Two new flavonoid glycosides from Semen Ziziphi Spinosae

Article abstract of DOI:10.1080/10286020.2011.637491

Two novel flavonoid glycosides, 6-dihydrophaseoylspinosin (1) and 6,6-diferuloylspinosin (2), were isolated from the MeOH extract of Semen Ziziphi Spinosae, together with six known flavonoids, isovitexin-2-O-β-(6-O-E- feruloyl)glucopyranoside (3), spinosin (4), isospinosin (5), 6-feruloylspinosin (6), swertisin (7), and isovitexin-2-O-β-d-glucopyranoside (8). The structures of 1 and 2 were elucidated by spectroscopic methods including UV, IR, ESI-TOF-MS, 1D NMR, and 2D NMR experiments.

Full text of DOI:10.1080/10286020.2011.637491

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Two new flavonoid glycosides from
Semen Ziziphi Spinosae
a
a
a
a
Lin Zhang , Zhi-Lin Xu , Chun-Fu Wu , Jing-Yu Yang
,
a
a
Yoshihiro Kano & Dan Yuan
a
Department of Traditional Chinese Medicines , Shenyang
Pharmaceutical University , Shenyang , 110016 , China
Published online: 01 Feb 2012.
To cite this article: Lin Zhang , Zhi-Lin Xu , Chun-Fu Wu , Jing-Yu Yang , Yoshihiro Kano & Dan Yuan
(2012) Two new flavonoid glycosides from Semen Ziziphi Spinosae , Journal of Asian Natural Products
Research, 14:2, 121-128, DOI: 10.1080/10286020.2011.637491
To link to this article: http://dx.doi.org/10.1080/10286020.2011.637491
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Journal of Asian Natural Products Research
Vol. 14, No. 2, February 2012, 121–128
Two new flavonoid glycosides from Semen Ziziphi Spinosae
Lin Zhang, Zhi-Lin Xu, Chun-Fu Wu, Jing-Yu Yang, Yoshihiro Kano and Dan Yuan*
Department of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang
1
10016, China
(
Received 22 July 2011; final version received 30 October 2011)
0
00
00 000
Two novel flavonoid glycosides, 6 -dihydrophaseoylspinosin (1) and 6 ,6 -
diferuloylspinosin (2), were isolated from the MeOH extract of Semen Ziziphi
00
Spinosae, together with six known flavonoids, isovitexin-2 -O-b-(6-O-E-feruloyl)glu-
000
copyranoside (3), spinosin (4), isospinosin (5), 6 -feruloylspinosin (6), swertisin (7),
0
elucidated by spectroscopic methods including UV, IR, ESI-TOF-MS, 1D NMR, and
0
and isovitexin-2 -O-b-D-glucopyranoside (8). The structures of 1 and 2 were
2D NMR experiments.
000
Keywords: Semen Ziziphi Spinosae; flavonoid glycosides; 6 -dihydrophaseoylspino-
000
0
0
sin; 6 ,6 -diferuloylspinosin
1
.
Introduction
reversed-phase packings led to two new
0
00
compounds 6 -dihydrophaseoylspinosin
0
Semen Ziziphi Spinosae is botanically
from the seeds of Ziziphus jujuba Mill
var. spinosa (Bunge) Huex. H.F. Chou,
which was used for tranquilizing mind,
nourishing liver, soothing nerves, and
arresting sweating in traditional Chinese
medical therapy. Previous phytochemical
studies on Semen Ziziphi Spinosae
reported the isolation of many flavonoids
0
000
(
1) and 6 ,6 -diferuloylspinosin (2),
together with six known flavonoids, iso-
0
0
vitexin-2 -O-b-(6-O-E-feruloyl)glucopyra-
0
00
noside (3), spinosin (4), isospinosin (5), 6 -
feruloylspinosin (6), swertisin (7), and
0
0
isovitexin-2 -O-b-D-glucopyranoside (8).
The structures of 1 and 2 were elucidated
by 1D and 2D NMR, UV, IR, and MS
techniques, and those of 3–8 were identified
by comparing their NMR and MS spectral
data with reported values [3,12].
[
[
1–6], saponins [6–9], and alkaloids
10,11]. In a continuing search for new
compounds from this plant, we obtained a
series of flavonoids including two new
flavonoid glycosides 1 and 2 and six
known compounds 3–8 (Figure 1). In this
paper, the isolation and structural elucida-
tion of these compounds are described.
Compound 1 was isolated as a yellow-
ish amorphous powder. The molecular
formula was determined to be C H O
3 52 19
4
2
from the [M 2 H] ion peak at m/z
71.3026 in the ESI-TOF-MS. The UV
8
absorption maxima at 219, 274, and
322 nm, together with a positive reaction
2
.
Results and discussion
The MeOH extract of the Semen Ziziphi
Spinosae was prepared by reflux method.
A series of column chromatography using
silica gel, Sephadex LH-20, and MDS-5
in AlCl reagent, revealed a flavone
3
nucleus. The IR spectrum showed absorp-
tion bands due to a hydroxyl group
2
(3398 cm ) and a carboxylic carbonyl
1
*Corresponding author. Email: yuandan_kampo@163.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.637491
http://www.tandfonline.com

1
22
L. Zhang et al.
OCH3
OH
OH
1
2''''
HO
4
'''' 3''''
2
''''
1
0''''
H CO
O
O
8''''
H CO
O
O
2''''
3
1''''
3
7
''''
9''''
O
1
''''
HO
14''''
5''''
O
3''''
9''''
6
''
11''''
O
5''''' 6'''' 7''''
6'''''
O 1''
OH
HO
O
OH
OH
8
''''
HO
6''''
15''''
O
4'''''
3'''''
5
''''
13''''
OH
2''
H
1''''' 8'''''
2'''''
7'''''
9'''''
OH
6'''
H
O
4''''
OH
OH
O
O
H CO
O
3
6'''
O
O
O
1'''
H
OH
1'''
OH
H
OH
OH
OH
OH
1
2
Figure 1. Chemical structures of compounds 1 and 2.
2
1705 cm ). The resonances for the
1
(
1.00/0.97, 3H, s, d 19.4/19.4), and an
C
3
sp -tertiary carbons (d 48.0/47.9). Thus,
carbons and protons located on the
flavonoid framework had a close resem-
blance to those of the known spinosin, and
they were assigned according to the
C
the substituent should be bicyclic moiety
according to the degrees of unsaturation.
The constitution of the substituent was
deduced from the HMBC spectrum
(Figure 2). The HMBC correlations of a
hexacyclic moiety were determined
1
13
literature values of the H and C NMR
spectral data for spinosin [3] as well as its
own HSQC spectrum. The spinosin moiety
accounted for a partial molecular formula
of C H O with 13 degrees of unsatura-
0
000
0000
0000
between H-12 and C-1 , C-2 , and
0
000
0000
0000
C-3 , between H -3 and C-4 , between
0000 0000 0000
H -5 and C-4 , and between H-13 and
b
2
8
32 15
b
tion, and thus, the remaining molecular
formula C H O consisted structurally
0
000
0000
0000
C-1 , C-5 , and C-6 . Those of a
pentacyclic moiety were shown between
1
5 21 4
of one substituent, accounting for six
degrees of unsaturation and being attached
to the outer glucopyranosyl moiety. The
resonances for the carbons and protons for
0
000
0000
0000
0000
0000
H-11 and C-1 , C-2 , C-3 , and C-6 ,
0
000
0000
and between H-12 and C-11 . Thus, a
bridge-cyclic moiety consisting of a
hexacyclic ring and a pentacyclic ring
was confirmed on the basis of the HMBC
correlations mentioned above. A chain
substituent (C H O ) was confirmed on
1
13
the substituent in the H and C NMR as
well as HSQC spectra included an ester
carbonyl group (dC 164.6/164.8), as
evidenced by the IR absorption band at
6
6 2
2
1
2
1
5
6
1
705 cm , three sp methines (dH
.51/5.50, 1H, s, dC 116.3/115.8; dH
the basis of the HMBC correlations
0
000
0000
between H-14 and C-10 , between
H-8 and C-7 , C-9 , C-10 , and C-
0000 0000 0000 0000
.42/6.40, 1H, d, J ¼ 15.6 Hz, d 135.6/
C
0
000
0000
0000
35.5; d 7.83/7.73, 1H, d, J ¼ 15.6 Hz,
14 , and between H-7 and C-8 and C-
9 . And an ester carbonyl group assigned
H
2
0000
d 129.1/129.0), a sp -tertiary carbon (d
C
C
3
29.1/129.0), four sp carbons that should
0000
1
be directly attached to oxygen, including
a methylene (dH 3.65, 3.58, 2H, m, dC
at C-14 was determined to be a,b-
unsaturated lactone by a significant high-
0000
field shift of C-14 [13]. Its location at
0
000
7
d 63.8/63.8), two sp -tertiary carbons (d
5.3/75.3) and a methine (d 3.92, 1H, m,
3
C-1 of the bridge-cyclic moiety was
deduced from the correlations between
H
C
C
0000 0000 0000
8
1.5/81.5; d 85.7/85.7), two methylenes
C
H-7 and C-1 , and between H-8 and
0000
C-1 . In addition, a hydroxyl group could
(
d 1.89, 1H, m, 1.64, 1H, m, d 45.4/45.4),
d 1.72, 1H, m, 1.54, 1H, m, d 43.8/43.8;
H
C
0000
be also attached to C-1 because of the
H
C
0000
HMBC correlation between 1 -OH and
a methyl (dH 1.86/1.91, 3H, s, dC
0.5/20.5) that should be directly attached
0000 0000
2
C-1 and C-7 . The geometry of the
0000
double bond was
2
0000
to sp carbon, two methyl groups (dH
.83/0.82, 3H, s, dC 16.0/16.0; dH
trans C-7 –C-8
confirmed on the basis of large coupling
0

Journal of Asian Natural Products Research
123
0
000
0000
0000
0000
constant 15.6 Hz, and that of the cis
1 S, 2 R, 4 S, 6 R absolute configur-
ation [14]. Thus, all of the protons and
functional groups were assigned and the
complete structure of 1 was elucidated,
0
000
0000
C-9 –C-14 double bond was confirmed
on the basis of the olefinic proton in a
highfield shift relative to the correspond-
ing signals of trans compounds [3]. Thus,
the structure of the substituent was
confirmed as a dihydrophaseoyl group
0
00
named as 6 -dihydrophaseoylspinosin.
1
1
The HMBC, H– H COSY, and NOESY
1
results of 1 led to assignments for the H
0
00
13
and C signals as shown in Table 1.
[
13]. It could be attached to C-6 for
000
the chemical shift of C-6 shifted
toward downfield by 1.3 ppm and that of
Compound 2 was isolated as a yellow-
ish amorphous powder. The molecular
formula was determined to be C H O
8 48 21
0
0
C-5 shifted toward upfield by 2.8 ppm
relative to the corresponding signals of
spinosin [3].
4
2
from the [M 2 H] ion peak at m/z
959.2604 in the ESI-TOF-MS. The UV
absorption maxima at 214, 258, and
327 nm, together with a positive reaction
in AlCl3 reagent, revealed a flavone
skeleton. The IR spectrum showed absorp-
tion bands due to a hydroxyl group
The relative configuration of 1 was
determined from the 2D NOESY corre-
lation (Figure 2). The strong NOESY
0000
correlations between a-H-4 and a-H-
1 , together with the rigid (2 ,6 )-
0000 0000 0000
1
bridged ring indicated that both 11 -CH
0000
21
(3407 cm ) and a carboxylic carbonyl
21
(1694 cm ). The resonances for the
2
0
000
and O-C (6 ) had a-orientation. The b-H-
0
000
5 at d 1.89 in the downfield region was
indicated by the strong NOESY corre-
carbons and protons of the flavonoids
framework had a close resemblance to
those of the known spinosin, except for
H
0000
0000
lations between b-H-5
Subsequently, the b-orientation of the
chain substituent (C H O ) at C-1 was
6 6 2
and H-13 .
0
0
00
000
those of C-5 , C-6 , and C-6 . They were
assigned according to the literature values
13
of the H and C NMR spectral data for
0000
1
confirmed by the strong NOESY corre-
0000
0
000
lations between b-H-5 and H-7 . The
absolute configuration at the C-4 pos-
ition was elucidated to be S according to
spinosin [3] as well as its own HSQC
00
0000
spectrum. The downfield shift of C-6 (d
0
C
00
64.3/64.3, þ 2.9) and C-6 (dC 62.5/
1
the literature values of the H NMR data
62.5, þ 1.9), and the upfield shift of
0
000
0000
000
for 4 S-dihydrophaseic acid and 4 R-
dihydrophaseic acid [14], and 1 possesses
C-5 (d 73.7/73.2, 22.5) [3], relative to
C
the corresponding signals of spinosin,
12''''
10''''
2''''
7''''
1''''
3''''
9''''
11''''
OH
8''''
14''''
O
4''''
6''''
HO
13''''
5''''
H
H
Figure 2. Selected HMBC (H ! C) and NOESY (H
H) correlations of compound 1.

1
24
L. Zhang et al.
b
a
a
a
a
b
b
a

Journal of Asian Natural Products Research
125
0
0
revealed the substitutions of C-6 and
0
00
C-6 . The resonances for the carbons
a
1
and protons of the substituents in the H
b
13
and C NMR as well as HSQC spectra
b
b
b
included two ABX aromatic proton
systems, two ester carbonyl groups, four
2
sp methines, and two methoxyl moieties.
The constitution of the substituents was
deduced to be two feruloyl moieties
from the HMBC spectrum (Figure 3),
which was also supported by the fragment
2
ion peaks at m/z 959 [M 2 H] , 783
[
2
M 2 feruloyl 2 H] , and 607 [M 2
2
feruloyl 2 H] in ESI-MS. The geome-
2
try of the trans C-7 –C-8 double bond
0
000
0000
0
0000
00000
and trans C-7 –C-8 double bond was
confirmed on the basis of large coupling
constant 15.6 Hz [3]. Hence, compound 2
0
0
000
was assigned as 6 ,6 -diferuloylspinosin.
1
The H and C NMR spectra of 1 and
13
2
showed the same phenomenon of
separate signals because of rotational
isomers which are produced by the
rotational barriers 7-OCH in flavone-6-
a
b
3
C-glycoside. It was proved that the
multiplicity collapsed to a first-order
spectrum when measuring at high tem-
perature [3]. Thus, structural elucidation of
this type of compounds will be easy when
measuring all spectra at high temperature
1
13
1
1
( H NMR, C NMR, H– H COSY,
HMQC, HMBC, and NOESY).
3
.
Experimental
3
.1 General experimental procedures
Optical rotations were measured with a
JASCO P-1010 polarimeter. UV spectrum
was obtained on a Shimadzu UV 1600
spectrophotometer. IR spectra were
recorded on a Bruker IFS-55 infrared
spectrometer. ESI-TOF-MS were obtained
on a Micro TOF Bruker Daltonics mass
spectrometer with a resolution of 25,000
b
a
a
b
(
10% Valley). 1D NMR and 2D NMR
were measured by a Bruker ARX-600
spectrometer with DMSO as the solvent
and tetramethylsilane as an internal
standard. HPLC analysis was carried out

1
26
L. Zhang et al.
OCH3
''''
3
OH
HO
4
2''''
''''
8
''''
O
O
H CO
3
5
''''
O
1
''''
6''
6
''''
9''''
5
'''''
7''''
O
O
HO
6
'''''
OH
4
'''''
H
OH
8
'''''
OH
3
'''''
O
O
H
H CO
1'''''
6'''
3
2'''''
9'''''
7'''''
O
O
OH
OH
OH
Figure 3. Selected HMBC correlations of compound 2 (H ! C).
on a Kaseisorb LC NH2-60-5 column
Tokyo Kasei Co., Ltd, Tokyo, Japan,
fraction (800 g), and a MeOH-soluble
fraction (470 g). The dried MeOH-soluble
fraction was subjected to silica gel
(
4
.6 mm i.d. £ 250 mm) with an LC-6AD
pump (Shimadzu, Kyoto, Japan), mon-
itored by an optical rotation detector
(CHCl –MeOH, 10:1 ! 0:1) to obtain
3
15 fractions (Frs 1–15). Fr. 7 (3.5 g) was
subjected to MDS-5 (MeOH–H O,
[Shodex OR-2 (Showa Denko Co., Ltd,
2
Tokyo, Japan)]. Silica gel (200–300 mesh)
was purchased from Qingdao Marine
Chemical Ltd (Qingdao, China), Sephadex
LH-20 from GE Healthcare (Stockholm,
Sweden), and MDS-5 RP (200–300 mesh)
from Beijing Medicine Technology Center
40:60 ! 100:0) to afford Frs 7.1–7.3. Fr.
7.2 (59.0 mg) was further subjected to
Sephadex LH-20 (MeOH) to give com-
pound 8 (36.5 mg). Fr. 9 (3.9 g) was
subjected to MDS-5 (MeOH–H O,
2
1:20 ! 100:0) to obtain compounds 2
(Beijing, China).
(7.0 mg) and 7 (8.0 mg). Compound 4
(
(
5.0 g) was recrystallized from Fr. 11
11.5 g) and the remaining Fr. 11 (6.5 g)
3
.2 Plant material
was subjected to silica gel (CHCl3–
MeOH, 7:1 ! 0:1) to give 6 (43.3 mg).
Fr. 11.2 (3.5 g) was further separated by
The specimens of Semen Ziziphi Spinosae
were collected from Chaoyang city of
Liaoning Province in China (May 2008)
and were identified by Prof. Dan Yuan,
Shenyang Pharmaceutical University,
China. A voucher specimen (No. 080518)
has been deposited at the Department of
Traditional Chinese Medicines, Shenyang
Pharmaceutical University.
MDS-5 (MeOH–H O, 1:4 ! 1:0) to pro-
2
vide compounds 1 (45.6 mg), 3 (22.6 mg),
and 5 (80.4 mg).
3
.3.1 Compound 1
A yellowish amorphous powder with a
2
0
D
molecular formula C H O , ½aꢀ
4
3
52 19
3
.3 Extraction and isolation
2
(log 1): 216 (1.62), 272 (1.52), 332 (1.29)
45.0 (c 0.1, MeOH). UV (MeOH) l
max
The air-dried, powdered Semen Ziziphi
Spinosae (6.0 kg) was successively
extracted with petroleum ether (48 liters)
and MeOH (48 liters) for 2 £ 1 h under
reflux to give a petroleum ether-soluble
2
nm. IR (KBr) nmax (cm ): 3398, 2927,
1
1706, 1654, 1607, 1512, 1490, 1448, 840,
1
and 779. For H and C NMR spectral
13
data see Table 1. ESI-TOF-MS: m/z

Journal of Asian Natural Products Research
127

1
28
L. Zhang et al.
2
71.3026 [M 2 H] (cacld for C H O ,
71.3025).
8
8
were dissolved in EtOAc, and partitioned
with water. The water solution was
analyzed and subjected to HPLC with
optical detector using a Kaseisorb LC
4
3 51 19
3
.3.2 Compound 2
NH -60-5 column (4.6 mm i.d. £ 250 nm)
2
A yellowish amorphous powder with a
molecular formula C H O , ½aꢀ 297.0
with CH CN–H O (v:v ¼ 85:15, flow:
3
2
2
D
0
4
8
48 21
0.8 ml/min) as the mobile phase. Identifi-
cation of D-glucose was carried out by
comparison of the retention time
(12.7 min) and OD (þ) with authentic
samples. Using the same method, the
monosaccharide from 2 was also identified
as D-glucose.
(
c 0.1, MeOH). UV (MeOH) l (log1):
14 (1.81), 275 (1.45), 326 (1.66) nm. IR
max
2
21
(
KBr) nmax (cm ): 3408, 2927, 1694, 1653,
605, 1514, 1491, 1449, 839, and 779. For
1
1
13
H and C NMR spectral data see Table 2.
2
ESI-MS: m/z 783 [M 2 feruloyl 2 H] ,
6
m/z 959.2604 [M 2 H]
2
07 [M 2 2feruloyl 2 H] . ESI-TOF-MS:
2
(cacld for
C H O , 959.2610).
48 48 21
References
[
1] L. Zeng, R.Y. Zhang, and X. Wang, Acta
Pharm. Sin. 22, 114 (1987).
[2] K.H. Shin, H.Y. Kim, and W.S. Woo,
Planta Med. 44, 94 (1982).
3] G. Cheng, Y.J. Bai, and Y.Y. Zhao,
Tetrahedron 56, 8915 (2000).
3
.4 Mild alkaline hydrolysis of 2
Solution of compound 2 (1 mg) in 0.05 N
NH OH–50% MeOH (2 ml) was stirred at
4
[
[
room temperature for 1 h. The reaction
mixture was neutralized with Dowex
4] S.M. Wang, Z.M. Bi, and P. Li, Chem.
Ind. Forest Prod. 25, 37 (2005).
þ
HCR-W2 (H form) and the resin was
removed by filtration. The hydrolysate was
extracted with EtOAc. After purification
w
of the extract by Varian BOND ELUT
[5] Q. Cao and K.W. Wang, J. Pharm. Pract.
7, 209 (2009).
[6] M. Yoshikawa, T. Murakami, and
A. Ikebata, Chem. Pharm. Bull. 45, 1186
(1997).
2
C18 column (1.0 £ 4.0 cm, GL Science,
Tokyo, Japan) eluting with a MeOH–H O
2
[7] H. Otsuka, T. Akiyama, and A. Ikebata,
Pytochemistry 6, 1349 (1978).
gradient (20% MeOH ! 50% MeOH !
MeOH), two aromatic compounds were
obtained, which were identified as spinosin
and ferulic acid by co-chromatography
with authentic samples.
[
8] H. Matsuda, T. Murakami, A. Ikebata,
J. Yamahara, and M. Yoshikawa, Chem.
Pharm. Bull. 47, 1744 (1999).
[
9] Q.C. Liu, B. Wang, H. Liang, Y.Y. Zhao,
and M.J. Liu, Acta Pharm. Sin. 39, 601
(2004).
10] B.H. Han, M.H. Park, and Y.N. Han,
Phytochemistry 29, 3315 (1990).
11] S.Z. Yin, B.Y. Jin, and S.G. Hong, Chin.
J. Chin. Mater. Med. 22, 692 (1997).
12] E. Pretsch, P. Buhlmann, and C. Affolter,
East Chin. Univ. Sci. Technol. Publ. 155,
238 (2002).
[
[
[
3
.5 Acid hydrolysis of 1 and 2
Compound 1 (1.0 mg) was dissolved in
MeOH (9 ml) containing HCl (1 ml) and
refluxed on a heated (808C) water bath for
3
h. After cooling, the reaction mixture
[
[
13] B.L. Cui, M. Nakamura, J. Kinjo, and
T. Nohara, Chem. Pharm. Bull. 40, 1943
was concentrated and dried under reduced
pressure, then was analyzed by TLC using
the solvent system CHCl –MeOH–H O
(
1992).
3
2
14] N. Hirai, S. Kondo, and H. Ohigashi,
Biosci. Biotechnol. Biochem. 67, 2408
(2003).
(20:10:1) for the identification of the
complete reaction. The dried residues