C-glucosyl flavones from the seeds of ziziphus jujuba var. spinosa

Article abstract of DOI:10.1007/s10600-011-9936-y

A new C-glucosyl flavone, 6'-vanilloylspinosin (1), was isolated from the seeds of Ziziphus jujuba var. spinosa, together with three known C-glucosyl flavones, spinosin (2), 6'-feruloylspinosin (3), and isospinosin (4). The structure of 1 was elucidated by spectral methods (1D NMR, ESI-MS, HR-MS, 2D NMR).

Full text of DOI:10.1007/s10600-011-9936-y

Chemistry of Natural Compounds, Vol. 47, No. 3, July, 2011 [Russian original No. 3, May–June, 2011]
C-GLUCOSYL FLAVONES FROM THE SEEDS
OF Ziziphus jujuba var. spinosa
1
1
1
2
Yi Wu, Feng He, Qin Pan, Yao Shi,
UDC 547.972
1
1*
Zhida Min, and Jingyu Liang
A new C-glucosyl flavone, 6ꢀꢀꢀ-vanilloylspinosin (1), was isolated from the seeds of Ziziphus jujuba var.
spinosa, together with three known C-glucosyl flavones, spinosin (2), 6ꢀꢀꢀ-feruloylspinosin (3), and isospinosin
(4). The structure of 1 was elucidated by spectral methods (1D NMR, ESI-MS, HR-MS, 2D NMR).
Keywords: 6ꢀꢀꢀ-vanilloylspinosin, flavone, Ziziphus jujuba var. spinosa.
Ziziphus jujuba var. spinosa (Bunge) Huex. H. F., a thorny rhamnaceous plant, is widely distributed in north China.
Its dried fruits have been used in traditional Chinese medicine as an immune system stimulant and antitumor. The seeds have
sedative, demulcent, and hypnotic effects, as well as hypotensive. In recent years, new saponins [1, 2], flavonoids [3], and
alkaloids [4, 5] have been reported from this plant. In our present research, a new C-glucosyl flavone 1, together with three
known compounds, spinosin (2) [3], 6ꢀꢀꢀ-feruloylspinosin (3) [3], and isospinosin (4) [3], were isolated from a 95% aqueous
EtOH extract of the seeds by repeated chromatography. The structure of 1 was assigned as apigenin 7-methyl-ether-6-[2-O-[6-
O-(4-hydroxyl-3-methoxybenzoyl)-C-ꢁ-D-glucopyranosyl]-C-ꢁ-D-glucopyranoside by spectroscopic methods, named as
6ꢀꢀꢀ-vanilloylspinosin.
6''
5''
1
O
HO
6'' H CO
3
HO
OH
O
O
7
1''
O
5''
3
OH
4
OH
5
1''
3''
2'
HO
1
OH
O
H CO
3
O
HO 3''
6'''
HO
5'''
OH
7
O
6'''
RO
O
3
6'
O
5'''
4
OH
1'''
5
OH
1'''
OH
O
HO 3'''
OH
HO 3'''
1 - 3
4
OH
OCH
OCH
3
3
3''''
O
3''''
7''''
1''''
7''''
1''''
O
OH
OH
1: R =
2: R = H
3:R =
5''''
9''''
5''''
1) China Pharmaceutical University, Nanjing 210009, P. R. China, 2) R&D Center, Tianjin Zhongxin Pharmaceuticals,
Tianjin 300457, P. R. China, e-mail: jyliang_08@yahoo.com. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 330–
332, May–June, 2011. Original article submitted April 20, 2010.
0009-3130/11/4703-0369 02011 Springer Science+Business Media, Inc.
369

13
TABLE 1. C NMR Data of Compounds 1–4 (DMSO-d )
6
C atom
1
2
3
4
2
3
4
5
6
7
8
9
10
1ꢀ
163.9, 164.1 (s)
102.5, 102.6 (d)
181.6, 182.1 (s)
159.5, 160.7 (s)
108.5, 108.7 (s)
163.4, 165.1 (s)
89.7, 90.4 (d)
157.0, 157.1 (s)
103.9, 104.4 (s)
123.1, 123.3 (s)
128.3, 128.5 (d)
116.0, 117.0 (d)
162.3, 162.3 (s)
56.0, 56.3 (q)
70.7, 71.0 (d)
79.7, 80.6 (d)
78.5, 78.9 (d)
70.3, 70.4 (d)
81.8, 81.9 (d)
61.4, 61.5 (t)
163.6, 163.7
102.8, 103.0
181.8, 182.2
159.6, 160.4
108.6, 108.6
163.8, 165.0
90.6, 90.9
156.8, 157.0
104.0, 104.4
121.0, 121.3
128.4, 128.5
115.6, 115.9
160.8, 161.2
55.9, 56.5
70.5, 71.0
80.4, 81.0
77.9, 78.2
70.2, 70.4
81.4, 81.7
61.4, 61.9
105.0, 105.2
74.5, 74.7
76.2, 76.3
163.8, 163.9
102.6, 102.9
182.2, 181.7
160.1, 160.9
108.7, 108.9
164.5, 164.9
90.1, 90.6
157.7, 158.1
104.3, 104.4
122.0, 121.4
128.6, 128.7
116.1, 116.2
161.4, 164.4
56.3, 56.4
71.1, 71.2
79.9, 80.1
78.6, 78.9
70.3, 70.5
81.9, 81.9
61.0, 61.3
104.6, 104.9
74.4, 74.5
76.0, 76.4
163.5
102.7
182.3
161.2
95.1
164.3
104.9
155.3
104.3
21.8
129.0
115.9
161.6
56.1
71.4
80.9
78.2
70.1
81.8
60.9
104.9
74.5
76.1
ꢀ ꢀ
2 ,6
ꢀ ꢀ
3 ,5
4ꢀ
OCH3
Glc-1ꢀꢀ
2ꢀꢀ
3ꢀꢀ
ꢀꢀ
4
ꢀꢀ
5
6ꢀꢀ
Glc-1ꢀꢀꢀ
2ꢀꢀꢀ
3ꢀꢀꢀ
4ꢀꢀꢀ
104.9, 105.5 (d)
74.4, 74.4 (d)
76.2, 76.2 (d)
68.9, 69.0 (d)
73.2, 73.3 (d)
62.1, 62.6 (t)
69.1, 69.4
76.3, 76.3
60.1, 60.6
68.7, 68.9
73.5, 73.8
62.4, 62.9
Feruloyl
69.3
76.2
60.3
ꢀꢀꢀ
ꢀꢀꢀ
5
6
Vanilloyl
119.8, 119.9 (s)
112.2, 112.4 (d)
147.2, 147.2 (s)
151.9, 152.1 (s)
114.9, 115.0 (d)
123.1, 123.3 (d)
165.0, 165.1 (s)
125.1, 125.5
111.8, 112.0
147.6, 147.8
149.9, 149.9
115.1, 115.4
123.4, 123.6
144.4, 144.9
114.0, 114.7
165.3, 165.4
55.5, 55.9
1ꢀꢀꢀꢀ
2ꢀꢀꢀꢀ
3ꢀꢀꢀꢀ
4ꢀꢀꢀꢀ
5ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
6
7ꢀꢀꢀꢀ
8ꢀꢀꢀꢀ
9ꢀꢀꢀꢀ
OCH3
55.1, 55.3 (q)
The UV spectrum (MeOH) absorptions maximum of 1 at 276 and 335 nm, were characteristic of flavonoids.
–1
–1
The IR spectrum implied the existence of hydroxyl (3340 cm ) and ester carbonyl (1725 and 1278 cm ) groups, as well as
–1
a benzene ring (1608, 1585, 1502, and 1483 cm ). The acid hydrolysis (2 M HCl) of compound 1 afforded swertisin,
D-glucose, and vanillic acid in the mixture, which were determined by gas-liquid chromatography [6]. As was introduced [3],
1
13
the multiplicity caused by rotational isomerism in 1 was also observed in its NMR spectra. The H and C NMR spectra
showed the presence of an apigenin 7-methyl ether moiety and two sugar residues which were in good agreement with those
of 6ꢀꢀꢀ-feruloylspinosin (3) [3]. This was further supported by the HMBC correlation peaks observed (Fig. 1). The correlations
between the anomeric proton of inner glucose at ꢂ 4.64, 4.65 (d, J = 10.0 Hz) (H-1ꢀꢀ) and the carbon at ꢂ 108.5, 108.7 (C-6)
showed the inner glucose group should be attached to C-6. The position of the outer glucose group was determined by the
correlations between the anomeric proton of outer glucose at ꢂ 4.10, 4.24 (d, J = 8.0 Hz) (H-1ꢀꢀꢀ) and the carbon at ꢂ 79.7, 80.6
(C-2ꢀꢀ). Then the correlations between H-6ꢀꢀꢀ at ꢂ 3.94, 3.95; 3.89, 3.99 (m) and the carbon at ꢂ 165.0, 165.1 (C-7ꢀꢀꢀꢀ) suggested
the acylation of the vanilloyl moiety and 6ꢀꢀꢀ-OH. Therefore, 1 was identified as apigenin 7-methyl-ether-6-[2-O-[6-O-(4-
hydroxy-3-methoxybenzoyl)]-ꢁ-D-glucopyranosyl]-C-ꢁ-D-glucopyranoside unambiguously, and named as 6ꢀꢀꢀ-vanilloylspinosin.
370

1
TABLE 2. H NMR Data of Compounds 1–4 (DMSO-d , J/Hz)
6
C atom
1
2
3
4
3
6
8
6.40, 6.61 (s)
6.83,6.84 (s)
6.83, 6.85 (s)
6.47 (s)
6.77 (s)
6.50, 6.60 (s)
7.77 (d, J = 8.8)
6.86 (d, J = 8.8)
6.67, 6.80 (s)
7.97 (d, J = 8.7)
6.95 (d, J = 8.7)
3.86 (s)
6.67, 6.77 (s)
7.81 (d, J = 7.9)
6.89 (d, J = 7.9)
3.86 (s)
8.03 (d, J = 8.4)
6.91 (d, J = 8.4)
3.86 (s)
2 ,6
ꢀ ꢀ
3 ,5
ꢀ ꢀ
3.77, 3.83 (s)
OCH3
4.64, 4.65 (d, J = 10.0)
4.22, 4.45 (m)
4.67, 4.69 (d, J = 9.8)
4.67, 4.70 (t, J = 9.3)
4.83 (d, J = 10.0)
Glc-1
2ꢀꢀ
ꢀꢀ
3.41, 3.41 (m)
3.18, 3.18 (m)
3.18; 3.19 (m)
3
4
5
6
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
3.36, 3.67; 3.34, 3.65 (m)
4.10, 4.24 (d, J = 8.0)
2.87, 2.91 (m)
4.15, 4.17 (d, J = 8.5)
4.23, 4.27 (t, J = 7.9)
4.04 (t, J = 8.8)
Glc-1ꢀꢀꢀ
2
ꢀꢀꢀ
3
ꢀꢀꢀ
4
ꢀꢀꢀ
5
ꢀꢀꢀ
6
ꢀꢀꢀ
3.07, 3.11 (m)
3.05, 3.11 (m)
2.95, 3.18 (m)
3.94, 3.95; 3.89, 3.99 (m)
Vanilloyl
Feruloyl
7.00, 7.10 (d, J = 1.6)
6.64, 6.73 (d, J = 8.0)
7.06, 7.13 (dd, J = 8.0, 1.6)
7.04, 7.17 (d, J = 2.0)
6.67 (d, J = 8.1)
6.72 (dd, J = 8.1, 2.0)
7.06, 7.18 (d, J = 15.7)
6.15, 6.23 (d, J = 15.7)
3.55, 3.65 (s)
2
5
6
7ꢀꢀꢀꢀ
8
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
3.56, 3.65 (s)
OCH3
H CO
3
O
O
HO
OH
O
OH
OH
H CO
3
HO
O
O
O
HO
OH
O
HO
1
OH
Fig. 1. Key HMBC correlations of compound 1.
EXPERIMENTAL
General Experimental Procedures. Melting points were determined by an X apparatus. UV spectra were taken in
4
1
13
MeOH by a UV-2401 PC spectrometer. H NMR and C NMR, HMQC, and HMBC spectra were recorded with a Bruker AV
400 instrument, with TMS as an internal standard. Mass spectra were obtained on an MS Agilent 1100 Series LC/MS Trap
mass spectrometer (ESI-MS) and a Micro Q-TOF MS (HR-ESI-MS), respectively. Silica gel (200~300 mesh) was purchased
from Qingdao Ocean Chemical Group Co. in China. Polyamide was bought from Taizhou, Zhejiang. Diaion HP-20 (Mitsubishi
Chemical), Sephadex LH-20 (Pharmacia), and RP-18 (ODS-A, 50 m, YMC) were used. TLC was performed on HPTLC plates
(Alltech), RP-18 F
precoated plate (Merck), and polyamide plate (from Tianjin).
254S
Plant Material. The seeds of Ziziphus jujuba var. spinosa (11.0 kg) were collected in November 2009 in Tianjin City
of China and were identified by Prof. Minjian Qin of the Department of Natural Resource, China Pharmaceutical University.
A voucher specimen has been deposited in China Pharmaceutical University (specimen No. Z-007).
371

Extraction and Isolation. The seeds of Ziziphus jujuba var. spinosa were extracted with 95% aqueous EtOH (3 ꢃ 20 L).
After removal of the solvent under reduced pressure, the extract was dissolved in water and partitioned with petroleum, ethyl
acetate, and n-BuOH, successively. The n-BuOH extract (252 g) was fractioned by HP-20 (2.5 kg) eluted with 20%, 50%, and
80% aqueous EtOH to give fractions A (20%), B (50%), and C (80%), respectively. Fractions B (30 g) was further fractionated
by a silica gel column (250 g) eluted with gradient CHCl –MeOH (10:1ꢄ8:2ꢄ6:4ꢄ2:8) to give four subfractions (B 1-4).
3
Subfraction B2 was subjected to column chromatography on polyamide eluted with gradient CHCl –MeOH to give fractions
3
B 2-1 to B 2-3. Fraction B 2-2 was further separated on a RP-18 column using gradient aqueous MeOH, followed by a
Sephadex LH-20 column eluted with aqueous MeOH to afford 1 (4 mg), 2 (85 mg), 3 (230 mg), and 4 (18 mg), respectively.
Hydrolysis and GC Chromatography. Two mg of compound 1 was refluxed with 5 mL 2M HCl for 2 h at 100ꢅC.
The reaction mixture was extracted with ethyl acetate. The residue was evaporated to dryness. The gas-liquid chromatography
experiment was finished using the reported method [6].
Compound 1 (6ꢀꢀꢀ-vanilloylspinosin), yellow powder, mp 225–228ꢅC. UV spectrum (MeOH,
, nm): 276, 335;
max
(NaOMe,
, nm): 276, 392; (NaOAc,
, nm): 276, 393; (AlCl ,
, nm): 284, 302, 351, 385; (AlCl -HCl,
, nm):
max
max
3
max
3
max
–1
284, 302, 351, 385. IR spectrum (KBr, ꢆ , cm ): 3340, 2928, 1725, 1608, 1585, 1502, 1483, 1380, 1345, 1278, 1200, 1175,
max
–
+
1093, 1078, 1021. ESI-MS m/z 757 [M – H] , HR-ESI-MS m/z 781.1953, calcd for C H O Na [M + Na] 781.1956. For
36 38 18
1
13
H NMR (400 MHz, DMSO-d ) (300 K) spectral data, see Table 2, and for C NMR (100 MHz, DMSO-d ) (300 K) spectral
6
6
data, see Table 1.
–
Compound 2 (spinosin), yellow powder, mp 237–240ꢅC. UV (MeOH,
, nm): 270, 334; ESI-MS m/z 607 [M – H] .
max
1
13
For H NMR (400 MHz, DMSO-d ) (300 K) spectral data, see Table 2, and for C NMR (100 MHz, DMSO-d ) (300 K)
6
6
spectral data, see Table 1.
Compound 3 (6ꢀꢀꢀ-feruloylspinosin), yellow powder, mp 224ꢅC (dec.). UV (MeOH,
, nm): 273, 328; ESI-MS m/z
max
13
–
1
783 [M – H] . For H NMR (400 MHz, DMSO-d ) (300 K) spectral data, see Table 2, and for C NMR (100 MHz, DMSO-d )
6
6
(300 K) spectral data, see Table 1.
Compound 4 (isospinosin), yellow powder, mp 215–216ꢅC. UV (MeOH,
, nm): 268, 330. ESI-MS m/z 607
max
–
1
13
[M – H] . For H NMR (400 MHz, DMSO-d ) (300 K) spectral data, see Table 2, and for C NMR (100 MHz, DMSO-d )
6
6
1
(300 K) spectral data, see Table 1. For comparison with the literature [3], we modified its H NMR spectral data.
ACKNOWLEDGMENT
The authors are grateful for the support of Tianjin Zhongxin Pharmaceuticals (China).
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