Studies on chemical constituents of Ophiopogon japonicus

Article abstract of DOI:10.1080/10286020.2014.935348

Two new and six known steroidal glucosides were isolated from the tuber of Ophiopogon japonicus. The new steroidal glucosides were established as (20R,25R)-26-O-β-d-glucopyranosyl-3β,26-dihydroxycholest-5-en-16,22-dioxo-3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside (1) and 26-O-β-d-glucopyranosyl-(25R)-furost-5-en-3β,14α,17α,22α,26-pentaol-3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside (3) on the basis of spectroscopic data as well as chemical evidence.

Full text of DOI:10.1080/10286020.2014.935348

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Studies on chemical constituents of
Ophiopogon japonicus
a
a
a
b
Yue Liu , Ling-Zhi Meng , Sheng-Xu Xie , Tun-Hai Xu , Lian-kun
c
b
a
a
Sun , Tong-Hua Liu , Ya-Juan Xu & Dong-Ming Xu
a
Key Laboratory of Effective Substances of Traditional Chinese
Medicine, Jilin Academy of Chinese Medicine Sciences, Changchun
1
30012, China
b
Department of Traditional Chinese Medicine Chemistry, School
of Traditional Chinese Medicine, Beijing University of Chinese
Medicine, Beijing 100102, China
c
College of Basic Medical Sciences, Jilin University, Changchun
130021, China
Published online: 01 Aug 2014.
To cite this article: Yue Liu, Ling-Zhi Meng, Sheng-Xu Xie, Tun-Hai Xu, Lian-kun Sun, Tong-Hua Liu,
Ya-Juan Xu & Dong-Ming Xu (2014): Studies on chemical constituents of Ophiopogon japonicus,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.935348
To link to this article: http://dx.doi.org/10.1080/10286020.2014.935348
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Journal of Asian Natural Products Research, 2014
http://dx.doi.org/10.1080/10286020.2014.935348
Studies on chemical constituents of Ophiopogon japonicus
a
Yue Liu , Ling-Zhi Meng , Sheng-Xu Xie , Tun-Hai Xu *, Lian-kun Sun ,
a
a
b
c
b
Tong-Hua Liu , Ya-Juan Xu * and Dong-Ming Xu
a
a
a
Key Laboratory of Effective Substances of Traditional Chinese Medicine, Jilin Academy of Chinese
Medicine Sciences, Changchun 130012, China; Department of Traditional Chinese Medicine
b
Chemistry, School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing
1
c
00102, China; College of Basic Medical Sciences, Jilin University, Changchun 130021, China
(
Received 6 August 2013; final version received 12 June 2014)
Two new and six known steroidal glucosides were isolated from the tuber of
Ophiopogon japonicus. The new steroidal glucosides were established as (20R,25R)-
26-O-b-D-glucopyranosyl-3b,26-dihydroxycholest-5-en-16,22-dioxo-3-O-a-L-rham-
nopyranosyl-(1 ! 2)-b-D-glucopyranoside (1) and 26-O-b-D-glucopyranosyl-(25R)-
furost-5-en-3b,14a,17a,22a,26-pentaol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glu-
copyranoside (3) on the basis of spectroscopic data as well as chemical evidence.
Keywords: Ophiopogon japonicus; Liliaceae; steroidal glucoside
–
peaks at m/z 899 [M–H] , 881 [M–H–
1
.
Introduction
–
H O] , and 735 [M–H–18–146] in
–
The tuber of Ophiopogon japonicus (L.f.)
Ker-Gawl., “maidong,” is a traditional
Chinese medicine and has various medical
functions for curing cardiovascular dis-
eases and bacterial infections. Phytochem-
ical studies on this plant were reported
previously [1]. In the previous papers, we
reported the isolation of steroidal gluco-
sides from the tuber of O. japonicus (L.f.)
Ker-Gawl [2,3]. Further examination led
to isolation of two new glucosides
2
negative ion mode and those at m/z 923
þ
þ
[
M þ Na] , 761 [M þ Na–162] , 777
þ
[M þ Na–146] , and 615 [M þ Na–
þ
146–162]
in positive ion mode,
suggesting the presence of two hexoses
1
and one deoxyhexose moiety. The
H
NMR spectrum of 1 showed diagnostic
proton signals of four methyl groups at d
0.63 (3H, s), 0.92 (3H, s), 0.86 (3H, d,
J ¼ 6.0 Hz), and 1.39 (3H, d, J ¼ 6.6 Hz),
one oxymethine at d 4.17 (1H, m), one
oxymethylene at d 3.45 (1H, dd, J ¼ 6.0,
(compounds 1 and 3) together with six
known steroidal glucosides. Their struc-
tures were determined by combination of
chemical methods with the analysis of
NMR, HR-MS, and physical data.
9
.6 Hz) and 3.91 (1H, m), one olefinic
group at d 5.18 (1H, br s), and three
anomeric protons at d 4.72 (1H, d,
J ¼ 7.8 Hz), 6.27 (1H, br s), and 4.93
(
1H, d, J ¼ 7.2 Hz), and a pentosyl H-6
1
3
2
.
Results and discussion
signal at d 1.66 (3H, d, J ¼ 6.0 Hz). In
C
NMR spectrum of 1, characteristic carbon
signals of four methyl groups at d 14.3,
19.4, 16.3, and 17.5, three anomeric
carbons at d 100.5, 102.2, and 105.1,
olefinic carbon signals at d 141.1 and
Compound 1 was obtained as a white
amorphous powder. Its molecular formula
was assigned as C H O on the positive
4
5 72 18
þ
ion HR-ESI-MS ([M þ Na] , m/z
23.4583). ESI-MS showed fragment ion
9
*Corresponding authors. Email: thxu@163.com; xyj6492@sohu.com
q 2014 Taylor & Francis

2
Y. Liu et al.
21.5, and two keto functions at d 216.9 carbonyl will be observed for 17-a-H (R),
1
and 212.6 were observed. The H and
1
13
C
NMR spectral data (Table 1) of 1 are
while its negative Cotton effect will be
significantly weakened for 17-b-H (S)
(Figure 3). Thus, it was inferred that C-17
was R-configuration (17-a-H) in com-
pounds 1 and 2. Compound 1 was the C-
20R epimer of compound 2. Besides, C-25
configuration of 1 was deduced to be R
based on the difference of chemical shifts
(Ddab ¼ d H – d H ) of the geminal
assigned unequivocally according to its
1
1
H– H COSY, HMQC, and HMBC
1
13
analysis. By comparing H and C NMR
data of compound 1 with those of
compound 2 (Figure 1) which was
identified as (20S,25R)-26-O-b-D-gluco-
pyranosyl-3b,26-dihydroxycholesten-5-
en-16,22-dioxo-3-O-a-L-rhamnopyrano-
syl-(1 ! 2)-b-D-glucopyranoside on the
basis of its identical spectral evidences as
the reference [4], except for almost
identical data of sugar moieties and most
of aglycone units, obvious differences lied
in the spectral data of C-12, C-13, C-14,
C-16, C-17, C-18, C-20, C-21, and C-22 in
a
b
protons at H-26 (d Ha
–
d H ¼
b
0.46 , 0.48). It had been described that
Ddab is usually .0.57 ppm in 25S
compounds and ,0.48 in 25R compounds
[7–9].
Acidic hydrolyzation of 1 with mineral
acid afforded rhamnose and glucose as the
sugar components identified on TLC by
comparison with authentic samples. The
1
3
C NMR spectrum, and H-17, H-18,
1
3
H-20, and H-21 in H NMR spectrum.
Significant upfield shifts of C-12, C-14,
C-16, C-17, and C-22 carbon signals by Dd
21.0, 20.8, 20.9, 22.4, and 20.5 ppm,
respectively; downfield shifts of C-13,
C-18, C-20, and C-21 carbon signals by Dd
large coupling constants ( J . 7.0 Hz)
1
,2
were consistent with the b-configuration
of glucose and the splitting (br s) of
anomeric proton signal inferred the
a-configuration of rhamnose [10–12].
The 3,26-bisdesmoside structure of 1 was
characterized by a HMBC experiment.
Long-range correlations were observed
between H-1 of Glc at d 4.93 and C-3 of
the aglycone at d 78.0, between H-1 of Rha
at d 6.27 and C-2 of Glc at d 79.8, and
0
.7, 1.3, 0.3, and 0.6; characteristic proton
signals at d 2.33 (1H, d, J ¼ 8.4 Hz, H-17),
.63 (3H, s, H-18), 2.71 (1H, m, H-20),
0
and 1.39 (3H, J ¼ 6.6 Hz, H-21) in 1; and
those at d 2.62 (1H, d, J ¼ 10.2 Hz, H-17),
0
0
0
.56 (3H, s, H-18), 2.81 (1H, m, H-20),
between H-1 of Glu at d 4.72 and C-26 of
the aglycone at d 75.3. Consequently,
compound 1 was elucidated to be
(20R,25R)-26-O-b-D-glucopyranosyl-3b,
26-dihydroxycholest-5-en-16,22-dioxo-
3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-
glucopyranoside.
and 0.92 (3H, J ¼ 6.6 Hz, H-21) in 2
inferred that compound 1 was the C-17 or
C-20 epimer of compound 2. In NOESY
spectrum, no correlation between H-18
(
d 0.63) and H-17 (d 2.33) and a weak
correlation between H-17 and H-9 (d 0.83)
were observed, inferring the a-orientation
of H-17. The correlations in NOESY
spectrum between H-18 (d 0.63) and H-
Compound 3 was obtained as a white
amorphous powder. The molecular for-
mula was assigned as C H O on the
4
5 74 20
2
1 (d 1.39), H-17 (d 2.33) and H-20 (d
.71) indicated the configuration of C-20
basis of the positive-ion HR-ESI-MS peak
þ
2
at m/z 957.4692 [M þ Na] . The negative
in 1 was R. For CD spectra (Figure 2),
compound 2 had a negative Cotton effect
at 292.8 nm and compound 1 had a more
stronger negative Cotton effect at
ion ESI-MS showed fragment ion peaks at
–
m/z 933 [M–H] , 915 [M–H–H O] , 777
–
2
–
[M–H–18–162] , and 463 [M–H–162–
–
146–162] , and the positive ion ESI-MS
2
97.4 nm. In accordance with the octant
rule of octahydro-2H-inden-2-one [5,6],
showed fragment ion peaks at m/z 957
þ
þ
[M þ Na] , 939 [M þ Na–H O] , 921
2
þ
negative Cotton effect of cyclopentanone
[M þ Na–2H O] , 775 [M þ Na–H O–
2
2

Journal of Asian Natural Products Research
3
d
d
d

4
Y. Liu et al.
d
d

Journal of Asian Natural Products Research
5
Figure 1. Structures of compounds 1–3.
þ
þ
1
1
46] , and 613 [M þ Na–H O–146–
6.27 (1H, br s), and 4.71 (1H, d,
0
2
62] , suggesting the presence of two
J ¼ 7.8 Hz, Glc -H-1), and an olefinic
hexoses and one deoxyhexose moieties.
proton signal at d 5.26 (1H, br s). These
data indicated that compound 3 contains
1
The H NMR spectrum of 3 showed two
methyl singlets at d 1.02 (3H, s) and 1.02
1
3
three sugar moieties. The
C NMR
(
3H, s), three methyl doublets at d 0.90
3H, d, J ¼ 6.6 Hz), 1.29 (3H, d,
spectrum showed 45 carbon signals, in
which the characteristic olefinic carbon
signals at d 140.4 and 122.4, hemiketal
carbon signal at d 111.3, and an
oxymethylene carbon signal at d 75.4
(
J ¼ 7.2 Hz), and 1.67 (3H, d,
J ¼ 6.0 Hz), three anomeric proton signals
at d 4.92 (1H, d, J ¼ 7.2 Hz, Glc-H-1),
Figure 2. UV and CD spectra of compounds 1 and 2.

6
Y. Liu et al.
1
Figure 3. Analysis for CD spectra of compounds 1 and 2 in accordance with the octant rule (R, R ,
and R : other part of the molecules).
2
were assigned to C-5, C-6, C-22, and C-26,
contained the same aglycone as ophiopo-
1
1
respectively, by H– H COSY, HSQC,
and HMBC experiments. The carbon
signals at d 75.4 (C-26), 111.3 (C-22),
gonin H. Thus, the aglycone of 3 was
identified as (25R)-furost-5-en-3b,14a,
17a,22a,26-pentaol.
1
3
1
40.4 (C-5), and 122.4 (C-6) indicated that
is a furostanol saponin with D5 (6) [13–
6].
Acidic hydrolysis of 3 with mineral
acid afforded glucose and rhamnose as the
sugar components identified on TLC by
comparison with authentic samples. The
In the HMBC spectrum, the proton
signals at d 1.29 (H-21), 1.02 (H-18), 2.51
H-20), 2.46 (H-15), and 4.99 (H-16)
3
large coupling constants ( J . 7.0 Hz)
,2
1
(
were consistent with the b-configuration
of glucoses and the splitting (br s) of
anomeric proton signal inferred the
a-configuration of rhamnose [10–12].
The 3,26-bisdesmoside structure of 3 was
characterized by a HMBC experiment, in
which correlations were observed between
H-1 of Glc at d 4.92 and C-3 of the
aglycone at d 78.0, between H-1 of Rha at
d 6.27 and C-2 of Glc at d 79.8, and
showed the long-range correlations with
the carbon signal at d 91.7, and the proton
signals at d 2.46 (H-15) and d 1.02 (H-18)
showed the long-range correlations with
the carbon signal at d 87.8. Upon the
combined analysis of these long-range
1
1
correlations and the H– H COSY and
HSQC spectra of 3, carbon signals at d
9
1.8 and 88.1 could be assigned to C-17
0
0
and C-14, respectively. Comparison of the
carbon signals of 3, especially for those at
d 87.8 (C-14), 90.9 (C-16), and 91.7
between H-1 of Glu at d 4.71 and C-26 of
the aglycone at d 75.4. Consequently,
compound 3 was determined to be 26-O-
b-D-glucopyranosyl-(25R)-furost-5-en-
3b,14a,17a,22a,26-pentaol-3-O-a-L-
rhamnopyranosyl-(1 ! 2)-b-D-glucopyra-
noside.
(C-17), with those of cixi-ophiopogons A
and B indicated the presence of a-
orientations for C-14 and C-17 hydroxy
groups [15,17]. The a-orientation of C-22
hydroxy group of the aglycone moiety was
deduced from the hemiketal carbon signal
at d 111.3, approximately 3–4 ppm higher
than that of b-configuration [18,19]. The
chemical shift difference between two
The other compounds were identified
as chonglouoside VI (4) [21], (25R)-5-en-
spirost-3b,14a,17a-trihydroxy-3-O-a-L-
rhamnopyranosyl-(1 ! 2)-b-D-glucopyra-
noside (5) [22], 25R-dracaenoside F (6)
[23], (25R)-spirost-5-en-3b,17a-dihy-
droxy-3-O-a-L-rhamnopyranosisyl-(1 !
2)-[b-D-xylopyranosyl-(1 ! 4)]-b-D-glu-
copyranoside (7) [24], and ophiopojaponin
C (8) [25], by comparison of their physical
proton signals of H-26 (Ddab
0
¼
.39 , 0.48) demonstrated the 25R-
configuration of 3 [7–9]. Comparison of
the NMR data of 3 with those of
ophiopogonin H [20] also indicated that 3

Journal of Asian Natural Products Research
7
and spectroscopic data with those reported
in the literature, respectively.
Materials in Chengdu, Sichuan Province,
China, and identified by Professor Minglu
Deng, Changchun University of Chinese
Medicine. A voucher specimen (No.
3
.
Experimental
2
009012) has been deposited in the
3
.1 General experimental procedures
Phytochemistry Laboratory of Jilin Acad-
emy of Chinese Medicine Sciences.
Melting points were determined on an X-6
microscope apparatus and are uncorrected
(Beijing Fukai Instrument Co., Ltd, Beij-
ing, China). Optical rotations were deter-
mined on a WZZ-2SS auto-polarimeter
3.3 Extraction and isolation
The dried and powdered tubers of O.
japonicus (10 kg) were exhaustively
extracted with 70% EtOH. The extract
was concentrated under vacuum to give a
water solution which was then chromato-
graphed on macroporous resin D21 (10 kg),
eluting with water until the eluate was
colorless and then with 80% EtOH (80 l).
The 80% EtOH solution was concentrated
and partitioned with EtOAc and n-BuOH,
successively, to yield EtOAc extract (11 g)
and n-BuOH layer extract (20 g). The
n-BuOH extract was chromatographed on
(Shanghai Jingke Scientific Instrument
Co., Ltd, Shanghai, China). UV and CD
spectra were obtained on a JASCO J-820
spectrometer (JASCO International Co.
Ltd, Tokyo, Japan). NMR spectra were
obtained on Bruker AV600 and AV400
instruments (Bruker Corporation, Rhein-
stetten, Switzerland), using tetramethylsi-
lane as the internal standard. The HR-ESI-
MS was recorded on an IonSpec HiResESI
FTICR instrument (IonSpec Co., Lake
Forest, CA, USA). ESI-MS were recorded
on Finnigan Mat LCQ mass spectrometry
silica gel eluted with CHCl :MeOH (11:1).
3
(Thermo Fisher Scientific, Inc., West Palm
Fr. 189–Fr. 196 was separated by HPLC
–1
Beach, FL, USA). High-performance
liquid chromatography (HPLC) (Shi-
madzu Corporation, Kyoto, Japan) was
carried out using an octadecylsilane-
bonded silica gel column (Megres-C ,
(
to yield 1 (17.8 mg, t : 15.465 min) and 2
v: 4.5 ml min ) with MeCN:H O (34:56)
2
R
(7.0 mg, t : 17.565 min). Fr. 291–Fr. 300
was separated by HPLC (v: 4.5 ml min
R
–1
)
with MeCN:H O (35:55) to yield 3 (70 mg,
1
8
2
2
50 mm £ 4.6 mm, 5 mm). Prep-HPLC
tR: 7.782 min). The EtOAc extract was
chromatographed on silica gel eluted with
(NP 7000, Hanbon Sci & Tech Co., Ltd,
Huaian, Jiangsu, China) was carried out
using an octadecylsilane-bonded silica gel
column (Megres-C , 250 mm £ 10 mm,
CHCl :MeOH (12:1) and ODS silica gel
3
with MeOH–H O (8:2). Fr. 33–Fr. 50 was
2
–1
separated by HPLC (v: 4.5 ml min ) with
1
8
5
mm). Column chromatography was car-
MeOH:H O (78:22) to yield 4 (72 mg, t :
R
2
ried out on macroporous resin D101 made
in Shandong Lu Neng Gel Co. (Shandong,
China), silica gel (200–300 mesh, Qing-
dao Oceanic Chemical Industry, Qingdao,
China), and reversed-phase silica gel
1
8.432 min), 5 (93 mg, t : 13.996 min), and
R
6
was separated by HPLC (v: 4.0 ml min
(98 mg, t : 20.132 min). Fr. 51–Fr. 61
–1
R
)
with MeOH:H O (78:22) to yield 7 (78 mg,
2
t : 23.848 min). Fr. 83–Fr. 95 was
R
(
50 mm, YMC Co., Ltd, Kyoto, Japan).
TLC was carried out on aluminum sheets
20 £ 20 cm, RP-18F254S, MERCK KGaA,
Darmstadt, Germany).
–1
separated by HPLC (v: 4.5 ml min
with MeOH:H O (75:25) to yield 8
)
2
(
(13.2 mg, t : 20.198 min).
R
3
.2 Plant material
3.3.1 Compound 1
The tubers of O. japonicus were purchased
from the Company of Chinese Medicinal
Amorphous powder; m.p. 131.1–132.38C;
2
7
½aꢀ 259.3 (c ¼ 0.30, 30% aqueous
D

8
Y. Liu et al.
–
4
solution of acetone); UV (c ¼ 7.11 £ 10
M, 70% aqueous solution of CH OH) l
2 h. Their reaction mixtures were neutral-
ized with NaHCO3 and extracted with
CHCl for three times. Each water phase
3
max
(
(
log 1): 216.7 (3.75), 274.0 (3.12) nm; CD
–
3
4
c ¼ 7.11 £ 10
M, 70% aqueous sol-
1
was chromatographed on the silica gel
HPTLC with the system of n-BuOH:i-
ution of CH OH): D1
3
26.1.
297.4nm
H
3
1
NMR (600 MHz, pyridine-d ) and
C
NMR (150 MHz, pyridine-d ) spectral
PrOH:H O (10:5:4, homogenous), then the
2
5
brown-colored spots were visualized by
heating after spraying with phenylamine-
ortho-benzene-dicarboxylic acid reagent.
Glucose and rhamnose were detected by
comparison with the authentic samples.
Each remaining aqueous layer was
desalted through 001 £ 7 (732) resin
column and concentrated to dryness. The
residue was dissolved in pyridine (1 ml)
and then L-cysteine methyl ester hydro-
chloride (2 mg) was added to the solution.
The mixture was heated at 608C for 2 h,
and an equal volume of acetic anhydride
was added, followed by heating at 908C for
5
data are given in Table 1. ESI-MS:
2
negative ion mode: m/z 899 [M–H] ,
2
8
1
81 [M–H–H O] , 735 [M–H–18–
2
2
46] ; positive ion mode: m/z 923
þ
þ
[
M þ Na] , 761 [M þ Na–162] , 777
þ
[
M þ Na–146] , 615 [M þ Na–146–
þ
1
62] . HR-ESI-MS: m/z 923.4583
þ
[
M þ Na] (calcd for C H O Na,
4
5
72 18
9
23.4616).
3
.3.2 Compound 2
3
UV (c ¼ 1.20 £ 10- M, 70% aqueous
solution of CH OH) l (log 1): 214.7
3
max
2
h. Then, the solution was concentrated to
–
(
3
3.52), 274.1 (2.75) nm; CD (c 1.20 £ 10
dryness and taken up in MeOH (0.5 ml),
which was then analyzed by Shimadzu
GC-2010 Plus gas chromatograph
M, 70% aqueous solution of CH OH):
3
1
D1292.8nm 23.5). H NMR (600 MHz,
1
pyridine-d ) and C NMR (150 MHz,
3
5
equipped with H flame ionization detector
2
pyridine-d ) spectral data are given in
5
(
Shimadzu Corporation, Kyoto, Japan).
DB-5 quartz capillary column: 30 m
0.25 mm, 0.25 mm; column temperature:
Table 1.
£
3
.3.3 Compound 3
160–2808C, programmed increase: 58C/
min; carrier gas N : 1.5 ml/min; injector
Amorphous powder; m.p. 179.1–180.0 8C;
2
2
7
D
and detector temperature: 2808C; injection
volume: 1 ml; split ratio: 10:1. The
monosaccharides of compounds were
confirmed by comparison of the retention
time of monosaccharides derivatives with
those of standard sugars (the standard
sugars were subjected to the same reac-
tion; the retention times of the derivatives
of L-rhamnose and D-glucose were 23.61
and 28.10 min, respectively).
½
aꢀ –58.0 (c ¼ 0.10, 30% aqueous sol-
1
ution of acetone). H NMR (600 MHz,
1
pyridine-d ) and C NMR (150 MHz,
3
5
pyridine-d ) spectral data are given in
5
Table 1. ESI-MS: negative ion mode: m/z
–
–
9
33 [M–H] , 915 [M–H–H O] , 777
2
–
M–H–18–162] , 463 [M–H–162–
[
–
46–162] ; positive ion mode: m/z 957
1
þ
þ
[
M þ Na] , 939 [M þ Na–H O] , 921
2
þ
[
M þ Na–2H O] , 775 [M þ Na–H O–
2
2
þ
þ
1
46] , 613 [M þ Na–H O–146–162] .
2
þ
HR-ESI-MS: m/z 957.4692 [M þ Na]
Acknowledgments
(calcd for C H O Na, 957.4671).
45 74 20
The authors were financially supported by the
International Science & Technology Cooperation
Program of Commission and the Innovative
Team of Beijing TCM University (no. 2011-
CXTD-19) and the programs of Jilin Province
Administration of Traditional Chinese Medicine
(Nos. 2010-pt036; 2011-zd15; YJS-0212).
3
.4 Acid hydrolysis
Compounds 1 and 3 (10 mg) were,
respectively, dissolved in 1 mol/l HCl in
MeOH:H O (1:1) and each refluxed for
2

Journal of Asian Natural Products Research
9
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