 79.1) supported
3
H
(
14) [5], 2-oxo-3,4-dehydroxyneomajucin (15)
[5], (1R*)-2-oxo-3,4-dehydroxyneomajucin (16)
5], pseudomajucin (17) [6], 7-O-β-D-glucopy-
the above deduction. The ROESY spectrum
showed cross-peaks between H -15 and H-10a/
3
[
H-3β and between H-2 and H-1/H-3α, which indi-
ranosylpseudomajucin (18) [6], abscisic acid (19)
[7], roseoside (20) [8], 1β,4β,7α-trihydroxyeudes-
cated that CH -15 and the benzoyloxy group were
3
β-oriented. The ROESY correlations of H -12 with
3
Zhang G et al. Sesquiterpenes from the… Planta Med 2013; 79: 1056–1062

Original Papers 1057
Fig. 1 Structures of compounds 1–8.
H-14b and of H-14a with H-10b confirmed the β-orientation of
CH -12 and the α-orientation of CH -13. Therefore, 1 was eluci-
dated as 2β-benzoyloxy-6α-hydroxypseudomajucin.
69.5). The glucose was deduced to be in the β-configuration from
its large coupling constant (8.0 Hz) of the anomeric proton, and it
was confirmed to be in the D-configuration by acid hydrolysis and
GC analysis of the derivatized sugar [14]. Thus, compound 4 was
deduced to be 14-O-β-D-glucopyranosylpseudomajucinone.
Compound 5 was obtained as a white powder. The molecular for-
3
3
Compound 2 was obtained as colorless crystals, and the molecu-
lar formula was determined to be C15H24O by its HR-ESIMS. Its
7
1
13
"
H and C NMR data (l Tables 1 and 2) were very similar to those
of (11)7,14-ortholactone-3α-hydroxyfloridanolide [12], except
that the position of the hydroxyl group is located at C-1 in 2 in-
stead of at C-3. This finding was confirmed by the HMBC correla-
tions from H-10 (δ 4.45)/H -15 (δ 1.63) to C-1 (δ 82.3). More-
mula C21H32O of 5 was indicated by the HR-ESIMS and NMR da-
9
ta. The NMR data suggested that 5 was a sesquiterpene glycoside.
The NMR data of its aglycone showed a close resemblance to
those of allo-cedrol [15], indicating that both possessed the allo-
cedrane skeleton. The HMBC correlations from H -15/H-4 (δ
H
3
H
C
over, the ROESY spectrum showed a correlation between H -15
3
3
H
and H-10, which confirmed the α-orientation of the hydroxyl
group at C-1. Thus, 2 was assigned as (11)7,14-ortholactone-1α-
hydroxyfloridanolide.
3.62) to C-2 (δ 73.9) suggested an additional hydroxyl group at
C
C-2. In addition, the HMBC correlations from H -7/H -10/H -12
2
2
3
to C-11 (δC 213.4) and from H-4/H -14 to C-13 (δC 174.9) re-
3
Compound 3 was isolated as a white powder, and its molecular
formula was determined to be C21H32O10 by HR-ESIMS. A com-
parison of the NMR data of 3 with those of cycloparviflorolide
vealed that there was a ketone group and an ester carbonyl lo-
cated at C-11 and C-13, respectively. The linkage of the sugar unit
at C-13 was supported by the HMBC correlation from H-1′ to C-
[13] revealed that both shared a pseudoanisatin sesquiterpene
13. In the ROESY spectrum, correlations between H-2/H -14 and
3
skeleton. The main differences were the absence of the hydroxyl
groups at C-6 and C-10 and the presence of an oxygen-bearing
substitute at C-8. In addition, there was a glucopyranosyl moiety
H-10a and between H-4 and H-7α/H‑8α/H -15 indicated that H-2
3
and CH -14 were β-oriented and that H-4 and CH -15 were α-
3
3
1
13
oriented. Based on the H and C NMR data, the sugar unit was
identified as a β-glucopyranosyl unit. The configuration of the
glucose unit was determined to be D after hydrolysis and GC anal-
ysis [14]. Furthermore, the absolute configuration for the agly-
cone of 5 was determined by analysis of its CD spectrum (Sup-
porting Information, Fig. 39S). The negative Cotton effect at
295 nm (Δε − 2.57) indicated that the absolute configuration of
the aglycone was 1R, 2S, 4S, 5R, 6R, and 9S based on the octant
rule for cyclohexanone [16]. Therefore, 5 was determined to be
(1R, 2S, 4S, 5R, 6R, 9S)-2-hydroxy-11-oxoallo-cedra-13-oic acid
13-O-β-D-glucopyranosyl ester.
(
δ 105.2, C-1′; δ 5.00, H-1′) connected to C-8 in 3. The observed
C H
HMBC correlations from H -12 (δ 1.28)/H -13 (δ 0.90) to C-6
3
H
3
H
(
δC 49.1), H-6 (δH 2.26)/H-1′ to C-8 (δ 81.3), and H-8 (δ 4.32)
C H
to C-10 (δ 32.1) supported the above deduction. In the ROESY
C
spectrum, the correlations between H-8/H -12 and H-10a indi-
3
cated the β-orientations of H-8 and CH -12. The large coupling
3
constant (8.0 Hz) of the anomeric proton revealed that glucose
was in the β-configuration, and the D-configuration of the moiety
was established by GC analysis [14]. Thus, compound 3 was elu-
cidated as 8-O-β-D-glucopyranosyl-8α-hydroxy-6,10-dideoxycy-
cloparviflorolide.
Compound 6 was isolated as a white powder. The molecular for-
Compound 4 was obtained as a white powder. The molecular for-
mula of 4 was established as C21H32O10 by the HR-ESIMS and
NMR data. The 13C NMR data (l Table 2) showed twenty-one car-
bon signals, of which fifteen were assigned to the aglycone and
six to the glucopyranosyl moiety. A comparison of the NMR data
of 4 with those of 2,14-diacetylpseudomajucinone [6] revealed
that they possessed the same skeleton. The difference was the
presence of a glucopyranosyl moiety at C-14 of the skeleton. This
was confirmed by the HMBC correlation from H-1′ to C-14 (δC
mula C21H32O was established by the HR-ESIMS and NMR data.
A comparison of the NMR data of 6 (l Tables 1 and 2) with those
8
"
"
of 5 indicated that the hydroxyl group at C-2 and the ester car-
boxyl group at C-13 were substituted by ketocarboxyl and hy-
droxymethyl, respectively. These substitutions were confirmed
by the HMBC correlations from H-1/H-3/H -15 to C-2 (δ 217.2)
3
C
and from H-4/H -14 to C-13 (δ 76.7). The glucose connected to
3
C
C-13 was established by the HMBC correlation from H-1′ (δH
4.62) to C-13. The ROESY correlations between H -15/H -14 and
3
3
Zhang G et al. Sesquiterpenes from the… Planta Med 2013; 79: 1056–1062

1
058 Original Papers
Table 1 1H NMR data for compounds 1–8.
No.
1a
2b
3b
4b
5b
6b
7c
8a
d
1
2.57 m
3.16 m
1.27 m
1.74 m
4.26 m
1.72 m
1.98 q (7.2)
1.94 m
2.12 m
d
2
2
3
α
β
α
5.35 t (4.5)
2.35 m
2.55 m
2.04 m
1.87 m
d
d
1.84 m
4.46 m
1.29 m
d
d
d
2.99 dd
1.84 m
2.21 dd
1.96 br dd
(12.5, 5.0)
2.21 d (10.6)
2.23 m
1.87 m
(15.0, 4.5)
(14.5, 3.5)
2.38 d (14.5)
d
d
d
d
3
4
6
7
β
2.18 d (15.0)
2.89 m
1.84 m
1.75 dd
2.21 d (10.6)
2.14 m
1.66 m
(
12.5, 7.0)
3.62 dd
13.0, 6.5)
d
2.41 t (10.6)
2.41 t (9.5)
1.64 m
(
2.26 q (7.5)
2.30 m
2.67 d (9.0)
2
.00 d (9.0)
d
d
4.19 br s
2.91 m
.29 m
1.17 m
1.14 m
1
1.64 m
1.64 m
d
d
8
8
9
α
β
2.10 d (13.5)
1.99 d (13.5)
3.11 br d (13.5)
1.91 br d (13.5)
3.50 d (18.0)
2.61 d (18.0)
1.88 m
1.32 m
1.17 m
1.14 m
2.40 m
d
d
4.32 s
1.17 m
1.14 m
1.93 m
d
1
.28 m
1
0 a
0 b
3.02 d (19.5)
2.76 d (19.5)
4.45 s
3.11 d (14.5)
2.73 d (14.5)
3.23 d (18.0)
2.69 d (18.0)
2.42 dd
2.49 d (18.6)
2.46 d (18.0)
2.17 d (18.0)
2.02 m
1.56 m
2.29 m
(
19.0, 2.5)
2.12 dd
19.0, 1.0)
d
1
2.19 d (18.6)
(
1
1
1
1
2
3
1.24 s
1.00 s
1.80 s
1.28 s
1.28 d (7.5)
0.90 s
1.26 d (7.0)
0.87 s
1.31 s
1.25 s
0.99 s
0.99 s
3.79 d (9.0)
3.84 d (9.0)
3.20 d (9.0)
0.76 s
3.65 br s
3
.19 d (9.0)
3.65 br s
d
d
14a
14b
15
3.77 d (9.5)
3.50 d (9.5)
1.00 d (7.5)
4.61 d (12.5)
3.63 d (12.5)
1.63 s
4.27 m
4.26 m
0.75 s
0.78 d (6.5)
d
4.00 m
3.52 d (9.0)
1.07 d (6.5)
4.64 d (8.0)
3.86 t (8.0)
4.13 t (9.0)
4.15 t (9.0)
3.79 m
1.04 d (6.5)
5.00 d (8.0)
1.06 d (7.5)
6.33 d (8.0)
4.15 t (9.0)
4.25 t (9.0)
4.27 t (9.0)
4.00 m
0.90 d (7.2)
4.62 d (7.8)
3.88 t (7.8)
4.19 t (9.0)
4.16 t (9.0)
3.92 br t (6.0)
0.87 d (6.5)
4.57 d (7.5)
3.83 t (9.0)
4.14 t (9.0)
4.00 t (9.0)
4.02 t (9.0)
0.89 d (4.0)
1
2
3
4
5
′
′
′
′
′
d
4.05 t (8.5)
7.90 d (8.5)
7.38 t (9.0)
7.52 t (7.5)
4.18 t (9.0)
4.10 t (9.0)
3.91 m
d
6
6
′a
′b
7.38 t (9.0)
4.55 br d (10.0)
4.41 br d (11.5)
4.40 dd
4.56 dd
4.72 m
(
12.0, 2.5)
4.30 dd
12.0, 2.5)
(11.4,1.8)
4.34 dd
(overlapped)
d
d
d
4.31 m
4.29 m
4.16 m
(
(11.4, 4.5)
(overlapped)
7
1
2
3
4
5
′
7.90 d (8.5)
′′
′′
′′
′′
′′
5.68 d (1.0)
4.83 m
4.76 t (6.0)
4.68 m
a 4.28 dd
(
12.0, 3.5)
b 4.14 dd
12.0, 5.0)
(
Data were recorded at a 500 MHz in CD3OD; b 500 MHz in pyridine-d5; c 600 MHz in pyridine-d5. d Signals overlapped
"
H -10 and between H-4 and H-1/H -13 revealed the β-orienta-
NMR spectra of 7 (l Tables 1 and 2) were similar to those of 6,
except for the presence of an α-L-arabinofuranosyl unit at the C-
6′ of the glucose. This observation was confirmed by 2D NMR ex-
periments and GC analysis of the hydrolysate of 7 [14]. In the
HMBC spectrum, the correlation from H-1′′ (δH 5.68) to C-6′ (δC
68.1) confirmed the location of the arabinose moiety. Thus, com-
pound 7 was assigned as (1S, 4S, 5R, 6R, 9S)-13-O-α-L-arabinofu-
ranosyl-(1 → 6)-β-D-glucopyranosyl-13-hydroxyallo-cedra-2,11-
dione.
2
2
tions of CH -14 and CH -15 and the α-orientation of H-4. The
3
3
CD spectrum (Supporting Information, Fig. 50S) of 6 showed a
negative Cotton effect at 293 nm (Δε − 5.58), which was stronger
than that of 5 at the same mol concentration, indicating that the
cyclohexanone and cyclopentanone moieties could contribute to
the Cotton effect. However, the additional contribution of the
cyclopentanone moiety showed no impact on the assignment of
the 1S, 4S, 5R, 6R, and 9S configurations for 6. Thus, 6 was eluci-
dated as (1S,4S,5R,6R,9S)-13-O-β-D-glucopyranosyl-13-hydroxy-
allo-cedra-2,11-dione.
Compound 8 was obtained as colorless needles. Its molecular
formula was determined to be C15H24O from the HR-ESIMS and
4
1
Compound 7 was obtained as a white powder with the molecular
formula C26H40O12 based on the HR-ESIMS and NMR data. The
NMR data. The IR absorption bands at 3436, 1736, and 1702 cm−
indicated the presence of hydroxyl and carbonyl functionalities.
Zhang G et al. Sesquiterpenes from the… Planta Med 2013; 79: 1056–1062

Original Papers 1059
Table 2 13C NMR data for compounds 1–8.
No.
1a
52.5
2b
82.3
3b
37.8
4b
52.1
5b
45.5
6c
53.3
7b
53.3
8a
1
2
3
4
5
6
7
8
9
44.7
27.8
29.2
39.3
54.3
47.3
216.3
45.9
33.9
31.6
52.5
NDd
80.0
43.8
41.3
32.8
91.1
48.2
77.5
78.7
27.7
53.5
75.4
113.6
21.8
15.0
67.9
23.1
30.0
23.7
92.8
44.5
49.1
107.9
81.3
56.6
32.1
174.6
9.3
72.7
44.8
97.7
45.9
47.1
211.1
51.9
49.7
41.8
178.2
9.1
73.9
35.1
45.9
48.3
51.0
28.4
30.1
44.6
47.7
213.4
15.4
174.9
17.9
10.2
96.7
74.2
78.8
71.0
79.5
62.2
217.2
36.8
44.2
40.7
48.8
27.8
19.5
43.0
49.2
218.5
14.3
76.7
15.7
8.1
217.2
36.7
44.0
40.6
48.7
27.6
19.5
42.8
49.1
218.5
14.3
76.6
15.7
8.0
103.7
51.5
79.1
107.0
50.2
49.6
1
1
1
1
1
1
0
1
2
3
4
5
1
2
3
4
5
6
7
1
2
3
4
5
41.6
179.2
17.9
12.7
24.1
69.3
15.8
105.2
75.3
78.2
71.3
78.8
62.9
20.0
69.5
9.8
65.4
17.2
14.7
71.7
10.1
′
167.6
131.2
130.7
129.5
134.2
129.5
130.7
103.8
75.0
78.5
70.9
78.2
62.4
105.1
75.6
77.8
71.6
78.7
62.8
105.7
75.4
77.6
71.7
77.0
68.1
′
′
′
′
′
′
′′
′′
′′
′′
′′
110.1
83.2
78.6
86.0
62.6
Data were recorded at a 125 MHz in CD3OD; b 125 MHz in pyridine-d5; c 150 MHz in pyridine-d5. d Signal not detected
Analysis of the 1D and 2D NMR data revealed that 8 was a sesqui-
1
1
terpene. In the H- H COSY spectrum, the correlations H-1/H -2/
2
H -3/H‑4/H -14 and H -15/H‑8/H -9/H -10 demonstrated the
2
3
3
2
2
presence of vicinal coupling systems. However, the absence of de-
finitive NMR signals prevented a determination of the structure.
We were fortunate to obtain small crystals of 8. The X‑ray assign-
ment of the planar structure and the relative configuration are
"
shown in l Fig. 2. The negative Cotton effect at 294 nm (Δε
−
0.32) in the CD spectrum (Supporting Information, Fig. 67S) in-
dicated that the absolute configuration of 8 was 1S, 4R, 5S, 8S, and
1R according to the n→π* transition of cyclohexanone [16].
1
Thus, compound 8 was deduced to be 1S-(2R-hydroxymethylace-
toxy)-4R, 8S-dimethyl spiro[4.5]dec-7-one [17].
Fig. 2 X‑ray crystal structure of compound 8.
To determine the efficacy and toxicity of this plant, the isolated
compounds were tested against five human tumor cell lines
[
18]. All of them were found to be inactive. However, this result
Materials and Methods
did not mean that the plant was not toxic as our previous inves-
tigations [2] showed high cytotoxicity against Vero cells by some
of the compounds isolated from the plant. Further, compound 10
was found to exhibit picrotoxin-like toxicity [19]. Based on the
reported literature [2,20], the isolates were also evaluated for
possible antiviral (against Coxsackie virus B3 [21]) and neuropro-
tective activities [22]. Compounds 4, 6, 7, 14, and 23 showed ac-
tivities against Coxsackie virus B3 with IC50 values of 40.50,
!
General experimental procedures
The melting points were measured on an XT5B (Beijing Keyi Elec-
tric Light Instrument Co., Ltd) melting instrument and were un-
corrected. The optical rotations were measured on a JASCO P-
2000 automatic polarimeter. The UV spectra were recorded on a
JASCO V-650 spectrophotometer. The CD spectra were obtained
on a JASCO J-815 spectropolarimeter. The IR spectra were re-
corded on a Nicolet 5700 FT‑IR microscope spectrophotometer
(FT‑IR Microscope Transmission). The NMR spectra were col-
lected on an INOVA-500 or a Mercury-300 spectrometer in pyri-
dine-d , acetone-d , or MeOH-d with solvent peaks as refer-
6
6.67, 91.07, 27.14, and 9.86 µmol/mL, respectively, and SI values
of 2.20–8.71 (the positive control, ribavirin, IC50 1.25 µmol/mL, SI
.84). However, none of the compounds showed neuroprotective
activity.
6
5
6
4
ences. The ESIMS data were collected on an Agilent 1100 Series
LC/MSD Trap/SL (Turbo Ionspray source) spectrometer. The HR-
Zhang G et al. Sesquiterpenes from the… Planta Med 2013; 79: 1056–1062

1
060 Original Papers
ESIMS data were measured using an Agilent 6250 Accurate-Mass
Q‑TOF LC/MS spectrometer. Silica gel (200–300 mesh; Qingdao
Marine Chemical, Inc.), Sephadex LH-20 (Pharmacia), polyamide
the eluent. Y4-3 (1.11 g) yielded six subfractions (Y4-3–1−Y4-3–
6) when chromatographed on Sephadex LH-20 with CH Cl /
2
2
CH OH (10:1, 2 L) as the eluent. Y4-3–2 (0.3 g) yielded com-
3
(30–60 mesh; Jiangsu Linjiang Chemical Reagents Factory), and
pounds 14 (17 mg, t = 15 min), 15 (56 mg, t = 35 min), 16
R
R
ODS (50 µm; YMC) were used for column chromatography. Pre-
parative HPLC was carried out on a Shimadzu LC-6AD instrument
with an SPD-20A or an RID detector using a YMC-Pack ODS‑A col-
umn (250 × 50 mm, 5 µm). TLC was carried out with precoated
silica gel GF254 glass plates (Qingdao Marine Chemical Inc.). The
spots were visualized under UV light or by spraying with 10%
H SO acid in EtOH followed by heating. The GC analysis was con-
(10 mg, t = 45 min), and 13 (60 mg, t = 48 min) when chromato-
R R
graphed on preparative HPLC with 5% CH CN/0.03% TFA‑H O
3
2
(7 mL/min) as the eluent. Y4-3–5 (0.05 g) was repeatedly crystal-
lized with CH Cl /CH OH (1:1, v/v) to yield compound 9 (40 mg).
2
2
3
Y4-4 (0.45 g) yielded two fractions (Y4-4–1−Y4-4–2) when chro-
matographed on Sephadex LH-20 with CH Cl /CH OH (1:1, 0.5 L)
2
2
3
as eluent. Y4-4–1 (0.15 g) yielded compound 17 (45 mg) when re-
peatedly crystallized with CH Cl /CH OH (1:1, v/v). Fraction Y4–
2
4
ducted on an Agilent 7890A instrument.
2
2
3
9
(0.7 g) yielded nine fractions (Y4–9–1−Y4–9–9) when chroma-
Plant material
tographed on Sephadex LH-20 column with 50% MeOH/H O (2 L)
2
The roots from I. jiadifengpi samples were collected in Guangxi
Province, China in August 2009 and were identified by Prof. Song-
ji Wei of the Guangxi Traditional Medical College. A voucher
specimen (No. 21976) was deposited at the Herbarium of the De-
partment of Medicinal Plants, Institute of Materia Medica, Chi-
nese Academy of Medical Sciences.
as the eluent. Y4–9–4 (0.09 g) yielded compound 1 (10 mg,
tR = 44 min) when chromatographed on preparative HPLC with
CH CN/0.03% TFA‑H O (35:65, v/v, 7 mL/min) as the eluent.
Fraction Y5 (4.7 g) yielded six subfractions (Y5-1−Y5–6) when
chromatographed on Sephadex LH-20 and then on ODS column
3
2
(40 × 3 cm, 340 g) with an MeOH/H O gradient (5:95, 1 L;
2
10:90, 1 L; 15:85, 1.5 L; 20:80, 3 L; 25:75, 3 L; 30:70, 3 L;
Extraction and isolation
35:65, 3 L; 45:55, 2 L; 50:50, 2 L; 55:45, 2 L; 60:40, 1.5 L;
65:35, 1.5 L; 70:30, 1 L; 80:20, 1 L; and 90:10, 1 L) as the eluent.
Y5-1 (0.25 g) yielded two fractions (Y5-1–1−Y5-1–2) when chro-
matographed on Sephadex LH-20 with CH Cl /CH OH (1:1, 0.3 L)
Roots of I. jiadifengpi (10 kg) were extracted three times with 95%
EtOH (50 L × 3) under reflux. The residue (900 g) obtained on
evaporation of the solvent was absorbed onto kieselguhr and ex-
tracted successively with petroleum ether, EtOAc, and MeOH. The
EtOAc extract (47 g) yielded five fractions (Y1−Y5) when chroma-
tographed on silica gel column (80 × 8 cm, 1000 g) with petro-
leum ether/acetone (100:0 to 100:25) and CH Cl /MeOH
2
2
3
as the eluent. Y5-1–1 (0.2 g) yielded compounds 11 (87 mg,
t = 37 min) and 12 (13 mg, t = 44 min) when chromatographed
R
R
on preparative HPLC with CH CN/0.03% TFA‑H O (5:95, v/v,
3
2
7 mL/min) as the eluent. Y5-2 (0.07 g) was further purified with
a Sephadex LH-20 and was repeatedly crystallized with CH Cl /
2
2
(100:4 to 100:25) as eluents.
2
2
Fraction Y3 (14.0 g) yielded five fractions (Y3-1−Y3–5) when
chromatographed on silica gel column (80 × 4 cm, 500 g) with pe-
troleum ether/acetone (6:1, 10 L) as the eluent. Fraction Y3–4
CH OH (1:1, v/v) to yield compound 2 (23 mg). Compound 23
(23 mg) was obtained by repeated chromatography on Sephadex
LH-20 from Y-5-5 (0.07 g).
3
(3.2 g) yielded six subfractions (Y3–4–1−Y3–4–6) when chroma-
The CH OH fraction (540 g) yielded three fractions when chro-
3
tographed on ODS column (40 × 3 cm, 340 g) with an MeOH/H O
matographed on a D101 macroporous adsorbent resin column
2
gradient (45:55, 1.5 L; 50:50, 1.5 L; 55:45, 2 L; 60:40, 2 L;
(120 × 15 cm, 2500 g) with H O (50 L), 50% EtOH (100 L), and
2
6
5:35, 2 L; 70:30, 3 L; 75:25, 1 L; 80:20, 1 L; 90:10, 1 L; and
95% EtOH (50 L) as eluents. The 50% fraction (200 g) yielded six
fractions (M1−M6) when chromatographed on silica gel column
(150 × 10 cm, 2000 g) with CH Cl /CH OH (100:0 to 100:50) as
100:0, 3 L) as the eluent. Y3–4–2 (0.45 g) yielded compound 19
(30 mg, t = 20 min) when chromatographed on Sephadex LH-20
R
2
2
3
(120 × 2.5 cm, 100 g) and then on preparative HPLC with 30%
the eluent.
CH CN/0.03% TFA‑H O (7 mL/min) as the eluent. Fraction Y3–5
M4 (7.0 g) yielded twenty fractions (M4-1−M4-20) when chro-
3
2
(6.5 g) yielded seven subfractions (Y3–5–1−Y3–5–7) when chro-
matographed on ODS column (40 × 3 cm, 340 g) with an MeOH/
matographed on Sephadex LH-20 (100 × 4 cm, 300 g) with CH Cl2
H O gradient (10:90, 1 L; 15:85, 1.5 L; 20:80, 1.5 L; 25:75,
2
2
(
5 L) and then on ODS column (40 × 3 cm, 340 g) with an MeOH/
1.5 L; 30:70, 1.5 L; 35:65, 1.5 L; 40:60, 1.5 L; 45:55, 3 L; 50:50,
3 L; 55:45, 3 L; 60:40, 2 L; 65:35, 1.5 L; and 70:30, 1 L) as the
H O gradient (20:80, 2 L; 25:75, 2 L; 30:70, 2 L; 35:65, 3 L;
2
40:60, 3 L; 45:55, 3 L; 50:50, 3 L; 55:45, 2 L; 60:40, 1.5 L;
eluent. M4-13 (0.16 g) yielded compound 3 (15 mg, t = 33 min)
R
65:35, 1.5 L; and 70:30, 1.5 L) as the eluent. Fraction Y3–5–2
when chromatographed on Sephadex LH-20 and on preparative
(
0.6 g) yielded compound 10 (400 mg, t = 45 min) when chroma-
HPLC with 15% CH CN/0.03% TFA‑H O (7 mL/min) as the eluent.
R
3
2
tographed on preparative HPLC with 20% CH CN/0.03% TFA‑H O
M4-17 (0.58 g) yielded seven fractions (M4-17–1−M4-17–7)
3
2
(7 mL/min) as the eluent. Fraction Y3–5–3 (80 mg) yielded com-
when chromatographed on Sephadex LH-20 with 30% CH OH/
3
pound 21 (32 mg) when chromatographed on silica gel column
H O (3 L) as the eluent. M4-17–1 (0.08 g) yielded compound 6
2
(
40 × 1.5 cm, 200–300 mesh, 15 g) with CH Cl /CH OH (10:1,
(10 mg, t = 39 min) when chromatographed on preparative HPLC
2
2
3
R
0.5 L) as eluent. Y3–5–4 (0.23 g) yielded compound 22 (20 mg)
with 20% CH CN/0.03% TFA‑H O (7 mL/min) as the eluent.
3
2
when chromatographed on Sephadex LH-20 with CH Cl /CH OH
M5 (32 g) yielded two fractions when chromatographed on poly-
2
2
3
(10:1) as the eluent.
amide column (100 × 8 cm, 500 g) with H O (12 L) and 95% EtOH
2
Fraction Y4 (7.5 g) yielded ten fractions (Y4-1−Y4-10) when chro-
(8 L) as eluents. The H O fraction (15 g) yielded eighteen fractions
2
matographed on ODS column (40 × 3 cm, 340 g) with an MeOH/
(M5-1−M5-18) when chromatographed on ODS column
H O gradient (5:95, 1 L; 10:90, 2 L; 15:85, 2 L; 20:80, 3 L;
2
(40 × 3 cm, 340 g) with an MeOH/H O gradient (3:97, 2 L; 7:93,
2
2
2
5:75, 3 L; 30:70, 3 L; 35:65, 3 L; 40:60, 2 L; 45:55, 2 L; 50:50,
L; 55:45, 2 L; 60:40, 1.5 L; 65:35, 1.5 L; and 70:30, 1 L) as the
2 L; 10:90, 3 L; 5:85, 3 L; 20:80, 2 L; 25:75, 2 L; 30:70, 2 L; and
40:60, 1 L) as the eluent. M5-5 (0.9 g) yielded seven fractions
(M5-5–1−M5-5–7) when chromatographed on Sephadex LH-20
with 10% CH OH/H O (1 L) as the eluent. M5-5–2 (0.32 g) yielded
eluent. Y4-1 (0.15 g) yielded compound 8 (32 mg) when chroma-
tographed on Sephadex LH-20 with CH Cl /CH OH (10:1, 2 L) as
2
2
3
3
2
Zhang G et al. Sesquiterpenes from the… Planta Med 2013; 79: 1056–1062

Original Papers 1061
compounds 18 (99 mg, t = 20 min) and 4 (28 mg, t = 37 min)
1S-(2R-Hydroxymethylacetoxy)-4R,8S-dimethyl spiro[4.5]dec-7-
R
R
20
when chromatographed on preparative HPLC with CH CN/0.03%
one (8): colorless needles, mp 148–150°C; [α]
CH OH); CD (CH OH) 293 (Δε − 0.32) nm; IR v 3436, 3233,
max
D
− 36.6 (c 0.083,
3
TFA‑H O (12:88, v/v, 7 mL/min) as the eluent. M5–6 (0.8 g)
2
3
3
−
1
1
yielded four fractions (M5–6–1−M5–6–4) when chromato-
2966, 2938, 1736, 1702, 1458, 1290, 1049, and 1017 cm ; H
NMR and C NMR, see l Tables 1 and 2; negative HR-ESIMS m/z
267.1586 [M – H] (calcd. for C15H23O , 267.1602).
1
3
"
graphed on Sephadex LH-20 with 10% CH OH/H O (1 L) as the
3
2
−
eluent. M5–6–1 (0.14 g) yielded compound 20 (28 mg, t =
R
4
3
5 min) when chromatographed on preparative HPLC with 12%
X‑ray crystallographic analysis of compound 8. C30H50O ,
M = 554.70, size 0.30 × 0.30 × 0.20 mm , orthorhombic, space
9
3
CH CN/0.03% TFA‑H O (7 mL/min) as the eluent. M5–9 (0.9 g)
3
2
yielded six fractions (M5–9–1−M5–9–6) when chromatographed
group P2(1)2(1)2(1); a = 6.6776(13) Å, b = 19.663(4) Å, c = 23.491
3
on Sephadex LH-20 with 10% CH OH/H O (1.5 L) as the eluent.
(5) Å, α = β = γ = 90°, V = 3084.4(11) Å , T = 293(2) K, Z = 4,
3
2
3
−1
M5–9–2 (0.45 g) yielded compound 5 (158 mg, t = 83 min) when
ρcalcd = 1.195 Mg/m , µ (Mo Ka) = 0.087 mm . The total number
of reflections measured was 3977, of which 1344 reflections
were observed (|F | 2 ≥ 2σ|F | 2), R1 = 0.0637, wR2 = 0.1416. The
crystal structure of 8 was solved by direct methods (SHELXS), ex-
panded using difference Fourier techniques and refined by the
program and full-matrix least-squares calculations. The crystal-
lographic data for the structure of 8 have been deposited in the
Cambridge Crystallographic Data Centre (deposition number
CCDC 893804). Copies of the data can be obtained free of charge
from the CCDC via www.ccdc.cam.ac.uk/data_request/cif.
R
chromatographed on preparative HPLC with 12.5% CH CN/0.03%
3
TFA‑H O (7 mL/min) as the eluent. M5-13 (0.65 g) yielded nine
2
fractions (M5-13–1−M5-13–9) when chromatographed on Se-
phadex LH-20 with 10% CH OH/H O (2 L) as the eluent. M5-13–
3
2
4
(0.1 g) yielded compound 7 (16 mg, t = 39 min) when chroma-
R
tographed on preparative HPLC with 17.5% CH CN/0.03%
3
TFA‑H O (7 mL/min) as the eluent.
2
The purities of the isolated compounds ranged from 95 to 99.5%
as determined by HPLC.
2
β-Benzoyloxy-6α-hydroxypseudomajucin (1): colorless crystals,
20
mp 198–200°C; [α]
D
− 67.2 (c 0.063, CH OH); UV (CH OH) λ
Acid hydrolysis and determination of the absolute
configuration of the monosaccharides [14]
3
3
max
(
1
log ε) 230 (4.02), 273 (2.86) nm; IR v
3498, 3441, 2984,
max
1
−
1
13
713, 1600, 1451, 1299, and 1279 cm ; H NMR and C NMR,
Compound 7 (2 mg) was dissolved in 2 M HCl−H O (3 mL) and
2
"
+
see l Tables 1 and 2; positive HR-ESIMS m/z 403.1760 [M + H]
heated at 70°C for 12 h. The reaction mixture was extracted with
EtOAc. The aqueous layer was evaporated under vacuum, diluted
+
(calcd. for C22H27O , 403.1751), m/z 425.1580 [M + Na] (calcd.
7
for C22H26O Na, 425.1571).
repeatedly with H O and evaporated in vacuo to furnish a neutral
7
2
(
11)7,14-Ortholactone-1α-hydroxyfloridanolide (2): colorless
residue. The residue was dissolved in anhydrous pyridine (1 mL),
to which 2 mg of L-cysteine methyl ester hydrochloride was
added. The mixture was stirred at 60°C for 2 h, and after evapo-
ration in vacuo to dryness, 0.2 mL of N-trimethylsilylimidazole
was added. The mixture was kept at 60°C for another 2 h. The re-
20
crystals, mp 260°C; [α]
3
NMR, see l Tables 1 and 2; positive HR-ESIMS m/z 339.1408 [M
+
D
− 3.5 (c 0.062, CH OH); IR v
304, 1721, 1459, 1383, 1353, and 798 cm ; H NMR and
3393,
3
max
−
1
1
13
C
"
+
Na] (calcd. for C15H24O Na, 339.1414).
7
8
-O-β-D-Glucopyranosyl-8α-hydroxy-6,10-dideoxycycloparviflor-
action mixture was partitioned between n-hexane and H O
2
20
olide (3): white powder, [α]
3
NMR, see l Tables 1 and 2; positive HR-ESIMS m/z 467.1886 [M
+
D
− 28.1 (c 0.059, CH OH); IR v
(2 mL each). The n-hexane extract was analyzed by GC under
the following conditions: capillary column, HP-5 (30 m ×
0.25 mm, with a 0.25 µm film; Dikma); detection, FID; detector
temperature, 280°C; injection temperature, 250°C; initial tem-
perature 100°C for 2 min, then raised to 280°C at 10°C/min, final
3
max
414, 2964, 2877, 1710, 1677, and 1076 cm ; H NMR and 13C
−
1
1
"
+
Na] (calcd. for C21H32O10Na, 467.1888).
14-O-β-D-Glucopyranosylpseudomajucinone (4): white powder,
2
0
[α]
D
− 2.7 (c 0.056, CH OH); IR v
3427, 3317, 2900, 1739,
temperature maintained for 10 min; and carrier, N gas. From the
3
max
13
2
−
1
1
"
1
2
691, and 1077 cm ; H NMR and C NMR, see l Tables 1 and
; positive HR-ESIMS m/z 467.1887 [M + Na]+ (calcd. for
acid hydrolysate of 7, D-glucose and L-arabinose were confirmed
by comparing the retention times of their derivatives with those
of authentic sugars derivatized in a similar way, which showed
retention times of 14.85 and 11.32 min, respectively. The constit-
uent sugars of compounds 3–6 were identified by the same
method as 7.
C21H32O10Na, 467.1888).
(1R,2S,4S,5R,6R,9S)-2-Hydroxy-11-oxoallo-cedra-13-oic acid 13-
2
0
O-β-D-glucopyranosyl ester (5): white powder, [α]
0
2
D
+ 3.5 (c
.059, CH OH); CD (CH OH) 293 (Δε − 2.57) nm; IR v 3377,
max
3
3
−1
1
13
"
935, 1717, and 1070 cm ; H NMR and C NMR, see l Tables 1
+
and 2; positive HR-ESIMS m/z 451.1943 [M + Na] (calcd. for
Anti-coxsackie virus B3 activity assay
C21H32O Na, 451.1939).
The antiviral activities against Coxsackie virus B3 in African green
monkey kidney cells (Vero cells) were determined using a cyto-
pathogenic effect (CPE) assay according to the literature [21]. Ri-
bavirin (Hubei Keyi Pharmaceutic Co., Ltd.; 95% purity) was used
as the positive control.
9
(1S,4S,5R,6R,9S)-13-O-β-D-Glucopyranosyl-13-hydroxyallo-cedra-
2
0
2,11-dione (6): white powder, [α]
D
− 108.5 (c 0.055, CH OH); CD
3
(
CH OH) 293 (Δε − 5.58) nm; IR v
3449, 2935, 1733, 1702,
3
max
−
1
1
13
"
1414, 1081, 1050, and 1018 cm ; H NMR and C NMR, see l Ta-
+
bles 1 and 2; positive HR-ESIMS m/z 413.2165 [M + H] (calcd. for
C21H33O , 413.217),m/z 435.1994 [M
+
Na]+ (calcd. for
Cytotoxicity assay
8
C21H32O Na, 435.1989).
Compounds 1–23 were tested for cytotoxicity against HT-29 (hu-
man colon cancer cell line), HePG2 (human hepatoma cancer cell
line), BGC-823 (human gastric cancer cell line), A549 (human
lung epithelial cell line), and A375 (human amelanotic melanoma
cell line) by means of the MTT method as described in the litera-
ture [18]. Camptothecin (Sigma; 95% purity) was used as the pos-
itive control.
8
(1S,4S,5R,6R,9S)-13-O-α-L-Arabinofuranosyl-(1→6)-β-D-glucopy-
2
0
ranosyl-13-hydroxyallo-cedra-2,11-dione (7): white powder, [α]
D
−
113.3 (c 0.060, CH OH); CD (CH OH) 293 (Δε − 3.54) nm; IR v
3 3 max
415, 2933, 1731, 1704, 1074, and 1049 cm ; H NMR and 13C
−
1
1
3
"
NMR, see l Tables 1 and 2; positive HR-ESIMS m/z 567.2415 [M
+
+
Na] (calcd. for C26H40O12Na, 567.2412).
Zhang G et al. Sesquiterpenes from the… Planta Med 2013; 79: 1056–1062

1
062 Original Papers
7
Powell RG, Weisleder D, Smith CR. Drummondones A and B: unique ab-
scisic acid catabolites incorporating a bicyclo[2.2.2]octane ring system.
J Org Chem 1986; 51: 1074–1076
Cui B, Nakamura M, Kinjo J, Nohara T. Chemical constituents of Astra-
gali semen. Chem Pharm Bull 1993; 41: 178–182
Neuroprotective activity assay
The cell culture and the cell viability assay were performed ac-
cording to previously reported procedures [22]. NGF (Sigma, pu-
rity > 97%) was used as the positive control.
8
9
Sung TV, Steffan B, Steglich W, Klebe G, Adam G. Sesquiterpenoids from
the roots of Homalomena aromatica. Phytochemistry 1992; 31: 3515–
Supporting information
Original spectra for compounds 1–8 are available as Supporting
Information.
3
520
1
1
1
0 Kuo YH, Chyu CF, Lin HC. Cadinane-type sesquiterpenes from the roots
of Taiwania cryptomerioides Hayata. Chem Pharm Bull 2003; 51: 986–
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1 Bai J, Chen H, Fang ZF, Yu SS, Wang WJ, Liu Y, Ma SG, Li Y, Qu J, Xu S, Liu JH,
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Acknowledgements
!
The work was supported by grants from the Natural Science
Foundation of China (No. 201072234, No. 21132009), the
National Science and Technology Project of China
1
1
(No. 2012ZX09301002–002), and PCSIRT (No. IRT1007).
2
136
Conflict of Interest
!
15 Nunez YO, Salabarria IS, Collado IG, Hernandez-Galan R. Sesquiterpenes
from the wood of Juniperus lucayana. Phytochemistry 2007; 68: 2409–
2
414
None of the authors have conflicts of interest in this study.
1
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methods, Volume 2. Stuttgart: Georg Thieme; 1977: 105–107
17 Song TF, Zhang WD, Xia XH, Shen YH, Liu CM, Lin S, Jin HZ, Li HL. Two new
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