Dammarane-type saponins from Ziziphus jujube

Article abstract of DOI:10.1080/10286020.2013.866098

Two new dammarane-type saponins jujubosides D and E, together with three known compounds, were isolated from the seeds of Ziziphus jujube. Their structures were elucidated on the basis of chemical and spectroscopic evidence. Compounds 1-5 showed lipoxygenase-inhibiting activity.

Full text of DOI:10.1080/10286020.2013.866098

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Dammarane-type saponins from
Ziziphus jujube
Zhi Yua, Xie-Shu Wanga & Ze-Hua Hub
a College of Horticulture and Forestry Science, Huazhong
Agricultural University, Wuhan, 430070, China
b School of Medicine, Hubei University for Nationalities, Enshi,
445000, China
Published online: 19 Dec 2013.
To cite this article: Zhi Yu, Xie-Shu Wang & Ze-Hua Hu (2014) Dammarane-type saponins
from Ziziphus jujube, Journal of Asian Natural Products Research, 16:2, 200-205, DOI:
10.1080/10286020.2013.866098
To link to this article: http://dx.doi.org/10.1080/10286020.2013.866098
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 2, 200–205, http://dx.doi.org/10.1080/10286020.2013.866098
Dammarane-type saponins from Ziziphus jujube
Zhi Yua*, Xie-Shu Wanga and Ze-Hua Hub
aCollege of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan 430070,
China; School of Medicine, Hubei University for Nationalities, Enshi 445000, China
b
(Received 8 June 2013; final version received 12 November 2013)
Two new dammarane-type saponins jujubosides D and E, together with three known
compounds, were isolated from the seeds of Ziziphus jujube. Their structures were
elucidated on the basis of chemical and spectroscopic evidence. Compounds 1–5
showed lipoxygenase-inhibiting activity.
Keywords: Ziziphus jujube; dammarane-type saponins; jujuboside D; jujuboside E;
lipoxygenase-inhibiting activity
1. Introduction
BuOH. The n-BuOH extract was separated
by repeated column chromatography using
silica gel, C18 silica gel, and HPLC to give
two new compounds jujuboside D (1) and
jujuboside E (2), together with three known
compounds, jujuboside A (3) [6], jujubo-
side C (4) [6], and jujuboside A1 (5) [5].
Their structures were elucidated on the
basis of chemical and spectroscopic
evidence.
Plants of the Ziziphus genus (Rhamnaceae)
are commonly used as edible or medicinal
plants in warm and tropical regions [1].
They are well known for its content of
triterpenes, saponins, and cyclopeptide
alkaloids [2]. 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 for treatment of insomnia. In
previous phytochemical investigation, a
series of dammarane saponins have been
isolated [3,4]. Some of them showed
inhibitory activity against histamine
release and anti-sweet activity [4,5]. In
the course of our studies on the bioactive
saponins of natural medicines, we inves-
tigated the chemical constituents and
biological activities of this plant. In this
article, we report the isolation and
structure elucidation of two new saponins
jujubosides D and E (Figure 1), as well as
their lipoxygenase-inhibiting activity.
Compound 1 was isolated as a white,
amorphous powder. The HR-ESI-MS
(positive-ion mode) experiment revealed
a pseudo-molecular ion peak [M þ Na]þ
at m/z 1391.6466, in agreement with the
molecular formula C64H104O31. The agly-
cone was assigned as jujubogenin on the
1
basis of the H and 13C NMR spectra and
homo- and heteronuclear correlations
observed in 1H–1H COSY, HSQC, and
HMBC experiments. Most of the 13C
NMR signals were assigned through 2JH–C
3
and JH–C couplings of the seven methyls
and are in agreement with literature data
[4–6] (Table 1). The downfield shifts of
C-3 at dC 89.8 suggested that 1 was a
monodesmosidic saponin. The ROESY
correlations between H-17, Me-21, and
H-22b and between H-22a and H-23 and
2. Results and discussion
The EtOH extract of dried seeds of Z.
jujube was partitioned between H2O and n-
*Corresponding author. Email: yuzhi198011@126.com
q 2013 Taylor & Francis

Journal of Asian Natural Products Research
201
22 a
H
22 b
H
HO
24
26
25
23
21
20
H
12
18
H
O
11
19
27
17
13
14
16
15
1
HO
HO
Glc-3
O
O
Glc-2
O
H
O
3
OH Ara-1
O
30
HO
Glc-1
HO
OH HO
O
O
H
28
O
HO
O
OH
29
HO
O
OR
1
O
HO
Rha-1
HO
OH
Xyl-1
O
HO
1 R =
1
HO
2 R = H
1
OH
R 1 = H
Figure 1. Structures of compounds 1 and 2.
the magnitude of the coupling constant
between H-13 and H-17 (J ¼ 7.0 Hz) are
in agreement with the configuration of
rings D–F of jujubogenin [6,7]. The
monosaccharides obtained after aqueous
acid hydrolysis of 1 were identified as
glucose, rhamnose, arabinose, and xylose
by TLC comparison with authentic
samples. The absolute configuration of
the monosaccharides was determined to
be D for glucose and xylose and L for
rhamnose and arabinose by GC analysis
of chiral derivatives of the monosacchar-
ides in the hydrolysate of each compound
(see Section 3). Complete assignment of
each sugar proton system was achieved by
with the carbon signals at dC 89.8
(Aglycone-C-3), 75.8 (Ara-1-C-2), 68.6
(Ara-1-C-4), 82.6 (Glc-1-C-2), 69.8 (Glc-
1-C-6), and 68.2 (Glc-2-C-6), respectively.
These signals provide evidence to deter-
mine the linkages between the sugars, and
between the sugar and the aglycone. From
the above evidence, the structure of 1 was
established as 3-O-b-D-glucopyranosyl-
(1 ! 6)-b-D-glucopyranosyl-(1 ! 6)-
[(1 ! 2)-b-D-xylopyranosyl)]-b-D-gluco-
pyranosyl-(1 ! 3)-[a-L-rhamnopyranosyl-
(1 ! 2)]-a-L-arabinopyranosyl jujubo-
genin, which has been named jujuboside D.
Compound 2 was isolated as a white,
amorphous powder. The HR-ESI-MS
(positive-ion mode) experiment revealed
a pseudo-molecular ion peak [M þ Na]þ
at m/z 1259.6041, in agreement with the
molecular formula C59H96O27. The
spectroscopic properties of 2 were
closely related to those of 1; however,
signals due to the xylopyranosyl moiety
linked to Glc-1-C-2 in 1 were absent for 2.
This observation was supported by a
relative upfield shift of Glc-1-C-2 of 2 at
dC 75.1 (Glc-1-C-2 of 1 at dC 82.6). Thus,
the structure of 2 was determined as 3-O-
b-D-glucopyranosyl-(1 ! 6)-b-D-gluco-
1
considering H–1H COSY and TOCSY
spectra, while glycosidic carbons were
assigned from the HSQC spectrum
(Tables 1 and 2). Evaluation of spin-spin
couplings and chemical shifts allowed the
identification of three b-glucopyranosyl
(Glc), one a-rhamnopyranosyl (Rha),
one b-xylopyranosyl (Xyl), and one a-
arabinopyranosyl moieties (Ara). In the
HMBC spectrum, the anomeric proton
signals at dH 4.42 (Ara-1), 5.12 (Rha-1),
4.50 (Glc-1), 4.62 (Xyl-2), 4.31 (Glc-2),
and 4.36 (Glc-3) showed cross-peaks

202
Z. Yu et al.
Table 1. 13C NMR spectral data for the aglycone and sugar moieties of compounds 1 and 2
(125 MHz, in CD3OD).
No.
1
2
No.
1
2
Aglycone
1
2
3
4
5
6
7
8
Ara-1
39.8
27.2
89.8
40.2
57.4
19.0
36.7
38.2
54.0
37.9
22.3
30.3
37.9
54.2
37.0
111.2
54.2
19.2
16.8
69.3
29.5
45.2
70.1
126.2
136.5
25.6
18.2
28.3
17.0
66.7
39.8
27.2
89.8
40.1
57.4
19.0
36.7
38.2
54.0
37.8
22.3
30.3
37.9
54.2
37.0
111.2
54.2
19.2
16.8
69.3
29.5
45.2
70.1
126.2
136.5
25.6
18.2
28.3
17.0
66.6
1
2
3
4
5
104.6
75.8
82.2
68.6
63.8
104.6
75.7
82.1
68.7
63.7
Rha-1
1
2
3
4
101.7
72.1
72.0
73.8
70.4
18.1
101.5
71.9
72.1
73.4
70.5
18.2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5
6
Glc-1
1
2
3
103.4
82.6
77.6
71.4
76.5
69.8
104.2
75.1
78.2
71.6
77.1
69.4
4
5
6
Xyl-1
1
2
3
105.6
75.3
78.0
70.8
67.9
4
5
Glc-2
1
2
3
104.6
75.2
77.9
71.2
76.5
68.2
104.5
75.2
77.9
71.1
76.4
68.1
4
5
6
Glc-3
1
2
3
4
5
6
104.8
74.8
78.0
71.2
78.0
62.7
104.8
74.9
78.1
71.1
78.0
62.6
pyranosyl-(1 ! 6)-b-D-glucopyranosyl-
(1 ! 3)-[a-L-rhamnopyranosyl-(1 ! 2)]-
a-L-arabinopyranosyl jujubogenin, which
These compounds are potent inhibitors of
lipoxygenase.
3. Experimental
has been named jujuboside E.
The inhibitory activity of 1–5 against
3.1 General experimental procedures
lipoxygenase was determined using the
method described in Section 3 (Table 3).
Optical rotations were determined on a
JASCO P-1020 digital polarimeter (JASCO

Journal of Asian Natural Products Research
203

204
Z. Yu et al.
Table 3. Inhibitory effects of compounds 1–5
against lipoxygenase.
passed through a Diaion HP-20 column,
successively eluting with 20% EtOH, 70%
EtOH, and 90% EtOH. The 70% EtOH
eluate fraction (224 g) was subjected to
silica gel column chromatography and
eluted with CHCl3–MeOH–H2O (10:1:0,
5:1:0.1, 1:1:0.2) to afford fractions 1–4
(41, 37, 66, and 55 g, respectively).
Fraction 2 was subjected to C18 silica gel
column chromatography and eluted with
MeOH–H2O in a gradient of MeOH
(MeOH–H2O, 10:90 ! 100:0%), to give
subfractions 2a–2d. Subfraction 2c (1.6 g)
was isolated by HPLC (MeOH–H2O,
32:68, 2.0 ml/min; tube temperature
1208C, gas flow 2.5 l/min) to yield
compounds 1 (r.t. 16.3 min, 51 mg) and 4
(r.t. 20.6 min, 27 mg). Subfraction 2d
(2.5 g) was isolated by HPLC (MeOH–
H2O, 34:66, 2.0 ml/min; tube temperature
1208C, gas flow 2.5 l/min) to yield
compounds 2 (r.t. 17.9 min, 61 mg), 3
(r.t. 19.5 min, 31 mg), and 5 (r.t. 21.7 min,
24 mg).
Compound
IC50 (mM)
1
2
3
4
56.7
53.9
61.2
48.9
71.3
21.4
5
Baicalein
Corporation, Tokyo, Japan). IR spectra were
recorded on an Avater-360 spectrometer
(Thermo Scientific, Waltham, MA, USA).
NMR spectra were recorded on a Bruker
INOVA 500 spectrometer (Bruker, Wal-
tham, MA, USA) with tetramethylsilane as
internal standard. The HR-TOF-MS were
acquired using an Agilent 6210 mass
spectrometer (Agilent, Santa Clara, CA,
USA). GC was employed on an Agilent
6890N gas chromatograph (Agilent). Chro-
matography was carried out on silica gel
(200–300 mesh, Qingdao Haiyang Chemi-
calCo.,Ltd, Qingdao, China),DiaionHP-20
(Mitsubishi Chemical, Tokyo, Japan), and
C18 silica gel (150–200 mesh, Merck,
Darmstadt, Germany), respectively. HPLC
separation was carried out on an ODS
column (250 £ 10mm, i.d. 5 mm, YMC,
Kyoto, Japan) with an Alltech ES 2000
evaporative light scattering detector (Grace,
Crutis Bay, MD, USA).
3.3.1 Jujuboside D (1)
20
White amorphous power, ½aꢀD 246,
(c ¼ 0.5, MeOH); IR(KBr) vmax (cm21):
1
3421, 1724, 1072; for H and 13C NMR
spectral data, see Tables 1 and 2. HR-ESI-
MS: m/z 1391.6466 [M þ Na]þ (calcd for
C64H104O31Na, 1391.6459).
3.3.2 Jujuboside E (2)
3.2 Plant material
20
White amorphous power, ½aꢀD 252
The roots and rhizomes of Z. jujube were
collected in August 2010 in Chengdu city,
Sichuan Province of China. Identification
of the plant was done by one of the authors
(Z. Yu). A voucher specimen (ZJ 4403) is
maintained in our laboratory.
(c ¼ 0.5, MeOH); IR(KBr) vmax (cm21):
3421, 1724, 1072; for H and 13C NMR
1
spectral data, see Tables 1 and 2. HR-ESI-
MS: m/z 1259.6041 [M þ Na]þ (calcd for
C59H96O27Na, 1259.6037).
3.3 Extraction and isolation
3.4 Acid hydrolysis
The dried seeds (4 kg) of Z. jujube were
extracted with EtOH. The EtOH extract
was concentrated under reduced pressure,
and the viscous concentrate (491 g) was
Compound 1 or Compound 2 (5.0mg each)
was heated in 5 ml of 10% HCl–dioxane
(1:1) at 808C for 4 h. After the dioxane was
removed, the solution was extracted with

Journal of Asian Natural Products Research
205
EtOAc (3 ml £ 3) to yield the aglycone and
the sugar, respectively. The aqueous layer
was evaporated and dissolved in anhydrous
pyridine (100ml), 0.1 M L-cysteinemethyl
ester hydrochloride (200ml) was added, and
the resultant mixture was warmed at 608C
for 1 h. The trimethylsilylation reagent
(hexamethyldisilazane–trimethylchlorosi-
lane–pyridine, 2:1:10; Acros Organics,
Geel, Belgium) was added and warmed at
608C for 30 min. To the mixture, the
thiazolidine derivatives were analyzed by
GC for sugar identification [8]. The reten-
tion times of L-arabinose (6.42 min), L-
rhamnose (7.83 min), D-xylose (8.68 min),
and D-glucose (11.36min) were confirmed
by comparison with those of authentic
standards.
followed for 10 min. Test compounds and
the control were dissolved in 50% EtOH.
All the reactions were carried out in
triplicate. The IC50 values were then
calculated using the EZ-Fit Enzyme
Kinetics program (Perrella Scientific,
Inc., Amherst, MA, USA). Baicalein was
used as a positive control.
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3.5 Assay of lipoxygenase inhibition
Lipoxygenase-inhibiting activity was con-
veniently measured by slightly modifying
the spectrometric method developed by
Tappel [9]. Lipoxygenase type I-B and
linoleic acid were purchased from Sigma
Chemicals (Saint Louis, MO, USA). A
mixture of 160 ml of 0.1 mM sodium
phosphate buffer (pH 7.0), 10 ml of test
compound solution, and 20 ml of lipox-
ygenase solution was incubated for 5 min
at 258C. The reaction was then initiated by
the addition of linoleic acid 10 ml (sub-
strate) solution, with the formation of (9Z,
11E, 13S)-13-hydroperoxyoctadeca-9,11-
dienoate. The change in absorbance was
[9] A.L. Tappel, Lipoxidase, Methods of
Enzymology (Academic Press, New York,
1962) Vol. 5, p. 539.