A novel spinosin derivative from Semen Ziziphi Spinosae

Article abstract of DOI:10.1080/10286020.2011.623128

A novel spinosin derivative, 6?-(4?-O-b-D-glucopyranosyl)- vanilloyl spinosin (1) was isolated from the methanol extract of Semen Ziziphi Spinosae, together with five known flavonoids, swertish (2), spinosin (3), 6?-feruloylspinosin (4), isospinosin (5) and kaempferol 3-O-a-L-rhamnopyranosyl-(1 → 2)-O-[O-a-L-rhamnopyranosyl-(1 → 6)]-b-D-glucopyranoside (6), and two alkanoids, zizyphusine (7) and 6-(2′,3′-dihydroxyl-4′-hydroxymethyl-tetrahydro-furan- 1′-yl)-cyclopentene[c]pyrrole-1,3-diol (8). The structure of compound 1 was elucidated by spectroscopic methods including UV, IR, ESI-TOF-MS, 1D, and 2D NMR techniques.

Full text of DOI:10.1080/10286020.2011.623128

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A novel spinosin derivative from Semen
Ziziphi Spinosae
a
a
a
a
Yuan-Yuan Xie , Zhi-Lin Xu , Hui Wang , Yoshihiro Kano & Dan
a
Yuan
a
Department of Traditional Chinese Medicines , Shenyang
Pharmaceutical University , Shenyang, 110016, China
Published online: 25 Nov 2011.
To cite this article: Yuan-Yuan Xie , Zhi-Lin Xu , Hui Wang , Yoshihiro Kano & Dan Yuan (2011) A
novel spinosin derivative from Semen Ziziphi Spinosae, Journal of Asian Natural Products Research,
13:12, 1151-1157, DOI: 10.1080/10286020.2011.623128
To link to this article: http://dx.doi.org/10.1080/10286020.2011.623128
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Journal of Asian Natural Products Research
Vol. 13, No. 12, December 2011, 1151–1157
NOTE
A novel spinosin derivative from Semen Ziziphi Spinosae
Yuan-Yuan Xie, Zhi-Lin Xu, Hui Wang, Yoshihiro Kano and Dan Yuan*
Department of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang
1
10016, China
(Received 21 March 2011; final version received 9 September 2011)
0
00
isolated from the methanol extract of Semen Ziziphi Spinosae, together with five
0000
A novel spinosin derivative, 6 -(4 -O-b-D-glucopyranosyl)-vanilloyl spinosin (1) was
000
known flavonoids, swertish (2), spinosin (3), 6 -feruloylspinosin (4), isospinosin (5)
and kaempferol 3-O-a-L-rhamnopyranosyl-(1 ! 2)-O-[O-a-L-rhamnopyranosyl-
0
0
(
1 ! 6)]-b-D-glucopyranoside (6), and two alkanoids, zizyphusine (7) and 6-(2 ,3 -
0
0
dihydroxyl-4 -hydroxymethyl-tetrahydro-furan-1 -yl)-cyclopentene[c]pyrrole-1,3-diol
8). The structure of compound 1 was elucidated by spectroscopic methods including
UV, IR, ESI-TOF-MS, 1D, and 2D NMR techniques.
(
Keywords: Semen Ziziphi Spinosae; flavone-C-glycoside; alkaloid; spectroscopic
methods
1
.
Introduction
the minor components have rarely been
studied yet.
Semen Ziziphi Spinosae, botanically from
the dried seeds of Ziziphus jujuba Mill.
var. spinosa (Bunge) Hues. H.F. Chou, has
been used as a sedative-hypnotic drug for
symptoms such as insomnia and dysphoria
for thousands of years in Chinese medical
practice. Chemical investigations on
jujuba seeds resulted in the discovery of
several groups of bioactive components
including flavonoids, triterpenoid sapo-
nins, and alkaloids [1–9]. Experimental
studies validated that they exhibited wide
pharmacological effects, such as the
inhibitory activities of the spontaneous
activity, facilitating the hypnotic action of
pentobarbital, and antagonizing the excit-
atory action of morphine and pentylenete-
trazole [10,11]. Although some flavone
C-glycosides such as spinosin, as well as
saponins of jujubogenin were isolated
from the seeds and leaves of this plant,
This study describes the isolation and
structural elucidation of a new spinosin
0
00
0000
derivative, 6 -(4 -O-b-D-glucopyrano-
syl)-vanilloyl spinosin (1), together with
seven known compounds including five
polyhydroxylated flavonoids (2–6) and
two alkaloids (7 and 8).
2. Results and discussion
The MeOH extract of the seeds of Z.
jujuba Mill. Var. spinosa was prepared by
reflux method after degreased by pet-
roleum ether. A new spinosin derivative,
0
00
0000
6 -(4 -O-b-D-glucopyranosyl)-vanilloyl
spinosin (1), was isolated from the MeOH
extract, together with seven known
compounds including five polyhydroxy-
lated flavonoids, swertish (2), spinosin (3),
0
00
6 -feruloylspinosin (4), isospinosin (5),
and kaempferol-3-O-a-L-rhamnopyrano-
*Corresponding author. Email: yuandan_kampo@163.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.623128
http://www.tandfonline.com

1
152
Y.-Y. Xie et al.
signals of two anomeric protons at d 4.67
syl-(1 ! 3)-O-[O-a-L-rhamnopyranosyl-
1 ! 6)]-b-D-glucopyranoside (6), and
two alkaloids, zizyphusine (7) and 6-
0
0
(
(1H, d, J ¼ 9.6 Hz, H-1 ) and 4.29 (1H, d,
J ¼ 7.8 Hz) and 10 protons at d 3.73–3.06
0
0
0
13
together with the C NMR spectral
(2 ,3 -dihydroxyl-4 -hydroxymethyl-tetra-
0
hydrofuran-1 -yl)-cyclopentene[c]pyrrole-
data indicated the presence of two b-D-
glucosyl moieties [16]. The linkage
1
,3-diol (8) (Figure 1). Among them,
00
compound 1 is a new one, whose structure
was elucidated using UV, IR, ESI-TOF-
MS, 1D, and 2D NMR techniques. The
known compounds were identified by
comparing their NMR and MS data with
the reported values [7,12–14].
between the anomeric C-1 and C-6 and
0
0
the interglucosidic linkage between C-2
0
00
and C-1 were determined by the HMBC
0
0
000
00
correlations of H-1 /C-6 and H-1 /C-2 .
The chemical shift value of the anomeric
0
0
carbon C-1 and HMBC correlation
0
0
Compound 1 was isolated as a yellow-
ish amorphous powder. The molecular
formula was determined to be C H O
between H-1 (d 4.67) and C-6
(d 108.8/108.7) indicated that the anome-
C
0
0
ric carbon (C-1 , d 70.8) of b-glucose was
C
4
2
48 23
2
from the [M 2 H] ion peak at m/z
19.2508 in the HR ESI-TOF-MS. The
UV absorption maxima at 216, 262, and
43 nm revealed a flavone skeleton.
connected to C-6 through a C-linkage.
The similar NMR phenomenon to that of
spinosin, i.e. the appearance of serial
separate signals, was observed. It is
proposed that rotational isomers produced
by the rotational barriers 7-OCH3 in
flavones-6-C-glycoside must exist in com-
9
3
A positive reaction in the AlCl reagent
3
suggested that compound 1 was a
hydroxyl-substituted flavonoid. The IR
spectrum showed absorption bands due to
0
00
pound 1 [7]. The downfield shift of C-6
(d 62.9, þ 2.2 ppm) and the upfield shift
2
1
hydroxyl group (3401 cm ), carbonyl
(
C
21
1652 and 1701 cm ), and aromatic ring
000
of C-5 (d 73.2, 23.6 ppm) relative to
C
2
1607, 1510, and 1443 cm ). The paper
1
(
chromatography (PC) analysis of the acid
hydrolysis of compound 1 exhibited only
the corresponding signals of spinosin
0
00
revealed the acylation of C-6 , which is
also supported by the corresponding signals
1
the presence of glucose. The H NMR
000
of 6 -feruloyspinosin (4) [7]. The spinosin
spectrum (600 MHz, DMSO-d ) of 1
6
moiety accounted for a partial molecular
formula of C H O . Thus, the remaining
molecular formula should be C H O as a
indicated six aromatic proton signals due
to aglycone moiety, i.e., a characteristic
singlet signal at d 6.67/6.64 due to the H-3
proton, a singlet at d 6.55/6.42 (1H, s, H-8)
indicating A-ring with three substituents,
2
8 31 15
1
4 17 8
substituent attached to the spinosin group at
0
00
C-6 . The resonances for the carbons and
13
1
protons of the substituent in the H and
C
NMR as well as HSQC spectra included an
0
0
an AA BB aromatic proton system
appearing at d 7.80/7.70 (d, J ¼ 8.4,
ABX aromatic proton system appearing atd
0000
0
0
9
.0 Hz, H-2 , 6 ) and 6.91/6.87 (d,
0
0
6.98 (1H, d, J ¼ 9.0 Hz, H-5 ;
0
J ¼ 8.4, 9.0 Hz, H-3 , 5 ) indicating a C-
dC 112.4/112.0), 7.10 (1H, dd, J ¼ 9.0,
0000
4
3
substituted B-ring [15], and a singlet at d
.86 (3H, s) due to methoxyl protons. The
1.2 Hz, H-6 ; d 122.7) and 7.20 (1H, d,
C
0
000
J ¼ 1.2 Hz, H-2 ; d 114.2/114.4), a
C
resonances for the carbons and protons of
the aglycone and the sugar moiety, except
methoxyl (d 3.66, 3H, s; d 55.4), and a
C
carbonyl (dC 165.3). The IR absorption
21
0
00
000
for those of C-5 and C-6 , as well as the
related protons (Table 1), had a close
resemblance to those of spinosin (3), and
bands at 1701 cm
also proved the
existence of the carbonyl group. These
data together with the HMBCs between
1
0000
0000
0000
0000
they were assigned according to the H and
1
C NMR spectral data [7] for spinosin as
well as its own HSQC spectrum. The
H-6 and C-7 , H-2 and C-7 as well
0000
3
as the proton of methoxyl and C-3
indicated the presence of a vanilloyl moiety

Journal of Asian Natural Products Research
1153
O
C
O
OH
C
HO
H CO
O
O
3
O
HO
OH
OH
O
OH
OH
H3CO
HO
OH
OH
Vanilloyl
R
OH
Feruloyl β-D-glucopyranosyl
OH
OR
6'''-(4''''-O-β-D-glucopyranosyl)-
6-(4-O-β-D-glc-vanilloyl)-glc
vanilloyl spinosin (1)
swertish (2)
H
spinosin (3)
-glc
6'''-feruloylspinosin (4)
6-feruloyl-glc
OH
HO
O
OH
HO
O
O
O
O
O
OH
O
OH
O
O
OH
H CO
3
OH
O
HO
O
O
OH
CH3
OH
OH
OH
OH
OH OH
OH
OH
O
CH3
OH OH
Kaempferol-3-O-α-L-rhamnpyranosyl-
Isospinosin (5)
(1
3)-O-[O-α-L-rhamnpyranosyl-(1 6)]
-β-D-glucopyranoside (6)
HO
HO
+
CH3
OH
N
N
H
HOH C
CH3
2
O
H CO
3
HO
H
OH
H CO
OH
3
H
Zizyphusine (7)
6-(2',3'-dihydroxyl-4'-hydroxyl-ethyl-
tetrahydrofuran)-cyclopentene [c]
pyrrole-1,3-diol (8)
Figure 1. Structures of compounds 1–8.
4-hydroxy-3-methoxy benzoyl) which
(
of an anomeric proton at d 5.05 (d,
0
0000
accounted for a partial molecular formula
of C H O [17]. In addition, the signals
J ¼ 7.2 Hz, H-1 ) and five protons at d
1
3.73–3.06 as well as the C NMR data
3
8
6 3

1
154
Y.-Y. Xie et al.
1 13
Table 1. H and C NMR spectroscopic data for compound 1 in DMSO-d6.
a
C
b
Position
d
H
d (J in Hz)
2
3
4
5
6
7
8
9
164.0 (163.6)
102.9
182.4 (181.8)
159.6
108.7 (108.9)
165.0 (164.8)
90.6 (90.0)
157.1 (156.8)
104.6 (104.0)
122.7 (122.5)
128.6 (128.4)
116.0
161.7 (160.9)
56.5 (56.2)
70.8
81.8
78.7
70.4
82.1
61.6
105.8 (105.1)
74.6
76.3
69.8
73.2
62.9 (62.5)
123.0 (122.8)
114.4 (114.2)
148.5 (148.4)
150.6 (150.5)
112.4 (112.0)
122.7 (122.5)
165.3
6.67/6.64, s
6.55/6.42, s
1
1
2
3
4
7
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
7
3
1
2
3
4
5
6
0
0
0
0
0
, 6
, 5
7.80/7.70, d (8.4)
6.91/6.87, d (8.4)
0
0
-OCH3
3.86, s (3.81, s)
4.67, d (9.6)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
4.29, d (7.8)
00
00
00
00
00
3.86, s (3.81, s)
7.20, d (1.2)
0000
0000
0000
0000
0000
0000
0000
0000
6.98, d (9.0)
7.10, dd (1.2, 9.0)
-OCH3
55.4 (55.2)
99.9 (99.7)
74.6
3.66, s (3.52, s)
5.05 (7.2)
00000
00000
00000
00000
00000
00000
77.3
70.8 (70.5)
78.9 (78.7)
62.9 (62.5)
a
Measured separately at 150 MHz.
Measured separately at 600 MHz.
b
0
000
indicated the presence of a b-D-glucosidic
firmed as 4 -O-b-D-glucopyranosyl-vanil-
loyl [18] (Figure 2).
After mild alkaline hydrolysis of 1 with
moiety in the substituent. The correlation
00000
between the proton at d 5.05 (H-1
)
0
.05 N NH OH in 50% MeOH, a mixture
4
0000
and the carbon at d 150.6/150.5 (C-4 )
C
of two aromatic compounds were recov-
ered from the ethyl acetate extract of the
hydrolysate. They were separated and one
of the hydrolysis products was identified as
spinosin. The above evidence suggested
in the HMBC experiment suggested that
the b-D-glucosyl group should be attached
0
000
to the C-4 position of the vanilloyl
moiety. Thus, the substituent was con-

Journal of Asian Natural Products Research
1155
3'
OH
2'
4
'
'
8
H CO
9 O
2
5
3
6
''
1'
HO
7
6
6'
5
''
3
O
1''
10
4
'' OH
4
5
O
OH3'' 2''
H
OH
O
7''''
O6''' 5
O
'''
2''''
1''''
O
4
'''
OH
1'''
3''''
H CO
6''''
3
OH 3''' 2'''
H
4''''
5''''
OH
6
''''
HO
O
5
'''''
O
4'''''
OH2'''''
1'''''
OH 3'''''
H
OH
Figure 2. Selected HMBC correlations of 1.
that compound 1 is a derivative of spinosin,
with 4-O-b-D-glucopyranosyl-vanilloyl
as a substituent attached to the sugar
moiety. The HMBC correlations between
Bruker Daltonics mass spectrometer with a
resolution of 25,000 (10% Valley). Pre-
parative HPLC was carried out using a
Shimadzu’s LC-8A solvent delivery pump
and Shimadzu’s SPD-10AVP detector.
Column chromatography was carried out
on silica gel (200–300 mesh, Qingdao
Haiyang Chem. Group Co., Qingdao,
China), Sephadex LH-20 (GE Healthcare,
Uppsala, Sweden), octadecylsilyl silica
(ODS) (YMC Co., Ltd, Kyoto, Japan),
MDS-5 reverse-phase packing (200–
300 mesh, Beijing Medicine Technology
Center, Beijing, China), and Waters C18
column (7.8 £ 250 mm, 6 mm, Waters Co.,
Ltd, Milford, MA, USA).
0
00
H-6 (d 3.84, m and d 4.02, m) and the
carbonyl carbon (dC 165.0/165.3)
suggested a linkage between the 4-O-b-D-
000
glucopyranosyl-vanilloyl moiety and C-6
of glucose. Hence, compound 1 was
0
00
0000
assigned as 6 -(4 -O-b-D-glucopyrano-
syl)-vanilloyl spinosin, a new flavonoid
glycoside. The possibility of 1 as an artifact
was ruled out by the fact that the HPLC
analysis confirmed its presence in the
extract from the outset.
3
.
Experimental
3
.1 General experimental procedures
3
.2 Plant material
Melting points were measured using a
micro melting point apparatus and are
uncorrected (Yanamoto Manufactory Co.,
Kyoto, Japan). Optical rotations were
recorded on a Perkin-Elmer 241MC
automatic polarimeter. UV spectra were
obtained on a Shimadzu UV-2201 spec-
trophotometer, and IR spectra were
obtained on a Bruker IFS-55 infrared
spectrometer. 1D and 2D NMR spectra
were recorded separately on a Bruker
ARX-600 or ARX-300 spectrometer. ESI-
TOF-MS were acquired on a Micro TOF
The seeds of Z. jujuba were obtained from
Chaoyang, Liaoning Province of China, in
May 2008, and authenticated by Prof. Dan
Yuan, Shenyang Pharmaceutical Univer-
sity. A voucher specimen (No. 080518)
has been maintained in the Department of
Traditional Chinese Medicines, Shenyang
Pharmaceutical University.
3.3 Extraction and isolation
The air-dried seeds (2.0 kg) of Z. jujuba
were ground into powder (particle size

1
156
Y.-Y. Xie et al.
2
0–40 mesh) and refluxed with 16 liters of
further subjected to a Sephadex LH-20
column eluting with MeOH to yield
subfractions F9-5-1–F9-5-10. F9-5-5
(55 mg) was further purified by ODS
open column chromatography eluted with
petroleum ether (60–908C) two times
each for 1 h). After removing the
(
petroleum ether under reduced pressure,
the residue was refluxed with 15 liters of
methanol twice to obtain the total extract
of jujuba seeds with a yield of 6.5%. The
condensed MeOH extract (130 g) was
subjected to silica gel column chromatog-
MeOH–H O (30:70) to afford compound
2
1 (10.4 mg). F9-5-10 (15 mg) was further
separated with preparative HPLC (Waters
C18 column, 7.8 £ 300 mm, 6 mm) eluting
raphy eluting with CHCl –MeOH in a
3
with MeOH–H O (30:70) at a flow rate of
2
stepwise manner to furnish five major
fractions: fraction 2 (CHCl –MeOH, 7:1,
2 ml/min to give compound 6 (9.4 mg),
and the detective wavelength was set at
254 nm.
3
1
0
1
3
2
.5 g), fraction 3 (CHCl –MeOH, 6:1,
3
.2 g), fraction 5 (CHCl –MeOH, 6:1,
3
.8 g), fraction 7 (CHCl –MeOH, 4:1–
3
0
00
0000
3
.3.1 6 -(4 -O-b-D-Glucopyranosyl)-
vanilloyl spinosin (1)
A yellowish amorphous powder,
:1, 3.0 g), and fraction 9 (CHCl –MeOH,
3
:1, 8 g). Fraction 2 (1.5 g) was passed
through a Sephadex LH-20 column eluted
with MeOH to give compound 8 (50 mg).
Fraction 3 (200 mg) was further separated
by open column chromatography using an
MDS-5 reverse-phase packing and eluting
2
4
m.p. 215–2188C. ½aꢀ 2 54.2 (c ¼ 0.2,
D
MeOH). UV (MeOH) l
nm: 217, 262,
1
max
2
and 343. IR (KBr) nmax cm : 3401, 2924,
1
13
1701, 1652, 1607, 1510. H and C NMR
spectral data, see Table 1. ESI-TOF-MS
2
(negative) m/z: 919.2508 [M 2 H] (cald
with a linear gradient of MeOH–H O
2
(
(
30:70–95:5) to yield four major fractions
F3-1, F3-2, F3-3, and F3-4). F3-4 (80 mg)
for C H O , 919.2502).
2 47 23
4
eluted with MeOH–H O (95:5) was
2
further subjected to Sephadex LH-20
column (1.0 £ 40 cm) and eluted with
MeOH to give compound 4 (50 mg).
Fraction 5 (5.3 g) was repeatedly recrys-
tallized with MeOH to give compound 3
3
.4 HPLC analysis
HPLC analysis was carried out on a
Waters 510 HPLC instrument equipped
with photodiode array detector. The
separation was carried out on a Hypersil
ODS2 column (250 £ 4.6 mm i.d., 5 mm,
Elite Analysis Instruments, Dalian,
China). The temperature of column was
set at 358C. The mobile phase consisted of
a gradient system of (A) water and (B)
acetonitrile with a gradient elution as
followed: linear gradient from eluent A–B
(100:0, v/v) to eluent A–B (50:50, v/v) in
60 min at the flow rate of 1.0 ml/min. The
detective wavelength was set at 334 nm.
(450 mg). The mother-liquor was sub-
jected to silica gel column chromatog-
raphy eluted with CHCl –MeOH gradient.
3
The fraction 7 (3.0 g) was further separated
with an open column chromatography
packed with MDS-5 reverse-phase pack-
ing particles eluted with MeOH–H O
2
(40:60) to yield compound 5 (20 mg)
and compound 2 (5.6 mg). Fraction 9 (8 g)
was subjected to MDS-5 open column
chromatography eluting with a linear
gradient of MeOH–H O (10:90–95:5) to
2
yield nine major fractions (F9-1–F9-8).
F9-3 (537 mg) eluted with MeOH–H O
3
.5 Mild alkaline hydrolysis of 1
2
(50:50) was purified using ODS open
column chromatography to give com-
Solution of compound 1 (1 mg) in
0.05 N NH OH-50%MeOH (2 ml) was
4
pound 7 (39 mg). Fraction 9-5 (153 mg)
eluted with MeOH–H O (60:40) was
stirred at room temperature for 1 h. The
reaction mixture was neutralized with
2

Journal of Asian Natural Products Research
1157
þ
Dowex HCR-W2 (H form) and the
resin was removed by filtration. The
hydrolysate was extracted with EtOAc.
After purification of the extract by Varian
[3] H. Otsuka, T. Akiyama, K. Kawai, S.
Shibata, Q. Inoue, and Y. Ogihara,
Phytochemistry 17, 1349 (1978).
[
4] M. Yoshikawa, T. Murakami, and A.
Ikebata, Chem. Pharm. Bull. 45, 1186
(1997).
w
BOND ELUT C column (1.0 £ 4.0 cm,
1
8
GL Science, Tokyo, Japan) eluting
with a MeOH–H O gradient (20%
[5] H. Matsuda, T. Murakami, A. Ikebata, J.
Yamahara, and M. Yoshikawa, Chem.
Pharm. Bull. 47, 1744 (1999).
2
MeOH ! 50% MeOH ! MeOH), an aro-
matic compound was obtained, which was
identified as spinosin by co-chromato-
[
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