Triterpenoid saponins from Dipsacus asper and their activities in vitro

Article abstract of DOI:10.1080/10286020.2011.598858

Two new triterpenoid saponins 1 and 2, along with six known saponins 3-8, were isolated from the roots of Dipsacus asper. The structures of new compounds were established as 3-O-β- d-glucopyranosyl-(1→4)-[α-l- rhamnopyranosyl-(1→3)]- β- d-glucopyranosyl-(1→3)-α- l-rhamnopyranosyl-(1→2)-α- l-arabinopyranosyl-hederagenin-28-O- β- d-glucopyranoside (dipsacus saponin J, 1) and 3-O-α- l-arabinopyranosyl-hederagenin-28-O-β- d-glucopyranosyl-(1→6)-β- d-glucopyranosyl-(1→6)-β- d-glucopyranoside (dipsacus saponin K, 2). The structures were determined by extensive analysis of their spectroscopic data. Compounds 6 and 7 could significantly stimulate UMR106 cell proliferation and increase alkaline phosphatase activities in UMR106 cell at the concentration of 4μM.

Full text of DOI:10.1080/10286020.2011.598858

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Triterpenoid saponins from Dipsacus
asper and their activities in vitro
Jing-Jing Liu a , Xin-Luan Wang b , Bao-Lin Guo a , Wen-Hua Huang
a , Pei-Gen Xiao a , Chao-Qing Huang a , Li-Zhen Zheng b , Ge Zhang
b , Ling Qin b & Guang-Zhong Tu c
a Key Laboratory of Bioactive Substances and Resources Utilization
of Chinese Herbal Medicine, Ministry of Education , Institute
of Medicinal Plants Development, Chinese Academy of Medical
Sciences and Peking Union Medical College , Beijing, 100193, China
b Musculoskeletal Research Laboratory, Department of
Orthopaedics & Traumatology , The Chinese University of Hong
Kong , Hong Kong SAR, China
c Beijing Institute of Microchemistry , Beijing, 100091, China
Published online: 10 Aug 2011.
To cite this article: Jing-Jing Liu , Xin-Luan Wang , Bao-Lin Guo , Wen-Hua Huang , Pei-Gen Xiao ,
Chao-Qing Huang , Li-Zhen Zheng , Ge Zhang , Ling Qin & Guang-Zhong Tu (2011) Triterpenoid
saponins from Dipsacus asper and their activities in vitro , Journal of Asian Natural Products
Research, 13:9, 851-860, DOI: 10.1080/10286020.2011.598858
To link to this article: http://dx.doi.org/10.1080/10286020.2011.598858
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Journal of Asian Natural Products Research
Vol. 13, No. 9, September 2011, 851–860
Triterpenoid saponins from Dipsacus asper and their activities in vitro
Jing-Jing Liua, Xin-Luan Wangb, Bao-Lin Guoa*, Wen-Hua Huanga, Pei-Gen Xiaoa,
Chao-Qing Huanga, Li-Zhen Zhengb, Ge Zhangb, Ling Qinb and Guang-Zhong Tuc
aKey Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine,
Ministry of Education, Institute of Medicinal Plants Development, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100193, China; Musculoskeletal Research
b
Laboratory, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong,
Hong Kong SAR, China; Beijing Institute of Microchemistry, Beijing 100091, China
c
(Received 1 April 2011; final version received 17 June 2011)
Two new triterpenoid saponins 1 and 2, along with six known saponins 3–8, were
isolated from the roots of Dipsacus asper. The structures of new compounds were
established as 3-O-b-D-glucopyranosyl-(1 ! 4)-[a-L-rhamnopyranosyl-(1 ! 3)]-b-D-
glucopyranosyl-(1 ! 3)-a-L-rhamnopyranosyl-(1 ! 2)-a-L-arabinopyranosyl-hedera-
genin-28-O-b-D-glucopyranoside (dipsacus saponin J, 1) and 3-O-a-L-arabinopyr-
anosyl-hederagenin-28-O-b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranosyl-(1 ! 6)-
b-D-glucopyranoside (dipsacus saponin K, 2). The structures were determined by
extensive analysis of their spectroscopic data. Compounds 6 and 7 could significantly
stimulate UMR106 cell proliferation and increase alkaline phosphatase activities in
UMR106 cell at the concentration of 4 mM.
Keywords: Dipsacus asper; triterpenoid saponin; anti-osteoporosis; UMR106 cell;
cytotoxicity
1. Introduction
nins have been shown to have potent
cytotoxicity [4]. In this paper, we describe
the isolation and structural elucidation of
eight triterpenoid saponins, including two
newsaponins, dipsacussaponinsJ(1)andK
(2), as well as six known compounds (3–8)
(Figure 1). The in vitro activities of the
isolated compounds were screened using
UMR106 cells for osteoprotective effect
and A375, BGC823, MCF7, SW1990, and
HEPG2 cells for cytotoxicity.
Dipsacus asper Wall. ex Henry1 (Dipsaca-
ceae) is widely distributed in southwest of
China. Theroots of D. asper have been used
as a tonic and analgesic agent in traditional
Chinese medicine for the treatment of low
backpain, kneepain, andbonefractures[1].
Previous studies have been performed on
the constituents of Radix Dipsaci
(Xuduan), including those on triterpene
glycosides, iridoids, caffeoylquinic acid,
and so on. Radix Dipsaci extract (RDE)
could prevent osteoporosis through
increasing bone mass and changing bone
histomorphology [2,3]. But few studies on
osteoprotective effect have been carried out
on monomeric compounds from RDE.
Furthermore, some of the dipsacus sapo-
2. Results and discussion
Compound 1 was obtained as a white
amorphous powder. The HR-ESI-MS
exhibited an ion at m/z 1405.6615
[M þ Na]þ, which was in accordance
with the molecular formula of C65H106
*Corresponding author. Email: guobaolin010@163.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.598858
http://www.informaworld.com

852
J.-J. Liu et al.
R1
glc1-4(rha1-3)glc1-3rha1-2ara-
ara- glc1-6glc1-6glc-
R2
30
29
21
1
2
3
4
5
6
7
8
glc-
19
18
20
12
26
ara-
ara-
glc-
glc1-6glc-
glc1-6glc-
glc1-6glc-
glc1-6glc-
glc-
22 O
C28
13
11
9
25
17
1
4
glc1-3rha1-2ara-
14
16
2
10
glc1-4(rha1-3)glc1-3rha1-2ara-
xyl1-4glc1-4(rha1-3)glc1-3rha1-2ara-
xyl1-4glc1-4(rha1-3)glc1-3rha1-2ara-
8
15
OR2
3
27
5
7
R1O
6
CH2OH
24
23
Figure 1. Structures of compounds 1–8.
O31. The IR spectrum revealed absorption
bands at 3412 (OH), 2939 (CZH), 1732
(CvO), 1637 (CvC), and 1074 (CZOZC)
constants of the anomeric protons from 6.5
to 8.0 Hz were deduced to be b-D-glucose
or a-L-arabinose, and single signals to be a-
L-rhamnose [6]. Furthermore, the a-
anomeric configuration of L-rhamnose
was confirmed by the chemical shift of C-
5 (dC 68.8 or 69.6) [7]. By comparing the
13C NMR spectral data of 1 with those of
hederagenin [5], C-3 (dC 81.0) was
observed to have a distinct downfield shift
and C-28 (dC 176.4) an upfield shift,
respectively, indicating that sugar moieties
were attached to those positions [8]. The
1H and 13C NMR spectral data (Table 2) of
the bisdesmosidic residues were assigned
starting from the anomeric protons by
means of the TOCSY, 1H–1H COSY,
HSQC, HMBC, and NOESY spectra
obtained for this compound. Linkage of
the sugar units at C-3 of the aglycone was
established from the following HMBC
correlations: H-1 (dH 5.01) of arabinose
with C-3 (dC 81.0) of the aglycone, H-1 (dH
6.22) of rhamnose with C-2 (dC 75.3) of
arabinose, H-1 (dH 5.35) of glucose with C-
3 (dC 82.8) of rhamnose, H-1 (dH 6.36) of
rhamnose’ with C-3 (dC 79.3) of glucose,
H-1 (dH 5.45) of glucose’ with C-4 (dC
75.3) of glucose (Figure 2). The sugar chain
at C-28 was established from the HMBC
correlations of H-1 (dH 6.31) of glucose’’
with C-28 (dC 176.4) of the aglycone.
1
cm21. The resonances in the H and 13C
NMR spectra for six methyls [dH/dC 0.86
(s)/23.7 (Me-30), dH/dC 0.87 (s)/33.1 (Me-
29), dH/dC 0.96 (s)/16.2 (Me-25), dH/dC
1.08 (s)/17.5 (Me-26), dH/dC 1.09 (s)/14.1
(Me-24), and dH/dC 1.15 (s)/26.1 (Me-27)],
an olefinic bond [dH/dC 5.40 (br s)/122.9
(C-12), 144.1(C-13)], andanestercarbonyl
[dC 176.4 (C-28)] suggested that 1 was an
olean-12-en-28-oic acid derivative. By
comparing the 13C NMR spectral data
with those reported [5], hederagenin was
identified as the aglycone (Table 1).
Six anomeric resonances at dH/dC 5.01
(d, J ¼ 6.5 Hz)/104.7, dH/dC 5.35
(d, J ¼ 8.0 Hz)/106.4, dH/dC 5.45 (d,
J ¼ 7.5 Hz)/104.4, dH/dC 6.22 (s)/101.4,
dH/dC 6.31 (d, J ¼ 7.5 Hz)/95.8, and dH/
dC 6.36 (s)/101.9 indicated the presence
of sugar moieties. The components of the
above sugar moieties were three hexoses,
two deoxyhexoses, and one pentose,
which were inferred from the ESI-MSn
data at m/z 1405 [M þ Na]þ, 1243
[M þ Na 2 162]þ, 1097 [M þ Na 2
162 2 146]þ, 773 [M þ Na 2 162 2
146 2 2 £ 162]þ, 495 [M þ Na 2
162 2 146 2 2 £ 162 2 146 2 132]þ,
and confirmed by the calculation of
relative molecular mass (1382 ¼ 472
aglycone þ 3 £ 162 þ 2 £ 146 þ 132).
Acid hydrolysis and GC-MS examination
of 1 suggested the existence of D-glucose,
L-rhamnose, and L-arabinose. The coupling
1
Careful investigation of the H and 13C
NMR spectral data of sugar chain at C-3
established that it was quite similar to those
reported for dipsacus saponins XI and XII
[9]. Evidence in ESI-MSn data also

Journal of Asian Natural Products Research
853
Table 1. 13C (150 MHz) and 1H (600 MHz) NMR spectral data for aglycone moieties of
compounds 1 and 2 (C5D5N, d in ppm and J in Hz).
1
2
a
dC
dH
dC
dH
1
2
3
4
5
6
7
8
39.1 (t)
26.4 (t)
81.0 (d)
43.6 (s)
47.6 (d)
18.1 (t)
32.7 (t)
39.9 (s)
48.2 (d)
36.9 (s)
23.9 (t)
122.9 (d)
144.1 (s)
42.1 (s)
28.3 (t)
23.4 (t)
47.0 (s)
41.7 (d)
46.2 (t)
30.8 (s)
34.0 (t)
32.6 (t)
64.0 (t)
14.1 (q)
16.2 (q)
17.5 (q)
26.1 (q)
176.4 (s)
33.1 (q)
23.7 (q)
1.05 (m), 1.54 (m)
1.99 (m), 2.19 (m)
4.26 (m)
38.8 (t)
26.1 (t)
81.9 (d)
43.5 (s)
47.6 (d)
18.3 (t)
32.8 (t)
40.0 (s)
48.2 (d)
37.0 (s)
23.7 (t)
122.9 (d)
144.2 (s)
42.1 (s)
28.3 (t)
23.4 (t)
47.0 (s)
41.7 (d)
46.2 (t)
30.7 (s)
34.0 (t)
32.6 (t)
64.5 (t)
13.6 (q)
16.2 (q)
17.6 (q)
26.1 (q)
176.5 (s)
33.1 (q)
23.7 (q)
1.03 (m), 1.56 (m)
2.20 (m), 2.26 (m)
4.24 (m)
–
1.65 (m)
1.33 (m), 1.65 (m)
1.58 (m), 1.74 (m)
–
–
1.70 (m)
1.26 (m), 1.64 (m)
1.30 (m), 1.51 (m)
–
9
1.72 (m)
1.74 (m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
–
–
1.91 (m), 2.01 (m)
5.40 (br s)
1.92 (m), 2.01 (m)
5.39 (br s)
–
–
–
–
1.05 (m), 2.27 (m)
1.91 (m), 2.01 (m)
–
3.16 (dd, J ¼ 14.0, 3.5)
1.21 (m), 1.70 (m)
–
1.06 (m), 1.31 (m)
1.73 (m), 1.80 (m)
3.84 (d, J ¼ 7.5), 4.22 (m)
1.09 (3H, s)
0.96 (3H, s)
1.08 (3H, s)
1.15 (3H, s)
–
1.07 (m), 2.27 (m)
1.87 (m), 1.91 (m)
–
3.15 (dd, J ¼ 13.8, 3.0)
1.19 (m), 1.69 (m)
–
1.09 (m), 1.29 (m)
1.72 (m), 1.87 (m)
3.67 (d, J ¼ 10.2), 4.26 (m)
0.91 (3H, s)
0.96 (3H, s)
1.10 (3H, s)
1.16 (3H, s)
–
0.87 (3H, s)
0.86 (3H, s)
0.81 (3H, s)
0.83 (3H, s)
Note: a Multiplicity determined by DEPT (1358) and HSQC.
supported this deduction. Finally, the
structure of compound 1 was elucidated
as 3-O-b-D-glucopyranosyl-(1 ! 4)-[a-L-
rhamnopyranosyl-(1 ! 3)]-b-D-glucopyr-
anosyl-(1 ! 3)-a-L-rhamnopyranosyl-
(1 ! 2)-a-L-arabinopyranosyl-hedera-
genin-28-O-b-D-glucopyranoside, named
dipsacus saponin J (Figure 1).
1653 (CvC), and 1076 (CZOZC) cm21
.
By comparing the 1H and 13C NMR
spectral data of compound 1 with those
reported in the literature [5], hederagenin
was identified as the aglycone (Table 1).
The sugar moieties were composed of three
hexoses and one pentose, which was
inferred from the calculation of relative
molecular mass (1090 ¼ 472 aglycone þ
3 £ 162 þ 132). Acid hydrolysis and GC-
MS examination of 2 suggested the
presence of D-glucose and L-arabinose.
The b-anomeric configuration for the
glucopyranose and a-configuration for the
arabinose were determined from their
Compound 2 was obtained as a white
amorphous powder. The HR-ESI-MS
exhibited an ion at m/z 1113.5419
[M þ Na]þ, which was in accordance
with the molecular formula of C53H86O23.
The IR spectrum revealed absorption bands
at 3399 (OH), 2927 (CZH), 1739 (CvO),

854
J.-J. Liu et al.
d
d
d
d
d

Journal of Asian Natural Products Research
855
coupling constants (3JH1,H2: 7.2–8.1 Hz).
The 13C NMR chemical shifts of C-3 (dC
81.9) and C-28 (dC 176.5) indicated that 2
was abidesmosidic saponin with glycosidic
linkages at C-3 and C-28. The 1H and 13
C
NMR spectral data (Table 2) of the
bisdesmosidic residues were assigned
starting from the anomeric protons by
means of the HSQC and HMBC spectra
obtained for this compound. The anomeric
proton signals were dH/dC 6.23 (d, J ¼
8.1 Hz)/95.6, dH/dC 5.01 (d, J ¼ 7.8 Hz)/
105.7, dH/dC 4.96 (d, J ¼ 7.2 Hz)/106.7,
and dH/dC 4.93 (d, J ¼ 7.8 Hz)/105.3,
according to the HSQC spectrum. The
long-range correlations between H-1
(dH 4.96) of arabinose and C-3 (dC 81.9)
of the aglycone indicated that Ara moiety
linked to the C-3 of hederagenin, and
correlations between H-1 (dH 6.23) of
glucose and C-28 (dC 176.5) of the
aglycone, between H-1 (dH 4.93) of glc0
and C-6 (dC 69.4) of glc, and between H-1
(dH 5.01) of glc00 and C-6 (dC 70.0) of glc0
indicated that the trisaccharide moiety
linked to C-28 of hederagenin was to be
glc(1 ! 6)glc(1 ! 6)glc. By comparing
the 13C NMR spectral data of compound 2
with those of dipsacus saponin VI [5,10],
the glycosylation shift of glc0-C-6 (dC 70.0)
and one more group of glucopyranose
signals (glc00) conformed this deduction
(Figure 2). Then, the structure of 2 can be
elucidated as 3-O-a-L-arabinopyranosyl-
hederagenin-28-O-b-D-glucopyranosyl-
(1 ! 6)-b-D-glucopyranosyl-(1 ! 6)-b-
D-glucopyranoside, named as dipsacus
saponin K (Figure 1).
Known compounds were identified as
HN saponin F (3) [10], dipsacus saponin
VI (4) [5,10], macranthoidin A (5) [10,11],
dipsacus saponin XII (6) [9], dipsacus
saponin X (7) [12], and 3-O-b-D-
xylopyranosyl(1 ! 4)-b-D-glucopyrano-
syl-(1 ! 4)]-[a-L-rhamnopyranosyl(1 !
3)]-b-D-glucopyranosyl(1 ! 3)-a-L-rha-
mnopyranosyl-(1 ! 2)-a-L-arabinopyra-
nosyl-hederagenin-28-O-b-D-glucopyra-
noside (8) [13].

856
J.-J. Liu et al.
O
C
O
OH
O
O
CH2OH
HO
OH
O
HO
O
H
OH
H
HO
O
HO
H
OH
O
O
OH
OH
O
O
OH
HMBC correlations
O
HO
HO
H
OH
H
Compound 1
O
H
HO
OH
HO
O
C
O
OH
O
O
CH2OH
O
HO
OH
OH
H
H
O
OH
HO
O
HO
H
O
H
OH
HO
HO
O
OH
Compound 2
HO
HO
Figure 2. Selected HMBC correlations for compounds 1 and 2.
As one of the best characterized clonal
osteoblastic cell lines, UMR106 was
used to study the mechanisms of drugs on
osteoporosis [14,15]. All isolated com-
pounds were tested on UMR106 cell lines
for their anti-osteoporosis activities,
except 1 and 3 due to their poor
dissolvability. The results given in Table 3
are expressed as relative percent prolifer-
ation of UMR106 cells at concentrations of
0.4, 4, and 40 mM. The six compounds
exerted a biphasic effect on cell prolifer-
ation, being slightly inhibitory at low
concentration (0.4 mM, except compound
7) and high concentration (40 mM, except
compound 2), and stimulation at middle
concentration (4 mM, except compound 4).
Compounds 6 and 7 displayed significant
promotion on UMR106 cells and revealed
anti-osteoporotic potential. Furthermore,
as one of the markers of osteoblastic
differentiation, alkaline phosphatase
(ALP) activity was measured in the
UMR106 cells. It can be seen from Table
3 that all tested compounds showed
significant increasing ALP activity at the
concentration of 4 mM.
The cytotoxic activities of the isolated
compounds were evaluated against A375,
BGC823, MCF7, SW1990, and HEPG2

Journal of Asian Natural Products Research
857
Table 3. Effect of isolates 2 and 4–8 on UMR106 cell proliferation and ALP activity.
Proliferation rate (%)ab
Relative increased
percentage of ALP
activity (%)c
Sample
0.4 mM
4 mM
40 mM
2
4
5
6
7
8
20.6 ^ 0.11
219.0 ^ 0.01
25.8 ^ 0.05
23.3 ^ 0.06
10.0 ^ 0.04
13.2 ^ 0.04
25.4 ^ 0.02
4.0 ^ 0.07
24.8 ^ 0.09
29.7 ^ 0.12
12.6 ^ 0.06
8.9 ^ 0.03
226.0 ^ 0.06
211.8 ^ 0.05
211.7 ^ 0.06
216.3 ^ 0.02
226.2 ^ 0.04
27.5
90.8
69.5 (0.056)
29.6
39.0 (0.093)
*
*
**
**
**
*
**
*
22.3 ^ 0.04
32.2 (0.077)
Notes: a The proliferation rate (%) is calculated as 100 £ (value of sample/value of control–1). Each value
represents the mean ^ SD of two individual experiments (n ¼ 3/experiments).
b Significantly different from the control group by t-test ( p , 0.05, p , 0.01).
*
**
c Tests were operated under the middle concentration of 4 mM. Relative increased percentage is calculated as
100 £ (value of sample/value of control–1) and exact values of p between 0.05 and 0.1 are shown in parentheses.
cell lines according to the MTT assay. The
result was that none of those compounds
expressed inhibition more than 50% at the
concentration from 0.1 to 80 mM. A study
by Tran Manh Hung showed that cytotoxic
saponins from D. asper had similar free
carboxyl at C-28, but the saponins with
sugar moiety linked at C-28 through an
ester bond exhibited IC50 values more than
30 mg/ml [4]. It suggested that bidesmosi-
dic triterpenoid saponins isolated from D.
asper had non-cytotoxic activities.
TLC were obtained from Qingdao Marine
Chemical Company (Qingdao, China).
Sephadex LH-20 from Mitsubishi Chemi-
cal Corporation (Tokyo, Japan) and macro-
porous resin D101 (20–40 mesh) from
Tianjin Nankai Hecheng S&T Co. Ltd
(Tianjin, China) were also used for column
chromatography. Preparative HPLC was
carried out using Waters 600 pump with
YMC-C18 column (250 £ 10 mm i.d.)
and 2487 detector. Gas chromatographic
analysis was carried out with an Agilent
6890 system equipped with a Mass
spectrograph 5973 detector (Agilent Tech-
nologies, Santa Clara, CA, USA). The
column was a HP-5 capillary column
3. Experimental
3.1 General experimental procedures
(30 m £ 0.25 mm £ 0.25 mm,
Agilent
Optical rotations were measured with a
Perkin-Elmer polarimeter. IR (KBr-disks)
spectra were recorded by a Shimadzu
FTIR-8400S spectrometer. Mass spectra
were obtained on a JEOL JMS-T100 CS
(HR-ESI-MS) and a Thermo Scientific
LTQ Orbitrap XL (ESI-MS). NMR exper-
iments were recorded in C5D5N on Bruker
AV-600 spectrometers. Solvents used were
of analytical grade and obtained from
Beijing Chemical Works (Beijing, China)
or chromatographic grade obtained from
Burdick & Jackson Company (Swedes-
boro, NJ, USA). Silica gel for column
chromatography and silica gel GF254 for
Technologies).
3.2 Plant material
The roots of D. asper were collected from
Chengdu, Sichuan Province of China, in
November 2007. It was identified by Prof.
Bao-Lin Guo, Insititute of Medicinal Plant
Development (IMPLAD), China Academy
of Medical Sciences, Beijing, China. The
voucher specimens had been deposited in
the Herbarium of IMPLAD (No.
2007101).

858
J.-J. Liu et al.
3.3 Extraction and isolation
3.3.1 Dipsacus saponin J (1)
Dried roots (4.2 kg) of D. asper were
powdered and extracted with 70% aqueous
ethanol (33 liters £ 3) under reflux. After
evaporation, the residue was separated by
column chromatography on macroporous
resin D101 with an EtOH/H2O (0:100,
30:70, 50:50, 95:5) gradient system to give
four fractions (XUD-1 to -4). Fraction
XUD-3 (453 g) was applied to a silica gel
column using a CHCl3/MeOH (v:v ¼ 9:1,
8:2, 7.5:2.5, 7:3, 5:5, 0:10) gradient system
to give six fractions (XUD-3-1 to -6). After
recrystallization from MeOH, 2 g of
compound 3 was obtained from fraction
XUD-3-2 (16.2 g). Fraction XUD-3-3
(10.1 g) was subjected to Sephadex LH-
20 column with MeOH as eluent and
compound 4 (100 mg) was obtained.
Fraction XUD-3-4 (18.7 g) was separated
by silica gel column (100 mesh) with
CHCl3/MeOH/H2O (7:3:0.2, 6.5:3.5:0.2,
6:4:0.2, 5.5:4.5:0.2) gradient system to
give four fractions (XUD-3-4-1 to -4).
After fraction XUD-3-4-2 was further
fractionated by silica gel (200–300
mesh) column with n-BuOH/EtOAc/H2O
(4:1:2, underlayer) as eluent and YMC-C18
column (MeOH/H2O, 57:43) through
HPLC purification, compounds 5 (93 mg)
and 2 (6 mg) were obtained. Compound 1
(49 mg) was obtained from fraction XUD-
3-4-3 by silica gel (H) chromatography
with CHCl3/MeOH/H2O (6.8:3.2:1, under-
layer) as eluent. Compound 8 (46 mg) was
gained from Fraction XUD-3-4-4 from
silica gel (H) column chromatography
with n-BuOH/EtOAc/H2O (4:1:2, under-
layer) as eluent and Sephadex LH-20
column eluted with Me2CO/MeOH (3:7).
Fraction XUD-3-5 (17.0 g) was subjected
to silica gel (H) column eluted with n-
BuOH/EtOAc/H2O (4:1:2, underlayer), to
yield compounds 6 (65 mg) and 7
(110 mg), respectively.
A white amorphous powder; mp 238–
20
2408C; ½aꢀD 20.57 (c ¼ 0.10, MeOH);
UV lmax (MeOH): 204 nm; IR nmax (KBr):
1
3412, 2939, 1732, 1637, 1074 cm21; H
and 13C NMR spectral data see Tables 1
and 2; HR-ESI-MS: m/z 1405.6615
[M þ Na]þ (calcd for C65H106O31Na,
1405.6616).
3.3.2 Dipsacus saponin K (2)
A white amorphous powder; mp 246–
20
2508C; ½aꢀD þ 5.2 (c ¼ 0.22, MeOH); IR
nmax (KBr): 3399, 2927, 1739, 1653,
1076 cm21 1H and 13C NMR spectral
;
data see Tables 1 and 2; HR-ESI-MS: m/z
1113.5419 [M þ Na]þ (calcd for
C53H86O23Na, 1113.5458).
3.4 Acid hydrolysis of compounds 1
and 2
Compounds 1 (6 mg) and 2 (3 mg) were
hydrolyzed with 2N TFA (5 ml) for 6 h at
boiling water bath. After extraction by
CH2Cl2 for three times, the remaining
aqueous layer was concentrated and
identified by TLC (CHCl3/MeOH/H2-
O ¼ 8:5:1) with authentic samples. Spots
were detected by spraying with 1%
anisaldehyde (in EtOH) followed by
heating.
3.5 Determination of sugar components
The absolute configuration of monosac-
charides from each aqueous layer was
determined by GC-MS of their trimethyl-
silylated derivatives. Column temperature:
180–2508C, programmed increase:
158C/min, carrier gas: N2 (1 ml/min);
injection temperature: 2508C, injection
volume: 1 ml, split ratio: 1/50. The
derivatives of L-arabinose, D-glucose, and
L-rhamnose were detected at tR: 13.02,
17.91, 16.26 min, respectively. By com-
paring with authentic samples, D-glucose,
L-rhamnose, and L-arabinose were

Journal of Asian Natural Products Research
859
detected from 1, and D-glucose and L-
arabinose from 2.
MCF-7 (human breast cancer), and
SW1990 (human pancreatic carcinoma)
were used for the MTT assay. Cells
(2.5 £ 103 cells/well) were incubated for
96 h with the test sample at final concen-
trations of 0.1, 1, 10, 40, and 80 mM/ml.
Then, the MTT solution (0.5 mg/ml) was
added, followed by further incubation for
4 h. The medium was then removed and the
resulting intracellular purple formazan
were dissolved in 180 ml of DMSO and
the absorbance was measured at 570 and
450 nm using a microplate reader. The
inhibition ratio ¼ 1 2 [OD (sample)
570 2 OD (sample) 450]/[OD (control)
570 2 OD (control) 450].
3.6 Colorimetric MTT (tetrazolium)
assay for cell proliferation
The UMR106 cell line was purchased from
the American Type Culture Collection
(Manassas, VA, USA), No. CRL-1661.
Cells were cultured in basal medium
constituted with Dulbecco’s modified
Eagle’s medium containing 10% (v/v)
fetal bovine serum and antibiotics for
incubation at 378C in a 5% CO2/95% air
humidified atmosphere [16]. UMR106
cells were placed into 96-well plate and
maintained for 24 h and then treated with
six saponins at the concentrations of 0.4, 4,
and 40 mM, and the medium with equiv-
alent vehicle as control. After 24 h,
50 ml/well of MTT solution (1 mg/ml) at
378C was added. The cells were incubated
for 4 h. The solution was then removed and
200 ml of DMSO was added into all wells.
The plates were then read using a
microplate reader system with a test
wavelength of 570 nm against a reference
wavelength of 650 nm [17].
Acknowledgements
This study was financially supported by
Guizhou Tongjitang Pharmaceutical Co. Ltd,
Guizhou, China.
Note
1. D. asper was sometimes named as
D. asperoides in China.
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