Complete assignment of 1H and 13C signals of a triterpene from ...

Complete assignment of 1 H and 13 C signals of a triterpene from Agrimonia pilosa Ledeb. by 2D NMR / Asian Journal of
Traditional Medicines, 2009, 4 (4)
Regular Articles
Complete assignment of 1 H and 13 C signals of a
triterpene from Agrimonia pilosa Ledeb. by 2D NMR
Ya Pan, Hongxia Liu, Liqin Ding, Yulei Zhuang, Feng Qiu *
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang
110016, China
Abstract
A known triterpene obtained from Agrimonia pilosa Ledeb. was identified as 1β, 2β, 3β, 19α-tetrahydroxyurs-12-en-28-oic acid. This
paper reports the complete assignment of the structure of this compound by the method of 2D NMR for the first time.
Key words: complete assignment; 2D-NMR; 1β, 2β, 3β, 19α-tetrahydroxyurs-12-en-28-oic acid
Introduction
Agrimonia pilosa Ledeb., a species of Agrimony
and widely distributed in China, is used as an antihaemorrhagic
anthelmintic and anti-inflammatory
agent in traditional Chinese medicine [1] . The
triterpenoids from this plant have been described
in detail and a known triterpene, 1β, 2β, 3β,
19α-tetrahydroxyurs-12-en-28-oic acid, was obtained
as a methylester from this plant [2] . In this paper, we
report the complete assignment of 1 H and 13 C signals
of 1β, 2β, 3β, 19α-tetrahydroxyurs-12-en-28-oic acid
on the basis of HSQC, HMBC and NOESY spectral
data for the first time.
Experimental
General experimental procedures
1D and 2D NMR spectra (Fig. 1-5) were measured
on a Bruker ARX-300 and a Bruker ARX-600
spectrometer with TMS as the internal standard.
ESI-MS were obtained on an Bruker esquire 2000
spectrometer. Silica gel (200-300 μ) used for CC and
silica GF 254
(10-40 μ) for TLC were supplied by the
Qingdao Marine Chemical Factory, China. Sephadex
LH-20 was obtained from Pharmacia Corporation.
Plant material
The material was obtained from the Northeast
Drug Store of Shenyang, and it was identified by
Prof. Qishi Sun. A voucher was deposited in the
Department of Traditional Chinese Medicine of
Shenyang Pharmaceutical University.
Extraction and isolation
* Author to whom correspondence should be addressed. Address:
School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang 110016, China; Tel:
+86-24-23986463; Email: fengqiu2000@tom.com
Received: 2008-05-03 Accepted: 2009-01-14
The dried material was extracted twice with 70 %
EtOH under reflux (85 °C). The EtOH extract was
concentrated and suspended in water. This suspension
was partitioned successively with cyclohexane, EtOAc
and n-BuOH. The EtOAc soluble fraction (108 g) was
147

Complete assignment
Complete
of 1 H
assignment
and 13 C signals
of
of
a triterpene
triterpene from
from
Agrimonia
Agrimonia
pilosa
pilosa
Ledeb.
Ledeb.
by 2D NMR / Asian Journal of
Traditional Medicines, 2009, 4 (4)
Figure Legends
1.00
1.00
0.95
0.97
1.04
1.11
1.04
1.04
1.07
5.26
3.07
3.26
3.10
5.45
5.53
6.16
3.12
0.97
8.73
7.58
7.22
5.76
4.49
4.48
4.48
3.62
3.60
3.59
3.46
3.46
3.25
3.22
3.18
3.18
3.16
3.16
3.14
3.13
3.09
2.69
2.67
2.67
2.66
2.64
2.41
2.39
2.39
2.37
2.36
2.20
2.19
2.18
2.17
2.16
2.15
2.12
2.10
2.09
1.80
1.61
9 8 7 6 5 4 3 2 1 0 ppm
Fig.1
1 H NMR spectrum of compound 1
Fig. 1. 1 H NMR spectrum of compound 1
Complete assignment of a triterpene from Agrimonia pilosa Ledeb.
181.13
150.65
139.29
136.22
130.14
124.22
81.17
77.83
76.64
73.13
55.03
54.09
49.54
48.79
44.15
42.79
42.69
41.51
39.39
38.94
34.30
30.77
29.79
28.29
27.55
27.40
26.94
25.31
18.91
18.36
18.05
5
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
Fig.2
13 C NMR spectrum of compound 1
Fig. 2. 13 C NMR spectrum of compound 1
subjected to silica gel chromatograph and eluted with
CHCl 3
-MeOH to give 9 fractions. Fraction 4 (20 g)
was isolated by repeated column chromatography using
sephadex LH-20 (MeOH) to give compound 1.
Results and discussion
Compound 1 was obtained ppm as an amorphous
powder from MeOH and gave a positive reaction to
20
the Liebermann-Burchard test. Its molecular formula
40
60
148
80
100

Complete assignment of 1 H and 13 C signals of a triterpene from Agrimonia pilosa Ledeb. by 2D NMR / Asian Journal of
Traditional Medicines, 2009, 4 (4)
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
Fig.2
13 C NMR spectrum of compound 1
ppm
20
40
60
80
100
120
140
160
180
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm
Fig.3 Fig. 3. HSQC HSQC spectrum of of compound 11
Complete assignment of a triterpene from Agrimonia pilosa Ledeb.
6
ppm
0
20
40
60
80
100
120
140
160
180
11 10 9 8 7 6 5 4 3 2 1 0 ppm
Fig.4 Fig. 4. HMBC HMBC spectrum of compound 11
was determined to be C 30
H 48
O 6
by the methods of ESI-
MS (m/z 503 [M-H]ˉ) and NMR spectral data. The 1 H
NMR spectrum (Fig. 1) of compound 1 showed seven
methyl proton signals at δ 1.25, 1.27, 1.39, 1.47, 1.61,
1.80 (s, each 3H) and δ 1.13 (d, J = 6.6 Hz, H-30), and
an olefinic proton signal at δ 5.75 (br.s, H-12), all of
which suggested 1 was a ursolic acid derivate. 1β, 2β,
3β OH substitution of this skeleton was evident from
149

ppm
1
Complete assignment of 1 H and 13 C signals of a triterpene from Agrimonia 2 pilosa Ledeb. by 2D NMR / Asian Journal of
Traditional Medicines, 2009, 4 (4)
Complete assignment of a triterpene from Agrimonia pilosa Ledeb.
3
4
5
ppm
1
6
2
3
7
4
8
5
6
9
9 8 7 6 5 4 3 2 1 0 ppm
Fig.5 NOESY spectrum of compound 1
9
9 8 7 6 5 4 3 2 1 0 ppm
Fig.5 NOESY spectrum of compound 1
Fig. 5. NOESY spectrum of compound 1
7
8
HO
HO
OH
HO
HO
HO
OH
COOH
HO
Fig.6 The structure and key correlations in the HMBC spectrum of compound 1
Fig. 7. Key NOE correlations in the NOESY spectrum of
compound 1
the chemical shift and the J value of three methine
protons signals at δ 3.59 (d, J = 4.0 Hz, H-1), 4.48
(t, J =3.9 Hz, H-2), 3.46 (d, J = 3.7 Hz, H-3). The
13 C NMR COOH spectrum (Fig. 2) of compound 1 showed
two olefinic carbon signals at δ 130.1 (C-12), 139.3
(C-13), a carbonyl group signal at δ 181.1 (C-28), and
four oxygenated methine signals at δ 81.2, 76.7, 77.8,
73.1. Comparison of the 1D NMR data of compound
1 with that of pomolic acid revealed that the structure
8
of compound 1 was similar to that of pomolic acid [3] .
Fig.6Fig. The 6. structure The structure and key correlations and key correlations in the HMBC in spectrum the HMBC of compound 1
Above all, the conclusions indicated that compound 1
spectrum of compound 1
Complete assignment of a triterpene from Agrimonia pilosa Ledeb. is 1β, 2β, 3β, 19α-tetrahydroxyurs-12-en-28-oic acid.
In the HMBC spectrum (Fig. 4 and Fig. 6), a
correlation between the methine proton signal at δ
8
3.59 (H-1) and the carbon signals at δ 49.5 (C-9), 44.1
HO
H
(C-10), 12.8 (C-25), a correlation between the methine
H
H
proton signal at δ 4.48 (H-2) and carbon signals at δ
HOH
HO
COOH 77.8 (C-3), 39.4 (C-4), 44.1 (C-10), and a correlation
H
between the methine proton signal at δ 3.46 (H-3) and
H
H
HO
H
the carbon signals at δ 39.4 (C-4), 20.7 (C-23), 18.3
H H
(C-24) were observed, the assignment of proton and
carbon signals of A ring were clear. In the NOESY
spectrum (Fig. 5 and Fig. 7), the proton signals
assignment of H-1, 2, 3 was further confirmed by the
Fig.7 Key NOE correlations in the NOESY spectrum of compound NOE 1correlations between proton signals at δ 3.59
150

Complete assignment of 1 H and 13 C signals of a triterpene from Agrimonia pilosa Ledeb. by 2D NMR / Asian Journal of
Traditional Medicines, 2009, 4 (4)
Table 1. 1 H and 13 C NMR data of compound 1 (pyridine-d 5
)
Position δ C
δ H
1 81.2 3.59 (1H, d, J=4.0 Hz)
2 76.7 4.48 (1H, t, J=3.9 Hz)
3 77.8 3.46 (1H, d, J=3.7 Hz)
4 39.4
5 54.1 0.95 (1H, br.d, J=6.9 Hz)
6 18.9 1.71 (2H, m)
7 34.3 1.71 (1H, m) (α)
8 41.5
1.50 (1H, m) (β)
9 49.5 2.19 (1H, dd, J=10.2 Hz, 3.6 Hz)
10 44.1
11 28.3 3.24 (1H, td, J=13.3 Hz, 4.2 Hz ) (α)
12 130.1 5.75 (1H, br.s)
13 139.3
14 42.7
15 29.8 1.35 (1H, m) (α)
2.66 (1H, ddd, J=13.3, 10.8 Hz, 3.3 Hz)
(β)
2.38 (1H, dt, J=13.6 Hz, 4.4 Hz) (β)
16 26.9 3.16 (1H, dt, J=13.2 Hz, 4.3 Hz) (α)
17 48.8
2.10 (1H, m ) (β)
18 55 3.09 (1H, br.s)
19 73.1
19-OH
4.97 (1H, brs)
20 42.8 1.52 (1H, m)
21 27.4 1.37 (1H, m) (α)
2.13 (1H, m) (β)
22 38.9 2.18 (1H, m ) (α)
2.12 (1H, m) (β)
23 30.7 1.27 (3H, s)
24 18.3 1.39 (3H, s)
25 12.8 1.61 (3H, s)
26 18 1.25 (3H, s)
27 25.3 1.80 (3H, s)
28 181.1
29 27.5 1.47 (3H, s)
30 17.2 1.13 (3H, d, J=6.6 Hz)
(H-1) and δ 4.48 (H-2), 3.46 (H-3), 0.95 (H-5), 2.19
(H-9), 3.24 (H-11α), the NOE correlation between
proton signals at δ 4.48 (H-2) and δ 3.59 (H-1), 3.46
(H-3), and the NOE correlation between proton
signals at δ 3.46 (H-3) and δ 3.59 (H-1), 4.48 (H-2),
0.95 (H-5), 1.27 (H-23). Moreover, a correlation
between the proton signal at δ 4.97 (19-OH) and the
carbons signals at δ 27.5 (C-29), 55.0 (C-18), 73.1
(C-19) was observed in the HMBC spectrum,and
in the NOESY spectrum there was a NOE correlation
between proton signals at δ 4.97 (19-OH) and δ 1.80
(H-27), 1.47 (H-29), 1.13 (H-30) which confirmed
the α orientation of 19-OH. The full assignment of
proton and carbon signals (Table 1) was based on
the analysis of 1D and 2D NMR (HSQC, HMBC,
NOESY) spectral data.
References
[1] The State Pharmacopeia Commission of P.R. of China:
Pharmacopoeia of the People’s Republic of China.
Beijing: Publishing House of Chemical Industry, 2005,
Vol. I: 67.
[2] Kouno I, Baba N, Ohni Y, Kawano N. Triterpenoids from
Agrimonia pilosa. Phytochemistry, 1988, 27(1): 297-9.
[3] Ju JH, Zhou L, Lin G, Liu D, Wang LW, Yang JS. Studies
on constitunts of triterpene acid from Eriobotrya japonica
and anti-inflammatory and antitussive effects. Chin Pharm
J, 2003, 38(10): 752-7.
151