Triterpenoids from the roots of Sanguisorba tenuifolia var. Alba

Article abstract of DOI:10.3390/molecules16064642

The ethyl acetate soluble fraction from the roots of Sanguisorba tenuifolia was found to have a hypoglucemic effect in alloxan-induced diabetic rats. Two new triterpenoids, identified as 2-oxo-3β,19α-dihydroxyolean-12-en- 28-oic acid β-D-gluco-pyranosyl ester (1) and 2a,19α-dihydroxy-3-oxo- 12-ursen-28-oic acid β-D-glucopyranosyl ester (4) were isolated from this fraction, along with thirteen known triterpenoids. Their structures were elucidated by chemical and spectroscopic methods. All these compounds demonstrated inhibitory activities against α-glucosidase with IC ₅₀ values in the 0.62-3.62 mM range.

Full text of DOI:10.3390/molecules16064642

Molecules 2011, 16, 4642-4651; doi:10.3390/molecules16064642
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Triterpenoids from the Roots of Sanguisorba tenuifolia
var. Alba
Hai-Xue Kuang *, Hong-Wei Li, Qiu-Hong Wang, Bing-You Yang, Zhi-Bin Wang and
Yong-Gang Xia
Key Laboratory of Chinese Materia Medica, Heilongjiang University of Chinese Medicine, Ministry of
Education, Harbin 150040, China
*
Author to whom correspondence should be addressed; E-Mail: hxkuang@hotmail.com;
Tel.: +86-451-82193001; Fax: +86-451-82110803.
Received: 15 April 2011; in revised form: 26 May 2011 / Accepted: 27 May 2011 /
Published: 3 June 2011
Abstract: The ethyl acetate soluble fraction from the roots of Sanguisorba tenuifolia
was found to have a hypoglucemic effect in alloxan-induced diabetic rats. Two
new triterpenoids, identified as 2-oxo-3β,19α-dihydroxyolean-12-en-28-oic acid
β-D-gluco-pyranosyl ester (1) and 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid
β-D-glucopyranosyl ester (4) were isolated from this fraction, along with thirteen known
triterpenoids. Their structures were elucidated by chemical and spectroscopic methods. All
these compounds demonstrated inhibitory activities against α-glucosidase with IC50 values
in the 0.62-3.62 mM range.
Keywords: Sanguisorba tenuifolia var. Alba; triterpenoids; α-glucosidase inhibitory
activity; diabetes mellitus
1
. Introduction
Diabetes mellitus (DM), considered a lifestyle related diseases, is a metabolic disease with
hyper-glycemia as a symptom and causes many complications [1]. Recently, DM is becoming a
serious problem around the World, and according to World Health Organization, it affects
approximately 171 million people worldwide and the number is expected to reach to 366 million over
the next 20 years [2]. Many researchers have enthusiastically studied the development of antidiabetic

Molecules 2011, 16
4643
agents, however, many potential therapeutics have a number of serious adverse effects [3,4], therefore
there is a growing trend toward using natural products as treatment [5]. China has a long history of
using herbs for the treatment of human diseases and several medicinal plants are used for the treatment
of diabetes. S. tenuifolia is one such plant.
S. tenuifolia (Rosaceae) is a perennial herb, which is widely distributed in China’s Heilongjiang,
Liaoning, and Jilin provinces and Inner Mongolia. The residents in Northeast China regard
S. tenuifolia as a substitute for S. officinalis, and apply its roots for the treatment of diarrhea, chronic
intestinal infections, duodenal ulcers, diabetes mellitus and bleeding [6,7]. Our studies indicated that
ethyl acetate fraction of a S. tenuifolia root ethanol extract contains plenty of triterpenes, which can
inhibit plasma glucose levels in alloxan-induced diabetic rats. α-Glucosidase inhibitors are oral
anti-diabetic drugs used for diabetes mellitus type 2. They can significantly delay the absorption of
carbohydrates from the small intestine and thus have a lowering effect on postprandial blood glucose
and insulin levels [8]. Based on a bioassay-guided isolation, a phytochemical study of S. tenuifolia was
performed and two new triterpenoids were isolated from its ethyl acetate fraction, along with thirteen
other known triterpenoids. The new compounds were identified as 2-oxo-3β,19α-dihydroxy-olean-12-
en-28-oic acid β-D-glucopyranosyl ester (1) and 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid
β-D-glucopyranosyl ester (4), respectively. In the present report, we describe the structural elucidation of
1
and 4, together with the α-glucosidase inhibitory activity data of all the compounds 1-15 (Figure 1).
Figure 1. Structures of 1-15.
R7 R6
R5
R4
2
21
1
9
1
25
26
1
3
COOR3
28
1
4
R1
R2
2
2
3
27
4 23
R
1
R
2
R
3
R
4
R
5
R
6
R
7
1
2
3
4
5
6
7
8
9
=O
β-OH
β-OH
β-OH
=O
β-D-Glc
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
=O
CH3
CH3
CH3
CH3
H
=O
β-D-Glc
β-D-Glc
H
H
α-OH
α-OH
α-OH
α-OH
α-OH
α-OH
α-OH
H
H
=O
H
=O
β-D-Glc
β-D-Glc
H
CH3
H
β-OH
α-OH
α-OH
β-OH
β-OH
β-O-coumaroyl
β-O-Ara
β-O-Ara
CH3
CH3
CH3
H
H
β-D-Glc
β-D-Glc
H
H
1
0
1
2
3
4
5
CH3
H
1
1
1
1
1
CH3
CH3
CH3
H
H
H
H
H
β-D-Glc
β-D-Glc
H
OH
H
H
=CH2
CH3
H
H
β-OH
OH
H

Molecules 2011, 16
. Results and Discussion
4644
2
Compound 1 was obtained as a white amorphous powder. The HR-ESI-MS data indicated a
+
molecular formula of C36
H
56
O
10, based on the [M + H] ion signal at m/z 649.3953 (calc. C36
H O
57 10
+
,
+
6
49.3952), [M + NH ] ion signal at m/z 666.4221 (calc. C H O N, 666.4217) and [M + Na] ion
4
36 60 10
56
signal at m/z 671.3779 (calc. C36H O10Na, 671.3771). The IR spectrum showed the presence of
−
1
−1
−1
hydroxyl groups (3,419.6 cm ), ester carbonyl (1,735.3 cm ), carbonyl (1,711.3 cm ) and double
−1
bond (1,640.8 cm ).
1
3
The C-NMR spectrum and DEPT of 1 showed seven methyl, nine methylene, eleven methine, and
nine quaternary carbon signals, including one ester carbonyl at δ
C
177.3, a quaternary olefinic carbonyl
1
at δ 144.5, one anomeric carbon signal at δ 95.9, a ketone carbonyl at δ 213.4. The H-NMR
C
C
C
spectrum exhibited seven singlet methyl signals at δ
anomeric proton signal at δ
H
1.21, 0.92, 1.01, 1.15, 1.52, 1.12 and 0.95, an
H
H
6.37 (d, J = 8.0 Hz), two methine proton signals at δ 3.89 (s) and 3.54 (d,
J = 2.8 Hz), and an olefinic proton signal at δ 5.45 (br s), which were characteristic of the oleanolic
H
acid skeleton. Comparison of the data 1 with those of oleanolic acid [9-10], suggested that the
aglycone of 1 was an oleanolic acid derivative with one hydroxyl group at the ring E portion, as well
as one ketone carbonyl group. The proton signal at δ
C-17, and C-28 in the HMBC spectrum, and was assigned to the H-18 (Figure 2). This proton had a
proton spin-coupling correlation with the signal at δ 3.54, which was associated with the carbon
signal at δ 81.0 (CH) in the HSQC spectrum. Thus, the presence of a hydroxyl group at C-19 was
H
3.50 showed long-rang correlations with C-13,
H
C
3
evident. The JH,H value of 2.8 Hz between H-18 and H-19, and NOE correlations from H-19 to Me-29
and Me-30 gave evidence for the C-19α hydroxy orientation [11]. There were long-range correlations
between protons and carbons: H-3 (δ
13.4); H-23 (δ 1.21), H-24 (δ 0.92)/C-3 (δ
ketone carbonyl group must be either at position C-1 or C-2. Furthermore, the long-range correlations
were observed between protons and carbons: H-3 (δ 3.89), H-25 (δ 1.01)/C-1 (δ 51.5), H-1 (δ 3.00,
.27)/ketone carbonyl (δ 213.4) in the HMBC spectrum (Figure 2). The ketone must be at the C-2
H
3.89)/C-23 (δ
C
27.6), C-24 (δ
C
21.7), and ketone carbonyl (δ
C
2
H
H
C
83.2) in the HMBC spectrum, which indicated that the
H
H
C
H
2
C
position based on comparison of the NMR spectral data for C-1, C-2 and C-3 of 1 with that of the
similar compound 2-oxopomolic acid (2) [C-1 (δ 53.6 δ 2.46, 2.15), C-2 (δ 211.2) and C-3 (δ 83.5;
C
H
C
C
δ
H
4.17 s)] and 2α,19α-dihydroxy-3-oxo-olean-12-en-28-oic acid β-D-glucopyranosyl ester (6) [C-2
216.5) and C-3 (δ 69.7; δ 4.80 s)] [12-13]. Based on these findings, the structure of the aglycone
(
δ
C
C
H
part of 1 was established to be 2-oxo-3β,19α-dihydroxyolean-12-en-28-oic acid, a new triterpene. The
configuration of the sugar unit was assigned after hydrolysis of 1 with 1 M HCl. The acid hydrolysis
gave D-glucose. The data of anomeric carbon signal at δ
C
95.9 and anomeric proton signal at δ
H
6.37
(
d, J = 8.0 Hz) indicated the glucose was in the β form and was bound to the aglycone by a glycosidic
linkage at C-28 in the HMBC spectrum (Figure 2). Therefore, the structure of compound 1 was
elucidated as 2-oxo-3β,19α-dihydroxy-olean-12-en-28- oic acid β-D-glucopyranosyl ester.
Compound 4 was obtained as a white amorphous powder. The HR-ESI-MS data indicated a molecular
+
formula of C H O based on the [M + H] ion signal at m/z 649.3945 (calc. 649.3952) in the. The IR
36
56 10
−1
−1
spectrum showed the presence of hydroxyl groups (3431.1 cm ), ester carbonyl (1,737.1 cm ), carbonyl
−
1
−1
(
1,714.3cm ), and double bond (1,644.6 cm ).

Molecules 2011, 16
Figure 2. Key HMBC and H- H COSY correlations of 1 and 4.
4645
1
1
1
3
The C-NMR spectrum shows seven methyl, nine methylene, eleven methine, and nine quaternary
carbon signals, including one ester carbonyl at δ 177.0, a quaternary olefinic carbonyl at δ 139.5,
C
C
1
one anomeric carbon signal at δ
C
C
95.9, a ketone carbonyl at δ 216.6. Its H-NMR spectrum shows the
presence of a hydroxymethine proton at δ
proton at (δ 5.50, br s), six singlets at δ
groups, one secondary methyl group (δ
of pomolic acid (δ 2.91, s), and one anomeric proton (δ
signal on ring E provides a most useful indicator for the presence of an urs-12-ene skeleton [10].
H
4.82 (1H, dd, J = 12.5, 6.3 Hz), one trisubstituted olefinic
H
H
1.19, 0.99, 1.18, 1.15, 1.59, 1.37 for six tertiary methyl
H
1.05, d, J = 6.6Hz), one methine proton characteristic of H-18
6.30 d, J = 8.0 Hz). The secondary methyl
H
H
1
3
Additionally, the signals in its C-NMR at δ
double bond in the ursene-type structure [14]. Acid hydrolysis of 4 with 1 M HCl (5 mL) gave a
D-glucose molecule and a triterpene (C30 , 5). The latter was identified as 2α,19α-dihydroxy-3-
oxo-12-ursen-28-oic acid by comparing its spectral and physical data with literature values [15]. When
C
128.0 and 139.5 are characteristic for a C-12/C-13
46 5
H O
1
13
the H- and C-NMR spectra of 4 were compared with those of 5, an upfield shift due to the glycoside
was detected at the C-28 signal at δ 177.0. The linked site of glycosyl group in 4 was further
C
established from correlations between the anomeric proton H-1' at δ
H
C
6.30 and C-28 at δ 177.0 in the
HMBC spectrum (Figure 2). Therefore, the structure of 4 was determined as 2α,19α-dihydroxy-3-oxo-
1
2-ursen-28-oic acid β-D-glucopyranosyl ester.
Table 1. NMR data of 1 and 4 in pyridine-d
5
(δ in ppm, J in Hz, recorded at 400 MHz and
100 MHz, respectively).
No.
1
4
δ
C
(DEPT)
δ
H
(J, Hz)
δ
C
(DEPT)
H
δ (J, Hz)
1
2
3
4
5
6
7
8
51.5 (CH
2
)
3.00 d (12.4 ), 2.27 d (12.4)
50.3 (CH
2
)
2.48 dd (12.5, 6.3), 1.37 m
4.82 dd (12.5, 6.3)
213.4 (C)
83.2 (CH)
42.2 (C)
69.8 (CH)
216.6 (C)
48.2 (C)
3.89 s
50.2 (CH)
2.02 m
57.7 (CH)
1.23 m
19.3 (CH2) 1.46 m, 1.31 m
33.2 (CH2) 1.43 m, 1.70 m
40.3 (C)
19.6 (CH2) 1.34 m, 1.29 m
33.2 (CH2) 1.43 m, 1.33 m
40.6 (C)

Molecules 2011, 16
4646
Table 1. Cont.
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
48.1 (CH)
42.8 (C)
1.83 m
47.4 (CH)
37.8 (C)
1.83 m
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
'
24.3 (CH2) 2.02 m
123.0 (CH) 5.45 br s
144.5 (C)
24.2 (CH2) 2.08 m
128.0 (CH) 5.50 br s
139.5 (C)
42.3 (C)
42.2 (C)
29.0 (CH2) 2.46 m, 1.20 m
27.9 (CH2) 2.81 m, 2.12 m
46.5 (C)
29.2 (CH2) 2.49 m, 1.22 m
26.1 (CH2) 3.09 m, 2.05 m
48.6 (C)
44.6 (CH)
81.0 (CH)
35.6 (CH)
3.50 d (2.8)
3.54 d (2.8)
54.4 (CH)
72.7 (CH)
42.2 (CH)
2.91 s
1.39 m
29.0 (CH2) 1.24 m, 2.35 m
33.0 (CH2) 2.04 m, 1.93 m
27.6 (CH3) 1.21 s
21.7 (CH3) 0.92 s
16.9 (CH3) 1.01 s
17.1 (CH3) 1.15 s
24.9 (CH3) 1.52 s
177.3 (C)
26.7 (CH2) 1.24 m, 2.02 m
37.9 (CH2) 2.03 m, 1.83 m
25.4 (CH3) 1.19 s
21.8 (CH3) 0.99 s
17.6 (CH3) 1.18 s
16.1 (CH3) 1.15 s
24.6 (CH3) 1.59 s
177.0 (C)
28.7 (CH3) 1.12 s
24.4 (CH3) 0.95 s
27.0 (CH3) 1.37 s
16.8 (CH3) 1.05 d (6.6)
95.9 (CH)
74.1 (CH)
79.3 (CH)
71.2 (CH)
79.0 (CH)
6.37 d (8.0)
4.22 t (8.4)
4.29 t (8.8)
4.38 t (9.0)
4.04 m
95.9 (CH)
74.1 (CH)
79.4 (CH)
71.2 (CH)
79.0 (CH)
6.30 d (8.0)
4.24 t (8.4)
4.34 t (8.7)
4.39 t (9.3)
4.05 (m)
'
'
'
'
'
62.2 (CH2) 4.42 br d (12.1), 4.46 dd (12.1, 3.8)
62.4 (CH2) 4.42 br d (11.7), 4.49 dd (11.7, 4.4)
The other compounds were characterized as 2-oxopomolic acid (2) [12], 2-oxopomolic
acid β-D-glucopyranoside (3) [12], 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid (5) [15],
α,19α-dihydroxy-3-oxo-olean-12-en-28-oic acid β-D-glucopyranosyl ester (6) [13], rosamutin (7) [16],
2
euscaphic acid (8) [17], kaji-ichigoside Fl (9) [16], 24-deoxysericoside (10) [17], ursolic acid (11) [10],
p-coumaroylursolic acid (12) [18], ziyu-glycoside I (13) [17], 3β-[(α-L-arabinopyranosyl) oxy]
urs-12,19(29)-dien-28-oic acid 28-β-D-glucopyranosyl ester (14) [11] and pomolic acid (15) [17] by
comparing their NMR spectroscopic data with the literature values. All these known compounds are
reported for the first time in S. tenuifolia.
We next evaluated the isolated compounds for their inhibitory activity against α-glucosidase since
some compounds are known α-glucosidase inhibitors [19]. The results are shown in Table 2, with
acarbose used as a positive control. Compounds 1-15 exhibited dose-dependent α-glucosidase
inhibitory activities with IC values of 0.62-3.62 mM. Compounds 8 and 12 showed the most potent
5
0
activity (IC50 0.67 and 0.62 mM, respectively), comparable with the positive control. Triterpenoids of

Molecules 2011, 16
4647
the EtOAc-soluble fraction may be the potential anti-hypoglycemic agents in this plant, as they have
been shown to induce an anti-diabetic effect.
Table 2. In vito α-glucosidase inhibitory assay.
Compound
IC50 (mM ± SEM, mM)
1.88 ± 0.28
1.35 ± 0.04
2.22 ± 0.06
1.56 ± 0.04
1.23 ± 0.09
2.01 ± 0.06
3.28 ± 0.08
0.67 ± 0.09
3.10 ± 0.24
3.52 ± 0.16
1.69 ± 0.04
0.62 ± 0.06
3.62 ± 0.21
2.87 ± 0.06
1.84 ± 0.12
0.79 ± 0.13
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
0
1
2
3
4
5
Acarbose
3. Experimental Section
3.1. General
Open column chromatography (CC) was carried out using silica gel (200-300 mesh, Qingdao
Marine Chemical Co., Qingdao, China) or octadecyl silica gel (ODS, 25-40 μm, Fuji) as stationary
phases. TLC employed precoated silica gel plates (5-7 μm, Qingdao Marine). Preparative HPLC was
carried out on a Waters 600 instument equipped with a Waters UV-2487 detector. A Waters Sunfire
prep C18 OBD (19 × 250 mm i.d.) column was used for this purpose. The IR spectra were recorded as
KBr pellets on a Jasco 302-A spectrometer. Optical rotation was recorded on a Jasco P-2000
polarimeter. HRESIMS were measured on a FTMS-7 instrument (Bruker Daltonics). Melting points
1
13
1
1
were determined on a Gallenkemp apparatus and are uncorrected. The H-, C- and 2D ( H- H COSY,
HSQC, HMBC, NOESY) NMR spectra were recorded on a Bruker AMX-400 spectrometer using
standard pulse sequences. Chemical shifts are reported in ppm (δ), and scalar coupling are reported in Hz.
GC analyses were carried out using a Fuli 9790 instrument equipped with a DM-5 column (0.25 μm,
30 m × 0.25 mm, Dikma, China). α-Glucosidase (EC.3.2.1.20) from Saccharomyces sp. was purchased
from Wako Pure Chemical Indutries Ltd. (Wako 076-02841). Other reagents were purchased from
various commercial sources.
3
.2. Plant Material
The roots of S. tenuifolia were collected in October 2008 from Fangzheng of Heilongjiang
Province, China, and identified by Zhenyue Wang, of Heilongjiang University of Chinese Medicine.

Molecules 2011, 16
4648
A voucher specimen (20081023) was deposited at the herbarium of Heilongjiang University of
Chinese Medicine, Harbin, China.
3.3. Extraction and Isolation
The dried roots of S. tenuifolia (5.0 kg) were extracted with 70% EtOH (3 × 10 L) to afford the
EtOH extract (1.3 kg) which was then suspended in water (10 L) and then extracted with petroleum
ether and ethyl acetate (EtOAc) (3 × 10 L each), yielding petroleum ether (10.2 g) and ethyl acetate
(
222.5 g) extracts. The EtOAc fraction (222.5 g) was subjected to silica gel column with a stepwise
CH Cl -MeOH gradient (30:1; 20:1; 10:1; 5:1, v/v), and finally with MeOH alone, to give five
2
2
fractions I-V. Fraction I (40.8 g) was separated using silica gel CC eluting with CH
2
Cl
2
-MeOH (50:1,
3
0:1, 10:1, v/v) to obtain three sub-fractions, I -I . Sub-fraction I (10.6 g) was further separated by
1
3
2
ODS silica gel CC with MeOH-H
2
O (9:1, v/v) and to 11 (33.2 mg), 12 (37.5 mg) and 15 (25.5 mg);
Cl -MeOH (30:1, 20:1, 10:1, v/v)
to afford four sub-fractions, II -II . Sub-fraction II (13.3 g) afforded compounds 2 (21.0 mg), 5 (44.5
Fraction II (38.3 g) was subjected to silica gel CC eluting with CH
2
2
1
4
1
mg) and 8 (24.6 mg, t
3:1, 3:2, v/v), followed by preparative HPLC with MeOH-H
subjected to silica gel CC eluting with CH Cl -MeOH (8:1, 5:1, 1:1, v/v) to afford four sub-fractions,
III -III . Sub-fraction III (7.3 g) afforded 4 (43.5 mg) and a mixture of 1, 3 and 6 by ODS silica gel
CC using MeOH-H O (2:1, v/v) as eluent. The mixture was separated into 1 (25.2 mg, t = 45.3 min),
(23.4 mg t = 43.2 min) and 6 (12.5 mg, t = 48.5 min) by preparative HPLC using MeOH-H O (3:2,
v/v). Fraction IV (43.1 g) was applied to a silica gel column which was eluted with CH Cl -MeOH-H
8:1:0.1, 6:1:0.1, 3:1:0.1, v/v) to afford four sub-fractions, IV -IV . Sub-fraction IV (16.3 g)
afforded a mixture of 7, 9 and 10, along with a few impurities, after ODS silica gel CC with
MeOH-H O (2:1, v/v). The mixture was separated by preparative HPLC using MeOH-H O (3:2, v/v)
into 7 (27.5 mg, t = 49.2 min), 9 (25.2 mg, t = 43.2 min) and 10 (30.4 mg, t = 45.2 min). Fraction V
40.1 g) was applied to a silica gel column eluted with CH Cl -MeOH-H O (6:1:0.1, 3:1:0.1, v/v) to
R
= 50.5 min), after subjecting it to ODS silica gel CC eluting with MeOH-H
2
O
(
2
O (4:1, v/v). Fraction III (31.3 g) was
2
2
1
4
2
2
R
3
R
R
2
2
2
2
O
(
1
4
2
2
2
R
R
R
(
2
2
2
afford three sub-fractions, V
1
-V
2
1
. Sub-fraction V (13.3 g) afforded 13 (60.8 mg) and 14 (19.4 mg) by
ODS silica gel CC eluting with MeOH-H
2
O (2:1, v/v).
2
-Oxo-3β,19α-dihydroxyolean-12-en-28-oic acid β-D-glucopyranosyl ester (1). White amorphous
2
0
powder. [α] + 16.5° (c 1.05, MeOH). IR (KBr): 3419.6, 1735.7, 1711.3, 1640.8, 1070.4, 1029.9,
D
-
1
+
+
9
93.3 cm . HR-ESI-MS m/z 671.3779 [M + Na] (calc. C36
H
56
O
10Na, 671.3771), 649.3953 [M + H]
+
1
13
(
(
calc. C36
H
57
O
10, 649.3952), 666.4221 [M + NH
4
] (calc. C36
H
60
O
10N, 666.4217); H- and C-NMR
pyridine-d
5
) data, see Table 1.
2
α,19α-Dihydroxy-3-oxo-12-ursen-28-oic acid β-D-glucopyranosyl ester (4). White amorphous
2
0
powder. [α] + 21.5° (c 1.25, MeOH). IR (KBr): 3431.1, 1737.1, 1714.3, 1644.6, 1070.4, 1029.9,
D
-1
+
1
13
9
91.3 cm . HR-ESI-MS m/z 649.3945 [M + H] (calc. C36
57
H O10, 649.3952); H- and C-NMR
(
pyridine-d ) data, see Table 1.
5

Molecules 2011, 16
4649
3
.3.1. Acid Hydrolysis of 1 and 4 and Determination of the Absolute Configuration of the
Mono-saccharides
1
2
(5 mg) in 1 M HCl (dioxane-H O, 1:1, 5 mL) was heated at 90 °C for 3 h under an Ar atmosphere.
After the dioxane was removed, the solution was extracted with EtOAc (3 mL × 3) to remove the
aglycone. The aqueous layer was neutralized by passing through an ion-exchange resin column
(Amberlite MB-3, Organo, Tokyo, Japan) and concentrated to dryness under reduced pressure to give the
sugar fraction. The residue was dissolved in pyridine (0.1 mL) to which 0.1 M L-cysteine methyl ester
hydrochloride in pyridine (0.1 mL) was added. The mixture was heated at 60 °C for 1 h. After the
reaction mixture was dried in vacuo, the residue was trimethylsilylated with l-trimethylsilylimidazole
(
0.2 mL) for 2 h. The mixture was partitioned between hexane and H O (0.6 mL, each), and the hexane
2
extracted was analyzed by GC under the following conditions: capillary column, DM-5 (0.25 mm × 30 m
0.25 μm); detector, FID; injector temperature, 280 °C, detector temperature, 280 °C; initial
×
temperature was maintained at 160 °C for 2 min and then raised to 195 °C at a rate of 10 °C/min;
carrier gas, N . In the acid hydrolysate of 1, D-glucose was confirmed by comparison of the retention
2
time of their derivatives with those of D-glucose and L-glucose derivatives prepared in a similar way,
which showed retention times of 28.56 and 27.72 min, respectively. The sugar from 4 (30 mg) was
also identified by the same method.
3
.3.2. α-Glucosidase Inhibition Assay
α-Glucosidase (EC.3.2.1.20) enzyme inhibition assay has been performed according to the literature [19].
α-Glucosidase (25 μL, 0.2 U/mL), various concentrations of samples (25 μL), and 67 mM phosphate
buffer (pH 6.8, 175 μL) were mixed at room temperature for 10 min. Reactions were initiated by the
addition of 23.2 mM p-nitrophenyl-α-D-glucopyranoside (25 μL). The reaction mixtures were
2 3
incubated at 37 °C for 15 min in a final volume of 250 μL, and then 1 M Na CO (50 μL) was added to
the incubation solution to stop the reaction. The activities of glucosidase were detected in a 96-well
plate, and the absorbance was read at 405 nm by a microplate spectrophotometer (Spectra Max,
Molecular Devices, USA). The negative control was prepared by adding phosphate buffer instead of
the sample in the same way as the test. Acarbose was utilized as the positive control. The blank was
prepared by adding phosphate buffer instead of α-glucosidase using the same method. The inhibition
rates (%) were calculated from the following formula:
[(ODnegative control − ODblank) − (ODtest − ODtest blank)] / (ODnegative blank − ODblank) × 100%
4
. Conclusions
Two new triterpenoids, 2-oxo-3β,19α-dihydroxyolean-12-en-28-oic acid β-D-glucopyranosyl ester (1)
and 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid β-D-glucopyranosyl ester (4) were isolated from an
ethyl acetate fraction of S. tenuifolia roots, along with thirteen known triterpenoids. All these
triterpenoids are reported for the first time in S. tenuifolia and demonstrated inhibitory activities
against α-glucosidase with IC50 values in the 0.62-3.62 mM range. Triterpenoids of the EtOAc-soluble
fraction may be the potential anti-hypoglycemic agents in this plant, as they have been shown to
induce an anti-diabetic effect.

Molecules 2011, 16
4650
Acknowledgments
Our work was supported by the Major Sate Basic Research Development Program of China (973
Program 2006CB504708), the National Natural Science Foundation of China (Nos. 30371736 and
3
0672633) and Special Fund Project of National Excellent Doctoral Dissertation of China (200980).
References and Notes
1
.
Rasmussen, L.M.; Ledet, T. Aortic atherosclerosis in diabetes mellitus is associated with an
insertion/deletion polymorphism in the angiotensin I-converting enzyme gene. No relation
between the polymorphism and aortic collagen content. Diabetologia 1996, 39, 696-700.
World Health Organization. Country and regional data. Available online: http://www.who.int/
diabetes/facts/world_figures/en/ (accessed on 30 May 2011).
2.
3.
4.
5.
6.
7.
8.
Bhatnagar, D. Lipid-lowering drugs in the management of hyperlipidemia. Pharmacol. Therapeut.
1
998, 79, 205-230.
May, L.; Lefkowitch, J.; Kram, M.; Rubin, D. Mixed hepatocelluar-cholestatic liver injury after
pioglitazone therapy. Ann. Intern. Med. 2002, 136, 449-452.
Jung, M.; Park, M.; Lee, H.C.; Kang, Y.H.; Kang, E.S.; Kim, S.K. Antidiabetic agents from
medicinal plants. Curr. Med. Chem. 2006, 13, 1203-1218.
Zhu, Y.C. Plantae Medicinal Chinae Boreali-Orientalis; Heilongjiang Science & Technology
Publishing House: Harbin, China, 1998; p. 553.
Jiangsu New Medical College. The Chinese Medicine Dictionary; People Publishing House:
Shanghai, China, 1997; p. 806.
Sou, S.; Mayumi, S.; Takahashi, H.; Yamasaki, R.; Kadoya, S.; Sodeoka, M.; Hashimoto, Y.
Novel alpha-glucosidase inhibitors with a tetrachlorophthalimide skeleton. Bioorg. Med. Chem.
Lett. 2000, 10, 1081-1084.
9
1
1
1
1
1
1
1
.
Kizu, H.; Shimana, H.; Tomimori, T. Studies on the constituents of Clematis species. VI1. The
constituents of Clematis stans SIEB. et ZUCC. Chem. Pharm. Bull. 1995, 43, 2187-2194.
0. Reher, G.; Budesinsky, M. Triterpenoids from plants of the Sanguisorbeae. Phytochemistry 1992,
1, 3909-3914.
3
1. Mimaki, Y.; Fukushima, M.; Yokosuka, A.; Sashida, Y.; Furuya, S.; Sakagami, H. Triterpene
glycosides from the roots of Sanguisorba officinalis. Phytochemistry 2001, 57, 773-779.
2. Jia, Z.J.; Liu, X.Q.; Liu, Z.M. Triterpenoids from Sanguisorba alpina. Phytochemistry 1993, 32,
1
55-159.
3. Chouksey, B.K.; Srivastava, S.K. New constituent from the roots of Terminalia arjuna:
Antifungal agent. Indian J. Chem. Sect. B: Org. Chem. Incl. Med. Chem. 2001, 40B, 354-356.
4. Durham, D.G.; Liu, X.; Richards, R.M. A triterpene from Rubus pinfaensis. Phytochemistry 1994,
3
6, 1469-1472.
5. Xu, H.X.; Zeng, F.Q.; Wan, M.; Sim, K.Y. Anti-HIV triterpene acids from Geum japonicum.
J. Nat. Prod. 1996, 59, 643-645.
6. Zhou, X.H.; Kasai, R.; Ohtani, K.; Tanaka, O.; Nie, R.L.; Yang, C.R.; Zhou, J.; Yamasaki, K.
Oleanane and ursane glucosides from Rubus species. Phytochemistry 1992, 31, 3642-3644.

Molecules 2011, 16
4651
1
1
7. Cheng, D.L.; Cao, X.P. Pomolic acid derivatives from the root of Sanguisorba officinalis.
Phytochemistry 1992, 31, 1317-1320.
8. Tanachatchairatana, T.; Bremner, B.J.; Chokchaisiri, R.; Suksamrarn, A. Antimycobacterial
activity of cinnamate-based esters of the triterpenes betulinic, oleanolic and ursolic acids. Chem.
Pharm. Bull. 2008, 56, 194-198.
1
9. Li, W.; Fu, H.W.; Bai, H.; Sasaki, T.; Kato, H.; Koike, K. Triterpenoid saponins from Rubus
ellipticus var. obcordatus. J. Nat. Prod. 2009, 72, 1755-1760.
Sample Availability: Samples of the compounds are available from the authors.
©
2011 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/).
(