Medicinal flowers. Part 29. Acylated oleanane-type triterpene bisdesmosides: Perennisaponins G, H, I, J, K, L, and M with pancreatic lipase inhibitory activity from the flowers of Bellis perennis

Article abstract of DOI:10.1002/hlca.200900258

The MeOH extract from the flowers of Bellis perennis was found to show pancreatic-lipase inhibitory activity (IC₅₀ 455 μg/ml). From the extract, seven new triterpene saponins named perennisaponins G (1; IC ₅₀ 163 μm), H (2; 137 μm),

Full text of DOI:10.1002/hlca.200900258

Helvetica Chimica Acta – Vol. 93 (2010)
573
Medicinal Flowers
Part 291)
Acylated Oleanane-Type Triterpene Bisdesmosides: Perennisaponins G, H, I, J, K, L,
and M with Pancreatic Lipase Inhibitory Activity from the Flowers of Bellis perennis
by Toshio Morikawaa), Xuezheng Lib), Eriko Nishidaa), Seikou Nakamurab), Kiyofumi Ninomiyaa),
Hisashi Matsudab), Yoshimi Odab), Osamu Muraokaa), and Masayuki Yoshikawa*a)b)
a) Pharmaceutical Research and Technology Institute, Kinki University, 3-4-1 Kowakae, Higashi-osaka,
Osaka 577-8502, Japan
b) Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
(phone: þ 81-75-5954633; fax: þ 81-75-5954768; e-mail: myoshika@mb.kyoto-phu.ac.jp)
The MeOH extract from the flowers of Bellis perennis was found to show pancreatic-lipase inhibitory
activity (IC50 455 mg/ml). From the extract, seven new triterpene saponins named perennisaponins G (1;
IC50 163 mm), H (2; 137 mm), I (3; 147 mm), J (4; 148 mm), K (5; 223 mm), L (6; 81.4 mm), and M (7;
195 mm) were isolated as pancreatic lipase inhibitors. The stereostructures of 1 – 7 were elucidated on the
basis of chemical and spectroscopic evidence.
Introduction. – The Asteraceae plant Bellis perennis is distributed widely in Europe
and North Africa, and the whole flowering plant has been used for bruises, bleeding,
muscular pain, purulent skin diseases, and rheumatism in European folk medicine
[2][3]. This herbal medicine is also well-known as an ornamental plant, and its flowers
and young leaves are edible as a salad. During the course of our studies on bioactive
constituents from medicinal flowers [1 – 20], we found that the MeOH extract from the
flowers of B. perennis was found to inhibit the increase of plasma triglyceride (TG)
levels in olive-oil-loaded mice [2]. From the MeOH extract, 13 acylated saponin
constituents, perennisosides I – VII [2] and perennisaponins A – F [3], were isolated,
together with eight saponins and two glycosides [2][3].
Our continuing search now led to the isolation and characterization of seven new
acylated oleanane-type triterpene oligoglycosides named perennisaponins G – M (1 –
7), which were obtained from the flowers of B. perennis. Furthermore, we examined
the inhibitory effects against pancreatic lipase of perennisaponins 1 – 7.
Results and Discussion. – The flowers of B. perennis cultivated in Albania were
treated with MeOH to give an extract (25.8% from the dried flowers), as described
previously [2]. The MeOH extract, which was found to inhibit pancreatic lipase activity
(IC50 455 mg/ml), was partitioned between AcOEt and H2O (1:1) to furnish an
1
)
For Part 28, see [1].
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

574
Helvetica Chimica Acta – Vol. 93 (2010)
AcOEt-soluble fraction (6.7%) and an aqueous phase. The aqueous phase was
subjected to column chromatography (Diaion HP-20, H2O ! MeOH) to give H2O-
and MeOH-eluted fractions (12.5 and 6.4%), respectively [2]. The MeOH-eluted
fraction was subjected to normal- and reversed-phase column chromatographies and
finally HPLC to give 1 (0.124%), 2 (0.139%), 3 (0.0038%), 4 (0.060%), 5 (0.027%), 6
(0.0028%), and 7 (0.065%) (Fig. 1).
Fig. 1. Perennisaponins G – M (1 – 7) isolated from Bellis perennis
Compound 1 was obtained as an amorphous powder with a negative optical
rotation. The IR spectrum of 1 showed absorption bands at 1736 and 1638 cmꢀ1
ascribable to ester C¼O and olefin moieties, and broad bands at 3450 and 1049 cmꢀ1,
suggestive of an oligoglycoside structure. In the positive- and negative-ion-mode FAB-
MS of 1, quasi-molecular-ion peaks were observed at m/z 1327 ([M þ Na]þ) and 1303
([M ꢀ H]ꢀ), respectively, and a positive-mode HR-FAB-MS analysis revealed the
molecular formula of 1 to be C63H100O28. On alkaline hydrolysis of 1 with 10% aqueous
KOH in H2O/1,4-dioxane 1:1, polygalacic acid 3-(a-l-rhamnopyranoside) (1a) [21] was
obtained together with AcOH, which was identified by HPLC analysis of the

Helvetica Chimica Acta – Vol. 93 (2010)
575
corresponding 4-nitrobenzyl derivative [3 – 7][18]. Treatment of 1 with 0.5% MeONa/
MeOH provided a deacyl derivative, bellissaponin BS1 (¼(2b,3b,4a,16a)-3-[(6-deoxy-
a-l-mannopyranosyl)oxy]-2,16,23-trihydroxyolean-12-en-28-oic acid O-6-deoxy-a-l-
mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-6-deoxy-a-l-mannopyrano-
syl-(1 ! 2)-6-deoxy-b-d-galactopyranosyl ester; 1b) [3]. The 1H- and 13C-NMR data of
1 (Tables 1 and 2), which were assigned by various NMR experiments (including
DEPT, DQF, HMQC, HMBC, and TOCSY) showed signals assignable to six Me s at
d(H) 0.93 (Me(29)), 1.03 (Me(30)), 1.17 (Me(26)), 1.23 (Me(24)), 1.58 (Me(25)), and
1.75 (Me(27)), an oxygenated CH2 group at d(H) 3.72 (2 br. s, CH2(23)), three
oxygenated CH groups at d(H) 4.39 (br. s, HꢀC(3)), 4.72 – 4.76 (m, HꢀC(2)), and 5.16
(br. s, HꢀC(16)), an olefinic H-atom at d(H) 5.64 (t-like, J ꢁ 3 Hz, HꢀC(12)), a
fucopyranosyl moiety at d(H) 1.24 (d, J ¼ 6.4 Hz, Me(6’’)) and 6.02 (d, J ¼ 8.3 Hz,
HꢀC(1’’)), three rhamnopyranosyl moieties at d(H) 1.60 (d, J ¼ 6.0 Hz, Me(6’)), 1.63
(d, J ¼ 6.0 Hz, Me(6’’’’’)), 1.75 (d, J ¼ 6.2 Hz, Me(6’’’)), 5.76 (br. s, HꢀC(1’)), 6.07 (br. s,
HꢀC(1’’’)), and 6.21 (br. s, HꢀC(1’’’’’)), and a xylopyranosyl moiety at d(H) 5.14 (d,
J ¼ 7.6 Hz, HꢀC(1’’’’)), together with two AcO groups at d(H) 1.98 and 1.99 (2 s,
2 Me). Comparison of the 13C-NMR data of 1 with those of the deacetyl derivative 1b
revealed acetylation shifts around the 4’’-position of the b-d-fucopyranosyl moiety (1:
d(C) 73.9 (C(3’’)), 74.4 (C(4’’)), and 70.4 (C(5’’)); 1b: d(C) 76.5 (C(3’’)), 73.1 (C(4’’)),
and 72.5 (C(5’’))) and around the 2’’’-position of the inner a-l-rhamnopyranosyl moiety
of the 28-O-sugars (1: d(C) 98.8 (C(1’’’)), 73.3 (C(2’’’)), and 70.0 (C(3’’’)); 1b: d(C)
101.4 (C(1’’’)), 71.9 (C(2’’’)), and 72.6 (C(3’’’))). The positions of the two AcO groups in
1
13
1 were clarified on the basis of an HMBC experiment, which showed H ! C long-
range correlations between the following pairs as shown in Fig. 2: d(H) 1.98 (Me of Ac)
and 5.51 (br. s, HꢀC(4’’) of Fuc)/d(C) 171.1 (C¼O of Ac), and d(H) 1.99 (Me of Ac)
and 5.99 – 6.01 (m, HꢀC(2’’’) of the inner Rha)/d(C) 170.4 (C¼O of Ac). On the basis
Fig. 2. Selected HMBCs (H ! C) of 1

576
Helvetica Chimica Acta – Vol. 93 (2010)
of the above evidence, the structure of perennisaponin G (1) was determined as 3-O-a-
l-rhamnopyranosylpolygalacic acid 28-[O-a-l-rhamnopyranosyl-(1 ! 3)-O-b-d-xylo-
pyranosyl-(1 ! 4)-O-2-O-acetyl-a-l-rhamnopyranosyl-(1 ! 2)-4-O-acetyl-b-d-fuco-
pyranosyl] ester.
Table 1. Selected 1H-NMR Data (600 MHz, 408, (D5)pyridine) of 1 – 3. d in ppm, J in Hz.
1
2
3
HꢀC(2)
HꢀC(3)
HꢀC(12)
HꢀC(16)
HꢀC(18)
CH2(23)
Me(24)
4.72 – 4.76 (m)
4.39 (br. s)
5.64 (t-like, J ꢁ 3)
5.16 (br. s)
3.39 (dd-like)
3.72 (br. s)
1.23 (s)
4.70 – 4.74 (m)
4.38 (d, J ¼ 2.8)
5.63 (t-like, J ꢁ 3)
5.16 (br. s)
3.41 (dd, J ¼ 3.4, 14.4)
3.70 (br. s)
1.26 (s)
4.72 – 4.76 (m)
4.40 (d, J ¼ 3.1)
5.65 (t-like, J ꢁ 3)
5.16 (br. s)
3.40 (dd-like)
3.71 (br. s)
1.24 (s)
Me(25)
1.58 (s)
1.55 (s)
1.59 (s)
Me(26)
1.17 (s)
1.14 (s)
1.19 (s)
Me(27)
1.75 (s)
1.75 (s)
1.76 (s)
Me(29)
0.93 (s)
0.92 (s)
0.93 (s)
Me(30)
1.03 (s)
1.03 (s)
1.03 (s)
3-O-Rha:
HꢀC(1’)
Me(6’)
28-O-Sugars:
Fuc:
5.76 (br. s)
1.60 (d, J ¼ 6.0)
5.76 (br. s)
1.60 (d, J ¼ 6.2)
5.78 (br. s)
1.62 (d, J ¼ 6.1)
HꢀC(1’’)
HꢀC(4’’)
Me(6’’)
AcOꢀC(4’’)
3HBOꢀC(4’’):
CH2(2)
6.02 (d, J ¼ 8.3)
5.51 (br. s)
6.06 (d, J ¼ 6.9)
5.47 (br. s)
6.03 (d, J ¼ 8.3)
5.59 (br. d, J ꢁ 4)
1.32 (d, J ¼ 6.4)
1.24 (d, J ¼ 6.4)
1.24 (d, J ¼ 6.2)
1.98 (s)
1.99 (s)
2.68 (dd, J ¼ 5.2, 14.4),
2.78 (dd, J ¼ 7.6, 14.4)
4.46 – 4.48 (m)
1.39 (d, J ¼ 6.4)
HꢀC(3)
Me(4)
Inner Rha:
HꢀC(1’’’)
HꢀC(2’’’)
HꢀC(3’’’)
Me(6’’’)
AcOꢀC(2’’’)
AcOꢀC(3’’’)
Xyl:
6.07 (br. s)
6.07 (br. s)
6.09 (d, J ¼ 1.5)
5.99 (dd, J ¼ 1.5, 3.4)
4.78 (dd, J ¼ 3.6, 9.8)
1.75 (d, J ¼ 6.1)
1.96 (s)
5.99 – 6.01 (m)
4.79 (dd, J ¼ 3.4, 9.8)
1.75 (d, J ¼ 6.2)
1.99 (s)
5.00 – 5.02 (m)
5.87 (dd, J ¼ 2.7, 9.6)
1.75 (d, J ¼ 6.2)
2.04 (s)
HꢀC(1’’’’)
Terminal Rha:
HꢀC(1’’’’’)
Me(6’’’’’)
5.14 (d, J ¼ 7.6)
4.98 (d, J ¼ 7.6)
5.15 (d, J ¼ 7.7)
6.21 (br. s)
1.63 (d, J ¼ 6.0)
6.21 (br. s)
1.66 (d, J ¼ 6.2)
6.17 (br. s)
1.64 (d, J ¼ 6.1)
Compound 2 was also obtained as an amorphous powder with a negative optical
rotation. The IR spectrum of 2 showed absorption bands at 3450, 1734, 1638, and
1049 cmꢀ1, ascribable to OH, ester C¼O, olefin, and ether functions. The molecular
formula of 2, C63H100O28, was determined from the positive- and negative-ion-mode

Helvetica Chimica Acta – Vol. 93 (2010)
577
Table 2. 13C-NMR Data of 1 – 3. At 150 MHz, 408, in (D5)pyridine; d in ppm.
1
2
3
1
2
3
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
44.8
71.1
81.4
43.0
47.6
18.3
33.2
40.2
47.5
37.2
24.1
122.8
144.2
42.3
36.2
74.1
49.7
41.6
47.3
30.7
35.9
32.0
65.3
14.9
17.7
17.7
27.1
176.1
33.1
24.5
44.8
71.1
81.4
43.0
47.7
18.3
33.2
40.2
47.5
37.1
24.1
122.8
144.2
42.2
36.1
74.1
49.7
41.6
47.3
30.8
35.9
31.9
65.2
15.0
17.7
17.6
27.1
176.2
33.1
24.6
44.8
71.1
81.5
43.0
47.6
18.3
33.3
40.3
47.5
37.2
24.1
122.8
144.2
42.3
36.2
74.1
49.5
41.6
47.3
30.8
36.0
32.0
65.3
15.0
17.8
17.7
27.1
176.2
33.1
24.6
28-O-Sugars:
Fuc:
C(1’’)
C(2’’)
C(3’’)
C(4’’)
C(5’’)
C(6’’)
AcOꢀC(4’’)
3HBOꢀC(4’’):
C(1)
C(2)
C(3)
C(4)
Inner Rha:
C(1’’’)
C(2’’’)
C(3’’’)
C(4’’’)
C(5’’’)
C(6’’’)
AcOꢀC(2’’’)
AcOꢀC(3’’’)
Xyl:
C(1’’’’)
C(2’’’’)
C(3’’’’)
C(4’’’’)
C(5’’’’)
Terminal Rha:
C(1’’’’’)
C(2’’’’’)
C(3’’’’’)
C(4’’’’’)
C(5’’’’’)
C(6’’’’’)
94.3
75.1
73.9
74.4
70.4
16.4
94.2
76.8
74.1
74.4
70.3
16.4
94.3
75.2
73.5
74.4
70.5
16.5
C(9)
171.1, 20.7
171.1, 20.7
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
C(23)
C(24)
C(25)
C(26)
C(27)
C(28)
C(29)
C(30)
3-O-Rha:
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
172.3
45.0
64.8
24.0
98.8
73.3
70.0
83.8
68.5
102.7
69.4
75.5
77.9
68.9
19.2
98.7
73.2
70.1
83.8
68.5
18.5
18.6
170.4, 20.9
170.4, 20.9
170.9, 21.4
106.8
76.2
83.3
69.2
67.3
105.8
75.0
83.3
69.8
67.1
106.8
76.2
83.4
69.3
67.3
102.6
72.5
72.6
73.9
69.9
18.5
102.6
72.5
72.7
73.9
69.8
18.6
102.6
72.5
72.6
74.0
70.0
18.6
104.2
72.4
72.7
73.2
70.3
18.6
104.1
72.4
72.7
73.5
70.3
18.6
104.2
72.5
72.8
74.0
70.3
18.6
FAB-MS (m/z 1327 ([M þ Na]þ) and 1303 ([M ꢀ H]ꢀ), resp.) and by positive-ion-
mode HR-FAB-MS, which was the same as that of 1. Alkaline hydrolysis of 2 with 10%
aqueous KOH in H2O/1,4-dioxane 1:1 liberated 1a together with AcOH. Treatment of
2 with 0.5% MeONa/MeOH yielded 1b. The 1H- and 13C-NMR spectra of 2 (Tables 1
and 2) indicated the presence of the following functions: a polygalacic acid part (six Me
s at d(H) 0.92 (Me(29)), 1.03 (Me(30)), 1.14 (Me(26)), 1.26 (Me(24)), 1.55 (Me(25)),
and 1.75 (Me(27)), an oxygenated CH2 group at d(H) 3.70 (br. s, CH2(23)), three
oxygenated CH groups at d(H) 4.38 (d, J ¼ 2.8 Hz, HꢀC(3)), 4.70 – 4.74 (m, HꢀC(2)),
and 5.16 (br. s, HꢀC(16)), and an olefinic H-atom at d(H) 5.63 (t-like, J ꢁ 3 Hz,

578
Helvetica Chimica Acta – Vol. 93 (2010)
HꢀC(12))), a fucopyranosyl moiety at d(H) 1.24 (d, J ¼ 6.2 Hz, Me(6’’)) and 6.06 (d,
J ¼ 6.9 Hz, HꢀC(1’’)), three rhamnopyranosyl moieties at d(H) 1.60 (d, J ¼ 6.2 Hz,
Me(6’)), 1.66 (d, J ¼ 6.2 Hz, Me(6’’’’’)), 1.75 (d, J ¼ 6.2 Hz, Me(6’’’)), 5.76 (br. s,
HꢀC(1’)), 6.07 (br. s, HꢀC(1’’’)), and 6.21 (br. s, HꢀC(1’’’’’)), and a xylopyranosyl
moiety at d(H) 4.98 (d, J ¼ 7.6 Hz, HꢀC(1’’’’)), together with two AcO groups at d(H)
1.99 and 2.04 (2s, 2 Me). The 1H- and 13C-NMR data of 2 were superimposable on those
of 1, except for the signals due to the part around the inner Rha unit of the 28-O-sugars.
The positions of the two AcO groups in 2 were determined by an HMBC experiment,
13
which showed the following 1H ! C long-range correlations: d(H) 1.99 (s, Me of Ac)
and 5.47 (br. s, HꢀC(4’’) of Fuc)/d(C) 171.1 (C¼O of Ac), and d(H) 2.04 (s, Me of Ac)
and 5.87 (dd, J ¼ 2.7, 9.6 Hz, HꢀC(3’’’) of the inner Rha)/d(C) 170.9 (C¼O of Ac).
Consequently, the structure of 2 was elucidated as 3-O-a-l-rhamnopyranosylpolyga-
lacic acid 28-[O-a-l-rhamnopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-3-O-
acetyl-a-l-rhamnopyranosyl-(1 ! 2)-4-O-acetyl-b-d-fucopyranosyl] ester.
Compound 3 was also obtained as an optically active amorphous powder. The
positive- and negative-ion-mode FAB-MS of 3 showed quasi-molecular-ion peaks at
m/z 1371 ([M þ Na]þ) and 1347 ([M ꢀ H]ꢀ), respectively. The positive-ion-mode HR-
FAB-MS of 3 revealed the molecular formula to be C65H104O29. Alkaline hydrolysis of
3 with 10% aqueous KOH in H2O/1,4-dioxane 1:1 liberated 1a and two organic acids,
AcOH and 3-hydroxybutanoic acid (3HBOH), which were identified by HPLC
analysis of the corresponding 4-nitrobenzyl derivatives. In addition, treatment of 3 with
0.5% MeONa/MeOH gave 1b, together with methyl (þ)-(3S)-3-hydroxybutanoate
[22], which was identified by HPLC and an optical-rotation detector [23 – 27]. The H-
1
and C-atom signals in the H- and 13C-NMR spectra of 3 (Tables 1 and 2, resp.) were
superimposable on those of 1, except for the signals due to the acyl groups: an AcO
group at d(H) 1.96 (s, Me of Ac) and a (3S)-3-hydroxybutanoyl (3HB) group at d(H)
1.39 (d, J ¼ 6.4 Hz, Me(4)), 2.68 (dd, J ¼ 5.2, 14.4 Hz, 1 HꢀC(2)), 2.78 (dd, J ¼ 7.6,
14.4 Hz, 1 HꢀC(2)), and 4.46 – 4.48 (m, HꢀC(3)). In the HMBC experiment with 3,
13
the following 1H ! C long-range correlations were observed: CH2(2) of 3HB,
HꢀC(3) of 3HB, and d(H) 5.59 (br. d, J ꢁ 4 Hz, HꢀC(4’’) of Fuc)/d(C) 172.3 (C¼O of
3HB); and d(H) 1.96 (s, Me of Ac) and 5.99 (dd, J ¼ 1.5, 3.4 Hz, HꢀC(2’’’) of the inner
Rha)/d(C) 170.4 (C¼O of Ac). Thus, the connectivities of the acyloxy groups in 3 were
elucidated. On the basis of above-mentioned evidence, the structure of 3 was
determined as 3-O-a-l-rhamnopyranosylpolygalacic acid 28-[O-a-l-rhamnopyranosyl-
(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-acetyl-a-l-rhamnopyranosyl-(1 ! 2)-4-O-
(3S)-3-hydroxybutanoyl-b-d-fucopyranosyl] ester.
Compounds 4 and 5 were obtained as amorphous powders with negative optical
rotations. The molecular formula of both 4 and 5, C67H106O30, was determined from the
positive- and negative-ion-mode FAB-MS at m/z 1413 ([M þ Na]þ) and 1389 ([M ꢀ
H]ꢀ) and by positive-ion-mode HR-FAB-MS measurement. Alkaline hydrolysis of 4
and 5 with 10% aqueous KOH in H2O/1,4-dioxane 1:1 provided 1a and two organic
acids, AcOH and 3HBOH, respectively. Treatment of each 4 and 5 with 0.5% MeONa/
MeOH gave 1b, together with methyl (þ)-(3S)-3-hydroxybutanoate, respectively. The
H- and C-atom signals in the 1H- and 13C-NMR spectra of 4 (Tables 3 and 4) indicated
the presence of an aglycon part, containing six Me s at d(H) 0.95 (Me(29)), 1.06
(Me(30)), 1.18 (Me(26)), 1.22 (Me(24)), 1.58 (Me(25)), and 1.75 (Me(27)), a CH2 and

Helvetica Chimica Acta – Vol. 93 (2010)
579
three CH groups bearing an O-atom function at d(H) 3.69 (br. s, CH2(23)), 4.35 (d, J ¼
3.5 Hz, HꢀC(3)), 4.68 – 4.72 (m, HꢀC(2)), and 5.12 (br. s, HꢀC(16)), an olefinic H-
atom at d(H) 5.65 (t-like, J ꢁ 3 Hz, HꢀC(12)), and a fucopyranosyl moiety at d(H) 1.25
(d, J ¼ 6.3 Hz, Me(6’’)) and 5.99 (d, J ¼ 8.0 Hz, HꢀC(1’’)), three rhamnopyranosyl
moieties at d(H) 1.60 (d, J ¼ 6.1 Hz, Me(6’)), 1.67 (d, J ¼ 6.3 Hz, Me(6’’’’’)), 1.75 (d, J ¼
6.4 Hz, Me(6’’’)), 5.71 (br. s, HꢀC(1’)), 6.04 (d, J ¼ 1.8 Hz, HꢀC(1’’’)), and 6.08 (br. s,
HꢀC(1’’’’’)), and a xylopyranosyl moiety at d(H) 5.10 (d, J ¼ 7.7 Hz, HꢀC(1’’’’)),
together with two AcO groups at d(H) 1.98 (2s, 2 Me) and a 3 HBO group at d(H) 1.30
(d, J ¼ 6.3 Hz, Me(4)), 2.68 (dd, J ¼ 5.5, 15.8 Hz, 1 HꢀC(2)), 2.78 (dd, J ¼ 7.5, 15.8 Hz,
1
1 HꢀC(2)), and 5.49 – 5.51 (m, HꢀC(3)). The H- and C-atom signals in the H- and
13C-NMR spectra of 4 resembled those of 3, except for the signals due to the additional
AcO group. Comparison of the 13C-NMR spectra of 4 with those of 3 revealed an
acetylation shift around the 3-position of the 3HBOꢀC(4’’) group (4: d(C) 40.8 (C(2)),
67.5 (C(3)), and 19.9 (C(4)); 3: d(C) 45.0 (C(2)), 64.8 (C(3)), and 24.0 (C(4))). The
connectivity of the above-mentioned AcO group in 4 was clarified by an HMBC
13
experiment, which showed 1H ! C long-range correlations between d(H) 1.98 (s, Me
of Ac) and HꢀC(3) of 3HBOꢀC(4’’) and d(C) 170.0 (C¼O of Ac). In turn, the H- and
C-atom signals in the 1H- and 13C-NMR spectra of 5 (Tables 3 and 4) resembled those
of 4, except for the signals due to the inner Rha of the 28-O-sugars. The position of an
AcO group at the inner Rha of 5 was established by an HMBC experiment, which
13
showed 1H ! C long-range correlations between d(H) 2.05 (s, Me of Ac) and 5.84 (dd,
J ¼ 2.5, 8.9 Hz, HꢀC(3’’’) of the inner Rha) and d(C) 170.9 (C¼O of Ac). On the basis
of the above-mentioned evidence, the structures of 4 and 5 were elucidated to be 3-O-
a-l-rhamnopyranosylpolygalacic acid 28-{O-a-l-rhamnopyranosyl-(1 ! 3)-O-b-d-xy-
lopyranosyl-(1 ! 4)-O-2-O-acetyl-a-l-rhamnopyranosyl-(1 ! 2)-4-O-[(3S)-3-(acetyl-
oxy)butanoyl]-b-d-fucopyranosyl} ester and 3-O-a-l-rhamnopyranosylpolygalacic acid
28-{O-a-l-rhamnopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-3-O-acetyl-a-l-
rhamnopyranosyl-(1 ! 2)-4-O-[(3S)-3-(acetyloxy)butanonyl]-b-d-fucopyranosyl} es-
ter, respectively.
The molecular formulas of compound 6, C69H110O31, and compound 7, C73H116O33,
were determined from their respective positive- and negative-ion-mode FAB-MS and
by positive-ion-mode HR-FAB-MS data. Alkaline hydrolysis of 6 and 7 with 10%
aqueous KOH in H2O/1,4-dioxane 1:1 provided 1a and two organic acids, AcOH and
3HBOH, respectively. Treatment of 6 and 7 with 0.5% MeONa/MeOH gave 1b,
together with (þ)-(3S)-3-hydroxybutanoic acid. The H- and C-atom signals in the 1H-
and 13C-NMR spectra of 6 (Tables 3 and 4) showed signals assignable to a bellissaponin
BS1 (1b) moiety, an AcO group at d(H) 1.99 (s, Me) and two 3HBO groups at d(H)
1.35 (d, J ¼ 6.2 Hz, Me(4)), 1.38 (d, J ¼ 6.9 Hz, Me(4’)), 2.61 – 2.65 and 2.71 – 2.75 (2 m,
CH2(2’)), 2.68 – 2.72 and 2.82 – 2.86 (2 m, CH2(2)), 4.53 – 4.57 (m, HꢀC(3’)), and 5.59 –
5.61 (m, HꢀC(3)). The connectivities of the acyl moieties in 6 were determined
13
through an HMBC experiment, which exhibited 1H ! C long-range correlations
between the following pairs: d(H) 1.99 (s, Me of Ac) and 6.01 – 6.03 (m, HꢀC(2’’’) of
the inner Rha)/d(C) 170.5 (C¼O of Ac), CH2(2) of 3HBOꢀC(4’’), HꢀC(3) of
3HBOꢀC(4’’), and d(H) 5.53 (br. d, J ꢁ 3 Hz, HꢀC(4’’) of Fuc)/d(C) 170.8 (C¼O of
3HBOꢀC(4’’)), and CH2(2’) of 3HBOꢀC(3), HꢀC(3’) of 3HBOꢀC(3), and
HꢀC(3) of 3HBOꢀC(4’’)/d(C) 171.3 (C¼O of 3HBOꢀC(3)). By comparison of

580
Helvetica Chimica Acta – Vol. 93 (2010)
Table 3. Selected H-NMR Data (600 MHz, 408, (D5)pyridine) of 4 – 7. d in ppm, J in Hz.
1
4
5
6
7
HꢀC(2)
HꢀC(3)
HꢀC(12)
HꢀC(16)
HꢀC(18)
CH2(23)
Me(24)
Me(25)
Me(26)
Me(27)
Me(29)
4.68 – 4.72 (m)
4.35 (d, J ¼ 3.5)
5.65 (t-like, J ꢁ 3)
5.12 (br. s)
4.71 (br. d, J ꢁ 3)
4.39 (d, J ¼ 2.9)
5.65 (t-like, J ꢁ 3)
5.14 (br. s)
4.74 – 4.78 (m)
4.41 (br. s)
4.70 (br. s)
4.36 (d, J ¼ 3.2)
5.63 (t-like, J ꢁ 3)
5.12 (br. s)
5.67 (t-like, J ꢁ 3)
5.16 (br. s)
3.39 (dd, J ¼ 4.3, 14.1) 3.40 (dd, J ¼ 3.4, 11.9) 3.41 (dd, J ¼ 4.2, 13.7) 3.40 (dd-like)
3.69 (br. s)
1.22 (s)
1.58 (s)
1.18 (s)
1.75 (s)
0.95 (s)
1.06 (s)
3.70 (br. s)
1.24 (s)
1.57 (s)
1.17 (s)
1.75 (s)
0.94 (s)
1.07 (s)
3.72 (br. s)
1.25 (s)
1.60 (s)
1.19 (s)
1.76 (s)
0.94 (s)
1.04 (s)
3.70 (br. s)
1.25 (s)
1.59 (s)
1.18 (s)
1.75 (s)
0.94 (s)
1.06 (s)
Me(30)
3-O-Rha:
HꢀC(1’)
Me(6’)
28-O-Sugars:
Fuc:
5.71 (br. s)
1.60 (d, J ¼ 6.1)
5.72 (br. s)
1.60 (d, J ¼ 6.0)
5.79 (br. s)
1.62 (d, J ¼ 6.2)
5.72 (br. s)
1.60 (d, J ¼ 6.0)
HꢀC(1’’)
HꢀC(4’’)
Me(6’’)
5.99 (d, J ¼ 8.0)
5.50 (br. d, J ꢁ 4)
1.25 (d, J ¼ 6.3)
6.03 (d, J ¼ 7.6)
5.48 (br. d, J ꢁ 3)
1.25 (d, J ¼ 6.0)
6.02 (d, J ¼ 8.2)
5.53 (br. d, J ꢁ 3)
1.27 (d, J ¼ 6.2)
6.00 (d, J ¼ 8.0)
5.50 (br. s)
1.25 (d, J ¼ 6.3)
3HBOꢀC(4’’):
CH2(2)
2.68 (dd, J ¼ 5.5, 15.8), 2.68 (dd, J ¼ 4.6, 13.1), 2.68 – 2.72 (m),
2.78 (dd, J ¼ 7.5, 15.8) 2.78 (dd, J ¼ 6.2, 13.1) 2.82 – 2.86 (m)
2.65 – 2.70 (m)a),
2.74 – 2.81 (m)b)
5.52 – 5.56 (m)c)
1.34 (d, J ¼ 6.3)d)
HꢀC(3)
5.49 – 5.51 (m)
1.30 (d, J ¼ 6.3)
1.98 (s)
5.50 – 5.52 (m)
1.30 (d, J ¼ 6.3)
2.00 (s)
5.59 – 5.61 (m)
1.35 (d, J ¼ 6.2)
Me(4)
AcOꢀC(3)
3HBOꢀC(3):
CH2(2’)
2.61 – 2.65 (m),
2.71 – 2.75 (m)
4.53 – 4.57 (m)
1.38 (d, J ¼ 6.9)
2.65 – 2.70 (m)a),
2.74 – 2.81 (m)b)
5.52 – 5.56 (m)c)
1.31 (d, J ¼ 6.3)d)
HꢀC(3’)
Me(4’)
3HBOꢀC(3’):
CH2(2’’)
2.60 (dd, J ¼ 5.2, 14.9),
2.74 – 2.81 (m)b)
4.52 – 4.54 (m)
1.38 (d, J ¼ 6.1)
HꢀC(3’’)
Me(4’’)
Inner Rha:
HꢀC(1’’’)
HꢀC(2’’’)
HꢀC(3’’’)
Me(6’’’)
AcOꢀC(2’’’)
AcOꢀC(3’’’)
Xyl:
6.04 (d, J ¼ 1.8)
6.03 (br. s)
6.10 (br. s)
6.01 – 6.03 (m)
6.05 (br. s)
5.94 (dd, J ¼ 1.8, 3.5) 4.95 – 4.97 (m)
5.94 (dd, J ¼ 1.5, 3.2)
4.74 (dd, J ¼ 3.2, 9.5)
1.75 (d, J ¼ 6.4)
1.99 (s)
4.73 (dd, J ¼ 3.5, 9.4) 5.84 (dd, J ¼ 2.5, 8.9) 4.80 – 4.84 (m)
1.75 (d, J ¼ 6.4)
1.98 (s)
1.74 (d, J ¼ 6.4)
1.78 (d, J ¼ 6.8)
1.99 (s)
2.05 (s)
HꢀC(1’’’’)
Terminal Rha:
HꢀC(1’’’’’)
Me(6’’’’’)
5.10 (d, J ¼ 7.7)
4.95 (d, J ¼ 7.6)
5.16 (d, J ¼ 6.8)
5.10 (d, J ¼ 7.7)
6.08 (br. s)
1.67 (d, J ¼ 6.3)
6.13 (br. s)
1.65 (d, J ¼ 6.2)
6.18 (br. s)
1.65 (d, J ¼ 6.2)
6.08 (br. s)
1.62 (d, J ¼ 6.3)
a)b)c) Overlapped. d) May be interchangeable within the same column.

Helvetica Chimica Acta – Vol. 93 (2010)
581
Table 4. 13C-NMR Data of 4 – 7. At 150 MHz, 408, in (D5)pyridine; d in ppm.
4
5
6
7
4
5
6
7
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
C(9)
C(10)
C(11)
44.8
71.0
81.6
43.0
47.7
18.4
33.3
40.3
47.6
37.2
24.1
44.8
71.0
81.6
43.0
47.7
18.3
33.2
40.3
47.6
37.2
24.1
44.9
71.1
81.4
43.0
47.6
18.3
33.3
40.3
47.6
37.2
24.1
44.8 28-O-Sugars:
71.0 Fuc:
81.6 C(1’’)
43.0 C(2’’)
47.7 C(3’’)
18.4 C(4’’)
33.3 C(5’’)
40.3 C(6’’)
47.6 3HBOꢀC(4’’):
37.2 C(1)
94.4
75.0
73.3
74.8
70.3
16.4
94.2
76.7
73.4
74.7
70.3
16.4
94.3
74.9
73.2
74.7
70.3
16.4
94.4
75.0
73.3
74.8
70.4
16.4
170.7
40.8
67.5
19.9
170.7
40.8
67.5
19.8
170.8
40.7
67.4
19.8
170.7
24.1 C(2)
40.7a)
67.9b)
19.8c)
C(12) 122.8 122.8 122.8 122.8 C(3)
C(13) 144.3 144.2 144.3 144.3 C(4)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
C(23)
C(24)
C(25)
C(26)
C(27)
42.4
36.2
74.1
49.6
41.7
47.4
30.8
36.0
31.9
65.4
15.0
17.8
17.7
27.1
42.2
36.1
74.2
49.5
41.6
47.4
30.7
36.0
31.7
65.3
15.0
17.7
17.7
27.1
42.4
36.2
74.1
49.5
41.6
47.3
30.8
36.0
32.0
65.3
15.0
17.8
17.7
27.1
42.4 AcOꢀC(3)
170.0, 21.1 170.1, 21.1
36.2
74.2
49.6
41.7
47.4
30.8
36.0
31.9
65.4
15.0
3HBOꢀC(3):
C(1’)
C(2’)
C(3’)
C(4’)
171.3
45.2
64.4
23.9
169.7
41.2a)
67.6b)
19.9c)
3HBOꢀC(3’):
C(1’’)
C(2’’)
C(3’’)
C(4’’)
171.4
45.1
64.4
23.9
17.7 Inner Rha:
17.7 C(1’’’)
27.1 C(2’’’)
98.8
73.3
102.5
69.5
75.5
77.9
68.9
19.1
98.7
73.2
70.1
83.7
68.6
18.5
98.7
73.3
70.0
83.9
68.6
18.5
C(28) 176.2 176.3 176.2 176.2 C(3’’’)
C(29)
C(30)
3-O-Rha:
70.1
33.1
24.7
33.1
24.7
33.1
24.5
33.1 C(4’’’)
24.7 C(5’’’)
C(6’’’)
84.0
68.6
18.5
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
104.1 104.0 104.2 104.1 AcOꢀC(2’’’)
170.4, 20.9
170.5, 20.9 170.4, 20.9
72.5
72.8
74.0
70.3
18.6
72.4
72.8
74.1
70.2
18.5
72.5
72.8
74.0
70.3
18.6
72.5 AcOꢀC(3’’’)
72.8 Xyl:
74.0 C(1’’’’)
70.4 C(2’’’’)
18.6 C(3’’’’)
C(4’’’’)
170.9, 21.3
106.8
76.1
83.7
69.3
67.3
105.7
75.0
83.6
69.8
67.1
106.7
76.2
83.3
69.3
67.3
106.8
76.1
83.7
69.3
67.3
C(5’’’’)
Terminal Rha:
C(1’’’’’)
C(2’’’’’)
C(3’’’’’)
C(4’’’’’)
102.6
72.4
72.6
74.0
70.0
18.5
102.7
72.4
72.7
74.0
69.8
18.5
102.6
72.4
72.6
74.0
70.0
18.6
102.6
72.4
72.6
74.0
70.0
18.5
C(5’’’’’)
C(6’’’’’)
a)b)c) Interchangeable within the same column.

582
Helvetica Chimica Acta – Vol. 93 (2010)
1
the H- and 13C-NMR spectra of 7 (Tables 3 and 4) with those of 6, 7 had one more
3HBO group than 6 at d(H) (1.31 (d, J ¼ 6.3 Hz), 1.34 (d, J ¼ 6.3 Hz), 1.38 (d, J ¼
6.1 Hz), Me(4’), Me(4), and Me(4’’)), 2.65 – 2.70 and 2.74 – 2.81 (2 m, 2 H each,
CH2(2) and CH2(2’)), 2.60 (dd, J ¼ 5.2, 14.9 Hz, 1 HꢀC(2’’)), 2.74 – 2.81 (m,
1 HꢀC(2’’)), 4.52 – 4.54 (m, HꢀC(3’’)), and 5.52 – 5.56 (m, HꢀC(3) and HꢀC(3’)).
1
13
In the HMBC experiment of 7, the following H ! C long-range correlations were
observed: CH2(2’’) of 3HBOꢀC(3’), HꢀC(3’’) of 3HBOꢀC(3’), and HꢀC(3’) of
3HBOꢀC(3)/d(C) 171.4 (C¼O of 3HBOꢀC(3’)). Consequently, the structures of 6
and 7 were elucidated to be 3-O-a-l-rhamnopyranosylpolygalacic acid 28-{O-a-l-
rhamnopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-acetyl-a-l-rhamnopyr-
anosyl-(1 ! 2)-4-O-{(3S)-3-[(3S)-3-(hydroxybutanoyl)oxy]butanoyl}-b-d-fucopyrano-
syl} ester and 3-O-a-l-rhamnopyranosylpolygalacic acid 28-{O-a-l-rhamnopyranosyl-
(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-acetyl-a-l-rhamnopyranosyl-(1 ! 2)-4-
O-{(3S)-3-{{(3S)-3-{[(3S)-3-hydroxybutanoyl]oxy}butanoyl}oxy}butanoyl}-b-d-fuco-
pyranosyl} ester, respectively.
Pancreatic lipase is well known to play an important role in lipid digestion.
Recently, inhibitory effects of saponins on pancreatic lipase were reported to be
involved in anti-obese effects of saponins (e.g., theasaponins, chikusetsusaponins) [28 –
31]. In the course of our characterization studies on anti-obese constituents from B.
perennis, inhibitory effects of the constituents on pancreatic lipase activity were
examined. Among the saponin constituents, perennisaponins G (1; IC50 163 mm), H (2;
137 mm), I (3;147 mm), J (4; 148 mm), K (5; 223 mm), L (6; 81.4 mm), and M (7; 195 mm)
were found to inhibit pancreatic lipase activity (Table 5). These saponins 1 – 7 are more
Table 5. Inhibitory Effects of the MeOH Extract and of 1 – 7 from the Flowers of B. perennis against
Pancreatic Lipase
Concentration [mg/ml]a)
100
IC50 [mg/ml]
0
200
400
800
MeOH Extract
0.0 ꢂ 0.6 8.3 ꢂ 1.8
21.4 ꢂ 2.1c) 43.8 ꢂ 1.9c) 71.6 ꢂ 4.5c) 455
Concentration [mm]a)
IC50 [mm]
0
50
100
200
400
Perennisaponin G (1) 0.0 ꢂ 2.8 ꢀ 18.8 ꢂ 10.4 21.3 ꢂ 5.8
Perennisaponin H (2) 0.0 ꢂ 1.5 ꢀ 33.5 ꢂ 12.2 14.7 ꢂ 9.3
Perennisaponin I (3) 0.0 ꢂ 1.1 ꢀ 17.0 ꢂ 18.9 27.5 ꢂ 8.9
58.9 ꢂ 3.7c) 85.5 ꢂ 1.7c) 163
71.9 ꢂ 3.1c) 87.4 ꢂ 1.3c) 137
60.2 ꢂ 2.4c) 85.2 ꢂ 4.1c) 147
Perennisaponin J (4) 0.0 ꢂ 3.0
Perennisaponin K (5) 0.0 ꢂ 3.8
Perennisaponin L (6) 0.0 ꢂ 4.2
Perennisaponin M (7) 0.0 ꢂ 5.7
16.6 ꢂ 13.6 38.3 ꢂ 10.0b) 55.9 ꢂ 5.1c) 55.2 ꢂ 8.6c) 148
35.0 ꢂ 5.0c) 47.1 ꢂ 5.0c) 43.1 ꢂ 10.1c) 55.6 ꢂ 6.5c) 223
26.9 ꢂ 7.2b) 55.3 ꢂ 2.8c) 52.7 ꢂ 5.1c) 68.5 ꢂ 6.9c) 81.4
22.6 ꢂ 12.0 28.5 ꢂ 11.3
53.9 ꢂ 3.8c) 67.2 ꢂ 5.6c) 195
Concentration [mm]a)
IC50 [mm]
0
100
200
400
800
Theasaponin E1
0.0 ꢂ 2.2
24.4 ꢂ 4.3b) 39.2 ꢂ 7.9c) 57.4 ꢂ 9.1c) 88.6 ꢂ 2.6c) 270
a) Values represent the means ꢂ s.e.m ( N ¼ 4). Significantly different from the control group: b) p < 0.05.
c) p < 0.01.

Helvetica Chimica Acta – Vol. 93 (2010)
583
efficient than theasaponin E1 (IC50 270 mm) [30], which was isolated from Camellia
sinensis [32], although their inhibitory activities were considerably weaker than that of
the lipase inhibitor orlistat (IC50 56 nm). On the basis of above in vitro results and our
previous in vivo evidence [2], the saponin constituents from the flowers of B. perennis
may be useful for the prevention of obesity.
O. M., T. M., and K. N. were supported by ꢂHigh-tech Research Centerꢃ Project for Private
Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) of Japan, 2007 – 2011, and by a Grant-in Aid for Scientific Research from MEXT.
M. Y., H. M., and S. N. were supported by the 21st COE Program, the Academic Frontier Project, and a
Grant-in Aid for Scientific Research from MEXT. H. M. was also supported by the Hoh-ansha
Foundation, Japan.
Experimental Part
General. Column chromatography (CC): normal-phase CC with silica gel BW-200 (SiO2; 150 – 300
mesh; Fuji Silysia Chemical, Ltd., Japan), reversed-phase CC with Chromatorex ODS DM1020T (100 –
200 mesh; Fuji Silysia Chemical, Ltd., Japan). HPLC: Shimadzu-RID-6A refractive-index, SPD-10A
UV/VIS, and Shodex-OR-2 optical-rotation detectors; Shimadzu-LC-6AD pump; Shimadzu-CTO-10A
column oven; Shimadzu-C-R6A chromatopac; Cosmosil-5C18-MS-II and -HILIC (Nacalai Tesque, Inc.),
Wakopak-Navi-C-30-5 (Wako Pure Chemical Industries Ltd.), and YMC-Pack-ODS-A and YMC-Pack-
ODS-AQ (YMC Co., Ltd.) columns; tR in min. Optical rotations: Horiba-SEPA-300 digital polarimeter
1
(l ¼ 5 cm). IR Spectra: Shimadzu-FTIR-8100 spectrometer; in cmꢀ1. H- and 13C-NMR Spectra: Jeol-
JNM-ECA600 spectrometer; at 600 and 150 MHz, resp.; d in ppm rel. to Me4Si, J in Hz. FAB- and HR-
FAB-MS: Jeol-JMS-SX-102A mass spectrometer; in m/z.
Plant Material. The flowers of B. perennis cultivated in Albania were purchased from Tochimoto
Tenkaido Co., Ltd., in November 2006, as described previously [2][3].
Extraction and Isolation. Compounds 1 – 7 were isolated from previously reported fractions [2][3],
Fr. 6.11 (4.850 g) and Fr. 6.12 (50.269 g), originally obtained from the MeOH-eluted fraction (6.4%,
140.0 g) of the MeOH extract from flowers of B. perennis. An aliquot of Fr. 6.11 (1820.0 mg) was purified
by HPLC (Wakopak-Navi-C30-5, 250 ꢃ 20 mm i.d., MeCN/MeOH/1% aq. AcOH 32 :16 :52; 9.0 ml/min)
to afford the following eight fractions: Fr. 6.11.1 (465.8 mg), Fr. 6.11.2 (272.1 mg), Fr. 6.11.3 (41.7 mg),
Fr. 6.11.4 (44.5 mg), Fr. 6.11.5 (263.0 mg), Fr. 6.11.6 (210.0 mg), Fr. 6.11.7 (137.9 mg), and Fr. 6.11.8
(88.6 mg). Fr. 6.11.5 (263.0 mg) was purified by HPLC (Cosmosil 5C18-MS-II, 250 ꢃ 20 mm i.d., MeCN/
MeOH/1% aq. AcOH 32 :16 :52; 9.0 ml/min): perennisaponin I (3; 33.1 mg, 0.0038%; tR 48.9). Fr. 6.11.7
(137.9 mg) was purified by HPLC (Cosmosil 5C18-MS-II, 250 ꢃ 20 mm i.d., MeCN/MeOH/1% aq. AcOH
32 :16 :52; 9.0 ml/min): perennisaponin G (1, 42.0 mg, 0.0048%; tR 64.1). Fr. 6.11.8 (88.6 mg) was purified
by HPLC (Cosmosil 5C18-MS-II, 250 ꢃ 20 mm i.d., MeCN/MeOH/1% aq. AcOH 32 :16 :52; 9.0 ml/min):
perennisaponin L (6, 24.3 mg, 0.0028%; tR 70.0). An aliquot of Fr. 6.12 (2015.0 mg) was further separated
by HPLC (Wakopak-Navi-C30-5, 250 ꢃ 20 mm i.d., MeCN/1% aq. AcOH 40 :60; 9.0 ml/min) to give the
following six fractions: Fr. 6.12.1 (893.3 mg), Fr. 6.12.2 (324.4 mg), Fr. 6.12.3 (131.3 mg), Fr. 6.12.4
(350.4 mg), Fr. 6.12.5 (48.8 mg), and Fr. 6.12.6 (135.0 mg). Fr. 6.12.1 (893.3 mg) was separated by HPLC
(Cosmosil 5C18-MS-II, 250 ꢃ 20 mm i.d., MeCN/MeOH/1% aq. AcOH 32 :16 :52; 9.0 ml/min) to furnish
the following fourteen fractions: Fr. 6.12.1.1 (13.0 mg), Fr. 6.12.1.2 (9.4 mg), Fr. 6.12.1.3 (34.5 mg),
Fr. 6.12.1.4 (10.5 mg), Fr. 6.12.1.5 (23.0 mg), Fr. 6.12.1.6 (80.8 mg), Fr. 6.12.1.7 (49.7 mg), Fr. 6.12.1.8
(86.8 mg), Fr. 6.12.10.9 (34.9 mg), Fr. 6.12.1.10 (125.8 mg, 0.139%; perennisaponin H (2); tR 58.2),
Fr. 6.12.1.11 (57.9 mg), Fr. 6.12.1.12 (109.2 mg, 0.119%; perennisaponin G (1); tR 64.0), Fr. 6.12.1.13
(56.0 mg), and Fr. 6.12.1.14 (22.2 mg). An aliquot of Fr. 6.12.2 (180.0 mg) was purified by HPLC
(Cosmosil HILIC, 250 ꢃ 20 mm i.d., MeCN/H2O 90 :10; 9.9 ml/min): perennisaponins J (4, 12.7 mg,
0.026%; tR 49.9) and M (7, 31.7 mg, 0.065%; tR 52.7) together with perennisaponins C (9.3 mg, 0.019%; tR
65.2), D (8.1 mg, 0.017%; tR 57.0), E (10.4 mg, 0.021%; tR 59.2), and F (28.5 mg, 0.059%; tR 55.7).

584
Helvetica Chimica Acta – Vol. 93 (2010)
Fr. 6.12.3 (131.3 mg) was purified by HPLC (Cosmosil HILIC, 250 ꢃ 20 mm i.d., MeCN/H2O 90 :10;
9.9 ml/min): perennisaponins J (4, 32.6 mg, 0.034%; tR 54.2) and K (5, 25.6 mg, 0.027%; tR 57.1).
Perennisaponin G (¼(2b,3b,4a,16a)-3-[(6-Deoxy-a-l-mannopyranosyl)oxy]-2,16,23-trihydroxy-
olean-12-en-28-oic Acid O-6-Deoxy-a-l-mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-
acetyl-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-4-O-acetyl-6-deoxy-b-d-galactopyranosyl Ester; 1): Amor-
1
phous powder. [a]2D5 ¼ ꢀ19.5 (c ¼ 4.48, MeOH). IR (KBr): 3450, 1736, 1638, 1049. H- and 13C-NMR:
Tables 1 and 2. FAB-MS (pos.): 1327 ([M þ Na]þ). FAB-MS (neg.): 1303 ([M ꢀ H]ꢀ), 1157 ([M ꢀ
C6H11O4]ꢀ). HR-FAB-MS (pos.): 1327.6296 ([M þ Na]þ, C63H100NaO2þ8 ; calc. 1327.6299).
Perennisaponin H (¼(2b,3b,4a,16a)-3-[(6-Deoxy-a-l-mannopyranosyl)oxy]-2,16,23-trihydroxy-
olean-12-en-28-oic Acid O-6-Deoxy-a-l-mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-
acetyl-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-4-O-acetyl-6-deoxy-b-d-galactopyranosyl Ester; 2): Amor-
1
phous powder. [a]2D5 ¼ ꢀ30.9 (c ¼ 4.43, MeOH). IR (KBr): 3450, 1734, 1638, 1049. H- and 13C-NMR:
Tables 1 and 2. FAB-MS (pos.): 1327 ([M þ Na]þ). FAB-MS (neg.): 1303 ([M ꢀ H]ꢀ), 1157 ([M ꢀ
C6H11O4]ꢀ), 649 ([M ꢀ C27H43O18]ꢀ). HR-FAB-MS (pos.): 1327.6305 ([M þ Na]þ, C63H100NaO2þ8 ; calc.
1327.6299).
Perennisaponin I (¼(2b,3b,4a,16a)-3-[(6-Deoxy-a-l-mannopyranosyl)oxy]-2,16,23-trihydroxy-
olean-12-en-28-oic Acid O-6-Deoxy-a-l-mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-
acetyl-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-6-deoxy-4-O-[(3S)-3-hydroxy-1-oxobutyl]-b-d-galactopyr-
anosyl Ester; 3): Amorphous powder. [a]2D5 ¼ ꢀ22.0 (c ¼ 2.28, MeOH). IR (KBr): 3450, 1738, 1638, 1049.
1H- and 13C-NMR: Tables 1 and 2. FAB-MS (pos.): 1371 ([M þ Na]þ). FAB-MS (neg.): 1347 ([M ꢀ
H]ꢀ), 1115 ([M ꢀ C10H17O6]ꢀ), 649 ([M ꢀ C29H47O19]ꢀ). HR-FAB-MS (pos.): 1371.6558 ([M þ Na]þ,
C65H104NaOþ29 ; calc. 1371.6561).
Perennisaponin J (¼(2b,3b,4a,16a)-3-[(6-Deoxy-a-l-mannopyranosyl)oxy]-2,16,23-trihydroxy-
olean-12-en-28-oic Acid O-6-Deoxy-a-l-mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-
acetyl-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-4-O-[(3S)-3-(acetyloxy)-1-oxobutyl]-6-deoxy-b-d-galacto-
pyranosyl Ester; 4): Amorphous powder. [a]2D5 ¼ ꢀ48.5 (c ¼ 1.20, MeOH). IR (KBr): 3445, 1734, 1638,
1049. 1H- and 13C-NMR: Tables 3 and 4. FAB-MS (pos.): 1413 ([M þ Na]þ). FAB-MS (neg.): 1389
([M ꢀ H]ꢀ), 649 ([M ꢀ C31H49O20]ꢀ). HR-FAB-MS (pos.): 1413.6664 ([M þ Na]þ, C67H106NaO3þ0 ; calc.
1413.6667).
Perennisaponin K (¼(2b,3b,4a,16a)-3-[(6-Deoxy-a-l-mannopyranosyl)oxy]-2,16,23-trihydroxy-
olean-12-en-28-oic Acid O-6-Deoxy-a-l-mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-3-O-
acetyl-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-4-O-[(3S)-3-(acetyloxy)-1-oxobutyl]-6-deoxy-b-d-galacto-
pyranosyl Ester; 5): Amorphous powder. [a]2D5 ¼ ꢀ47.1 (c ¼ 1.00, MeOH). IR (KBr): 3445, 1734, 1638,
1049. 1H- and 13C-NMR: Tables 3 and 4. FAB-MS (pos.): 1413 ([M þ Na]þ). FAB-MS (neg.): 1389
([M ꢀ H]ꢀ), 649 ([M ꢀ C31H49O20]ꢀ). HR-FAB-MS (pos.): 1413.6674 ([M þ Na]þ, C67H106NaO3þ0 ; calc.
1413.6667).
Perennisaponin L (¼(2b,3b,4a,16a)-3-[(6-Deoxy-a-l-mannopyranosyl)oxy]-2,16,23-trihydroxy-
olean-12-en-28-oic Acid O-6-Deoxy-a-l-mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-
acetyl-6-deoxy-a-l-mannopyranosyl-(1 ! 2)-6-deoxy-4-O-{(3S)-3-{[(3S)-3-hydroxy-1-oxobutyl]oxy}-1-
oxobutyl}-b-d-galactopyranosyl Ester; 6): Amorphous powder. [a]2D6 ¼ ꢀ22.4 (c ¼ 1.64, MeOH). IR
1
(KBr): 3450, 1736, 1655, 1049. H- and 13C-NMR: Tables 3 and 4. FAB-MS (pos.): 1457 ([M þ Na]þ).
FAB-MS (neg.): 1433 ([M ꢀ H]ꢀ), 1115 ([M ꢀ C14H23O8]ꢀ), 649 ([M ꢀ C33H53O21]ꢀ), 503 ([M ꢀ
C39H62O25]ꢀ). HR-FAB-MS (pos.): 1457.6937 ([M þ Na]þ, C69H110NaO3þ1 ; calc. 1457.6929).
Perennisaponin M (¼(2b,3b,4a,16a)-3-[(6-Deoxy-a-l-mannopyranosyl)oxy]-2,16,23-trihydroxy-
olean-12-en-28-oic Acid O-6-Deoxy-a-l-mannopyranosyl-(1 ! 3)-O-b-d-xylopyranosyl-(1 ! 4)-O-2-O-
acetyl-6-deoxy-a-l-mannopyranosyl-(1! 2)-6-deoxy-4-O-{(3S)-3-{{(3S)-3-{[(3S)-3-hydroxy-1-oxybutyl]-
oxy}-1-oxobutyl}oxy}-1-oxobutyl}-b-d-galactopyranosyl Ester; 7): Amorphous powder. [a]2D6 ¼ ꢀ24.2
(c ¼ 1.42, MeOH). IR (KBr): 3445, 1736, 1655, 1049. 1H- and 13C-NMR: Tables 3 and 4. FAB-MS (pos.):
1543 ([M þ Na]þ). FAB-MS (neg.): 1519 ([M ꢀ H]ꢀ), 649 ([M ꢀ C37H59O23]ꢀ). HR-FAB-MS (pos.):
1543.7290 ([M þ Na]þ, C73H116NaO3þ3 ; calc. 1543.7297).
Alkaline Hydrolysis of 1 – 7. A soln. of each perennisaponins 1 – 7 (6 mg) in H2O/1,4-dioxane 1:1
(1 ml) was treated with 10% aq. KOH soln. (1 ml), and the mixture was stirred at 408 for 12 h. The
mixture was neutralized over Dowex-HCR-W2 resin (Hþ form), which was then removed by filtration.

Helvetica Chimica Acta – Vol. 93 (2010)
585
The filtrate was concentrated, and the resulting product subjected to reversed-phase CC (2 g of ODS;
H2O ! MeOH) to afford H2O- and MeOH-eluted fractions, resp. The H2O-eluted fraction was dissolved
in ClCH2CH2Cl (2 ml). This soln. was treated with N,N’-diisopropyl O-(4-nitrobenzyl)-isourea (¼(4-
nitrophenyl)methyl N,N’-bis(1-methylethyl)carbamimidate; 10 mg) and stirred at 808 for 1 h. The
mixture was then subjected to HPLC (YMC-Pack ODS-A, 250 ꢃ 4.6 mm i.d., MeOH/H2O 65 :35; 0.8 ml/
min, UV detection at 254 nm): 4-nitrobenzyl esters of 3-hydroxybutanoic acid (a; tR 7.0) from 3 – 7 and of
AcOH (b; tR 8.3) from 1 – 7. The MeOH-eluted fraction was subjected to normal-phase CC (2 g of SiO2,
CHCl3/MeOH/H2O 10 :3 :1, lower layer) to give (2b,3b,4a,16a)-3-[(6-deoxy-a-l-mannopyranosyl)oxy]-
2,16,23-trihydroxyolean-12-en-28-oic acid (1a; 2 mg each from 1 – 7).
Deacylation of 1 – 7. A soln. of each perennisaponin 1 – 7 (2 mg) in 0.5% MeONa/MeOH (1 ml) was
stirred at r.t. for 3 h. An aliquot of the mixture was subjected to HPLC (YMC-Pack ODS-AQ, 250 ꢃ
4.6 mm i.d.; MeOH/H2O 20 :80; 0.7 ml/min, optical-rotation detector): methyl (þ)-(3S)-3-hydroxybu-
tanoate (tR 9.2, pos. optical rotation) from 3 – 7. The rest of each mixture was neutralized over Dowex-
HCR-W2 resin (Hþ form), which was then removed by filtration. The filtrate was concentrated and the
resulting product purified by HPLC (Cosmosil 5C18-MS-II, 250 ꢃ 20 mm i.d., MeCN/MeOH/H2O
32 :16 :52; 9.0 ml/min): bellissaponin BS1 (1b; 1.5 mg each from 1 – 7).
Effect on Pancreatic Lipase Activity. A suspension of triolein (80 mg), phosphatidylcholine (10 mg),
and sodium taurocholate (5 mg) in 9 ml of 0.1m Tris · HCl buffer (pH 7.0) containing 0.1m NaCl was
homogeneously emulsified with a homogenizer (strait Teflon pestle with strait glass tube, volume 20 ml).
The substrate suspension (0.1 ml) in a test tube was preincubated with 5 ml of test sample in DMSO and
95 ml of Tris · HCl buffer for 3 min at 378. An aliquot of porcine pancreatic lipase (250 mg/ml, type II,
Sigma – Aldrich, Inc.; 50 ml) or Tris · HCl buffer (50 ml) as a blank test was then added to start the
reaction. After 30 min of incubation, the test tube was immediately immersed in boiling water for 2 min
to stop the reaction and then cooled with water. Free fatty acid concentration was determined by a
commercial kit (Wako NEFA C test, Wako Pure Chemical Industries, Ltd.). Theasaponin E1 was isolated
from Camellia sinensis [32] and used as a reference compound [28]. IC50 was determined graphically
(N ¼ 4).
Statistics. Values were expressed as means ꢂ s.e.m. For statistical analysis, one-way analysis of
variance followed by Dunnettꢃs test was used. Probability (p) values less than 0.05 were considered
significant.
REFERENCES
[1] L.-B. Wang, T. Morikawa, S. Nakamura, K. Ninomiya, H. Matsuda, O. Muraoka, L.-J. Wu, M.
Yoshikawa, Heterocycles 2009, 78, 1235.
[2] T. Morikawa, X. Li, E. Nishida, Y. Ito, H. Matsuda, S. Nakamura, O. Muraoka, M. Yoshikawa, J. Nat.
Prod. 2008, 71, 828.
[3] M. Yoshikawa, X. Li, E. Nishida, S. Nakamura, H. Matsuda, O. Muraoka, T. Morikawa, Chem.
Pharm. Bull. 2008, 56, 559.
[4] H. Matsuda, T. Morikawa, T. Ohgushi, T. Ishiwada, N. Nishida, M. Yoshikawa, Chem. Pharm. Bull.
2005, 53, 387.
[5] M. Yoshikawa, T. Morikawa, K. Yamamoto, Y. Kato, A. Nagatomo, H. Matsuda, J. Nat. Prod. 2005,
68, 1360.
[6] M. Yoshikawa, S. Sugimoto, S. Nakamura, H. Matsuda, Chem. Pharm. Bull. 2007, 55, 571.
[7] M. Yoshikawa, F. Xu, T. Morikawa, Y. Pongpiriyadacha, S. Nakamura, Y. Asao, H. Matsuda, Chem.
Pharm. Bull. 2007, 55, 308.
[8] S. Nakamura, S. Sugimoto, H. Matsuda, M. Yoshikawa, Heterocycles 2007, 71, 577.
[9] M. Yoshikawa, S. Nakamura, Y. Kato, K. Matsuhira, H. Matsuda, Chem. Pharm. Bull. 2007, 55, 598.
[10] M. Yoshikawa, T. Morikawa, Y. Asao, E. Fujiwara, S. Nakamura, H. Matsuda, Chem. Pharm. Bull.
2007, 55, 606.
[11] M. Yoshikawa, S. Sugimoto, S. Nakamura, H. Sakumae, H. Matsuda, Chem. Pharm. Bull. 2007, 55,
1034.

586
Helvetica Chimica Acta – Vol. 93 (2010)
[12] S. Nakamura, S. Sugimoto, H. Matsuda, M. Yoshikawa, Chem. Pharm. Bull. 2007, 55, 1342.
[13] M. Yoshikawa, T. Wang, S. Sugimoto, S. Nakamura, A. Nagatomo, H. Matsuda, S. Harima,
Yakugaku Zasshi 2008, 128, 141.
[14] M. Yoshikawa, S. Sugimoto, Y. Kato, S. Nakamura, T. Wang, C. Yamashita, H. Matsuda, Chem.
Biodiversity 2009, 6, 903.
[15] M. Yoshikawa, S. Sugimoto, S. Nakamura, H. Matsuda, Chem. Pharm. Bull. 2008, 56, 1297.
[16] Y. Xie, T. Morikawa, K. Ninomiya, K. Imura, O. Muraoka, D. Yuan, M. Yoshikawa, Chem. Pharm.
Bull. 2008, 56, 1628.
[17] S. Nakamura, Y. Okazaki, K. Ninomiya, T. Morikawa, H. Matsuda, M. Yoshikawa, Chem. Pharm.
Bull. 2008, 56, 1704.
[18] S. Sugimoto, M. Yoshikawa, S. Nakamura, H. Matsuda, Heterocycles 2009, 78, 1023.
[19] S. Sugimoto, G. Chi, Y. Kato, S. Nakamura, H. Matsuda, M. Yoshikawa, Chem. Pharm. Bull. 2009, 57,
269.
[20] T. Morikawa, L.-B. Wang, S. Nakamura, K. Ninomiya, E. Yokoyama, H. Matsuda, O. Muraoka, L.-J.
Wu, M. Yoshikawa, Chem. Pharm. Bull. 2009, 57, 361.
[21] T. Schçpke, C. Al-Tawaha, V. Wray, M. Nimiz, K. Hiller, Phytochemistry 1997, 45, 125.
[22] M. J. Burk, T. G. P. Harper, C. S. Kalberg, J. Am. Chem. Soc. 1995, 117, 4423.
[23] T. Morikawa, H. Matsuda, T. Ohgushi, N. Nishida, T. Ishiwada, M. Yoshikawa, Heterocycles 2004, 63,
2211.
[24] M. Yoshikawa, T. Wang, T. Morikawa, H. Xie, H. Matsuda, Chem. Pharm. Bull. 2007, 55, 1308.
[25] K. Ninomiya, T. Morikawa, H. Xie, H. Matsuda, M. Yoshikawa, Heterocycles 2008, 75, 1983.
[26] T. Morikawa, H. Xie, T. Wang, H. Matsuda, M. Yoshikawa, Chem. Pharm. Bull. 2008, 56, 1438.
[27] T. Morikawa, H. Xie, T. Wang, H. Matsuda, M. Yoshikawa, Chem. Biodiversity 2009, 6, 411.
[28] L.-K. Han, Y. Kimura, M. Kawashima, T. Takaku, T. Taniyama, T. Hayashi, Y.-N. Zheng, H. Okuda,
Int. J. Obes. 2001, 25, 1459.
[29] L.-K. Han, Y.-N. Zheng, M. Yoshikawa, H. Okuda, Y. Kimura, BMC Complem. Altern. Med. 2005, 5,
9.
[30] T. Morikawa, Y. Xie, Y. Asao, M. Okamoto, C. Yamashita, O. Muraoka, H. Matsuda, Y.
Pongpiriyadacha, D. Yuan, M. Yoshikawa, Phytochemistry 2009, 70, 1166.
[31] Y. Asao, T. Morikawa, Y. Xie, M. Okamoto, M. Hamao, M. Matsuda, O. Muraoka, D. Yuan, M.
Yoshikawa, Chem. Pharm. Bull. 2009, 57, 198.
[32] I. Kitagawa, K. Hori, T. Motozawa, T. Murakami, M. Yoshikawa, Chem. Pharm. Bull. 1998, 46, 1901.
Received July 9, 2009