New Biologically Active Triterpenoid Saponins from Scabiosa tschiliensis

Article abstract of DOI:10.1021/np0304722

Eleven new triterpenoid saponins, scabiosaponins A-K (1-11), and hookerosides A (12) and B (13) were isolated from the whole plants of Scabiosa tschiliensis. The structures of the new compounds were established on the basis of extensive NMR (DEPT, DQF-COSY, HETCOR, TOCSY, HMQC, HMQC-TOCSY, HMBC, and NOESY) and MS studies coupled with chemical degradations. The biological activity of compounds 1-10, 12, and 13 and prosapogenin 1b were examined against pancreatic lipase. Scabiosaponins E, F, G, I (5, 6, 7, 9), hookerosides A, B (12, 13), and prosapogenin 1b all exhibited strong inhibition of pancreatic lipase in vitro.

Full text of DOI:10.1021/np0304722

604
J . Nat. Prod. 2004, 67, 604-613
New Biologica lly Active Tr iter p en oid Sa p on in s fr om Sca biosa tsch ilien sis
Quan Zheng,† Kazuo Koike,† Li-Kun Han,‡ Hiromichi Okuda,‡ and Tamotsu Nikaido*,†
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama,
Funabashi, Chiba 274-8510, J apan, and Department of Environmental and Symbiotic Sciences,
Prefectural University of Kumamoto, Tsukide 3-1-100, Kumamoto City, Kumamoto 862-8502, J apan
Received October 22, 2003
Eleven new triterpenoid saponins, scabiosaponins A-K (1-11), and hookerosides A (12) and B (13) were
isolated from the whole plants of Scabiosa tschiliensis. The structures of the new compounds were
established on the basis of extensive NMR (DEPT, DQF-COSY, HETCOR, TOCSY, HMQC, HMQC-
TOCSY, HMBC, and NOESY) and MS studies coupled with chemical degradations. The biological activity
of compounds 1-10, 12, and 13 and prosapogenin 1b were examined against pancreatic lipase.
Scabiosaponins E, F, G, I (5, 6, 7, 9), hookerosides A, B (12, 13), and prosapogenin 1b all exhibited strong
inhibition of pancreatic lipase in vitro.
Scabiosa tschiliensis Grun. (Dipsacaceae) is a perennial
herb widely distributed in the northeast region of the Inner
Mongolia autonomy district and the west of Hebei Province
of the People’s Republic of China. S. tschiliensis is called
“Meng Gu Shan Luo Bo”, and the flowers have long been
used as an herbal medicine for the treatment of headache,
fever, cough, and jaundice in Inner Mongolia.1 Scabiosa
species have also been used as a remedy for the skin
disease mange in Europe.2 However, there has been no
chemical or biological study reported on this plant. In this
paper, we report the isolation and structure elucidation of
11 new triterpenoid saponins (1-11) from the whole plant
of S. tschiliensis along with the pancreatic lipase inhibitory
activity of these compounds (1-10, 12, and 13).
with n-hexane, EtOAc, and n-BuOH. The n-BuOH-soluble
fraction, on chromatographic purification over Diaion HP-
20, followed by repeated medium-pressure liquid chroma-
tography (MPLC) and HPLC purification, afforded 11 new
compounds (1-11), along with two known saponins, hoo-
keroside A (12) and B (13). All these compounds were found
to possess gentiobiose, that is, a â-D-glucopyranosyl-(1f6)-
â-D-glucopyranoside unit directly linked to C-28 of the
aglycons.
Scabiosaponin A (1) had a molecular formula of C64H104O30
determined from its matrix-assisted laser desorption ion-
ization time-of-flight (MALDI-TOF) mass spectra (m/z 1375
[M + Na]+, 1391 [M + K]+) and from positive HRFABMS
data for the [M + Na]+ ion at m/z 1375 (measured m/z
1375.6511, calculated m/z 1375.6510) as well as 13C DEPT
NMR spectra. The spectral features and physicochemical
properties suggested 1 to be a triterpenoid saponin. The
IR spectrum exhibited absorptions at 3419 cm-1 (-OH),
1742 cm-1 (ester carbonyl), and 1644 cm-1 (double bond).
The seven tertiary methyl groups (δ 0.90 × 3, 1.10, 1.14,
and 1.27 × 2) and one trisubstituted olefinic proton (δ 5.42,
1
br s) observed in the H NMR spectrum coupled with the
information from the 13C NMR spectrum (seven sp3 carbons
at δ 15.7, 33.1, 23.7, 17.5, 17.2, 28.2, and 26.1 and two sp2
olefinic carbons at δ 122.9 and 144.2) indicated that the
aglycon possessed an olean-12-ene skeleton. After an
extensive 2D NMR study, the aglycon was identified as
oleanolic acid. The chemical shifts of C-3 (δ 88.7) and C-28
(δ 176.5) revealed that 1 was a bisdesmosidic glycoside.
Of the 64 carbon signals observed in the 13C NMR spectrum
of 1 (Tables 1 and 2), 30 were assigned to the aglycon and
34 to the oligosaccharide moieties. The 1H and 13C NMR
spectra of compound 1 exhibited six sugar anomeric protons
at δ 4.86 (d, J ) 6.0 Hz), 5.04 (d, J ) 7.6 Hz), 5.05 (d, J )
7.8 Hz), 5.28 (d, J ) 7.8 Hz), 6.24 (br s), and 6.27 (d, J )
8.0 Hz) and carbons at δ 95.7, 101.5, 103.7, 105.1, 105.3,
and 107.2 (Tables 2 and 3). The methyl carbon signal at δ
18.5 and the doublet methyl proton signal at δ 1.55 (3H,
d, J ) 6.0 Hz) indicated the presence of a 6-deoxy sugar.
The identities of the monosaccharides and the oligosac-
charide sequence were determined by a combination of
DQF-COSY, TOCSY, DEPT, HMQC, HMQC-TOCSY,
HMBC, and phase-sensitive NOESY NMR experiments.
Acid hydrolysis afforded oleanolic acid, and the monosac-
charides L-arabinose, L-rhamnose, D-xylose, and D-glucose
(1:1:1:3) by HPLC analysis following conversion to the
Resu lts a n d Discu ssion
A methanolic extract of the whole plant of S. tschiliensis
was suspended in H2O and then partitioned successively
* To whom correspondence should be addressed. Tel: 81-47-472-1391.
Fax: 81-47-472-1404. E-mail: nikaido@phar.toho-u.ac.jp.
† Toho University.
‡ Prefectural University of Kumamoto.
10.1021/np0304722 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/12/2004

Triterpenoid Saponins from Scabiosa
J ournal of Natural Products, 2004, Vol. 67, No. 4 605
Ta ble 1. 13C NMR Spectroscopic Data (δ) for the Aglycon Moieties of 1-13, 1a , 1b, and 1c (125 MHz in pyridine-d5)a
position
1
1a
1b
1c
2
3
4
5
6
7
8
9
10
11
1
39.0
26.7
88.7
39.6
56.0
18.6
33.1
39.9
48.1
37.1
23.8
122.9
144.2
42.1
28.3
23.4
47.1
41.7
46.3
30.8
34.0
32.6
28.2
17.2
15.7
17.5
26.1
176.5
33.1
23.7
38.8
26.5
88.5
39.5
55.9
18.4
33.0
39.8
48.0
36.9
23.3
122.7
144.0
42.0
28.1
23.7
46.9
41.6
46.1
30.7
33.9
32.4
28.1
17.1
15.5
17.3
26.0
176.3
33.1
23.5
38.9
26.6
88.7
39.6
56.0
18.5
33.2
39.7
48.0
37.0
23.7
122.5
144.8
42.2
28.3
23.8
46.7
42.0
46.5
30.9
34.2
33.2
28.2
17.1
15.5
17.4
26.2
180.2
33.3
23.7
38.6
26.6
88.5
39.4
55.7
18.4
33.1
39.6
47.9
36.9
23.6
122.4
144.7
42.0
28.2
23.7
46.5
41.9
46.3
30.8
34.1
33.1
28.1
16.8
15.4
17.3
26.0
180.0
33.1
23.6
39.0
26.6
88.8
39.6
56.0
18.6
33.1
39.9
48.1
37.1
23.8
122.9
144.2
42.2
28.3
23.4
47.1
41.7
46.3
30.8
34.0
32.6
28.2
17.2
15.7
17.5
26.1
176.5
33.1
23.7
39.0
26.7
88.7
39.6
56.1
18.6
33.1
40.0
48.1
37.1
23.8
122.9
144.2
42.2
28.3
23.4
47.1
41.7
46.3
30.8
34.1
32.6
28.2
17.2
15.7
17.5
26.1
176.5
33.1
23.7
39.0
26.6
88.8
39.5
56.1
18.6
33.2
40.0
48.1
37.1
23.9
122.9
144.2
42.2
28.3
23.5
47.1
41.8
46.3
30.8
34.1
32.6
28.1
17.0
15.7
17.6
26.1
176.6
33.1
23.7
39.0
26.9
88.6
39.7
56.1
18.5
33.1
39.9
48.1
37.1
23.8
123.1
144.2
42.2
28.3
23.4
47.1
41.7
46.3
30.8
34.0
32.6
28.2
17.4
15.7
17.5
26.1
176.5
33.1
23.7
39.1
26.9
88.7
39.7
56.2
18.6
33.2
40.0
48.1
37.1
23.8
122.9
144.2
42.2
28.3
23.5
47.1
41.7
46.3
30.8
34.1
32.6
28.3
17.3
15.7
17.5
26.1
176.5
33.2
23.7
39.0
26.9
88.5
39.7
56.1
18.5
33.0
39.9
48.1
37.1
23.8
122.9
144.2
42.2
28.2
23.4
47.1
41.7
46.3
30.8
34.0
32.6
28.2
17.4
15.7
17.5
26.1
176.5
33.1
23.7
39.0
26.7
88.8
39.6
56.1
18.7
33.5
40.5
47.8
37.0
24.0
128.3
139.3
42.1
29.3
26.1
48.7
54.3
72.7
42.0
26.7
37.8
28.2
17.2
15.7
17.4
24.6
177.0
27.0
16.7
39.0
26.6
88.9
39.5
56.0
18.7
33.4
40.5
47.7
37.0
24.0
128.4
139.2
42.1
29.3
26.1
48.7
54.3
72.7
42.0
26.6
37.7
28.2
17.1
15.7
17.4
24.5
177.0
27.0
16.6
38.8
26.6
88.8
39.6
56.1
18.7
33.2
40.2
48.3
37.2
24.1
123.0
144.3
42.1
29.0
28.0
46.5
44.5
81.1
35.5
29.0
33.0
28.2
17.0
15.6
17.5
24.8
177.3
28.7
24.8
38.8
26.7
88.8
39.6
56.2
18.8
33.2
40.2
48.4
37.2
24.2
123.7
144.4
42.1
29.1
28.0
46.6
44.6
81.1
35.6
29.0
33.1
28.2
17.2
15.6
17.6
24.9
177.3
28.8
24.8
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a
Assignments were based on DQF-COSY, TOCSY, DEPT, HETCOR, NOESY, and HMBC experiments.
1-[(S)-N-acetyl-R-methylbenzylamino]-1-deoxyalditol ac-
etate derivatives.3,4 All the monosaccharides were in the
pyranose forms, as determined from their 13C NMR data.
The â-anomeric configurations for the glucose and xylose
pletely methylated by means of the Hakomori method10,11
and had a retention time identical with authentic methy-
lated gentiobiose by GLC. Thus, the sugar sequence of C-28
of the aglycon was determined to be â-D-gentiobiose (â-D-
glucopyranosyl-(1f6)-â-D-glucopyranoside). On the basis
of all the foregoing evidence, scabiosaponin A (1) was
elucidated as 3-O-â-D-glucopyranosyl-(1f4)-â-D-xylopyra-
nosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyra-
nosyloleanolic acid 28-O-â-D-glucopyranosyl-(1f6)-â-D-
glucopyranoside.
3
units were determined from their J H1,H2 coupling constants
(7.6-8.0 Hz), and the arabinose unit was determined to
3
have an R-configuration on the basis of the J H1,H2 (6.0 Hz)
values observed in the 4C1 forms. These configurations were
also confirmed from the NOE relationships between H-1
and H-3 and between H-1 and H-5. For the rhamnose
moiety, the anomeric proton was observed as a singlet. The
1H nonsplitting pattern and the three-bond strong HMBC
correlations from the anomeric proton to C-3 and C-5 (the
dihedral angles between H-1 and C-3 and H-1 and C-5
about 180°) indicated that the anomeric proton was equa-
torial, thus possessing an R-configuration in the 1C4 form.5
The sequence of the glycan part connected to C-3 of the
aglycon was deduced from the following HMBC correla-
tions: H-1 (δ 5.05) of glucose with C-4 (δ 77.9) of xylose,
H-1 (δ 5.28) of xylose with C-3 (δ 83.1) of rhamnose, H-1
(δ 6.24) of rhamnose with C-2 (δ 75.3) of arabinose, H-1 (δ
4.86) of arabinose with C-3 (δ 88.7) of the aglycon (Figure
1). The disaccharide part at C-28 was established by the
following HMBC information: the correlations between H-1
(δ 5.04) of glucose (terminal) and C-6 (δ 69.4) of glucose
(inner) and between the H-1 (δ 6.27) of glucose (inner) and
C-28 (δ 176.5) of the aglycon (Figure 1). The same conclu-
sion with regard to the sugar sequence was also drawn
from the NOESY experiments (Figure 1). Further support-
ing information concerning the sugar sequence was fur-
nished by enzymatic degradation. Hydrolysis of 1 with
naringinase for 10 h afforded prosapogenin 1a and glucose,
indicating that both C-3 and C-28 sugar chains possessed
terminal â-D-glucoses. Hydrolysis of 1 with naringinase for
2 weeks afforded prosapogenin 1c,6 which upon acid
hydrolysis yielded oleanolic acid and L-arabinose. On
selective cleavage of the ester-glycoside linkage with
anhydrous LiI and 2,6-lutidine in anhydrous methanol,7,8
compound 1 afforded prosapogenin 1b,9 which was com-
Scabiosaponin B (2) had a molecular formula of C69H112O34
determined from its positive ion HRFAB mass spectrum
1
and from 13C DEPT NMR data. Its H and 13C NMR spectra
indicated that compound 2 possessed the same aglycon as
that of 1 but differed in the sugar moiety (Tables 2 and 3).
The chemical shifts of C-3 and C-28 revealed that 2 was a
1
bisdesmosidic glycoside. The H and 13C NMR spectra of 2
exhibited seven anomeric protons and carbons (Tables 2
and 3). Acid hydrolysis afforded oleanolic acid and the
component sugars, which were identified as L-arabinose,
1
L-rhamnose, D-xylose, and D-glucose (1:1:2:3). The H and
13C NMR chemical shift assignments were accomplished
by a combination of DQF-COSY, TOCSY, DEPT, HMQC,

606 J ournal of Natural Products, 2004, Vol. 67, No. 4
Zheng et al.
Ta ble 2. 13C NMR Spectroscopic Data (δ) for the Sugar Moieties of 1-13, 1a , 1b, and 1c (125 MHz in pyridine-d5)a
1
1a
1b
1c
2
3
4
5
6
7
8
9
10
11
12b
13b
3-O-sugar
arabinose
(xylose)
1
105.1 105.2 105.1 107.4 105.0 105.1 104.8 106.1 106.3 106.2 105.2 105.2 105.3 105.3 106.1 106.0
2
3
4
5
75.3
74.5
69.2
65.5
75.0
74.6
69.2
65.6
75.5
74.4
69.1
65.4
74.5
72.8
69.4
66.6
75.5
74.2
69.0
65.3
75.9
74.3
79.8
65.0
76.5
73.7
79.3
64.2
77.0
79.8
71.6
67.1
77.2
79.5
71.6
67.0
77.0
79.6
71.5
67.0
75.5
74.4
69.2
65.6
75.7
74.2
69.1
65.4
75.6
74.3
69.2
65.6
75.3
74.6
69.3
65.6
77.2
79.6
71.5
67.0
77.4
79.5
71.5
67.0
rhamnose
1
101.5 101.3 101.5
101.5 101.1 101.8 101.5 101.6 101.5 101.5 101.5 101.6 101.5 101.5 101.5
2
3
4
5
71.9
83.1
73.0
69.7
18.5
71.9
82.9
72.9
69.6
18.3
71.9
83.1
73.0
69.6
18.4
71.9
83.1
73.0
69.7
18.4
71.7
83.1
72.9
69.7
18.5
72.3
72.5
74.1
69.9
18.6
72.0
83.1
73.0
69.7
18.6
71.6
83.4
73.0
69.8
18.6
71.7
83.7
73.1
69.7
18.6
71.7
83.5
73.0
69.7
18.4
71.6
83.3
72.9
69.7
18.4
71.7
83.3
73.0
69.8
18.4
72.0
83.2
73.0
69.7
18.5
71.8
83.2
72.9
69.6
18.6
71.8
83.1
72.9
69.7
18.6
6
xylose
1
2
3
4
107.2 107.4 107.1
107.1 107.1
107.6
75.7
78.5
71.0
67.5
107.2 107.2 107.1
75.3
76.4
77.9
64.9
75.5
78.4
71.0
67.4
75.3
76.3
77.9
64.9
75.3
76.2
77.6
64.8
75.2
76.3
77.9
64.9
75.3
76.4
78.0
65.0
75.3
76.4
77.9
65.0
75.3
76.2
77.7
64.9
5
glucose
1
2
3
4
5
6
103.7
74.3
78.2
71.7
78.8
62.6
103.6
74.3
78.2
71.7
78.8
62.6
103.2 103.6 106.3
106.9 106.7 106.5 106.7 106.8 103.7 103.7 103.2
74.0
76.1
80.7
76.9
61.6
74.3
78.2
71.7
78.8
62.7
75.5
78.6
71.4
78.8
62.7
76.0
78.6
71.6
78.6
62.6
75.5
76.8
81.1
76.8
61.1
75.5
76.6
81.1
76.6
61.7
75.9
78.7
71.5
78.5
62.5
75.9
78.4
71.5
78.5
62.5
74.3
78.2
71.7
78.9
62.7
74.2
78.7
71.6
78.8
62.7
74.0
76.1
80.7
76.9
61.7
glucose′
(xylose′)
1
2
3
4
5
6
105.5 106.4
105.0 104.9
105.5
74.9
78.3
70.8
67.4
74.9
78.3
70.8
67.4
75.5
78.5
71.4
78.7
62.6
74.8
78.8
71.5
78.5
62.4
74.7
78.8
71.5
78.4
62.4
28-O-sugar
glucose (inner)
1
2
3
4
5
6
95.7
73.9
78.8
70.9
78.0
69.4
95.6
74.0
79.2
71.0
78.8
62.1
95.7
73.9
78.7
71.0
78.0
69.5
95.7
73.9
78.7
71.0
78.0
69.5
95.7
74.0
78.8
71.1
78.0
69.5
95.7
73.9
78.5
71.2
78.0
69.5
95.7
74.0
78.4
71.1
78.0
60.5
95.7
74.0
78.2
71.0
78.0
69.5
95.7
73.8
78.4
71.1
77.9
69.6
95.7
73.8
78.4
71.1
77.9
69.6
95.7
73.9
78.3
71.0
77.9
69.4
95.8
74.0
78.4
71.0
78.0
69.4
95.7
73.9
78.2
71.0
78.0
69.5
95.7
74.0
78.7
71.1
78.0
69.5
glucose
(terminal)
1
2
3
4
5
6
105.3
75.2
78.4
71.5
78.5
62.6
105.2 105.2 105.3 105.3 105.3 105.3 105.3 105.2 105.2 105.4 105.2 105.2
75.2
78.4
71.6
78.4
62.7
75.2
78.4
71.6
78.4
62.7
75.2
78.5
71.7
78.5
62.8
75.2
78.8
71.6
78.4
62.7
75.2
78.8
71.6
78.4
62.7
75.2
78.4
71.5
78.5
62.7
75.2
78.2
71.6
78.4
62.7
75.1
78.3
71.6
78.4
62.7
75.1
78.7
71.5
78.4
62.7
75.2
78.4
71.6
78.5
62.6
75.2
78.4
71.7
78.4
62.7
75.2
78.4
71.6
78.4
62.7
a
Assignments were based on DQF-COSY, TOCSY, DEPT, HETCOR, HMQC, HMQC-TOCSY, NOESY, and HMBC experiments. b Earlier
assignments due to sugar moieties (ref 15) were corrected.
HMQC-TOCSY, HMBC, and phase-sensitive NOESY ex-
acid) as that in 1 and 2. It contained seven sugars, one
more hexose (δ 106.4, δ 5.11, d, J ) 7.8 Hz) than 1 (Tables
2 and 3). Acid hydrolysis afforded oleanolic acid and the
component sugars, which were identified as L-arabinose,
L-rhamnose, D-xylose, and D-glucose (1:1:1:4). Detailed
NMR analysis indicated that 3 has the same glycan parts,
a gentiobiose at C-28 and an arabinose directly linked to
C-3. Most of its remaining 13C NMR signals were almost
the same as that in 1 except for C-4 of arabinose, being
downfield shifted to δ 79.8 and showing one more set of
signals attributed to a terminal glucose. The above infor-
mation indicated that 3 had a branched glycan part at C-3.
Thus, scabiosaponin C (3) was elucidated as 3-O-[â-D-
glucopyranosyl-(1f4)-â-D-xylopyranosyl-(1f3)-R-L-rham-
nopyranosy-(1f2)][â-D-glucopyranosyl-(1f4)]-R-L-arabinopy-
ranosyloleanolic acid 28-O-â-D-glucopyranosyl-(1f6)-â-D-
glucopyranoside.
1
periments. From its H and 13C NMR data (Tables 2 and
3), it was evident that the sugar structure at C-28 was the
same as that in 1 and the remaining five sugars at C-3
were of a linear structure with a terminal xylose linked to
C-4 (δ 80.7) of the inner glucose. On the basis of these
results, scabiosaponin B (2) was established as 3-O-â-D-
xylopyranosyl-(1f4)-â-D-glucopyranosyl-(1f4)-â-D-xylopy-
ranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopy-
ranosyloleanolic acid 28-O-â-D-glucopyranosyl-(1f6)-â-D-
glucopyranoside.
Scabiosaponin C (3) had a molecular formula of C70H114O35
determined from its positive ion HRFAB mass spectrum
and from 13C DEPT NMR data. The overall structure
assignment was accomplished using the same protocol as
in 1. Its 1H and 13C NMR data revealed that 3 was a
bisdesmosidic glycoside with the same aglycon (oleanolic

Triterpenoid Saponins from Scabiosa
J ournal of Natural Products, 2004, Vol. 67, No. 4 607
Ta ble 3. 1H NMR Spectroscopic Data (δ) for the Sugar Moieties of 1, 1a , 1b, 1c, and 2 (500 MHz in pyridine-d5)a
1
1a
1b
1c
2
3-O-sugar
arabinose(xylose)
1
2
3
4
5
4.86 d (6.0)
4.58 t (7.1)
4.26 t (5.7)
4.25 m
4.32 dd (10.3, 4.3)
3.82 d (10.3)
4.87 d (5.9)
4.60 d (7.5)
4.26 m
4.23 m
4.29 dd (10.4, 4.3)
3.83 d (10.4)
4.88 d (5.9)
4.56 t (8.2)
4.25 d (8.5)
4.24 m
4.32 dd (10.0, 5.7)
3.84 d (10.0)
4.78 d (7.1)
4.43 t (8.5)
4.17 dd (8.7, 3.2)
4.31 m
4.33 dd (13.0, 2.5)
3.84 m
4.86 d (6.0)
4.54 t (6.7)
4.25 m
4.25 m
4.29 d (10.0)
3.81 d (10.4)
rhamnose
1
6.24 br s
6.32 br s
6.24 br s
6.17 br s
2
3
4
5
4.88 t (5.5)
4.93 m
4.86 t (5.9)
4.86 t (6.0)
4.66 dd (9.4, 2.8)
4.45 d (9.4)
4.59 dq (9.4, 6.0)
1.55 d (5.9)
4.69 dd (9.6, 2.7)
4.47 dd (9.6, 3.2)
4.62 dq (9.4, 6.2)
1.55 d (6.0)
4.76 dd (9.6, 3.0)
4.48 d (9.6)
4.63 t (7.1)
1.55 d (6.0)
4.68 dd (9.4, 3.0)
4.47 dd (9.4, 5.0)
4.63 dq (9.4, 9.4)
1.57 d (6.2)
6
xylose
1
2
3
4
5
5.28 d (7.8)
5.38 d (7.5)
4.07 t (8.3)
4.15 t (8.3)
4.22 m
4.33 dd (10.1, 5.6)
3.71 d (10.1)
5.30 d (7.8)
5.25 d (7.6)
4.02 t (8.4)
4.16 t (8.4)
4.26 m
4.38 dd (11.7, 5.2)
3.63 t (11.2)
4.04 dd (8.5, 7.6)
4.19 dd (8.7, 2.8)
4.30 m
4.41 dd (11.5, 5.3)
3.64 t (11.5)
4.03 dd (7.8, 2.5)
4.17 dd (9.0, 3.0)
4.28 m
4.39 dd (11.4, 5.1)
3.65 d (11.4)
glucose
1
2
3
4
5
6
5.05 d (7.8)
4.04 dd (8.5, 7.6)
4.22 m
4.18 dd (8.7, 3.2)
3.99 m
4.55 dd (9.6, 2.5)
4.30 m
5.06 d (8.1)
4.98 d (7.8)
4.02 t (8.4)
4.18 t (8.4)
4.25 m
3.90 m
4.47 d (9.6)
4.35 m
4.02 dd (6.2, 2.5)
4.20 dd (6.2, 3.0)
4.17 dd (9.0, 3.0)
3.98 m
4.54 dd (11.7, 2.1)
4.30 m
glucose′ (xylose′)
1
5.06 d (7.7)
2
3
3.99 d (8.0)
4.07 d (8.7)
4
5
6
4.15 ddd (8.7, 5.7, 2.5)
4.24 dd (10.3, 5.7)
3.65 d (10.3)
28-O-sugar
glucose (inner)
1
2
3
4
5
6
6.27 d (8.0)
4.14 dd (8.9, 8.0)
4.23 m
4.36 m
4.12 m
4.72 br s
4.37 d (9.6, 2.5)
6.36 d (8.0)
4.21 m
4.05 m
4.35 m
4.28 m
4.49 d (9.6)
4.78 d (9.6, 3.0)
6.24 d (8.3)
4.13 m
4.20 t (8.7)
4.31 dd (8.9, 8.7)
4.11 m
4.70 d (9.6)
4.35 m
glucose (terminal)
1
2
3
4
5
6
5.04 d (7.6)
4.02 dd (9.4, 8.0)
4.19 m
4.22 m
3.89 m
4.47 dd (9.4, 3.2)
4.35 m
5.02 d (7.8)
3.98 m
4.18 dd (8.7, 3.2)
4.19 t (8.7)
3.87 m
4.44 d (9.6)
4.34 m
a
Assignment were based on DQF-COSY, TOCSY, DEPT, HETCOR, HMQC, HMQC-TOCSY, NOESY, and HMBC experiments.
F igu r e 1. Key HMBC and NOE correlations for the sugar of scabiosaponin A (1).
Scabiosaponin D (4) had a molecular formula of C59H96O26
determined from its positive ion HRFAB mass spectrum
and from 13C DEPT NMR data. Its proton and carbon NMR
data also indicated that 4 was a bisdesmosidic glycoside
based on oleanolic acid and contained five sugars. Acid
hydrolysis yielded oleanolic acid and the monosaccharides
L-arabinose, L-rhamnose, and D-glucose (1:1:3). Complete
1H and 13C NMR assignments were accomplished by a
combination of DQF-COSY, TOCSY, DEPT, HMQC, HMBC,
and phase-sensitive NOESY experiments. As with com-

608 J ournal of Natural Products, 2004, Vol. 67, No. 4
Zheng et al.
Ta ble 4. 1H NMR Spectroscopic Data (δ) for the Sugar Moieties of 3-8 (500 MHz in pyridine-d5)a
3
4
5
6
7
8
3-O-sugar
arabinose
(xylose)
1
2
4.74 d (6.2)
4.48 t (7.8)
4.79 d (5.7)
4.48 m
4.83 d (7.6)
4.21 m
4.80 d (7.2)
4.18 m
4.81 d (7.3)
4.23 m
4.82 d (5.9)
4.53 dd (13.1,
6.0)
3
4
5
4.21 m
4.25 m
4.40 dd (10.8,
4.7)
4.26 m
4.29 m
4.41 dd (12.1,
3.9)
4.19 m
4.19 m
4.35 dd (10.1,
4.8)
4.14 m
4.12 m
4.30 dd (11.0, 5.0)
4.16 dd (5.5,6.1)
4.22 m
4.33 dd (10.7, 5.1)
4.20 dd (5.2)
4.22 m
4.29 dd (11.6,
3.6)
3.78 d (10.8)
3.82 d (10.3)
3.71 d (10.1)
3.67 dd (11.0,
10.7)
3.70 dd (10.4, 10.7)
3.79 d (10.5)
rhamnose
1
2
3
4
6.18 br s
4.13 br s
4.65 dd (9.6, 2.7)
4.44 d (9.6)
6.11 br s
6.59 d (1.2)
5.00 br s
4.82 dd (9.6, 3.2)
4.54 dd (9.4, 2.5)
6.46 br s
5.02 br s
4.85 dd (9.3, 2.7)
4.50 d (9.4)
6.55 br s
6.18 br s
4.91 br s
4.75 m
4.48 dd (9.0,
2.6)
4.71 dd (8.9, 4.3)
4.58 dd (9.1, 3.0)
4.27 m
5.02 dd (5.0, 3.0)
4.84 dd (9.4, 3.0)
4.55 dd (9.6, 9.1)
5
4.61 dq (9.4, 6.2)
1.56 d (6.2)
4.59 dq (9.4,
6.3)
1.64 d (6.2)
4.78 dq (9.4, 6.2)
1.65 d (6.2)
4.73 dq (9.4, 6.2)
1.62 d (5.9)
4.78 dq (9.4, 6.2)
1.65 d (6.2)
4.61 dq (9.2, 6.2)
6
1.54 d (6.2)
xylose
1
2
3
4
5
5.25 d (7.8)
5.39 d (7.5)
4.09 t (8.2)
4.17 m
4.23 m
4.35 dd (10.1, 4.8)
3.73 d (10.1)
3.98 dd (8.7, 3.3)
4.15 dd (9.7, 3.0)
4.26 m
4.41 dd (11.7, 4.2)
3.66 t (11.7)
glucose
1
2
3
5.01 d (7.8)
4.00 t (8.5)
4.18 m
5.13 d (7.8)
4.03 t (9.1)
4.18 m
5.47 d (7.8)
4.09 m
4.24 t (8.5)
5.48 d (7.8)
4.12 dd (9.4, 8.0)
4.29 dd (9.0, 9.0)
5.41 d (8.0)
4.09 dd (9.2)
4.27 dd (10.1,
9.0)
4
5
4.13 dd (9.7, 3.0)
3.96 m
4.23 m
3.89 m
4.21 t (9.8)
3.95 m
4.38 dd (10.5, 2.7)
3.95 ddd (9.4, 6.0,
3.0)
4.37 t (9.1)
3.91 m
6
4.52 dd (11.5, 1.9)
4.27 m
4.48 m
4.36 dd (9.8, 3.2)
4.45 dd (9.2, 6.0)
4.37 dd (9.2, 4.1)
4.57 dd (9.4, 6.0)
4.42 dd (9.4, 3.6)
4.53 dd (9.1, 5.3)
4.38 dd (9.1, 2.3)
glucose′
(xylose′)
1
2
3
4
5
5.11 d (7.8)
4.01 d (8.5)
4.15 dd (8.7, 3.0)
4.20 m
5.22 d (7.8)
4.08 dd (8.4, 8.0)
4.24 m
4.20 dd (8.8, 5.7)
4.00 ddd (11.4, 4.1,
2.4)
5.18 d (7.8)
4.05 dd (8.2, 7.8)
4.24 m
4.19 dd (8.8, 5.2)
3.99 m
3.88 m
6
4.47 m
4.34 dd (11.4,
4.6)
4.52 dd (7.5, 2.5)
4.31 m
4.51 dd (7.3, 2.6)
4.27 dd (7.3, 5.2)
28-O-sugar
glucose
(inner)
1
2
3
4
5
6.24 d (8.3)
4.12 t (8.7)
4.20 m
4.30 t (9.9)
4.09 m
6.25 d (8.0)
4.13 m
4.19 m
4.31 m
4.11 m
6.26 d (8.3)
4.15 m
4.19 m
4.27 t (8.3)
4.12 m
6.24 d (8.0)
4.12 m
4.17 m
4.30 dd (9.1, 5.9)
4.11 m
6.28 d (8.0)
4.14 dd (9.4, 8.2)
4.22 dd (8.7, 8.8)
4.35 m
6.23 d (8.0)
4.15 m
4.21 m
4.31 m
4.15 m
4.14 m
6
4.69 d (11.4)
4.70 d (8.9, 4.3)
4.72 d (9.6)
4.69 dd (9.2, 6.7)
4.73 d (9.6)
4.72 dd (9.1, 2.7)
glucose
(terminal)
1
2
3
4
5
6
5.02 d (7.8)
4.02 t (8.5)
4.16 m
4.18 m
3.86 m
5.03 d (7.8)
3.99 dd (8.2, 7.8)
4.17 m
4.18 m
3.88 m
5.04 d (7.8)
4.00 t (8.0)
4.17 m
4.18 m
3.87 m
5.02 d (7.8)
3.99 dd (8.5, 8.0)
4.20 m
4.11 m
3.88 m
5.05 d (7.8)
4.01 t (7.1)
4.20 dd (8.8, 5.7)
4.22 dd (8.7, 8.8)
3.89 m
5.06 d (7.8)
4.02 t (6.9)
4.19 m
4.25 dd (8.4, 8.0)
3.89 m
4.45 d (11.5)
4.45 dd (12.1,
3.9)
4.47 d (11.2)
4.45 d (9.6)
4.48 dd (9.6, 2.3)
4.49 dd (9.0, 2.6)
4.32 dd (11.4,
4.6)
4.35 m
4.33 dd (11.2, 4.6)
4.33 dd (9.6, 5.3)
4.37 dd (9.6, 5.3)
4.36 dd (9.0, 5.0)
a
Assignment were based on DQF-COSY, TOCSY, DEPT, HETCOR, HMQC, HMQC-TOCSY, NOESY, and HMBC experiments.
pounds 1-3, 4 also possessed a gentiobiose unit at C-28
and an arabinose directly linked to C-3 of the oleanolic acid.
The remaining two sugars, L-rhamnose and D-glucose, were
determined to be linked to C-2 (δ 76.5) and C-4 (δ 79.3) of
the arabinose, the same substitution pattern as in 3. On
the basis of all the foregoing evidence, scabiosaponin D (4)
was elucidated as 3-O-[R-L-rhamnopyranosyl-(1f2)][â-D-
glucopyranosyl-(1f4)]-R-L-arabinopyranosyloleanolic acid
28-O-â-D-glucopyranosyl-(1f6)-â-D-glucopyranoside.
Scabiosaponin E (5) had a molecular formula of C58H94O25
determined from its positive ion HRFAB mass spectrum.
The aglycon was identified as oleanolic acid. Chemical
shifts of C-3 (δ 88.6) and C-28 (δ 176.5) revealed that 5
was a bisdesmosidic glycoside. The 1H and 13C NMR
spectra of 5 exhibited five sugar anomeric protons and
carbons (Tables 2 and 4). The identity and absolute
configurations of these sugars were determined to be
L-rhamnose, D-xylose, and D-glucose (1:2:2) by HPLC

Triterpenoid Saponins from Scabiosa
J ournal of Natural Products, 2004, Vol. 67, No. 4 609
F igu r e 2. Key NOE correlations of scabiosaponin H (8).
analysis following conversion to the 1-[(S)-N-acetyl-R-
methylbenzylamino]-1-deoxyalditol acetate derivatives.3,4
As a common feature of the saponins from this source, 5
also possessed a gentiobiose unit at C-28. Unlike the
previous saponins, 5 possessed a xylose linked to C-3 of
the aglycon instead of an arabinose. From the long-range
HMBC couplings, it was apparent that C-2 (δ 77.0) of the
xylose was substituted by an L-rhamnose. The remaining
xylose was determined to be linked at C-3 of the rhamnose.
Thus, scabiosaponin E (5) was elucidated as 3-O-â-D-
xylopyranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-â-D-xy-
lopyranosyloleanolic acid 28-O-â-D-glucopyranosyl-(1f6)-
â-D-glucopyranoside.
Scabiosaponin H (8) had a molecular formula of C65H106-
O32 determined from its positive ion HRFAB mass spec-
trum and from 13C DEPT NMR data. The IR spectrum
exhibited absorptions at 3399 cm-1 (-OH), 1731 cm-1 (ester
carbonyl), and 1646 cm-1 (double bond). The seven tertiary
methyl groups and one trisubstituted olefinic proton (δ
1
5.54, br s) observed in the H NMR spectrum coupled with
the information from the 13C NMR spectrum (seven sp3
carbons and two sp2 olefinic carbons at δ 128.3 for C-12
and 139.3 for C-13) indicated that the aglycon possessed
an urs-12-ene skeleton. From the chemical shifts of C-18
(δ 54.3) and C-22 (δ 37.8) of the aglycon, the C-30 methyl
group was determined to be in R-orientation.12 Further-
more, the strong NOE correlation between the C-29 methyl
and H-18 (â-orientated) of the aglycon in the NOESY
experiment (Figure 2) indicated the C-29 methyl group on
the â-face. The C-19 resonated at δ 72.7, indicating this
carbon was hydroxylated. Thus, the aglycon was identified
as pomolic acid,12 which was further confirmed after
hydrolysis with cellulase and NMR analysis.12 The chemi-
cal shifts of C-3 and C-28 revealed that 8 was a bisdesmo-
sidic glycoside. Six sugar anomeric protons and carbons
were displayed in its 1H and 13C NMR spectra. Acid
hydrolysis afforded L-arabinose, L-rhamnose, and D-glucose
(1:1:4). Detailed NMR analysis indicated that 8 also
contained a gentiobiose unit at C-28 and an arabinose at
C-3 of the aglycon. The arabinose was substituted at C-2
by a rhamnose, which, in turn, was glycoslyted by a glucose
at its C-3 position. The C-4 of the glucose resonated at δ
81.8, indicating that the remaining glucose was connected
to this position. Thus, scabiosaponin H (8) was elucidated
as 3-O-â-D-glucopyranosyl-(1f4)-â-D-glucopyranosyl-(1f3)-
R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyranosylpomol-
ic acid 28-O-â-D-glucopyranosyl-(1f6)-â-D-glucopyranoside.
Scabiosaponin F (6) had a molecular formula of C59H96O26
determined from its positive ion HRFAB mass spectrum
and from 13C DEPT NMR data. Five anomeric protons and
carbons were displayed in its 1H and 13C NMR spectra. Acid
hydrolysis afforded oleanolic acid and the component
sugars L-rhamnose, D-xylose, and D-glucose (1:1:3). Com-
plete 1H and 13C NMR assignments were furnished by a
combination of 2D-NMR experiments. The spectral features
indicated that compound 6 was closely related to 5, in
which a gentiobiose unit was linked to C-28 and a xylose
attached directly to C-3 of the aglycon. As in 5, the
rhamnose was also linked to C-2 of the xylose, as indicated
from the long-range coupling from an HMBC experiment.
The remaining glucose was found linked to C-3 of the
rhamnose. On the basis of the foregoing evidence, scabio-
saponin F (6) was elucidated as 3-O-â-D-glucopyranosyl-
(1f3)-R-L-rhamnopyranosyl-(1f2)-â-D-xylopyranosylolean-
olic acid 28-O-â-D-glucopyranosyl-(1f6)-â-D-glucopyranoside.
Scabiosaponin G (7) had a molecular formula of C65H106O31
determined from its positive ion HRFAB mass spectrum
1
and from 13C DEPT NMR data. H and 13C NMR spectra
indicated that 7 possessed the same aglycon as 6 but
differed in the sugar parts. Six sugar units were indicated,
as there were six anomeric protons and carbons displayed
in their 1H and 13C NMR spectra. Acid hydrolysis yielded
oleanolic acid and D-xylose, L-rhamnose, and D-glucose (1:
1:4). The linkage positions were established using the
HMBC and NOESY correlations. As in compound 6, 7 also
contained a gentiobiose unit at C-28 and a xylose directly
linked to C-3 of its aglycon. The xylose was substituted at
C-2 by a rhamnose, which, in turn, was glycosylated by a
glucose at its C-3 position. The C-4 of the glucose resonated
at δ 81.8, indicating that the remaining glucose was
connected to this position. Accordingly, scabiosaponin G (7)
was elucidated as 3-O-â-D-glucopyranosyl-(1f4)-â-D-glu-
copyranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-â-D-xylopy-
ranosyloleanolic acid 28-O-â-D-glucopyranosyl-(1f6)-â-D-
glucopyranoside.
Scabiosaponin I (9) had a molecular formula of C59H96O27
determined from its positive ion HRFAB mass spectrum
and from 13C DEPT NMR data. The overall structure
assignment was made using the same protocol as for 8.
From its completely assigned NMR data, it was apparent
that 9 possessed the same aglycon as 8 but differed in the
sugar parts. Most of the C-13 resonances were the same
except that the signals attributed to the terminal glucose
in 8 disappeared. Hydrolysis of 9 with cellulase afforded
the aglycon, identified as pomolic acid. The sugars were
determined to be L-arabinose, L-rhamnose, and D-glucose
(1:1:3) by HPLC analysis. The sequences of the sugar
chains were further confirmed from the HMBC and NOE-
SY correlations. On the basis of all the foregoing evidence,
scabiosaponin I (9) was elucidated as 3-O-â-D-glucopyra-
nosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyra-

610 J ournal of Natural Products, 2004, Vol. 67, No. 4
Zheng et al.
nosylpomolic acid 28-O-â-D-glucopyranosyl-(1f6)-â-D-glu-
copyranoside.
of pancreatic lipase.16 Pancreatic lipase is a key enzyme
for lipid breakdown to absorb fatty acids. The inhibition of
dietary fat absorption has been reported to be one effective
way of managing obesity. Therefore, the application of a
lipase inhibitor such as orlistat17 has proven its usefulness
for the treatment of obesity.
The pancreatic lipase inhibitory action of scabiosaponins
A-J (1-10) and hookerosides A and B (12, 13) was
evaluated in an assay system using triolein emulsified with
phosphatidylcholine. As shown in Figure 3, scabiosaponins
E, F, G, and I (5, 6, 7, 9) and hookerosides A and B (12,
13) exhibited strong inhibitory activity on pancreatic lipase.
Moreover, prosapogenin 1b showed the strongest inhibitory
activity against pancreatic lipase among the compounds
tested (Figure 4). The inhibitory activity of prosapogenin
1b against pancreatic lipase at 0.12 mg/mL was similar to
that of orlistat at 0.005 mg/mL. Experiments are now in
progress to clarify the antiobesity action of the pure
saponins isolated from S. tschiliensis in mice fed a high-
fat diet.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
measured using a Yanaco microscope apparatus and are
uncorrected. IR spectra were determined using a J ASCO 300E
FTIR spectrometer. Optical rotations were measured using a
J ASCO DIP-370 digital polarimeter. 1H and 13C NMR were
recorded using a J EOL ECP-500 NMR spectrometer. Chemical
shifts were expressed in δ (ppm) referring to TMS. MALDI-
TOF MS and HRFABMS were conducted using a PerSeptive
Biosystems Voyager DE-STR and J EOL J MS-700 mass spec-
trometer, respectively. Diaion HP-20 (ion-exchange resin,
Mitsubishi Chemical Corporation, Tokyo, J apan), silica gel
(silica gel 60, Merck), and ODS (Chromatorex 1020TM, 100-
200 mesh, Fuji Sylisia Chemical, Ltd., Aichi, J apan) were used
for column chromatography. Preparative HPLC was performed
using ODS columns (PEGASIL ODS, Senshu Pak, 10 mm i.d.
× 250 mm, Senshu Scientific Co., Ltd., Tokyo, J apan, and
ODS-A, YMC-Pack, 20 mm i.d. × 250 mm, YMC Co., Ltd.
Kyoto, J apan, detector, UV 210 nm). GLC, Shimadzu GC-7A;
column, silicone OV-17 on Uniport HP (80-100 mesh), 3 mm
i.d. × 2.1 m; column temperature, 122 °C; carrier gas, N2; flow
rate, 40 mL/min.
Scabiosaponin J (10) had a molecular formula of C59H96O27
determined from its positive ion HRFAB mass spectrum
and from 13C DEPT NMR data. The IR spectrum exhibited
absorptions at 3428 cm-1 (-OH), 1728 cm-1 (ester carbo-
nyl), and 1646 cm-1 (double bond). The seven tertiary
methyl groups and one trisubstituted olefinic proton ob-
1
served in the H NMR spectrum coupled with the informa-
tion from the 13C NMR spectrum (seven sp3 carbons and
two sp2 olefinic carbons at δ 123.0 and 144.3) indicated that
the aglycon possessed an olean-12-ene skeleton. After an
extensive 2D NMR study, the aglycon was identified as
siaresinolic acid.13 Hydrolysis of 10 with cellulase yielded
the aglycon, which showed NMR data identical to those of
an authentic sample.14 Acid hydrolysis of 10 afforded
L-arabinose, L-rhamnose, and D-glucose (1:1:3) by HPLC
1
analysis. The complete H and 13C NMR data assignment
was achieved by a combination of 2D-NMR experiments.
From its1H and 13C NMR data, it was apparent that 10
possessed the same sugar structures as that in 9 (Tables
2 and 5). Thus, scabiosaponin J (10) was elucidated as 3-O-
â-D-glucopyranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-R-L-
arabinopyranosylsiaresinolic acid 28-O-â-D-glucopyranosyl-
(1f6)-â-D-glucopyranoside.
P la n t Ma ter ia l. Scabiosa tschiliensis was collected from
Kaiyuan City, Liaoning Province, People’s Republic of China,
in August 1999, and identified by Prof. C. Chen (Liaoning
Normal University). A voucher specimen (TOHO-ST1999121)
has been deposited at the Department of Pharmacognosy, Toho
University.
Scabiosaponin K (11) had a molecular formula of
C
64H104O31 determined from its positive ion HRFAB mass
spectrum and from 13C DEPT NMR data. Hydrolysis of 11
with cellulase afforded siaresinolic acid. Compound 11
possessed the same oligosaccharide chains as 1, as indi-
Extr a ction a n d Isola tion . The air-dried and finely cut
whole plants of S. tschiliensis (4.8 kg) were extracted with
MeOH three times under reflux for 2 h. The combined MeOH
extracts were concentrated (760 g), suspended in H2O, and
then partitioned successively between n-hexane (85 g), EtOAc
(71 g), and n-BuOH (270 g). The n-BuOH-soluble fraction was
applied to a column of Diaion HP-20 and eluted with H2O and
40, 60, 80, and 100% MeOH. The fractions eluted with 80%
MeOH were combined and repeatedly chromatographed over
silica gel, ODS open columns, and MPLC to give several
saponin fractions. Further HPLC purification (50-68% MeOH
in H2O, UV detector, 210 nm) afforded 1 (250 mg), 2 (49 mg),
3 (28 mg), 4 (10.4 mg), 5 (25 mg), 6 (16 mg), 7 (12.5 mg), 8
(110 mg), 9 (120 mg), 10 (33 mg), 11 (6.8 mg), 12 (50 mg), and
13 (23 mg), respectively.
cated by the complete agreement of their H and 13C NMR
1
data (Tables 2 and 5). Thus, scabiosaponin K (11) was
established as 3-O-â-D-glucopyranosyl-(1f4)-â-D-xylopy-
ranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopy-
ranosylsiaresinolic acid 28-O-â-D-glucopyranosyl-(1f6)-â-
D-glucopyranoside.
Additionally, the previously reported compounds hook-
erosides A (12) and B (13)15 were also isolated and
identified by detailed NMR analyses.
According to the theory of traditional Chinese Medicine,
S. tschiliensis has the ability to clear the “heat” from inside
the body. Too much “heat” or rather energy accumulated
inside the body could lead to obesity or other kinds of
illnesses. On the basis of this notion, we decided to conduct
a pancreatic lipase inhibitory test on the saponins isolated.
It is well known that dietary fat is not directly absorbed
from the intestine unless it has been subjected to the action
Sca biosa p on in A (1): amorphous solid; mp 216-218 °C;
[R]20 -6.6° (c 0.10, MeOH); IR (KBr) νmax 3419, 2935, 1742,
D
1066 cm-1; H NMR (pyridine-d5, 500 MHz) δ 5.42 (1H, br s,
1
H-12), 3.29 (1H, dd, J ) 11.7, 4.1 Hz, H-3), 3.20 (1H, dd, J )
13.5, 3.7 Hz, H-18), 1.27 (6H, s, H3 of C-23, C-27), 1.14 (3H, s,
H3 of C-24), 1.10 (3H, s, H3 of C-26), 0.90 (9H, s, H3 of C-25,
C-29, C-30); other NMR data, see Tables 1-3; MALDI-TOF

Triterpenoid Saponins from Scabiosa
J ournal of Natural Products, 2004, Vol. 67, No. 4 611
Ta ble 5. 1H NMR Spectroscopic Data (δ) for the Sugar Moieties of 9-13 (500 MHz in pyridine-d5)a
9
10
11
12b
13b
3-O-sugar
arabinose (xylose)
1
2
3
4
5
4.82 d (5.9)
4.49 dd (11.5, 6.2)
4.19 m
4.22 m
4.27 dd (8.3, 2.9)
3.78 d (10.3)
4.83 d (6.0)
4.50 dd (12.1, 6.0)
4.19 m
4.22 m
4.28 dd (10.1, 3.7)
3.80 d (10.1)
4.87 d (6.0)
4.59 dd (10.0, 6.0)
4.26 m
4.25 m
4.35 dd (11.2, 5.7)
3.84 d (10.1)
4.82 d (7.3)
4.20 m
4.15 m
4.15 m
4.33 dd (11.0, 4.8)
3.69 d (11.0)
4.81 d (7.1)
4.19 dd (8.5, 7.7)
4.14 m
4.13 m
4.31 dd (11.0, 4.4)
3.67 d (11.0)
rhamnose
1
6.12 br s
6.15 d (1.2)
6.26 br s
6.51 br s
6.47 br s
2
3
4
5
4.95 m
4.78 dd (9.4, 2.9)
4.47 m
4.59 dq (9.4, 6.2)
1.54 d (5.9)
4.95 br s
4.79 dd (9.3, 3.2)
4.46 d (9.3)
4.61 dq (9.4, 6.2)
1.54 d (6.0)
4.89 m
4.93 m
4.93 br s
4.71 m
4.46 m
4.72 dq (9.4, 2.3)
1.63 d (6.2)
4.68 dd (9.3, 3.0)
4.48 dd (9.4, 2.5)
4.64 dq (9.3, 6.1)
1.55 d (6.2)
4.72 dd (9.3, 3.0)
4.48 dd (9.6, 2.5)
4.74 dq (9.8, 6.2)
1.64 d (6.2)
6
xylose
1
2
3
4
5
5.28 d (7.8)
4.03 dd (8.4,8.0)
4.22 m
4.30 m
4.42 dd (10.7, 5.0)
3.66 t (10.8)
5.28 d (7.6)
4.04 dd (8.1, 7.7)
4.18 m
4.29 m
4.40 dd (10.5, 5.0)
3.66 t (10.5)
5.26 d (7.6)
4.02 dd (8.3, 8.2)
4.14 m
4.25 m
4.39 dd (11.5, 5.2)
3.64 dd (11.5, 2.3)
glucose
1
2
3
4
5
6
5.43 d (7.8)
4.08 m
4.22 m
4.24 m
3.93 m
4.43 m
4.32 m
5.44 d (7.8)
4.08 m
4.26 m
4.26 m
3.94 m
4.44 dd (11.2, 2.3)
4.36 dd (11.2, 4.8)
5.05 d (7.8)
4.04 dd (8.4, 8.0)
4.21 m
4.20 m
4.00 ddd (10.7, 5.7, 2.8)
4.55 br s
5.02 d (7.8)
4.03 dd (8.1, 7.7)
4.20 m
4.17 m
3.96 m
4.52 dd (9.2, 6.0)
4.29 m
4.97 d (8.1)
4.00 dd (8.2, 8.0)
4.18 m
4.22 m
3.88 m
4.45 m
4.33 dd (9.6, 4.4)
4.32 dd (8.7, 8.7)
glucose′ (xylose′)
1
2
5.05 d (7.7)
3.97 m
3
4
4.07 t (8.7)
4.13 m
5
6
4.23 dd (7.3, 5.2)
3.63 dd (7.3, 2.3)
28-O-sugar
glucose (inner)
1
2
3
4
5
6
6.21 d (8.0)
4.15 m
4.21 m
4.28 dd (8.3, 2.9)
4.13 m
4.72 d (9.8)
4.35 dd (9.8, 6.4)
6.28 d (8.0)
4.13 dd (8.2, 8.2)
4.19 m
4.30 m
4.10 m
4.68 d (11.2)
4.34 dd (11.2, 4.8)
6.32 d (8.3)
4.15 dd (8.4, 8.3)
4.23 m
4.35 dd (11.2, 5.7)
4.13 ddd (13.7, 6.2, 2.2)
4.72 dd (9.6, 6.9)
4.35 dd (9.6, 8.5)
6.24 d (8.2)
4.14 m
4.18 m
4.28 m
4.12 m
4.70 dd (12.6, 2.3)
4.34 dd (12.6, 4.3)
6.21 d (8.2)
4.11 m
4.18 m
4.28 dd (9.2, 8.2)
4.09 m
4.68 dd (9.6, 2.7)
4.33 dd (9.6, 4.4)
glucose (terminal)
1
2
3
4
5
6
5.05 d (7.6)
4.00 t (6.9)
4.19 m
4.18 m
3.91 m
4.47 dd (11.2, 2.3)
4.36 dd (11.2, 5.3)
5.02 d (8.0)
3.98 t (6.6)
4.20 m
4.16 dd (8.2, 8.2)
3.88 m
4.45 dd (11.5, 2.3)
4.35 dd (11.5, 5.1)
5.04 d (7.6)
4.02 dd (8.4, 7.7)
4.20 m
5.02 d (7.8)
4.02 dd (8.1, 7.7)
4.16 m
4.18 m
3.87 m
4.45 d (9.8)
4.32 dd (9.8, 4.3)
5.00 d (7.8)
3.96 m
4.15 m
4.16 m
3.85 m
4.43 d (9.7)
4.31 dd (11.0, 4.4)
4.23 m
3.90 ddd (10.7, 5.6, 2.5)
4.48 dd (9.4, 2.5)
4.37 dd (9.6, 8.5)
a
Assignment were based on DQF-COSY, TOCSY, DEPT, HETCOR, HMQC, HMQC-TOCSY, NOESY, and HMBC experiments. b Earlier
assignments due to sugar moieties (ref 15) were corrected.
F igu r e 4. Effects of prosapogenin 1b and orlistat on pancreatic lipase
activity. Results are expressed as means ( SEM of three experiments.
(C: control; final concentration: mg/mL).
F igu r e 3. Effects of various saponins isolated from Scabiosa tschil-
iensis on pancreatic lipase activity. Results are expressed as means (
SEM of three experiments. (C: control; 1-10: scabiosaponins A-J ;
Sca biosa p on in B (2): amorphous solid; mp 230-231 °C;
12, 13: hookerosides A, B; final concentration: 1 mg/mL).
[R]25 -28.9° (c 0.30, MeOH); IR (KBr) νmax 3422, 2929, 1737,
D
1638, 1047 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.42 (1H,
MS (positive ion mode) m/z 1375 [M + Na]+, 1391 [M + K]+;
br s, H-12), 3.29 (1H, dd, J ) 11.5, 4.2 Hz, H-3), 3.20 (1H, dd,
HRFABMS (positive ion mode) m/z 1375.6511 [M + Na]+ (calcd
for C64H104O30, 1375.6510).
J ) 13.5, 3.9 Hz, H-18), 1.26 (6H, s, H3 of C-23, C-27), 1.12
(3H, s, H3 of C-24), 1.08 (3H, s, H3 of C-26), 0.90 (9H, s, H3 of

612 J ournal of Natural Products, 2004, Vol. 67, No. 4
Zheng et al.
C-25, C-29, C-30); other NMR data, see Tables 1-3; HR-
FABMS (positive ion mode) m/z 1507.6930 [M + Na]+ (calcd
for C69H112O34, 1507.6933).
(1H, dd, J ) 11.7, 4.1 Hz, H-3), 1.63, 1.32, 1.15, 1.13, 1.13,
1.02, 0.91 (each 3H, s, H3 of C-27, C-23, C-24, C-26, C-29, C-30,
C-25); other NMR data, see Tables 1, 2, and 5; MALDI-TOF
MS (positive ion mode) m/z 1259 [M + Na]+, 1275 [M + K]+;
HRFABMS (positive ion mode) m/z 1259.6016 [M + Na]+ (calcd
for C59H96O27, 1259.6037).
Sca biosa p on in C (3): amorphous solid; mp 219-220 °C;
[R]25 -19.3° (c 0.20, MeOH); IR (KBr) νmax 3420, 2926, 1736,
D
1638, 1068 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.42 (1H,
br s, H-12), 3.25 (1H, dd, J ) 11.4, 3.9 Hz, H-3), 3.20 (1H, dd,
J ) 13.7, 3.6 Hz, H-18), 1.27 (6H, s, H3 of C-23, C-27), 1.12
(3H, s, H3 of C-24), 1.09 (3H, s, H3 of C-26), 0.90 (9H, s, H3 of
C-25, C-29, C-30); other NMR data, see Tables 1, 2, and 4;
HRFABMS (positive ion mode) m/z 1537.6992 [M + Na]+ (calcd
for C70H114O35, 1537.7038).
Sca biosa p on in K (11): amorphous solid; mp 220-222 °C;
[R]20 -36.0° (c 0.10, MeOH); IR (KBr) νmax 3430, 2933, 1739,
D
1067 cm-1 ;1H NMR (pyridine-d5, 500 MHz) δ 5.51 (1H, br s,
H-12), 3.57 (1H, t-like, H-19), 3.54 (1H, br s, H-18), 3.29 (1H,
dd, J ) 11.7, 4.1 Hz, H-3), 1.65, 1.28, 1.17, 1.02, 0.93 (each
3H, s, H3 of C-27, C-23, C-24, C-30, C-25), 1.14 (6H, s, H3 of
C-26, C-29); other NMR data, see Tables 1, 2, and 5; MALDI-
TOF MS (positive ion mode) m/z 1391 [M + Na]+, 1407 [M +
K]+; HRFABMS (positive ion mode) m/z 1391.6508 [M + Na]+
(calcd for C64H104O31, 1391.6460).
Sca biosa p on in D (4): amorphous solid; mp 228-229 °C;
[R]25 -11.8° (c 0.17, MeOH); IR (KBr) νmax 3423, 2923,1743,
D
1646, 1247, 1030 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.42
(1H, br s, H-12), 3.22 (1H, H-3), 3.20 (1H, H-18), 1.25 (6H, s,
H3 of C-23, C-27), 1.17 (3H, s, H3 of C-24), 1.10 (3H, s, H3 of
C-26), 0.90 (9H, s, H3 of C-25, C-29, C-30); other NMR data,
see Tables 1, 2, and 4; HRFABMS (positive ion mode) m/z
1243.6066 [M + Na]+ (calcd for C59H96O26, 1243.6088).
Sca biosa p on in E (5): amorphous solid; mp 208-210 °C;
Acid Hyd r olysis a n d Deter m in a tion of th e Absolu te
Con figu r a tion of th e Mon osa cch a r id es. A solution of 1 (10
mg) in 1 M HCl (dioxane-H2O, 1:1, 2 mL) was heated at 100
°C for 2 h. After dioxane was removed, the solution was
extracted with EtOAc (2 mL × 3). The extract was washed
with H2O, dried over MgSO4, and evaporated to give oleanolic
acid (3 mg). The H2O layer was neutralized by passing through
an ion-exchange resin (Amberlite MB-3) column and concen-
trated to furnish a monosaccharide residue. The sugar residue
was dissolved in H2O (1 mL), to which a solution of (S)-(-)-
1-phenylethylamine (8 mg) and Na[BH3CN] (16 mg) in EtOH
(1 mL) were added. The mixture was allowed to stand
overnight, then acidified by addition of glacial HOAc acid (0.3
mL) and evaporated to dryness. The resulting solid was
acetylated with Ac2O anhydride (0.5 mL) in pyridine (0.3 mL)
at 100 °C for 1 h. After co-distillation with toluene, H2O (2
mL) was added to the residue, and the crude mixture was
passed through a Sep-pak Plus C18 cartridge (Waters) and
washed with H2O-MeCN (4:1, 1:1, each 5 mL). The H2O-
MeCN (1:1) eluate contained a mixture of the 1-[(S)-N-acetyl-
R-phenylethylamino]-1-deoxyalditol acetate derivatives of the
monosaccharides, which were identified by co-HPLC analysis
with standard sugars prepared under the same conditions.
HPLC conditions: column, PEGASIL ODS, 4.6 × 150 mm;
solvent, MeCN-H2O (33:67); flow rate, 0.8 mL/min; detection,
UV 230 nm; temperature, 40 °C. The derivatives of d-glucose,
D-xylose, L-rhamnose, and L-arabinose were detected with
retention time of 39.1, 26.4, 44.8, and 25.0 min, respectively.
Sugars in compounds 2-11 were also identified by the same
method.
[R]20 -22.9° (c 0.56, MeOH); IR (KBr) νmax 3295, 2945, 2745,
D
1353 cm-1; H NMR (pyridine-d5, 500 MHz) δ 5.41 (1H, br t,
1
J ) 3.3 Hz, H-12), 3.35 (1H, dd, J ) 11.7, 4.1 Hz, H-3), 3.20
(1H, dd, J ) 13.5, 3.9 Hz, H-18), 1.39 (3H, s, H3 of C-23), 1.26
(3H, s, H3 of C-27), 1.24 (3H, s, H3 of C-24), 1.09 (3H, s, H3 of
C-26), 0.89 (9H, s, H3 of C-25, C-29, C-30); other NMR data,
see Tables 1, 2, and 4; MALDI-TOF MS (positive ion mode)
m/z 1213 [M + Na]+, 1229 [M + K]+; HRFABMS (positive ion
mode) m/z 1213.5944 [M + Na]+ (calcd for C58H94O25, 1213.5982).
Sca biosa p on in F (6): amorphous solid; mp 218-219 °C;
[R]25 -11.1° (c 0.10, MeOH); IR (KBr) νmax 3410, 2926, 1646,
D
1533, 1378, 1043 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.40
(1H, br s, H-12), 3.32 (1H, dd, J ) 11.4, 4.1 Hz, H-3), 3.18
(1H, dd, J ) 13.3, 3.2 Hz, H-18), 1.38 (3H, s, H3 of C-23), 1.25
(3H, s, H3 of C-27), 1.21 (3H, s, H3 of C-24), 1.08 (3H, s, H3 of
C-26), 0.89 (9H, s, H3 of C-25, C-29, C-30); other NMR data,
see Tables 1, 2, and 4; HRFABMS (positive ion mode) m/z
1243.6066 [M + Na]+ (calcd for C59H96O26, 1243.6088).
Sca biosa p on in G (7): amorphous solid; mp 228-230 °C;
[R]21 -20.3° (c 1.28, MeOH); IR (KBr) νmax 3418, 2930,1736,
D
1643, 1380, 1058 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.41
(1H, br t-like, H-12), 3.33 (1H, dd, J ) 11.7, 4.1 Hz, H-3), 3.20
(1H, dd, J ) 13.8, 3.7 Hz, H-18), 1.37 (3H, s, H3 of C-23), 1.26
(3H, s, H3 of C-27), 1.23 (3H, s, H3 of C-24), 1.09 (3H, s, H3 of
C-26), 0.89 (9H, s, H3 of C-25, C-29, C-30); other NMR data,
see Tables 1, 2, and 4; MALDI-TOF MS (positive ion mode)
m/z 1405 [M + Na]+, 1421 [M + K]+; HRFABMS (positive ion
Olea n olic a cid : amorphous solid; mp 275-276 °C; [R]21
D
+82° (c 0.10, MeOH); IR (KBr) νmax 3456, 2940, 1696, 1036
cm-1; spectral data consistent with literature values.18
En zym a tic Hyd r olysis of Sca biosa p on in A (1). 1 (16
mg) dissolved in 0.2 M acetate buffer (pH 4.0, 5 mL) was
incubated with naringinase (Sigma Chemical Co., 32 mg). The
reaction mixture was stirred at 40 °C for 10 h. Then a small
amount of EtOH was added. After concentration, the residue
was purified by silica gel column chromatography (CHCl3-
MeOH-H2O, 10:5:1) to give prosapogenin 1a (8 mg) and
D-glucose. The prosapogenin 1c (2 mg) was obtained when the
incubation was prolonged for 2 weeks.
mode) m/z 1405.6616 [M + Na]+ (calcd for C65H106O31
1405.6613).
,
Sca biosa p on in H (8): amorphous solid; mp 230-232 °C;
[R]20 -11.3° (c 0.10, MeOH); IR (KBr) νmax 3399, 2928, 1731,
D
1646, 1378, 1071 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.54
(1H, br s, H-12), 3.28 (1H, dd, J ) 11.4, 3.9 Hz, H-3), 2.93
(1H, s, H-18), 1.33 (1H, m, H-20), 1.04 (3H, d, J ) 6.4 Hz, H3
of C-30), 1.70, 1.36, 1.29, 1.18, 1.16, 0.94 (each 3H, s, H3 of
C-27, C-29, C-23, C-26, C-24, C-25); other NMR data, see
Tables 1, 2, and 4; MALDI-TOF MS (positive ion mode) m/z
1421 [M + Na]+, 1437 [M + K]+; HRFABMS (positive ion
P r osa p ogen in (1a ): amorphous solid; mp 198-200 °C;
mode) m/z 1421.6602 [M + Na]+ (calcd for C65H106O32
1421.6565).
,
[R]20 -12.3° (c 0.78, MeOH); IR (KBr) νmax 3415, 2938, 1729,
D
1068 cm-1; H NMR (pyridine-d5, 500 MHz) δ 5.44 (1H, br s,
1
Sca biosa p on in I (9): amorphous solid; mp 213-215 °C;
H-12), 3.31 (1H, dd, J ) 11.7, 3.9 Hz, H-3), 3.22 (1H, dd, J )
13.3, 3.7 Hz, H-18), 1.33, 1.29, 1.19, 1.11, 0.93, 0.91, 0.89 (each
3H, s, H3 of C-23, C-27, C-24, C-26, C-29, C-30, C-25); other
NMR data, see Tables 1-3; MALDI-TOF MS (positive ion
mode) m/z 1051 [M + Na]+, 1067 [M + K]+.
[R]20 -15.0° (c 0.10, MeOH); IR (KBr) νmax 3429, 2928, 1730,
D
1070 cm-1 ; 1H NMR (pyridine-d5, 500 MHz) δ 5.55 (1H, br s,
H-12), 3.28 (1H, dd, J ) 11.7, 4.1 Hz, H-3), 2.92 (1H, s, H-18),
1.36 (1H, m, H-20), 1.03 (3H, d, J ) 6.6 Hz, H3 of C-30), 1.69,
1.36, 1.30, 1.17, 1.14, 0.94 (each 3H, s, H3 of C-27, C-29, C-23,
C-26, C-24, C-25); other NMR data, see Tables 1, 2, and 5;
MALDI-TOF MS (positive ion mode) m/z 1259 [M + Na]+, 1275
[M + K]+; HRFABMS (positive ion mode) m/z 1259.6053 [M
+ Na]+ (calcd for C59H96O27, 1259.6036).
Absolu te Con figu r a tion of th e Glu cose by HP LC. The
glucose obtained from compound 1 was determined to be of
D-type ([R]25 +37.8°, c 0.09, 24 h after dissolution in H2O) in
D
comparison with an authentic sample under the following
HPLC conditions: pump, J ASCO-980; detection, Shodex RI-
71; column, Asahi Pak NH2P-50, 4.6 × 150 mm; solvent,
MeCN-H2O (75:25); flow rate, 1.0 mL/min; tR, 11.2 min.
P r osa p ogen in (1c): amorphous solid; mp 249-252 °C;
Sca biosa p on in J (10): amorphous solid; mp 212-214 °C;
[R]20 -6.5° (c 0.10, MeOH); IR (KBr) νmax 3429, 2934, 1729,
D
1646, 1066 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.50 (1H,
s, H-12), 3.56 (1H, t-like, H-19), 3.53 (1H, br s, H-18), 3.28
[R]16 +64.3° (c 0.20, MeOH); IR (KBr) νmax 3427, 2939, 1701,
D

Triterpenoid Saponins from Scabiosa
J ournal of Natural Products, 2004, Vol. 67, No. 4 613
1650, 1459, 1380, 1074 cm-1; 1H NMR (pyridine-d5, 500 MHz)
δ 5.48 (1H, t-like, H-12), 3.35 (1H, dd, J ) 11.9, 4.3 Hz, H-3),
3.30 (1H, dd, J ) 13.5, 3.7 Hz, H-18), 1.30, 1.28, 1.03, 1.00,
0.96, 0.95, 0.85 (each 3H, s, H3 of C-23, C-27, C-24, C-26, C-29,
C-30, C-25); other NMR data, see Tables 1-3; MALDI-TOF
MS (positive ion mode) m/z 611 [M + Na]+, 627 [M + K]+.
Acid Hyd r olysis of 1c. Compound 1c (1 mg) was heated
in 1 mL of 1 M HCl (dioxane-H2O, 1:1) at 100 °C for 2 h. After
dioxane was removed, the solution was extracted with EtOAc
(2 mL × 3). The extract was washed with H2O and then
concentrated to give oleanolic acid. The monosaccharide por-
tion was neutralized by passing through an ion-exchange resin
(Amberlite MB-3) column, concentrated (dried overnight), then
treated with 1-(trimethylsilyl)imidazole. The TMSi derivative
of the monosaccharide was identified as arabinose by co-GLC
analysis with standard arabinose.
Selective Clea va ge of Ester -Glycosid e Lin k a ge of
Sca biosa p on in A (1). A solution of 1 (40 mg), anhydrous LiI
(40 mg), and 2,6-lutidine (3 mL) in anhydrous MeOH (1 mL)
was refluxed for 16 h at 140 °C. A solution of 50% EtOH (2
mL) was added to stop the reaction. The reaction mixture was
deionized with Amberlite MB-3 resin and concentrated to
dryness. The residue was chromatographed on Si gel [CHCl3-
MeOH-H2O (25:10:1, 15:10:1)] to give prosapogenin 1b (9 mg)
and methyl gentiobiose (4 mg).
in 9 mL of 0.1 M N-tris(hydroxymethyl)methyl-2-amino-
etheanesulfonic acid (TES) buffer (pH 7.0) containing 0.1 M
NaCl was sonicated for 5 min. This sonicated substrate
suspension (100 µL) was incubated with 50 µL (10 units) of
pancreatic lipase and 100 µL of various sample solutions for
30 min at 37 °C in a final volume of 250 µL. The amount of
released oleic acid was determined by the method of Zapf et
al.19 with a slight modification.20 The incubation mixture was
added to 3 mL aliquots of a 1:1 (v/v) mixture of CHCl3 and
n-heptane containing 2% (v/v) MeOH and extracted by shaking
the tubes horizontally for 10 min in a shaker. The mixture
was centrifuged at 2000g for 10 min, and the upper aqueous
phase was removed by suction. A copper reagent (1 mL) was
then added to the lower organic phase. The tube was shaken
for 10 min, the mixture was centrifuged at 2000g for 10 min,
and 0.5 mL of the upper organic phase, which contained copper
salts of the extracted free fatty acids, was treated with 0.5
mL of 0.1% (w/v) bathocuproine in CHCl3 containing 0.05%
(w/v) 3(2)-tert-butyl-4-hydroxyanisole. The absorbance was
then measured at 480 nm. Lipase activity was expressed as
micromoles of oleic acid released per milliliter of reaction
mixture per hour.
Ack n ow led gm en t. The authors are grateful to Dr. Zhong-
hua J ia (CAS) and Dr. Yangfang Su (Tianjin University,
China) for their help and Dr. Y. Asada (Kitasato University,
J apan) for HRFABMS measurements.
P r osa p ogen in (1b): amorphous solid; mp 214-216 °C;
[R]20 -18.4° (c 0.37, MeOH); IR (KBr) νmax 3430, 2936, 1695,
D
1379, 1074 cm-1; 1H NMR (pyridine-d5, 500 MHz) δ 5.49 (1H,
br s, H-12), 3.22 (1H, m, H-3), 3.22 (1H, m, H-18), 1.33, 1.30,
1.14, 1.03, 1.00, 0.95, 0.86 (each 3H, s, H3 of C-23, C-27, C-24,
C-30, C-26, C-29, C-25); other NMR data, see Tables 1-3;
MALDI-TOF MS (positive ion mode) m/z 1051 [M + Na]+, 1067
[M + K]+.
Refer en ces a n d Notes
(1) J iangsu New Medical College. Zhong Yao Da Ci Dian; Shanghai
People’s Public Health Publishing House: Shanghai, P. R. China,
1977; p 2464.
(2) Hotta, M.; Ogata, K.; Nitta, A.; Hosikawa, K.; Yanagi, M.; Yamazaki,
K. Useful Plants of the World; Heibonsha: Tokyo, J apan, 1989; p 959.
(3) Oshima, R.; Kumanotani, J . Chem. Lett. 1981, 943-946.
(4) Oshima, R.; Yamauchi, Y.; Kumanotani, J . Carbohydr. Res. 1982, 107,
169-176.
(5) J ia, Z.; Koike, K.; Nikaido, T. J . Nat. Prod. 1998, 61, 1368-1373.
(6) Higuchi, R.; Kawasaki, T. Chem. Pharm. Bull. 1976, 24, 1021-1032.
(7) Ohtani, K.; Mizutani, K.; Kasai, R.; Tanaka, O. Tetrahedron Lett.
1984, 25, 4537-4540.
(8) Wu, F.; Koike, K.; Ohmoto, T.; Chen, W. Chem. Pharm. Bull. 1989,
37, 2445-2447.
(9) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1982, 30, 859-865.
(10) Hakomori, S. J . Biochem. 1964, 58, 205-208.
(11) Needs, P. W.; Selvendran R. R. Carbohydr. Res. 1993, 245, 1-10.
(12) Kakuno, T.; Yoshikawa, K.; Arihara, S. Phytochemistry 1992, 31,
3553-3557.
(13) Aplin, R.; Hui, W.; Ho, C.; Yee, C. J . Chem. Soc., (C) 1971, 1067.
(14) Hata, C.; Kakuno, M.; Yoshikawa, K.; Arihara, S. Chem. Pharm. Bull.
1992, 40, 1990-1992.
(15) Tian, J .; Wu, F.; Qiu, M.; Nie, R. Phytochemistry 1993, 32, 1535-
1538.
Met h yla t ion of Met h yl Gen t ib iose via H a k om or i
Meth od . Compound 1d was identified as methyl gentiobiose
through comparison of the permethyl ether and an authentic
sample by GLC.10,11
En zym a tic Hyd r olysis of 8 a n d 10. Compound 8 (20 mg)
dissolved in EtOH-H2O (1:9, 3 mL) and 0.01 M NaH2PO4
buffer (pH 4.0, 3 mL) was incubated with cellulase (35 mg,
Tokyo Kasei) at 37 °C for 2 weeks. EtOH (2 mL) was added to
stop the reaction, and the solution was concentrated to
dryness. The residue was chromatographed on a silica gel
column with CHCl3-MeOH-H2O (250:40:1) to afford pomolic
acid (4 mg). Using the same method, 10 (10 mg) yielded
siaresinolic acid (2 mg).
P om olic a cid : mp 298-300 °C; [R]20D +55.5° (c 0.85, THF);
IR (KBr) νmax 3400, 1690, 1045, 1025 cm-1; MALDI-TOF MS
(positive ion mode) m/z 495 [M + Na]+; 1H and 13C NMR data
consistent with literature values.12
(16) Verger, R. Pancreatic lipase. In Lipase; Borgstrom, B., Brockman,
H. L., Eds.; Elsevier: Amsterdam, 1984; pp 83-150.
(17) Drent, M. L.; Larsson, I.; William, O. T.; Quaade, F.; Czubayko, F.;
von Bergmann, K.; Strobel, W.; van der Veen, E. A. Int. J . Obes. 1995,
19, 221-226.
Sia r esin olic a cid : mp 277-279 °C; [R]19 +33.3° (c 0.20,
D
MeOH); IR (KBr) νmax 3625, 1190, 1165 cm-1; MALDI-TOF MS
(positive ion mode) m/z 495 [M + Na]+; 1H and 13C NMR data
consistent with literature values.14
(18) Zhang, Z.; Koike, K.; J ia, Z.; Nikaido, T.; Guo, D.; Zheng, J . Chem.
Pharm. Bull. 1999, 47, 388-393.
(19) Zapf, J .; Schoenle, E.; Waldvoge, M.; Sand, M.; Foresch, E. R. Eur. J .
Biochem. 1981, 133, 605-609.
Mea su r em en t of P a n cr ea tic Lip a se Activity. Lipase
activity was determined by measuring the rate of release of
oleic acid from triolein. Briefly, a suspension of triolein (80
mg), phosphatidylcholine (10 mg), and taurocholic acid (5 mg)
(20) Tsujita, T.; Okuda, H. Eur. J . Biochem. 1983, 133, 215-220.
NP0304722