Dammarane-type saponins from Ziziphus jujube and their inhibitory effects against TNF-α release in LPS-induced RAW 246.7 macrophages

Article abstract of DOI:10.1016/j.phytol.2016.04.010

Four new dammarane-type saponins jujubosides F-J, together with six known compounds were isolated from the seeds of Ziziphus jujube. Their structures were elucidated on the basis of chemical and spectroscopic evidences. Compounds 1-10 showed moderate inhibitory effects against pro-inflammatory cytokine TNF-α release in LPS-induced RAW 246.7 macrophages.

Full text of DOI:10.1016/j.phytol.2016.04.010

Phytochemistry Letters 16 (2016) 169–173
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Dammarane-type saponins from Ziziphus jujube and their inhibitory
effects against TNF-
a release in LPS-induced RAW 246.7 macrophages
a
a
a
b,
Qiang Fu , Hai-Mei Yuan , Jiang Chen , Jian-You Shi *
a
School of Pharmacy and Bioengineering, Chengdu University, Chengdu 610106, China
Individualized Medication Key Laboratory of Sichuan Province, Sichuan Academy of Medical Science & Sichuan Provincial People’s Hospital, Chinese
b
Academy of Sciences Sichuan Translational Medicine Research Hospital, School of Medicine, Center for Information in Medicine, University of Electronic
Science and Technology of China, Chengdu 610072, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 19 February 2016
Received in revised form 7 April 2016
Accepted 14 April 2016
Available online xxx
Four new dammarane-type saponins jujubosides F–J, together with six known compounds were isolated
from the seeds of Ziziphus jujube. Their structures were elucidated on the basis of chemical and
spectroscopic evidences. Compounds 1–10 showed moderate inhibitory effects against pro-inflamma-
tory cytokine TNF-a release in LPS-induced RAW 246.7 macrophages.
ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Ziziphus jujube
Dammarane-type saponins
Jujubosides F–J
TNF-a
Inhibiting activity
1. Introduction
natural medicines, four new saponins jujubosides F–J (Fig. 1) and
six known saponins were isolated from the seeds of Z. jujube.
Ziziphus jujube Mill. is a thorny rhamnaceous plant which is
widely distributed in northern China. Its seeds have been used as a
traditional Chinese medicine, which is prescribed for tonic and
sedative purposes and treatment of insomnia (Yoshikawa et al.,
Herein, we report the isolation and structure elucidation, and anti-
inflammatory activity of these compounds.
2. Results and discussion
1
997). Up to now, many chemical components have been isolated
from this plant, including triterpene saponins (Yoshikawa et al.,
997; Matsuda et al., 1999), flavonoids (Cheng et al., 2000), and
The EtOH extract of dried seeds of Z. jujube was subjected to
silica gel column chromatography and prep-HPLC to obtain four
new compounds (1–4), together with six known structures, (5)
1
alkaloids (Tripathia et al., 2001). In previous phytochemical
investigation, a series of dammarane saponins with a sugar chain
linked to C-3 of aglycone have been isolated. Some of them showed
inhibitory activities against histamine release and lipoxygenase
1
jujuboside I (Wang et al., 2013), (6) jujuboside A (Yoshikawa et al.,
1997), (7) jujuboside A (Renault et al., 1997), (8) jujuboside B
(Okamura et al., 1981), (9) jujuboside C (Yoshikawa et al., 1997),
(10) jujubasaponin IV (Yoshikawa et al., 1992),
(Yu et al., 2013; Yoshikawa et al., 1997), and 3-glycoside moiety is
important to show immunological adjuvant activity (Matsuda
et al., 1999). In the course of our studies on the anti-inflammation
saponins of natural medicines, we have isolated twenty four
triterpene saponins from Clematis chinensis (Ranunculaceae) and
three dimeric monoterpene glycosides from Paeonia lactiflora
Their structures were elucidated on the basis of spectroscopic
evidence and hydrolysis products. The inhibitory effects against
pro-inflammatory cytokine TNF-
a release in LPS-induced RAW
246.7 macrophages of all compounds were also evaluated.
Compound 1 was isolated as white, amorphous powder. The
HRESIMS (positive-ion mode) experiment reveled a pseudo-
(
Ranunculaceae); these compouns were found to show inhibitory
+
activities against COX-1/COX-2 enzymes and nitric oxide release in
LPS-induced RAW 246.7 macrophages (Fu et al., 2010, 2015). As a
continuing part of our screening for anti-inflammatory saponins of
molecular ion peak [M + Na] at m/z 1141.5765, in agreement with
90
the molecular formula C55H O23. The aglycone of 1 was identified
1
13
as jujubogenin by comparison of the H and C NMR data obtained
in 2D NMR experiments with literature values (Wang et al., 2013).
The ROESY correlations between H-17, Me-21, and H-22b and
between H-22a and H-23 and the magnitude of the coupling
*
Corresponding author.
E-mail address: Shijy1982@tom.com (J.-Y. Shi).
http://dx.doi.org/10.1016/j.phytol.2016.04.010
874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1

170
Q. Fu et al. / Phytochemistry Letters 16 (2016) 169–173
Fig. 1. The structures of compounds 1–4.
constant between H-13 and H-17 (J = 7.0 Hz) are in agreement with
the configuration of rings D-F of jujubogenin (Wang et al., 2013; Yu
et al., 2013). The downfield shifts of C-3 at d 88.6 suggested that 1
C
galactose, glucose, and rhamnose, which were detected by TLC
comparison with authentic samples. The absolute configuration of
the monosaccharides was determined to be D for glucose and
galactose and L for rhamnose by GC analysis of chiral derivatives of
the monosaccharides in the hydrolysate of each compound (see
experimental section). The relatively large coupling constants (7.0–
was a monodesmosidic saponin.
The 1H NMR spectrum of 1 exhibited 4 anomeric proton
resonances at
d 6.40 (1H, brs), 5.29 (1H, d, J = 7.5 Hz), 5.16 (1H, d,
1
J = 7.5 Hz), and 4.81 (1H, d, J = 7.0 Hz). Acid hydrolysis of 1 yielded
7.5 Hz) for the anomeric protons in the H NMR spectra (see
Table 1
1
H NMR and 13C NMR spectral data for the aglycone moieties of compounds 1–4. (500 MHz for 1H NMR, 125 MHz for 13C NMR, in Pyridine-d
).
5
1
2
3
4
No.
1
d
H
d
c
d
H
d
c
d
H
d
c
d
H
d
c
1.52 m
39.2
27.2
1.53 m
0.75 m
2.12 m
1.88 m
39.0
27.1
1.52 m
0.76 m
2.12 m
1.86 m
39.2
27.2
1.51 m
0.74 m
2.13 m
1.87 m
39.1
27.2
0
.75 m
2.12 m
.86 m
2
1
3
4
5
6
3.23 dd (12.0, 4.0)
88.6
40.1
56.8
18.9
3.22 dd (12.0, 4.5)
88.6
40.0
56.7
18.8
3.23 dd (12.0, 4.0)
88.6
40.1
56.7
18.8
3.22 dd (12.0, 4.5)
88.5
40.0
56.7
18.8
0.64 d (11.0)
1.44 m
0.66 d (10.5)
1.44 m
0.64 d (10.5)
1.43 m
0.65 d (11.0)
1.45 m
1
.34 m
1.49 m
.38 m
1.33 m
1.48 m
1.38 m
1.36 m
1.48 m
1.38 m
1.36 m
1.48 m
1.39 m
7
36.4
36.4
36.4
36.3
1
8
9
38.1
53.3
37.7
21.9
38.0
53.2
37.7
21.8
38.0
53.3
37.7
21.8
37.9
53.2
37.7
21.8
0.82 dd (12.0, 2.0)
0.81 dd (12.0, 2.0)
0.81 dd (12.0, 2.0)
0.82 dd (12.0, 2.0)
1
0
11
1.52 m
1.53 m
1.32 m
1.90 m
1.79 m
2.82 m
1.52 m
1.33 m
1.91 m
1.80 m
2.81 m
1.54 m
1.33 m
1.92 m
1.81 m
2.82 m
1
.31 m
1.90 m
.79 m
12
28.9
28.9
28.8
28.8
1
1
3
2.81 m
37.8
54.2
37.5
37.8
54.1
37.4
37.8
54.2
37.4
37.8
54.2
37.4
1
4
15
2.48 d (8.0)
2.48 d (8.0)
1.53 m
2.47 d (8.0)
1.50 m
2.48 d (8.0)
1.52 m
1.52 m
1
6
111.2
54.4
19.5
17.0
69.1
30.5
46.1
111.1
54.4
19.4
17.0
69.0
30.5
46.2
111.2
54.4
19.4
17.1
69.2
30.5
46.2
111.1
54.3
19.4
17.0
69.1
30.5
46.1
1
7
1.41 d 7.0
1.07 s
0.75 s
1.41 d 7.0
1.07 s
0.76 s
1.42 d 7.0
1.06 s
0.75 s
1.42 d 7.0
1.06 s
0.74 s
1
8
1
9
2
0
2
1
1.38 s
1.75 d (12.0)
1.39 s
1.74 d (12.0)
1.67 m
1.38 s
1.75 d (12.0)
1.67 m
1.38 s
1.76 d (11.5)
1.68 m
2
2
1.66 m
23
24
25
26
27
28
29
30
5.20 m
5.52 br d (7.5)
69.0
127.5
134.6
26.1
18.8
28.4
17.4
5.20 m
5.53 br d (7.5)
69.1
127.4
134.5
26.1
18.8
28.4
17.3
5.21 m
5.53 br d (7.0)
69.0
127.4
134.5
26.2
18.8
28.4
17.4
5.22 m
5.53 br d (7.5)
69.0
127.4
134.4
26.1
18.7
28.3
17.3
1.67 s
1.69 s
1.23 s
1.15 s
4.27 m
1.66 s
1.69 s
1.24 s
1.14 s
4.26 m
4.21 m
1.67 s
1.68 s
1.24 s
1.16 s
4.28 m
4.21 m
1.67 s
1.69 s
1.24 s
1.16 s
4.28 m
4.21 m
66.2
66.1
66.1
66.1
4
.20 m

Q. Fu et al. / Phytochemistry Letters 16 (2016) 169–173
171
Experimental Section) of 1 suggested that the galactopyranosyl
and glucopyranosyl moieties have a -configuration. The -con-
figuration of the rhamnopyranosyl moiety was determined from
the broad singlet observed for the anomeric proton.
peaks with the carbon signals at
d
C
88.6 (Aglycone-C-3), 74.9 (Gal-
b
a
C-2), 85.8 (Gal-C-3), and 85.1 (Glc-C-2), respectively. These signals
provide evidence to determine the linkages between the sugars,
and the sugar and the aglycone. These linkages were also
confirmed by NOESY correlations between Aglycone-H-3/Gal-H-
1, Gal-H-2/Rha-H-1, Gal-H-3/Glc-H-1, and Glc-H-2/Glc -H-1 (see
Tables 1 and 2). From the above evidence, the structure of 1 was
The spin–spin coupling system of individual monosaccharide
units was identified by analysis of 1D TOCSY and 2D NMR spectra.
0
1
H NMR spectral data of individual monosaccharide units were
obtained by selective irradiation of the anomeric protons or methyl
groups of rhamnopyranosyl units in a series of 1D TOCSY
experiments. Analysis of the 1H– H COSY spectrum resulted in
sequential assignment of all proton resonances of the 4 monosac-
charide units, including identification of most of their multiple
splitting patterns and coupling constants, as shown in Table 2. In
the HSQC experiment, proton resonances were correlated with
those of the corresponding carbons, and associated anomeric
protons were correlated with their respective carbon atoms from
HSQC-TOCSY data, leading to unambiguous assignments of the
carbons in each monosaccharide unit (see Table 2). Taking into
established as 3-O-
b
-
D
-glucopyranosyl-(1 !2)-
b-D-glucopyrano-
-galactopyranosyl
syl-(1 !3)-[
a
-
L
-rhamnopyranosyl-(1 !2)]-
b-D
1
jujubogenin, which has been named jujuboside F.
Compound 2 was isolated as white, amorphous powder. The
HRESIMS (positive-ion mode) experiment revealed a pseudo-
molecular ion peak [M + Na] at m/z 1303.6303, in agreement with
+
the molecular formula C61
100
H O28. The spectroscopic properties of
2 were closely related to those of 1, except for the appearance of
0
0
signals due to Glc connected to Glc-C-6 in 2. This observation was
supported by a relative downfield shift of Glc-C-6 of 2 at c 70.2
(Glc-C-6 of 1 at c 62.8), and confirmed by HMBC correlation
between Glc -H-1 and Glc-C-6. Thus, the structure of 2 was
determined as 3-O- -glucopyra-
-glucopyranosyl-(1 !6)-[
nosyl-(1 !2)]- -glucopyranosyl-(1 !3)-[ -rhamnopyrano-
syl-(1 !2)]- -galactopyranosyl jujubogenin, which has been
named jujuboside G.
d
d
00
account the known effects of O-glycosylation, 1 contains one
galactopyranosyl unit (Gal), one - rhamnopyranosyl unit (Rha),
and two -glucopyranosyl units (Glc).
D-
L
b
-
D
b-D
D
b-D
a-L
In the HMBC spectrum, the anomeric proton signals at
Gal-1), 6.40 (Rha-1), 5.16 (Glc-1), and 5.29 (Glc -2) showed cross-
d
H
4.81
b-D
0
(
Table 2
1
H NMR and 13C NMR spectral data for the sugar moieties of compounds 1–4. (500 MHz for 1H NMR, 125 MHz for 13C NMR, in Pyridine-d
).
5
1
2
3
4
No.
d
H
d
c
d
H
d
c
d
H
d
c
d
H
d
c
Gal
Gal
Gal
Gal
1
2
3
4
5
6
4.81 d (7.0)
4.65 dd (9.5, 7.0)
4.25 m
4.82 m
4.10 m
105.9
74.9
85.8
70.5
76.7
62.6
4.80 d (7.0)
4.63 dd (9.0, 7.0)
4.26 m
4.82 m
4.12 m
105.8
74.9
85.8
70.5
76.6
62.5
4.81 d (7.0)
4.65 dd (9.5, 7.0)
4.25 m
4.82 m
4.10 m
105.8
74.8
85.8
70.5
76.6
62.5
4.82 d (7.0)
4.64 dd (9.0, 7.0)
4.24 m
4.81 m
4.11 m
105.8
74.8
85.7
70.4
76.5
62.6
4.30 dd (12.0, 6.0)
4.33 dd (12.0, 6.0)
4.42 dd (12.0, 2.0)
4.30 dd (12.0, 6.0)
4.41 dd (12.0, 2.0)
4.32 dd (12.0, 6.0)
4.43 dd (12.0, 2.0)
4
.41 dd (12.0, 2.0)
Rha
Rha
Rha
Rha
1
2
3
4
5
6
6.40 br s
4.71 br s
4.58 dd (9.0, 3.0)
4.28 m
4.72m
1.69 d (6.5)
101.7
73.0
73.2
74.4
70.4
18.8
6.41 br s
4.72 br s
4.58 dd (9.0, 2.5)
4.29 m
4.73m
1.70 d (6.0)
101.7
73.0
73.2
74.4
70.4
18.8
6.40 br s
4.71 br s
4.58 dd (9.0, 3.0)
4.28 m
4.72m
101.6
73.0
73.2
74.4
70.4
18.8
6.42 br s
4.71 br s
4.60 dd (9.0, 2.5)
4.28 m
4.72m
101.6
73.1
73.3
74.4
70.3
18.8
1.69 d (6.5)
1.70 d (6.0)
Glc
Glc
Glc
Glc
1
2
3
4
5
6
5.16 d (7.5)
4.01 dd (9.0, 7.5)
4.28 m
4.12 m
3.90 m
103.5
85.1
78.8
71.2
78.7
62.8
5.07 d (7.5)
3.98 dd (9.0, 7.5)
4.20 m
3.88 m
3.98 m
103.8
84.9
78.7
71.3
76.2
70.2
5.18 d (7.5)
4.04 dd (9.0, 7.5)
4.27m
4.14 m
3.91 m
103.7
83.2
78.6
71.2
78.6
62.7
5.05 d (7.0)
4.02 dd (9.0, 7.5)
4.21 m
3.89 m
3.98 m
103.9
83.5
78.6
71.3
76.2
70.e
4.49 dd (12.0, 2.0)
4.88 dd (12.0, 2.0)
3.92 dd (12.0,6.0)
4.47 dd (12.0, 2.0)
4.34 dd (12.0, 6.0)
4.89 dd (12.0, 2.0)
3.90 dd (12.0,6.0)
4
.32 dd (12.0, 6.0)
0
0
Glc
Glc
Xyl
Xyl
1
2
3
4
5
6
5.29 d (7.5)
4.24 dd (9.0, 7.5)
4.17 t (9.0)
4.32 m
3.82 m
4.41 dd (12.0, 2.0)
106.8
76.4
78.6
70.8
79.2
62.4
5.28 d (7.0)
4.24 dd (9.0, 7.0)
4.16 t (9.0)
4.30 m
3.81 m
106.8
76.4
78.5
70.8
79.1
62.3
5.35 d (7.0)
4.19 dd (9.0, 7.0)
4.16 m
4.22 m
4.50 m
106.8
76.4
78.5
71.2
5.34 d (7.0)
4.20 dd (9.0, 7.0)
4.17 m
4.21 m
4.50 m
106.8
76.3
78.4
71.2
68.2
68.3
4.40 dd (12.0, 1.5)
4.32 dd (12.0, 6.0)
3.76 t (11.0)
3.78 t (11.0)
4
.34 dd (12.0, 6.0)
0
0
0
Glc
Glc
1
2
3
4
5
6
4.88 d (7.5)
4.04 d (9.0, 7.5)
4.14 m
4.22 m
3.86 m
105.3
75.8
78.3
71.4
78.6
62.8
4.87 d (7.5)
4.03 d (9.0, 7.5)
4.13 m
4.21 m
3.84 m
105.2
75.7
78.2
71.3
78.7
62.8
4.48 dd (12.0, 1.5)
4.47 dd (12.0, 2.0)
4.32 dd (12.0, 6.0)
4
.34 dd (12.0, 6.0)

172
Q. Fu et al. / Phytochemistry Letters 16 (2016) 169–173
Compound 3 was isolated as white, amorphous powder. The
herbarium of School of Pharmacy and Bioengineering, Chengdu
University.
HRESIMS (positive-ion mode) experiment revealed a pseudo-
+
molecular ion peak [M + Na] at m/z 1111.5668, in agreement with
the molecular formula C54
H
88
O22. Acid hydrolysis of 3 yielded
3.3. Extraction and isolation
galactose, glucose, xylose, and rhamnose, which were detected by
TLC comparison with authentic samples. The absolute configura-
tion of the xylose was determined to be D by GC analysis of chiral
derivatives. The coupling constants (7.0 Hz) for the anomeric
protons of xylopyranosyl (Xyl) suggested that the Xyl moiety has a
The dried seeds (9.6 kg) of Z. jujube were refluxed two times
with EtOH, each for 2 h. After concentrated in vacuo, the residue
(1332 g) was suspended in water, and partitioned with ethyl
acetate and n-butanol successively. The n-butanol-soluble fraction
(337 g) was further chromatographed over a silica gel column
b
-configuration. A comparison of the 13C NMR spectra of 3 with
that of 1 revealed the similar aglycone. The only difference was that
the signals for Glc in 1 were replaced by those for Xyl in 3. The
chromatography using CHCl
to give fractions 1–7(8.7, 22.3, 151.7, 38.4, 23.2, 18.8 and 14.9 g,
3
-MeOH (100:1 !10:90) as an eluent
0
correlation observed between Xyl-H-1 and the Glc-C-2 in the
HMBC spectrum indicated that the Xyl is linked to the C-2 of Glc.
From the above evidence, the structure of 3 was determined as 3-
respectively). Fraction 2 (10.5 g) was subjected to ODS open
2
column chromatography (MeOH ꢀꢀ H O, 10:90 ! 95:5) to afford
sub-fractions 1–5 (2.2, 1.5, 1.8, 2.4 and 1.6 g, respectively). Sub-
O-
b
-
D
-xylopyranosyl-(1 !2)-
b
-
D
-glucopyranosyl-(1 !3)-[
a
-
L
-
fraction 3 (1.8 g) was separated by prep-HPLC (MeOH ꢀꢀ H
2
O, 38:62,
ꢂ
rhamnopyranosyl-(1 !2)]-
b
-D galactopyranosyl jujubogenin,
2.0 ml/min, tube temperature 120 C, gas flow 2.5 l/min) to yield
which has been named jujuboside H.
R
compound 10 (42 mg, t 18.1 min). Fraction 3 (11.2 g) was subjected
The molecular formula of 4 was determined as C60
H
98
O
27 by
+
to C18 silica gel chromatography (MeOH ꢀꢀ H O, 25:75 ! 60:40) to
2
HRESIMS, which showed a pseudomolecular ion peak [M + Na] at
m/z 1273.6188. The spectroscopic properties of 4 were closely
related to those of 3, except for the appearance of signals due to
afford sub-fractions 1–8 (0.9, 1.5, 1.3, 0.6, 0.4, 1.8, 2.4 and 1.5 g,
respectively). Sub-fraction 2 (1.5 g) was separated by prep-HPLC
ꢂ
(ACN ꢀꢀ H
2.5 l/min), affording compound 5 (51 mg, t
23.2 min). Sub-fraction 3 (1.3 g) was separated by prep-HPLC
2
O, 32:68, 2.0 ml/min, tube temperature 120 C, gas flow
0
Glc connected to Glc-C-6 in 4. This observation was supported by a
R
19.4 min) and 8 (37 mg,
relative downfield shift of Glc-C-6 of 4 at
d
c 70.3 (Glc-C-6 of 3 at
d
c
t
R
0
ꢂ
6
2.7), and confirmed by HMBC correlation between Glc -H-1 and
(ACN ꢀꢀ H
2.5 l/min), affording compound 6 (71 mg, t
24.6 min), 3 (21 mg, t 27.3 min), and 1 (19 mg, t
R
2
O, 30:70, 2.0 ml/min, tube temperature 120 C, gas flow
22.7 min), 7 (69 mg, t
33.2 min). Sub-
Glc-C-6. Thus, the structure of 4 was determined as 3-O-
glucopyranosyl-(1 !6)-[ -xylopyranosyl ꢀ(1 !2)]- -gluco-
-galacto-
b-D
-
R
R
b-
D
b
-
D
R
pyranosyl-(1 !3)-[
a
-
L
-rhamnopyranosyl-(1 !2)]-
b
-
D
fraction 4 (0.6 g) was separated by prep-HPLC (ACN ꢀꢀ H
2
O, 28:72,
ꢂ
pyranosyl jujubogenin, which has been named jujuboside J.
All isolates were evaluated for inhibitory activity against LPS-
2.0 ml/min, tube temperature 120 C, gas flow 2.5 l/min) to yield
R R
compound 9 (73 mg, t 18.7 min), 4 (23 mg, t 21.7 min), and 2
induced TNF-
Experimental). Curcumin was used as positive control (72.3%
inhibition rate, 10 M). Those compounds showed moderate
inhibitory effects against TNF- production with the inhibition
ratios of 33.3%, 29.1%, 27.2%, 18.8%, 42.6%, 17.4%, 18.5%, 26.9%, 29.1%
and 19.5% at 50 M, respectively. No cytotoxicity was observed in
compounds 1–10 treated cells (cell viability >90%).
a
production in RAW 246.7 macrophages (see
(28 mg, t 24.2 min).
R
m
3.3.1. Jujuboside F (1)
White amorphous power, [
a
a
]20
D
ꢀ42, (c 0.5, MeOH); IR(KBr)
13
ꢀ1
1
v
max (cm ): 3430, 1647, 1046; H and C NMR spectral data, see
+
m
Tables 1 and 2. HRESIMS: m/z 1141.5765 [M + Na] (calcd for
23Na, 1141.5771).
55 90
C H O
3
. Experimental
3.3.2. Jujuboside G (2)
White amorphous power, [
a
]20
D
ꢀ56 (c 0.5, MeOH); IR(KBr)
13
ꢀ
1
1
3.1. General experimental procedures
vmax (cm ): 3422, 1645, 1036; H and C NMR spectral data, see
+
Tables 1 and 2. HRESIMS: m/z 1303.6303 [M + Na] (calcd for
28Na, 1303.6299).
Optical rotations were measured on a JASCO P-1020 digital
61 100
C H O
polarimeter (Jasco, Tokyo, Japan). Spots were visualized by
spraying 10% H SO –EtOH followed by heating. IR spectra were
2
4
3.3.3. Jujuboside H (3)
obtained on a Bruker IFS-55 plus spectrometer (Bruker, Ettlingen,
German). NMR spectra were recorded on an Inova 500 spectrome-
ter with TMS as an internal standard, operating at 500 MHz for
White amorphous power, [
a
]20
D
ꢀ44, (c 0.5, MeOH); IR(KBr)
13
ꢀ
1
1
v
max (cm ): 3418, 1636, 1072; H and C NMR spectral data, see
+
Tables 1 and 2. HRESIMS: m/z 1111.5668 [M + Na] (calcd for
54 88
C H O22Na, 1111.5665).
1
13
Hand 125 MHz for C (Bruker, Waltham, MA, USA). HR-ESI–MS
data were obtained on a Bruker-Daltonics APES-III 7.0 TESLA FTMS
spectrometer (Bruker, Billerica, MA, USA). GC was obtained on a
SHIMADZU GC-14D. Precoated silica gel GF254 plates (Qingdao
Haiyang Chemical Co., Qingdao, China) were employed for thin
layer chromatography. Column chromatography was performed
with silica gel (Merck, Darmstadt, Germany) and C18 silica gel
3.3.4. Jujuboside J (4)
White amorphous power, [
a
]20
D
ꢀ59 (c 0.5, MeOH); IR(KBr)
13
ꢀ1
1
v
max (cm ): 3424, 1729, 1049; H and C NMR spectral data, see
+
Tables 1 and 2. HRESIMS: m/z 1273.6188 [M + Na] (calcd for
27Na, 1273.6193).
60 98
C H O
(
(
(
150–200 mesh, Merck). High performance liquid chromatography
HPLC) separation was carried out on an octadecylsilanized column
3.4. Acid hydrolysis
YMC-pack ODS-A, 250 ꢁ10 mm, i.d. 5
mm, YMC, Kyoto, Japan)
ꢂ
with an Alltech ES 2000 evaporative light scattering detector
Grace, Crutis Bay, MD, USA).
Each compound (5 mg) was heated in 0.5 ml of 2 M HCl at 95 C
for 10 h in a sealed tube. After filtration of the reaction mixture, the
(
2
filtrate was evaporated under vacuum. After addition of H O, the
3
.2. Plant material
acidic solution was evaporated again to remove HCl. This
procedure was repeated until a neutral solution was obtained,
which was finally evaporated and dried in vacuo to furnish a
monosaccharide residue. The sugar components obtained after
acid hydrolysis of 1–10 were analyzed by GC analysis of the methyl
The seeds of Z. jujube were collected in May 2015 in Chengdu
city, Sichuan Province of China, and identified by author (Qiang
Fu). A voucher specimen (ZJ 201505) is maintained in the

Q. Fu et al. / Phytochemistry Letters 16 (2016) 169–173
173
sugar peracetates. The monosaccharide residue was dissolved in
anhydrous pyridine (100 ml), 0.1 M -cysteinemethyl ester hydro-
Acknowledgement
L
chloride (200 ml) was added, and the resultant was warmed at
This work was supported by the National Natural Science fund
of China (Grant No. 81503234).
ꢂ
60 C for 2 h. The trimethylsilylation reagent HMDS–TMCS
(
hexamethyldisilazane–trimethylchlorosilane–pyridine, 2:1:10;
ꢂ
Acros Organics, Geel, Belgium) was added and warmed at 60 C
for 30 min. To the mixture, the thiazolidine derivatives were
analyzed by GC for sugar identification. Separations were carried
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