Terpene glycosides from the roots of sanguisorba officinalis l. and their hemostatic activities

Article abstract of DOI:10.3390/molecules17077629

Guided by a hemostasis bioassay, seven terpene glycosides were isolated from the roots of Sanguisorba officinalis L. by silica gel column chromatography and preparative HPLC. On the grounds of chemical and spectroscopic methods, their structures were identified as citronellol-1-O-α-L-arabinofuranosyl- (1→6)-β-D-glucopyranoside (1), geraniol-1-O-α-L- arabinofuranosyl-(1→6)-β-D-glucopyranoside (2), geraniol-1-O-α- Larabinopyranosyl-( 1→6)-β-D-glucopyranoside (3), 3β-[(α-L- arabinopyranosyl)oxy]-19α- hydroxyolean-12-en-28-oic acid 28-β-D-glucopyranoside (4), 3β-[(α-L-arabinopyranosyl)- oxy]-19α-hydroxyurs-12-en-28-oic acid 28-β-D-glucopyranoside (ziyu-glycoside I, 5), 3β,19α-hydroxyolean-12-en-28-oic acid 28-β-D-glucopyranoside (6) and 3β,19α-dihydroxyurs- 12-en-28-oic acid 28-β-D-glucopyranoside (7). Compound 1 is a new mono-terpene glycoside and compounds 2, 3 and 5 were isolated from the Sanguisorba genus for the first time. Compounds 1-7 were assayed for their hemostatic activities with a Goat Anti-Human α2-plasmin inhibitor ELISA kit, and ziyu-glycoside I (5) showed the strongest hemostatic activity among the seven terpene glycosides. This is the first report that ziyu-glycoside ? has strong hemostatic activity.

Full text of DOI:10.3390/molecules17077629

Molecules 2012, 17, 7629-7636; doi:10.3390/molecules17077629
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Terpene Glycosides from the Roots of Sanguisorba officinalis L.
and Their Hemostatic Activities
Wei Sun, Zi-Long Zhang, Xin Liu, Shuang Zhang, Lu He, Zhe Wang and Guang-Shu Wang *
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
*
Author to whom correspondence should be addressed; E-Mail: wgs@jlu.edu.cn;
Tel.: +86-431-8561-9706.
Received: 15 May 2012; in revised form: 13 June 2012 / Accepted: 15 June 2012 /
Published: 25 June 2012
Abstract: Guided by a hemostasis bioassay, seven terpene glycosides were isolated from
the roots of Sanguisorba officinalis L. by silica gel column chromatography and
preparative HPLC. On the grounds of chemical and spectroscopic methods, their structures
were identified as citronellol-1-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside (1),
geraniol-1-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside (2), geraniol-1-O-α-L-
arabinopyranosyl-(1→6)-β-D-glucopyranoside (3), 3β-[(α-L-arabinopyranosyl)oxy]-19α-
hydroxyolean-12-en-28-oic acid 28-β-D-glucopyranoside (4), 3β-[(α-L-arabinopyranosyl)-
oxy]-19α-hydroxyurs-12-en-28-oic acid 28-β-D-glucopyranoside (ziyu-glycoside I, 5),
3
β,19α-hydroxyolean-12-en-28-oic acid 28-β-D-glucopyranoside (6) and 3β,19α-di-
hydroxyurs-12-en-28-oic acid 28-β-D-glucopyranoside (7). Compound 1 is a new
mono-terpene glycoside and compounds 2, 3 and 5 were isolated from the Sanguisorba
genus for the first time. Compounds 1–7 were assayed for their hemostatic activities with a
Goat Anti-Human α2-plasmin inhibitor ELISA kit, and ziyu-glycoside I (5) showed the
strongest hemostatic activity among the seven terpene glycosides. This is the first report
that ziyu-glycoside Ι has strong hemostatic activity.
Keywords: Sanguisorba officinalis; ziyu-glycoside Ι; citronellol; geraniol; hemostasis
1
. Introduction
Sanguisorba officinalis L. (Rosaceae) is a perennial plant widely distributed in China, and its roots
have been used as a traditional Chinese medicine for the treatment of hemostasis and inflammation [1].

Molecules 2012, 17
7630
A variety of chemical constituents, including tannins, triterpenoids, flavonoids, anthraquinones,
steroids were isolated from S. officinalis L., and pharmacological studies on its hemostatic and anti-
inflammatory properties have been reported [2,3], but the molecular level mechanisms of these
activities have not been reported until now. In order to study the mechanism of hemostasis, we have
carried out the bioassay-guided isolation and identification of hemostatic constituents of the roots of
S. officinalis L. In the present study, we report the isolation and identification of a new monoterpene
glycoside 1, together with two known monoterpene glycosides 2, 3 and four known triterpenoids 4–7,
and their hemostatic activities.
2
. Results and Discussion
Compound 1, a colorless amorphous powder, produced a positive reaction to Molish reagent, and
+
had the molecular formula C H O as determined by HRESIMS ([M+Na] m/z 473.2369). Its IR
2
1
38 10
−1
spectrum indicated the presence of hydroxyl (3450 cm ) groups. Acid hydrolysis of compound 1 gave
1
13
D-glucose and L-arabinose. The H-, C- and DEPT-NMR spectrum (DMSO-d
6
) of compound 1
showed signals of a monoterpene moiety consisting of two singlets and one double methyl groups
[
δ
H
1.64 (3H, s, H-8), 1.56 (3H, s, H-9), and 0.85 (3H, d, J = 6.4 Hz H-10); δ
C-8), 17.5 (C-9), and 19.3 (C-10)], four methylenes [δ 3.41 and 3.76 (each 1H, m, H-1), 1.32 and
.56 (each 1H, m, H-2), 1.12 and 1.29 (each 1H, m, H-4), and 1.93 (2H, m, H-5); δ 66.9 (C-1), 36.3
C-2), 36.8 (C-4), and 24.9 (C-5)], one olefinic methine [δ 1.52 (1H, m, H-3); δ 28.9 (C-3)], and one
carbon-carbon double bond [δ 5.09 (1H, t-like, J = 7.2 Hz, H-6); δ 124.7 (C-6) and 130.4
C-4)]. Based on the 2D NMR data of H-H COSY, HMQC and HMBC experiments, the monoterpene
C
25.5
(
H
1
C
(
H
C
H
C
(
moiety was identified as citronellol [4]. The coupling constant of the anomeric proton of glucose at
δ 4.11 (d, 1H, J = 8.0 Hz) indicated that glucose moiety was in a -configuration. The HMBC
correlation signal of the anomeric proton of glucose, 1'-H (δ
glucose was linked to C-1 of the alycone. The downfield shift by about 5 ppm of the signal of C-6' of
glucose (δ 67.2) showed that arabinose was linked to C-6' of the glucose, which was further
confirmed by the HMBC correlations of 6'-H (δ 3.39 and 3.85) to C-1" (δ 108.5) of arabinose and
67.2). The C-NMR signals of compound 1 assignable to the arabinose
moiety [δ 82.0 (C-2"), 83.8 (C-2")] and the coupling constant of the anomeric proton of arabinose at
H C
4.11) to C-1 (δ 66.9) showed that
C
H
C
1
3
1
H C
"-H (δ 4.79) to C-6' (δ
C
δ
H
4.79 (d, 1H, J = 1.6 Hz) indicated that arabinose moiety was an α- L -arabinofuranose moiety [5],
which was further confirmed by the HMBC correlation of 1"-H (δ 4.79) to C-4" (δ 83.8). The
complete assignment of the signals of compound 1 was based on DEPT C-NMR and 2D-NMR H-H
H
C
1
3
1
13
COSY, HMQC and HMBC data. For all the H-, C-, and HMBC NMR data of compound 1 see Table 1,
and for the structure of compound 1, see Figure 1. Therefore, the structure of compound 1 was
elucidated as citronellol-1-O-α-L-arabinofuranosyl-(1→6)-α-D-glucopyranoside.
Using similar methods as described above, compounds 2–7 were identified as geraniol-1-O-α-L-
arabinofuranosyl-(1→6)-β-D-glucopyranoside (2) [5], geraniol-1-O-α-L-arabinopyranosyl-(1→6)-β-D-
glucopyranoside (3) [5], 3β-[(α-L-arabinopyranosyl)oxy]-19α-hydroxyolean-12-en-28-oic acid 28-β-D-
glucopyranoside (4) [6], 3β-[(α-L-arabinopyranosyl)oxy]-19α-hydroxyurs-12-en-28-oic acid 28-β-D-
glucopyranoside (ziyu-glycoside І, 5) [7], 3β,19α-hydroxyolean-12-en-28-oic acid 28-β-D-gluco-
pyranoside (6) [8], 3β,19α-dihydroxyurs-12-en-28-oic acid 28-β-D-glucopyranoside (7) [8], respectively.

Molecules 2012, 17
7631
1
13
Table 1. H-NMR (400 MHz), C-NMR (100 MHz), HMQC and HMBC data of
compound 1 (DMSO-d ,  ppm).
6
No.
C
H
HMBC(H→C) No.
glc
1' 102.8 4.11 (d, 1H, J = 8.0 Hz) 66.9, 75.4, 76.7
C
H
HMBC(H→C)
aglycone
1
66.9
36.3
3.41, 3.76
m, each 1H)
1.32, 1.56
28.9, 102.8
19.3, 36.8,
(
(
2
2'
73.4
2.93 (t-like, 1H,
m, each 1H)
J = 8.0 Hz)
3
4
28.9
36.8
1.52 (m, 1H)
1.12, 1.29
66.9
3'
4'
76.7 3.13 (t, 1H, J = 8.8 Hz)
19.3, 36.3,
124.7
70.4
75.4
67.2
2.98 (t-like, 1H,
J = 8.4 Hz)
73.4
102.8
108.5
(
m, each 1H)
5
6
24.9
1.93 (m, 2H)
28.9, 130.4
5'
3.28 (t-like, 1H,
J = 8.4 Hz)
124.7
5.09 (t-like, 1H,
17.5, 25.5, 36.8 6'
3.85 (d-like, 1H,
J = 10.8 Hz), 3.39 (dd,
J = 7.2 Hz)
1
H, J = 10.8, 8.4 Hz)
7
8
9
130.4
25.5
17.5
19.3
ara(f)
1" 108.5 4.79 (d, 1H, J = 1.6 Hz) 67.2, 77.2, 83.8
1.64 (s, 3H)
1.56 (s, 3H)
0.85 (d, 3H,
J = 6.4 Hz)
17.5, 124.7
25.5, 124.7
36.3, 36.8
2"
3"
82.0
77.2
3.79 (m, 1H)
3.62 (m, 1H)
1
0
4
5
"
"
83.8
61.4
3.72 (m, 1H)
3.55 (dd, 1H, J = 11.6,
2
.0 Hz), 3.40 (m, 1H)
All assignments based on extensive 1D and 2D NMR experiments (HMQC, HMBC, 1H-1H COSY).
Figure 1. The key HMBC correlations of compound 1.
HO
OH
3
"
OH
1"
4
"
O
O
6
'
5
'
O
HO
HO
6
8
1
OH
'
O
3
'
3
7
1
1
0
9
Note: Arrows point from proton to carbon.
Compounds 1–7 were next assayed for hemostatic activity with a Goat Anti-Human α2-plasmin
inhibitor ELISA kit, and the results are shown in Table 2. The data proved that ziyu-glycoside I
showed strongest hemostasis activity within 7 kinds of terpene glycosides. It is the first report that
ziyu-glycoside І (5) has strong hemostatic activity.

Molecules 2012, 17
Table 2. The hemostasis assay data of the separated fractions and the isolated compounds.
7632
3
0%
70%
EtOH
0.131 ±
0.014
95%
EtOH
0.051 ±
0.006
Control
blank
Standard
(10 g/L)
0.828 ±
0.031
Fractions
OD value
H
2
O
I
II
III
IV
EtOH
0.053 ±
0.002
0.056 ±
0.011
0.051 ±
0.005
0.051 ±
0.006
0.490 ± 0.061 ±
0.028 ±
0.004
(
n = 3)
0.017
57.8
0.004
4.1
Percent
3
.5
3.1
12.8
2.9
2.9
2.9
inhibition
Control
blank
Standard
(10 g/L)
0.828 ±
0.031
Compounds
1
2
3
4
5
6
7
OD value
0.138 ±
0.016
0.111 ±
0.009
0.122 ±
0.003
0.260 ±
0.013
0.741 ±
0.012
0.227 ±
0.010
0.214 ±
0.015
0.060 ±
0.004
(
n = 3)
Percent
1
0.2
6.6
8.1
26.0
88.7
21.7
20.1
inhibition
Note: Percent inhibition = [(ODsample − ODblank) ÷ (ODstandard − ODblank)] × 100; the OD value is directly proportional the
concentration of α2-plasmin inhibitor present in the samples.
3. Experimental
3.1. General
IR spectra were recorded on a FT-IR 5DX Nicolet/Nicolet Magna IR-560 spectrometer (Thermo
1
13
Scientific, Osaka, Japan). H- and C-NMR spectra were recorded on a Bruker AV-400 spectrometer
Zürich, Switzerland). HR-ESI-MS were recorded on a Bruker microOTOF-Q II mass spectrometer.
Prep. HPLC was performed on a Shimadzu LC-10A equipped with a SPD-10A detector and Gemini
μm C18 110A column (250 mm × 10.00 mm, 5 μm, flow rate: 3.0 mL/min). The bioactivities were
(
5
measured on a DG5033A Enzyme immunoassay spectrophotometer (Nanjing, China), using Goat
Anti-Human α2-plasmin inhibitor ELISA kit [96/48-wells microtiter plates, plastic cover, standard
(40 g/L), blank control, standard diluent, biotinylated anti-α2-plasmin inhibitor, streptavidin-HRP,
washing buffer, substrate A, substrate B, stopping solution, sample diluent)] (R&D Systems,
Minneapolis, MN, USA). The roots of S. officinalis L. were collected in Tong-Hua County in Jilin
Province, China. They were identified by Prof. Jing-min Zhang of School of Pharmaceutical Sciences,
Jilin University.
3.2. Extraction and Isolation
The air-dried the roots of S. officinalis (4.0 kg) were extracted with hot 70% EtOH (ca. 20 L, 24 h,
45 °C). The EtOH extract was concentrated under reduced pressure, and the viscous concentrate (420 g)
2
was passed through a D101 polyporous resin column eluting successively with H O, 30% EtOH, 70%
EtOH, and 95% EtOH, and by vacuum distillation recovery, four fractions (I–IV) were obtained. The
bioassay experiments suggested that the 70% EtOH portion had hemostatic activity. Therefore the 70%
EtOH fraction was subjected to silica gel column chromatography eluted with a stepwise gradient
mixture of CHCl
were obtained. Fraction III having hemostasis activity was further subjected to a silica gel column
eluted with CHCl –MeOH–EtOAc–H O (6.5:5:4:1.7), and three fractions (A, B, C) were obtained.
3
–MeOH (9:1; 6:1; 3:1), and finally with MeOH alone, and four fractionons (I–IV)
3
2

Molecules 2012, 17
7633
Fraction B was applied to a ODS-A (50 μm, 12 nm, YMC, Kyoto, Japan) column eluted with a
stepwise gradient mixture of MeOH–H O (2:3; 3:2; 4:1), and thus compound 5 (230 mg) and Fraction
D were obtained. Fractions A, C, and D were separated by preparative HPLC using MeOH–H
60:40, 70:30, 65:35, respectively), and compound 1 (22 mg), 2 (25 mg), and 3 (27 mg) were isolated
2
2
O
(
from fraction A, compound 6 (30 mg) and 7 (33 mg) from fraction C, and compound 4 (30 mg) from
Fraction B.
24
Compound 1: Colorless amorphous powder, [α] : −21.0° (c 0.6, MeOH). HRESIMS, m/z: 473.2369
D
+
−1 1
13
(
[M+Na] ; calcd for C21
H
38
O
10Na, 473.2363). IR (KBr) max: 3450 cm . H and C-NMR: See Table 1.
+
Compound 2: Colorless amorphous powder. HRESIMS, m/z: 471.2209 ([M+Na] ; calcd for
−1 1
C
21
H
36
O
6
10Na, 471.2206). IR (KBr) max: 3440 cm . H-NMR (DMSO-d ) δ: 1.57 (s, 3H, H-9), 1.61 (s,
3
H, H-10), 1.64 (s, 3H, H-8), 1.99 (2H, t, J = 6.8 Hz, H-4), 2.05 (2H, m, H-5), 4.08 (1H, dd, J = 11.9,
.6 Hz, H-1a), 4.19(1H, dd, J = 11.9, 6.1 Hz, H-1b), 5.07 (1H, t-like, J = 6.8 Hz, H-6), 5.26 (1H, m,
7
H-2); 4.11 (1H, d, J = 7.8 Hz, glc-H1), 2.95 (1H, t-like, J = 8.0 Hz, glc-H2), 3.12 (1H, t-like, J = 8.8 Hz,
glc-H3), 2.98 (1H, t-like, J = 8.8 Hz, glc-H4), 3.25 (1H, t-like, J = 8.4 Hz, glc-H5), 3.86 (1H, d-like,
J = 9.9 Hz, glc-H6a), 3.39 (1H, m, glc-H6b); 4.79 (1H, d, J = 1.6 Hz, ara-H1), 3.79 (1H, m, ara-H2),
3
.62 (1H, m, ara-H3), 3.72 (1H, m, ara-H4), 3.52 (1H, d-like, J = 13.4Hz, ara-H5a),3.40 (m, 1H,
13
ara-H5b). C-NMR (DMSO-d ) δ: 16.1(C-10), 17.6 (C-9), 25.6 (C-8), 25.9 (C-5), 39.4(C-4), 64.2
6
(
C-1), 120.6 (C-2), 123.9 (C-6), 130.4 (C-7), 139.4 (C-3); 101.3 (glc-C1), 73.3 (glc-C2), 76.7 (glc-C3),
0.5 (glc-C4), 75.5 (glc-C5), 67.2 (glc-C6); 108.5 (ara(f)-C1), 82.1 (ara(f)-C2), 77.3 (ara(f)-C3), 83.8
ara(f)-C4), 61.4 (ara(f)-C4).
7
(
+
Compound 3: Colorless amorphous powder. HRESIMS, m/z: 471.2208 ([M+Na] ; calcd for
−
1 1
C
21
H
36
O
10Na, 471.2206). IR (KBr) max: 3445 cm . H-NMR (DMSO-d
H, H-10), 1.64 (s, 3H, H-8), 1.99 (2H, t, J = 6.8 Hz, H-4), 2.05 (2H, t, J = 6.8 Hz, H-5), 4.06 (1H, dd,
J = 12.7, 7.3 Hz, H-1a), 4.22(1H, dd, J = 12.7, 6.0 Hz, H-1b), 5.08(1H, t-like, J = 6.5 Hz, H-6), 5.28
6
) δ: 1.57 (s, 3H, H-9), 1.61 (s,
3
1
3
(
1H, J = 6.5 Hz, H-2); 4.12 (1H, d, J = 7.8 Hz, glc-H1), 4.20 (1H, d, J = 6.0 Hz, ara-H1). C-NMR
DMSO-d ) δ: 16.1 (C-10), 17.6 (C-9), 25.5 (C-8), 25.8 (C-5), 39.3 (C-4), 64.4 (C-1), 120.7 (C-2),
23.9 (C-6), 130.9 (C-7), 139.1 (C-3); 101.5 (glc-C1), 73.3 (glc-C2), 76.6 (glc-C3), 70.5 (glc-C4), 75.6
(
6
1
(
glc-C5), 67.2 (glc-C6); 103.4 (ara(p)-C1), 70.1 (ara(p)-C2), 72.5 (ara(p)-C3), 68.5(ara(p)-C4), 64.7
ara(p)-C4).
(
+
Compound 4: Colorless amorphous powder. HRESIMS, m/z: 789.4404 ([M+Na] ; calcd for
−
1
1
C H O Na, 789.4401). IR (KBr)  : 3440, 1720 cm . H-NMR (DMSO-d ) δ: 5.24 (1H, d,
4
1
66 13
max
6
J = 7.8 Hz, glc-H1), 5.23(1H, br.s, H-12), 4.45 (1H, d, J = 7.0Hz, ara-H1), 3.11 (1H, br.s, H-19), 3.01
(1H, dd, J = 11.5, 3.9 Hz, H-3), 2.91(1H, br.s, H-18), 1.23 (3H, s, Me-27), 0.97 (3H, s, Me-23), 0.87
(
3H × 2, s, Me-26 and Me-29), 0.85 (3H, s, Me-30), 0.76 (3H, s, Me-24), 0.65(3H, s, Me-25).
1
3
6
C-NMR (DMSO-d ) δ: 38.0(C-1), 25.6 (C-2), 87.7 (C-3), 38.8 (C-4), 55.1 (C-5), 17.9 (C-6), 32.3
(C-7), 40.2 (C-8), 47.3 (C-9), 36.4 (C-10); 23.2 (C-11), 122.3 (C-12), 143.2 (C-13), 41.1 (C-14), 28.3
(C-15), 27.0 (C-16); 45.2 (C-17), 43.2 (C-18), 80.0 (C-19), 34.8 (C-20), 27.8 (C-21), 31.8 (C-22), 27.6
(C-23), 16.4 (C-24), 15.1 (C-25), 16.6 (C-26); 24.5 (C-27), 175.8 (C-28), 28.0 (C-29), 24.0 (C-30);

Molecules 2012, 17
7634
9
4.1 (glc-C1), 72.4 (glc-C2), 76.7 (glc-C3), 69.5 (glc-C4), 77.7 (glc-C5), 60.6 (glc-C6); 105.8 (ara(p)-
C1), 71.0 (ara(p)-C2), 72.7 (ara(p)-C3), 67.6 (ara-C4), 65.1 (ara-C4).
+
Compound 5: Colorless amorphous powder. HRESIMS, m/z: 789.4403 ([M+Na] ; calcd for
−
1
1
C H O Na, 789.4401). IR (KBr)  : 3475, 1740 cm . H-NMR (DMSO-d ) δ: 5.16 (1H, d,
4
1
66 13
max
6
J = 7.8 Hz, glc-H1), 5.17(1H, br.s, H-12), 4.12 (1H, d, J = 6.0Hz, ara-H1), 3.11 (1H, br.s, H-19), 3.01
(
1H, dd, J = 11.1, 3.9 Hz, H-3), 2.36(1H, br.s, H-18), 1.23 (3H, s, Me-27), 1.09 (3H, s, Me-29), 0.97
3H, s, Me-23), 0.88 (3H, s, Me-26), 0.84(3H, d, J = 6.6 Hz, Me-30), 0.76 (3H, s, Me-25), 0.67 (3H, s,
(
1
3
Me-24). C-NMR (DMSO-d ) δ: 38.2(C-1), 25.7 (C-2), 87.8 (C-3), 38.7 (C-4), 55.0 (C-5), 17.9 (C-6),
6
32.5 (C-7), 39.4 (C-8), 46.7 (C-9), 36.2 (C-10); 23.2 (C-11), 127.0 (C-12), 138.2 (C-13), 41.2 (C-14),
28.1 (C-15), 25.1 (C-16); 47.3 (C-17), 53.2 (C-18), 71.6 (C-19), 41.0 (C-20), 25.8 (C-21), 36.6 (C-22),
27.6 (C-23), 16.4 (C-24), 15.2 (C-25), 16.4 (C-26); 23.8 (C-27), 175.5 (C-28), 26.4 (C-29), 16.2
(
C-30); 94.1 (glc-C1), 72.2 (glc-C2), 76.7 (glc-C3), 69.5 (glc-C4), 77.6 (glc-C5), 60.6 (glc-C6); 105.8
ara(p)-C1), 71.0 (ara(p)-C2), 72.7 (ara(p)-C3), 67.6 (ara-C4), 65.1 (ara-C4).
(
+
Compound 6: Colorless amorphous powder. HRESIMS, m/z: 657.39783 ([M+Na] ; calcd for
−
1
1
C
36
H
58
O
9
6
Na, 657.39785). IR (KBr) max: 3440, 1720 cm . H-NMR (DMSO-d ) δ: 5.23 (1H, d,
J = 7.6 Hz, glc-H1), 5.23(1H, br.s, H-12), 3.11 (1H, br.s, H-19), 3.00 (1H, dd, J = 11.2, 3.9 Hz,
H-3), 2.91(1H, br.s, H-18), 1.23 (3H, s, Me-27), 0.89 (3H, s, Me-23), 0.87 (3H, s, Me-29), 0.84
13
(
3H × 2, s, Me-25 and Me-30), 0.68 (3H, s, Me-24), 0.64(3H, s, Me-25). C-NMR (DMSO-d
C-1), 26.9 (C-2), 77.7 (C-3), 38.4 (C-4), 54.9 (C-5), 18.1 (C-6), 32.3 (C-7), 40.2 (C-8), 47.3 (C-9),
6.7 (C-10); 23.2 (C-11), 122.3 (C-12), 143.2 (C-13), 41.1 (C-14), 28.3 (C-15), 27.0 (C-16); 45.2
6
) δ: 38.0
(
3
(C-17), 43.1 (C-18), 80.0 (C-19), 34.8 (C-20), 27.8 (C-21), 31.8 (C-22), 28.3 (C-23), 15.9 (C-24), 15.0
(C-25), 16.6 (C-26); 24.1 (C-27), 175.8 (C-28), 28.0 (C-29), 24.5 (C-30); 94.1 (glc-C1), 72.4
(glc-C2), 76.7 (glc-C3), 69.5 (glc-C4), 76.9 (glc-C5), 60.6 (glc-C6).
+
Compound 7: Colorless amorphous powder. HRESIMS, m/z: 657.39784 ([M+Na] ; calcd for
−
1
1
C H O Na, 657.39785). IR (KBr)  : 3475, 1740 cm . H-NMR (DMSO-d ) δ: 5.16 (1H, d,
3
6
58
9
max
6
J = 8.0 Hz, glc-H1), 5.17(1H, br.s, H-12), 4.12 (1H, d, J = 6.0Hz, ara-H1), 3.11 (1H, br.s, H-19), 3.00
(
1H, dd, J = 11.0, 3.9 Hz, H-3), 2.37(1H, br.s, H-18), 1.27 (3H, s, Me-27), 1.08 (3H, s, Me-29), 0.89
3H, s, Me-23), 0.85 (3H, s, Me-26), 0.84 (3H, d, J = 6.6 Hz, Me-30), (3H, s,), 0.67 (3H × 2, s, Me-25
(
1
3
and Me-24). C-NMR (DMSO-d
6
) δ: 38.2 (C-1), 27.0 (C-2), 77.6 (C-3), 38.4 (C-4), 54.8 (C-5), 18.1
(
(
(
C-6), 32.6 (C-7), 39.2 (C-8), 46.7 (C-9), 36.5 (C-10); 23.2 (C-11), 127.0 (C-12), 138.1 (C-13), 41.2
C-14), 28.1 (C-15), 25.1 (C-16); 47.3 (C-17), 53.2 (C-18), 71.6 (C-19), 41.1 (C-20), 25.4 (C-21), 36.6
C-22), 28.2 (C-23), 16.4 (C-24), 15.1 (C-25), 16.2 (C-26); 23.8 (C-27), 175.5 (C-28), 26.4 (C-29),
1
6.0 (C-30); 94.0 (glc-C1), 72.2 (glc-C2), 76.7 (glc-C3), 69.5 (glc-C4), 76.9 (glc-C5), 60.6 (glc-C6).
3
.3. Acid Hydrolysis of 1–7
2 4
Solutions of 1–7 (each 1.0 mg) in 0.5 M H SO (2.0 mL) were heated under reflux for 3 h. After
cooling, each reaction mixture was diluted with H O, neutralized with BaCO , then filtered. The
2
3
solution was partitioned with EtOAc to give two layers. The aqueous layer was evaporated and then
subjected to TLC analysis with authentic sugar samples using n-BuOH–MeOH–CHCl –HOAc
3

Molecules 2012, 17
7635
(
12.5:4.5:9:1.5:1, detection with aniline-phthalic acid). Compounds 1–5 afforded D-glucose (R
f
= 0.30)
and L-arabinose (R = 0.36), and 6–7 gave D-glucose (R = 0.30).
f
f
3.4. Bioactivity Assay
The hemostasis assay was carried out by using a Goat Anti-Human 2-plasmin inhibitor kit which
is a solid phase sandwich enzyme-linked quantitative immunoabsorbent assay (ELISA) with a purified
antibody specific for α2-plasmin inhibitors. The above separated fractions and compounds were
dissolved in dimethylsulfoxide (DMSO) (0.188 mg/μL for the fractions; 0.094 mg/μL for compounds),
and were diluted with sample diluent (1:1). After an aliquot (50 μL) of the above samples or standards
was added to each microplate well, a portion of biotinylated anti-α2-plasmin inhibitor (50 μL) was
immediately added to each well, and the microplate was incubated for 1 h at 37 °C. After a wash with
washing buffer, streptavidin-HRP was added, and the microplate was incubated for 30 min at 37 °C.
Again, after a wash with washing buffer, substrate A and substrate B (50 μL each) were added to each
well, and the microplate was incubated for 10 min at 37 °C. The enzyme-substrate reaction was
stopped by quickly pipetting stopping solution (50 μL) into each well. The optical density absorbance
(
OD value) of each well was measured at 450 nm wavelength on a DG5033A enzyme immunoassay
spectrophotometer. All the separated fractions and isolated compounds were tested for their hemostasis
activities, and the results are summarized in Table 2.
The correlation of calibration curve test data is shown in Table 3. By using OD value as Y-axis(Y)
and standards cocentration as X-axis (X), the linear regression equation, y = 0.0687x + 0.0317,
r = 0.9997, was obtained. The results showed that the linear relation between OD value and
concentration of standards were good and linearity domain of the measure is 0~40g/L. Therefore the
OD value is directly proportional the concentration of α2-plasmin inhibitor present in the samples.
Table 3. The correlation of calibration curve test data.
Standards
Concentration (g/L)
OD
Control
blank
4
0
20
10
5.0
2.5
1.25
2.9404
1.5171
0.828
0.4905
0.2955
0.0895
0.0214
Note: Y = 0.0687x + 0.0317, r = 0.9997, linearity domain: 0~40 g/L; the OD value is directly proportional the
concentration of α2-plasmin inhibitor present in the samples.
4
. Conclusions
Compound 1 is a new monoterpene glycoside and compounds 2, 3 and 5 were isolated from the
Sanguisorba genus for the first time. Compounds 1–7 were assayed for their hemostatic activities
with a Goat Anti-Human α2-plasmin inhibitor ELISA kit, and the hemostatic constituent of
S. officinalis wasidentified as ziyu-glycoside I (5). This is the first report that ziyu-glycoside I has
strong hemostatic activity.

Molecules 2012, 17
7636
Acknowledgments
This work was supported by National Science and Technology Major Project of China
2009ZX09502-011). The authors gratefully thank Jing-Min Zhang (School of Pharmaceutical
(
Sciences, Jilin University) for the identification of plants.
References and Notes
1
.
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3.
4.
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6.
7.
8.
Ji, C.J.; Tan, N.H.; Fu, J.; Zhang, Y.M.; He, M. Monoterpene disaccharide glycosides from
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Sample Availability: Samples of the compounds 5 are available from the authors.
©
2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(