A study of the chemical constituents of the leaves of Crataegus ...

A study of the chemical constituents of the leaves of Crataegus pinnatifida
/ Asian Journal of Traditional Medicines, 2008, 3 ( 2 )
Notes
A study of the chemical constituents of the leaves of
Crataegus pinnatifida
Jia Chen, Shaojiang Song * , Jun He, Suixu Xu
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
Abstract
A new biphenyl glucoside, Shanyenoside A (5, 4'-dimethoxy-biphenyl- 4-ol-3-O-β-D–glucoside), together with five known compounds,
ursolic acid, vitexin, 2"-O-rhamnosyl vitexin, quercetin and β-sitosterol, were isolated from the Chinese Hawthorn Leaf. The
structure of the new biphenyl glucoside was assigned by 2D-NMR spectral data.
Key words: structure identification; NMR; C. pinnatifida Bge.; biphenyl glucoside
Introduction
The Chinese Hawthorn Leaf is the leaf of
Crataegus pinnatifida Bge., and its flavones and
triterpenes are known for their beneficial effects in
increasing cardiac contractility and improving cardiac
efficiency, for preventing the leakage of intracellular
enzymes following ischemic injury and for protecting
against arrhythmia [1, 2] .
A new biphenyl glucoside compound, named as
Shanyenoside A (5, 4'-dimethoxy-biphenyl-4-ol-3-Oβ-D-glucoside,
1), has been isolated from the Chinese
Hawthorn Leaf. In addition to the new compound,
five known compounds, ursolic acid (2), vitexin
(3), 2″-O-rhamnosyl vitexin (4), quercetin (5) and
β-sitosterol (6), were also isolated. In this article we
will describe the isolation and structural elucidation of
these six compounds.
* Author to whom correspondence should be addressed. Address:
School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, 103 Wunhua Road , Shenyang 110016,
China; Tel.: +86-24-23986510; E-mail: songsj99@yahoo.com.cn
Received: 2007-06-26 Accepted: 2007-12-09
Materials and methods
General
1
H NMR, 13 C NMR and also HMQC and HMBC
experiments were carried out using a Bruker ARX-600
spectrometer at 600 MHz, with C 5
D 5
N as a solvent
and TMS as an internal standard. The low resolution
ESI-MS was obtained on a spectrometer, using a
Bruker Daltonics Inc. APEX II FT-ICRMS instrument.
Adsorption flash chromatography and column
chromatography were performed with Silica gel 60
(Qing Dao Haiyang Chemical Co. Ltd, 200-300 mesh)
and polyamide (Zhe Jiang Plastics Plant, 200-300
mesh).
Plant material
The leaves of Crataegus pinnatifida Bge. were
bought from the Liaoning Province Medical Material
Corporation, and were identified by Professor Jincai
Lu of Shenyang Pharmaceutical University. Samples
were kept in the Department of Natural Products,
Shenyang Pharmaceutical University.
Extraction and isolation
80

A study of the chemical constituents of the leaves of Crataegus pinnatifida
/ Asian Journal of Traditional Medicines, 2008, 3 ( 2 )
The dry powdered sample of the leaves (5 kg)
was extracted 3-times for 2-hour periods with 80 %
alcohol-water at 70 ºC and concentrated under reduced
pressure to obtain the crude extract which was then
treated with macroporous resin and the portion eluted
with 55 % alcohol-water was collected. The eluted
portion was concentrated under reduced pressure, and
the extract (100 g) was a brownish red powder. The
extract was gradient eluted with CHCl 3
-MeOH on a
silica gel column. Several fractions (50 ml each) were
collected, and then analyzed by TLC and combined.
The CHCl 3
-MeOH (10:1) eluates were subjected to
repeated silica gel column chromatography (petroleum
ether- acetoacetate; 1:1) to afford β-sitosterol (8.3
mg) and ursolic acid (5.6 mg). The CHCl 3
-MeOH
(8:1) eluates were purified by ODS reverse phase
column chromatography (H 2
O-MeOH; 1:1) to obtain5,
4'-dimethoxy-biphenyl-4-ol-3-O-β-D–glucoside
(25.2 mg). Also, the CHCl 3
-MeOH (5:1) eluates
were subjected to repeated column chromatography
followed by polyamide column chromatography (H 2
O-
MeOH; 10:1-1:1), then further purified by Sephadex
LH-20 column chromatography (H 2
O-MeOH; 1:1) to
obtain the other two compounds, vitexin (19.8 mg)
and 2″-O-rhamnosyl vitexin (10.6 mg).
Results and discussion
Six compounds were isolated from the 80 % alcohol
extract. These were: 5,4'-dimethoxy- biphenyl-4-
ol-3-O-β-D-glucoside (1), ursolic acid (2), vitexin
(3), 2″-O-rhamnosyl vitexin (4), quercetin (5)
and β-sitosterol (6). The structures of the known
compounds were identified by comparing their
properties (m.p., MS, IR, 1 H NMR and 13 C NMR) with
the reported values in the literature or by comparison
with authentic samples. The structure of the new
compound was deduced as follows.
Compound 1 was obtained in the usual manner
as described in the experimental section. The
structure of compound 1 was elucidated by ESI mass
spectrometry, 1 H and 13 C NMR spectra and 2D-NMR
techniques (HMQC and HMBC).
Compound 1, white power, ESI-MS showed an
ion peak at m/z 409.0 [M+H] + ,431.0 [M+Na] + ,
838.7 [2M+Na] + , corresponding to the formula
C 20
H 24
O 9
. Based on 1 H NMR spectra (Table 1),
compound 1 had two methoxyl groups, two benzene
rings and a sugar moiety. The proton singlets at 3.85
and 3.67 due to the methoxyl group were attached to
the benzene ring. The two doublets at δ 7.55 (d, J =
1.75 Hz) and δ 7.13 (d, J = 1.75 Hz) were assigned to
H
OH
HO
4
6
5
H
O
HO
3
H
2
O
OH 1 3 2
2 3
H
H
HO
1 1
4
4
OCH 3
H 3 CO
5 6
6
5
Fig. 1. 5, 4'-dimethoxy-biphenyl-4-ol-3-O-β-D- glucoside
81

A study of the chemical constituents of the leaves of Crataegus pinnatifida
/ Asian Journal of Traditional Medicines, 2008, 3 ( 2 )
H-2 and H-6 of a 1, 3, 4, 5-tetra-substitued benzene
ring, respectively. The two doublets at δ 7.66 (2H,
d, J = 8.67 Hz) and δ 7.02 (2H, d, J = 8.67 Hz) were
assigned to H-2′, H-6′and H-3′, H-5′ of the other
aromatic ring. An anomeric proton at 5.61 (d, J=6.0
Hz), indicating the anomeric configuration of the sugar
moiety was determined to be β- on the basis of the J H-H
value. The 13 C NMR spectrum of compound 1 showed
signals of two methoxyl group carbons at 56.46 and
55.24, and an anomeric carbon of a sugar at 104.86.
The 1 H NMR and 13 C NMR spectral data (Table 1)
showed that compound 1 was a biphenyl with one
sugar moiety. This sugar moiety was identified as
glucose by co-TLC with authentic samples after acid
hydrolysis. The sugar linkage was determined on the
basis of the HMBC spectrum. The HMBC correlation
was observed between the proton signal at 5.61 (Glc-
H -1",d, J=6.0 Hz) and the carbon signal at 147.59
(C-3) of the aglycone moiety. In the HMBC spectra,
the proton at δ 7.02 showed correlations with δ 134.20
(C-1′) and δ 159.25 (C-4′), while the proton at δ 7.66
showed correlations with δ 131.74 (C-1) and δ 159.25
(C-4′). The proton at δ 7.55 showed correlations with
δ 106.80 (C-6), δ 138.60 (C-4), δ 134.20 (C-1′) and
δ 147.59 (C-3), while the proton at δ 7.13 showed
correlations with δ 110.93 (C-2), δ 134.20 (C-1′), δ
138.60 (C-4) and δ 149.90 (C-5), indicating that the
two benzene rings were linked at positions 1 and 1′.
Comparing the literature [4] and the HMQC and HMBC
spectral (Fig. 2) data, the structure of compound 1 was
confirmed as 5, 4'-dimethoxy-biphenyl-4-ol-3-O-β-Dglucoside.
Table 1. 1 H and 13 C NMR (300HMz, pyridine-d 5
) spectra data of compound 1
No. 1
H NMR (in pyr-d 5
)
13
C NMR (in pyr-d 5
)
1 131.74
2 7.55(d, J=1.75 Hz) 110.93
3 147.59
4 138.60
5 149.90
6 7.13(d, J=1.75 Hz) 106.80
1′ 134.20
2′ 7.66(d, J=8.67 Hz) 128.17
3′ 7.02(d, J=8.67 Hz) 114.65
4′ 159.25
5′ 7.02(d, J=8.67 Hz) 114.65
6′ 7.66(d, J=8.67 Hz) 128.17
5 -OCH 3
3.85(s) 56.46
4′-OCH 3
3.67(s) 55.24
Glc -1" 5.61(d, J=6.0 Hz) 104.86
2" 4.37 75.15
3" 4.08 79.19
4" 4.32 71.30
5" 4.41 78.52
6" 4.55 62.36
82

A study of the chemical constituents of the leaves of Crataegus pinnatifida
/ Asian Journal of Traditional Medicines, 2008, 3 ( 2 )
H
HO 4''
HO
OH
6''
H
5''
H 2''
3''
H
O
OH 1''
H
HO
O
4
3
2
H
1
H
1'
2' 3'
H
4'
OCH 3
H 3 CO
5 6
H
H
6'
5'
H
show HMBC correlation
Fig. 2. the key HMBC correlation of compound 1
References
[1] Handel AM. Crataegus. Toxikologie and pharmakologie
teil II: pharmkodynamik. Plant Med, 1981: 43(3): 209-39.
[2] Kashnikova MV. Sheichenko VI acetylytexin - a new
flavonoid from the flowers of crataegus sanguines. Khim
Prir Soedin, 1984: 1: 108-9.
[3] Chen J, Song SJ. Research progress in Crataegus
pinnatifida Bge. Research and Information on Traditional
Chinese Medicine, 2005: 7: 20-23, 26.
[4] Teanette GG. Atlas of Spectra Data and physical Constance
for organic compounds, 2nd, 1975: 284.
83