Chemical constituents of the bulbs of scilla peruviana and their pancreatic lipase inhibitory activity

Article abstract of DOI:10.3390/ijms22031262

Scilla species are used as medicinal plants and contain lanosterol-type triterpene glyco-sides. The phytochemical investigation of the bulbs of Scilla peruviana led to the isolation of 17 com-pounds, including three new rearranged pentacyclic-lanosterol glycosides (1?3) and two new ho-moisoflavanone glycosides (12 and 13). The structures of the undescribed compounds were deter-mined by extensive spectroscopic analyses, including two-dimensional (2D) NMR. Among the triterpene glycosides, 2, 3, and 6 showed significant pancreatic lipase inhibitory activity in a concen-tration-dependent manner in vitro. The oral administration of scillascilloside D-2 (6) reduced serum triglyceride levels in a dose-dependent manner in soybean oil-loaded mice.

Full text of DOI:10.3390/ijms22031262

International Journal of
Molecular Sciences
Article
Chemical Constituents of the Bulbs of Scilla peruviana and
Their Pancreatic Lipase Inhibitory Activity
Yukiko Matsuo *, Asuka Yamashiro, Kanae Ootomo, Mika Nakagawa, Hiroko Tsuchihashi, Niro Inaba and
Yoshihiro Mimaki
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji,
Tokyo 192-0392, Japan; y104219@toyaku.ac.jp (A.Y.); y114037@toyaku.ac.jp (K.O.); y134172@toyaku.ac.jp (M.N.);
154150@toyaku.ac.jp (H.T.); ninaba@toyaku.ac.jp (N.I.); mimakiy@toyaku.ac.jp (Y.M.)
* Correspondence: matsuoy@toyaku.ac.jp; Tel.: +81-42-676-4577
Abstract: Scilla species are used as medicinal plants and contain lanosterol-type triterpene glycosides.
The phytochemical investigation of the bulbs of Scilla peruviana led to the isolation of 17 compounds,
including three new rearranged pentacyclic-lanosterol glycosides (1–3) and two new homoisofla-
vanone glycosides (12 and 13). The structures of the undescribed compounds were determined by
extensive spectroscopic analyses, including two-dimensional (2D) NMR. Among the triterpene glyco-
sides, 2, 3, and 6 showed significant pancreatic lipase inhibitory activity in a concentration-dependent
manner in vitro. The oral administration of scillascilloside D-2 (6) reduced serum triglyceride levels
in a dose-dependent manner in soybean oil-loaded mice.
Keywords: Scilla peruviana; Asparagaceae; triterpene; lanosterol glycoside; homoisoflavanone; pan-
creatic lipase
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Matsuo, Y.; Yamashiro, A.;
Ootomo, K.; Nakagawa, M.;
1. Introduction
Tsuchihashi, H.; Inaba, N.; Mimaki, Y.
Chemical Constituents of the Bulbs of
Scilla peruviana and Their Pancreatic
Lipase Inhibitory Activity. Int. J. Mol.
Sci. 2021, 22, 1262. https://doi.org/
10.3390/ijms22031262
Scilla, Eucomis, Chionodoxa, and Muscari species belong to the Asparagaceae (formerly
Liliaceae) family and are rich sources of lanosterol-type triterpene glycosides [1–4]. Al-
though the bulbs of Scilla scilloides (Lindl.) Druces, which contain homoisoflavones and
triterpenes, has been used as a traditional medicine for the treatment of dermal inflam-
mation [
conducted phytochemical examinations of the bulbs of S. peruviana and characterized two
rearranged pentacyclic-lanosterol glycosides, peruvianosides A and B [ ], a pentacyclic-
norlanosterol glycoside, scillascilloside E-1 [ ], and four hexacyclic-lanosterol glycosides
with a modified spirocyclic side chain, including scillascilloside D-2 and scillasaponin
B [10 11]. Peruvianoside A and scillasaponin B were found to show cyclic adenosine
monophosphatase (cAMP) phosphodiesterase inhibitory activity [10 11]. We performed
further phytochemical analyses of the bulbs of S. peruviana that resulted in the isolation
of 17 compounds ( 17), including three undescribed rearranged pentacyclic-lanosterol
glycosides ( ) and two undescribed homoisoflavanone glycosides (12 and 13). The
5,6]. S. peruviana L. are widely used as an ornamental plant. Previously, we
Academic Editor: Charles Brennan
Received: 13 January 2021
Accepted: 26 January 2021
Published: 27 January 2021
7,8
9
,
,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1–
1–3
structures of the undescribed compounds were determined by extensive spectroscopic
analyses, including two-dimensional (2D) NMR. Recently, some triterpenes and triterpene
glycosides have been reported to exhibit potent pancreatic lipase inhibitory activity and
reduce serum triglyceride (TG) levels in mice via pancreatic lipase inhibition, such as
Copyright:
© 2021 by the authors.
oleanane-type triterpene bisdesmosides (IC50 81.4–223
µM). Pancreatic lipase plays a key
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
role in the digestion of TG and is one of the targets for anti-obesity treatment [12
–
14]. Our
previous study revealed that oral administration of cymbopogonol, a lupine-type triterpene,
from Cymbopogon citratus reduced serum TG levels in mice via pancreatic lipase inhibitory
activity [15]. On the other hand, homoisoflavanones have not been reported previously
as pancreatic lipase inhibitors in natural products polyphenols [16]. In this study, we
Int. J. Mol. Sci. 2021, 22, 1262. https://doi.org/10.3390/ijms22031262
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 1262
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evaluated the pancreatic lipase inhibitory activity of the isolated lanosterol-type triterpene
glycosides (1–11) and the effect of 6 on serum TG levels in soybean oil-loaded mice.
2. Results and Discussion
2.1. Structural Elucidation
The bulbs of S. peruviana (11.2 kg dry weight) were treated with MeOH. After removing
the solvent, the MeOH extract (981 g) was applied to a porous-polymer polystyrene resin
(Diaion HP-20) column. The MeOH-eluted fraction was passed through column chromatog-
raphy (CC) on silica gel and octadecylsilanized (ODS) silica gel, producing
Compounds 11 and 14 17 were identified as peruvianoside A ( ) [ ], peruvianoside B
) [ ], (23S,25R)-3 ,31-dihydroxy-17 ,23-epoxy-5 -lanost-8-en-23,26-olactone3 -O-[O- -L-
rhamnopyranosyl-(1 2)-O- -D-gluco-pyranosyl-(1 2)-O- -L-arabinopyranosyl-(1 6)-O-
-D-glucopyranoside] (scillascilloside D-2) ( ) [10], (23S,25R)-3 ,30,31-trihydroxy-17 ,23-
-L-rhamno-pyranosyl-(1 2)-O- -D-glucopyr-
6)-O- -D-glucopyranoside] ( ) [10], (23S,25R)-
-lanost-8-en-23,26-olactone 3 -O-[O- -D-glucopyranosyl-
-L-rhamnopyranosyl-(1 2)]-O- -D-glucopyranosyl-(1 2)-O- -L-arabinopyra-
6)-O- -D-glucopyranoside] (scillanoside L-2) ( ) [17], 15-deoxy-eucosterol3 -[O-
-D-glucopyranosyl-(1 3)-O-[ -L-rhamnopyranosyl-(1 2)]- -D-glucopyranosyl-(1 2)-
-L-arabinopyranosyl-(1 6)-O- -D-glucopyranoside] (scillascilloside E-1) ( ) [ ], 15-
-[O- -D-glucopyranosyl-(1 3)-O-[ -L-rhamnopyranosyl-
2)-O- -L-arabinopyranosyl-(1 6)-O- -D-glucopyranosi-
4–11 and 14–17.
4–
–
4 7
(5
7
β
α
α
β
α
→
β
→
α
→
β
6
β
α
epoxy-5
anosyl-(1
α
-lanost-5-en-23,26-olactone3-O-[O-
2)-O- -L-arabinopyranosyl-(1
,31-dihydroxy-17 ,23-epoxy-5
3)-O-[
nosyl-(1
α
→
β
→
α
→
β
7
3β
α
α
β
β
(1
→
α
→
β
→
α
→
β
8
→
β
β
→
α
β
→
O-
α
→
β
9 9
deoxy-30-hydroxyeucosterol3
β
β
→
α
(1 2)]- -D-glucopyranosyl-(1
→
β
→
α
→
β
de] (scillanoside L-1) (10) [17], scillanostaside G (11) [18], (3R)-5,7-dihydroxy-6-methoxy-3-
(40-methoxybenzyl)-chroman-4-one (14) [19], (3R)-5,7-dihydroxy-3-(40-hydroxybenzyl)-6-
0
methoxy-chroman-4-one (15) [20], and (3S)-6,7-dimethoxy-5-hydroxy-3-(4 -hydroxybenzyl)-
chroman-4-one (16) [21], and (3R)-5,7-dihydroxy-3-(40-methoxybenzyl)-chroman-4-one
(
17) [22] (Figure 1). This is the first report on the isolation of
S. peruviana.
Compound
8, 10, 11, and 14–17 from
1
was obtained as an amorphous solid having the molecular formula
C49H74O22, which was determined by high-resolution electrospray ionization time-of-flight
mass spectrometry (HR-ESI-TOF-MS, m/z 1037.4563 [M + Na]+, calculated for C49H74NaO22:
1037.4569) and 13C NMR (49 carbon signals) data. The IR spectrum of
1
showed absorption
bands of hydroxy groups at 3398 cm−1 and carbonyl groups at 1699, 1670, and 1587 cm−1
.
The 1H and 13C NMR spectra showed signals assignable to seven methyl groups [δH 1.99 (d,
J = 1.2 Hz, Me-27), 1.84 (s, Me-32), 1.37 (s, Me-19), 1.30 (s, Me-30), 1.28 (s, Me-18), 1.16 (s,
Me-31), and 1.07 (d, J = 6.9 Hz, Me-21); δC 30.7 (Me-32), 27.9 (Me-30), 21.1 (Me-18), 17.4 (Me-
19), 16.5 (Me-31), 13.3 (Me-27), and 11.9 (Me-21)], two olefinic groups [δC 151.2 (C-8) and
151.6 (C-9) and δH 6.96 (dd, J = 10.6, 1.2 Hz, H-24); δC 143.5 (C-24) and 130.3 (C-25)], two
oxymethine groups [δH 3.35 (dd, J = 11.8, 4.7 Hz, H-3); δC 88.4 (C-3), and δH 5.10 (dd, J = 7.4
,
6.8 Hz, H-16); δC 79.4 (C-16)], an hemiacetalic proton and carbon [δH 5.24 (d, J = 6.5 Hz
,
H-23); δC 97.6 (C-23)], a tertiary carbon bearing a hydroxy group [δC 82.1 (C-17)], a methyl
ester group [δC 168.3 (C-26) and δH 3.71(3H, s); δC 51.6], and three anomeric protons and
carbons [δH 6.38 (br s, H-1000), 5.83 (d, J = 6.9 Hz, H-100), and 4.91 (d, J = 7.7 Hz, H-10);
δC 105.2 (C-10), 102.1 (C-1000), and 101.9 (C-100)] (Tables 1 and 2). These 1H and 13C NMR
spectral features of 1 were similar to those of 4 [5], except for the signals arising from the
B and C-rings parts of the aglycone moiety. The two methylene signals at δC 26.5 (C-7)
and 21.1 (C-11) observed in the 13C NMR spectrum of
4
were displaced by two conjugated
carbonyl signals at δC 201.9 (C-7) and 203.0 (C-11) in
1. Furthermore, an olefinic carbon
signals of
and observed at δC 151.2 (C-8) and 151.6 (C-9) in
was a 7,11-dioxo derivate of . This was confirmed by long-range correlations between
4
at δC 135.9 (C-8) and 134.6 (C-9) were shifted downfield by 15.3 and 17.0 ppm
1
, respectively. These data indicated that
1
4
H2-12 at δH 3.75 and 2.77 and C-11 at δC 203.0, and between H-5 at δH 1.69/H2-6 at
δH 2.60 and 2.30 and C-7 at δC 201.9 in the heteronuclear multiple bond correlation (HMBC)
spectrum of 1 (Figure 2). The configuration of the C-23 hydroxy group was confirmed to

Int. J. Mol. Sci. 2021, 22, 1262
3 of 12
be
α
based on the spin-coupling constants of 3JH-20,H-22 = 11.8 Hz, 3JH-22,H-23 = 6.5 Hz, and
3JH-22,H-24 = 10.6 Hz, and NOE correlations between H-23 and H-24/H-20 [10]. Enzymatic
hydrolysis of
1 gave D-glucose (Glc) and L-rhamnose (Rha), which were identified by
comparison of their refractive index and optical rotations with those of authentic samples
using HPLC. The anomeric conformation of the Glc and Rha groups was ascertained by the
J values of their anomeric protons, respectively. In the HMBC spectrum of
1
, long-range
000
00
00
correlations were observed between H-1 of Rha at δH 6.38 and C-2 of Glc at δC 78.7, H-1
of Glc at δH 5.83 and C-20 of Glc at δC 78.7, and between H-10 of Glc at δH 4.91 and C-3 of
the aglycone at δC 88.4. Accordingly,
1 was determined to be 7,11-dioxo-peruvianoside A.
Figure 1. Isolated compounds from Scilla peruviana.

Int. J. Mol. Sci. 2021, 22, 1262
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1
Table 1. H and 13C NMR (500 and 125 MHz, C5D5N) spectroscopic assignments of the aglycone moieties of 1–3.
1
2
3
C
δH
J (Hz)
δC
δH
J (Hz)
δC
δH
J (Hz)
δC
1a
b
2a
b
3
4
5
6a
b
3.00
1.21
2.28
1.92
3.35
—
1.69
2.60
2.30
—
dt
br dd
m
m
dd
13.8, 3.3
13.8, 3.4
34.4
1.54
1.10
2.26
1.85
3.33
—
1.15
1.70
1.51
2.08
1.51
—
—
—
2.11
2.00
2.45
1.58
—
m
m
m
m
dd
35.8
1.62
1.19
2.26
1.84
3.36
—
1.11
1.71
1.50
2.10
1.39
—
—
—
2.14
2.06
2.45
1.63
—
m
m
m
m
dd
35.8
26.9
27.1
27.1
11.8, 4.7
88.4
36.9
50.7
36.5
11.3, 4.2
89.7
39.8
51.0
18.4
11.7, 4.2
89.7
39.8
51.1
18.4
br d
br d
m
7.8
8.7
br d
dd
m
m
m
7.3
12.1, 6.2
br d
dd
m
m
m
7.8
12.0, 6.1
7
201.9
26.6
26.6
8
9
10
11
—
—
—
—
151.2
151.6
39.9
135.9
135.0
37.0
135.9
135.1
37.1
203.0
m
m
dt
20.8
m
m
m
20.8
12a
b
13
14
15a
b
16
17
18
19
20
21
22
23
24
25
26
27
3.75
2.77
—
d
d
15.9
15.9
48.9
13.0, 9.2
13.0, 4.9
25.4
25.7
br dd
br dd
13.0, 3.4
12.2, 7.8
52.4
47.4
42.5
48.5
48.1
44.4
48.6
48.3
44.4
—
—
—
3.36
2.50
5.10
—
1.28
1.37
2.29
1.07
3.38
5.24
6.96
—
dd
dd
dd
12.9, 7.4
12.9, 6.8
7.4, 6.8
2.22
2.03
5.06
—
1.20
0.99
3.31
1.15
3.98
5.26
7.22
—
m
m
dd
2.23
2.03
4.52
—
1.24
1.00
3.76
1.34
3.27
5.05
7.48
—
dd
m
m
79.4
82.1
21.1
17.4
36.2
11.9
44.3
97.6
143.5
130.3
168.3
13.3
—
9.2, 6.2
80.2
99.3
18.2
19.3
43.3
13.0
49.5
110.4
140.8
130.8
168.1
13.4
—
81.6
98.8
19.0
19.3
46.6
13.2
39.8
103.9
139.8
129.6
168.3
13.5
—
s
s
dq
d
ddd
d
dd
s
s
dq
d
ddd
d
dd
s
s
dq
d
ddd
d
dd
11.8, 6.9
6.9
11.8, 10.6, 6.5
6.5
10.0, 6.9
6.9
10.9, 10.0, 6.3
6.3
8.6, 6.1
6.1
8.7, 8.6, 5.8
5.8
10.6, 1.2
10.9, 1.1
8.7, 1.2
—
1.99
—
—
2.03
—
—
2.11
—
d
1.2
d
1.1
d
1.2
28
29
30
31
32
—
—
—
—
—
—
1.30
1.16
1.84
s
s
s
27.9
16.5
30.7
1.40
1.17
1.46
s
s
s
28.3
16.7
27.2
1.41
1.17
1.43
s
s
s
28.3
16.7
27.0
23-
OMe
26-
OMe
—
—
3.49
s
56.4
3.42
s
55.1
3.71
s
51.6
3.71
s
51.7
3.67
s
51.6
1
Table 2. H and 13C NMR (500 and 125 MHz, C5D5N) spectroscopic assignments of the sugar moieties of 1–3.
1
2
3
C
δH
J (Hz)
δC
δH
J (Hz)
δC
δH
J (Hz)
δC
Glc
Glc
10
20
4.91
4.42
4.54
4.06
3.89
4.51
4.35
5.83
4.24
4.23
4.06
3.85
d
dd
t
7.7
9.0, 7.7
9.0
105.2
78.7
79.5
71.9
77.9
62.8
4.97
4.43
4.56
4.10
3.94
4.55
4.39
5.85
4.31
4.25
4.09
3.85
d
dd
t
7.6
9.2, 7.6
9.2
105.2
79.0
79.5
72.0
77.9
62.8
4.97
4.44
4.56
4.10
3.93
4.53
4.34
5.86
4.33
4.25
4.09
3.86
d
dd
t
8.3
9.1, 8.3
9.1
105.2
79.0
79.5
71.9
77.9
62.8
30
40
t
9.0
t
9.2
t
9.1
50
ddd
dd
dd
d
dd
t
9.0, 5.4, 2.7
11.9, 2.7
11.9, 5.4
6.9
8.9, 6.9
8.9
m
dd
dd
d
dd
t
m
br d
dd
d
dd
t
60a
b
11.7, 3.4
11.7, 5.3
7.5
8.9, 7.5
8.9
8.9
8.9, 5.3, 2.6
11.4
11.4, 5.5
7.4
8.9, 7.4
8.9
8.9
8.9, 5.8, 3.1
100
200
300
400
500
101.9
78.7
79.4
72.9
77.6
102.0
78.6
79.4
72.9
77.5
102.0
78.6
79.4
72.9
77.5
t
8.9
8.9, 5.7, 3.0
t
t
ddd
ddd
ddd

Int. J. Mol. Sci. 2021, 22, 1262
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Table 2. Cont.
1
2
3
C
δH
J (Hz)
δC
δH
J (Hz)
δC
δH
J (Hz)
δC
600a
b
4.49
4.29
6.38
4.76
4.67
4.32
5.01
1.74
dd
dd
br s
br d
dd
t
10.8, 3.0
10.8, 5.7
63.5
4.49
4.31
6.41
4.76
4.69
4.32
5.06
1.80
dd
dd
br s
br d
dd
t
11.3, 2.6
11.3, 5.3
63.5
4.49
4.31
6.41
4.76
4.69
4.33
5.07
1.80
dd
dd
br s
br d
dd
t
11.5, 3.1
11.5, 5.8
63.5
Rha
1000
2000
3000
4000
5000
6000
102.1
72.4
72.7
74.3
69.5
19.0
102.1
72.4
72.7
74.3
69.6
18.9
102.1
72.4
72.7
74.3
69.6
19.0
3.3
9.3, 3.3
9.3
9.3, 6.3
6.3
3.3
9.2, 3.3
9.2
9.2, 6.2
6.2
3.3
9.3, 3.3
9.3
9.3, 6.2
6.2
dq
d
dq
d
dq
d
Figure 2. Key HMBC correlations of the aglycone moiety of 1.
1
The H and 13C NMR spectral features of
2
(C50H80O20) and
. However, when the 13C NMR spectra of
, the signal assignable to the hemiacetal carbon at δC 97.7 (C-23) in
was shifted downfield by 12.7 ppm in and 6.2 ppm in , and observed at δC 110.4 in
and at δC 103.9 in and displayed a deshielded methyl singlet
. The 1H NMR spectra of
, the methyl
singlet at δH 3.49 showed a long-range correlation with C-23 at δC 110.4. Additionally, the
methyl singlet at δH 3.42 exhibited an HMBC correlation with C-23 at δC 103.9 in . These
data indicated that the hemiacetal moiety of was changed to the methyl acetal in and
. In the NOESY spectrum of , NOE correlations between H-23 and H-24 and between
H-22 and C23-OMe/Me-27, and a spin-coupling constant of 3JH-22,H-23 = 6.3 Hz showed
the configuration at C-23 to be in . On the other hand, NOE correlations between C23-
3
(C50H80O20) were
closely related to those of
compared with that of
4
2
and were
3
4
4
2
2
3
3
2
3
signal at δH 3.49 and δH 3.42, respectively (Table 1). In the HMBC spectrum of
2
3
4
2
3
2
α
2
OMe and H-20/H-24 and between H-23 and H-22/Me-27, and a spin-coupling constant
3
of JH-22,H-23 = 5.8 Hz gave evidence that
3
2
2
was the C-23 epimer of
and was ascertained to be the same as that
and . Therefore, and were identified as
β
-methyl-peruvianoside A. The 1H NMR spectrum of
2. The triglycoside
moiety attached to C-3 of the aglycone of
4
α
3
of
by the analysis of the HMBC spectra of
-methyl-peruvianoside A and 23
3
2
3
23
12 (C30H38O15) was essentially analogous to that of the homoisoflavanone (14), showing
signals for two methylene groups at δH 4.36 (dd, J = 11.5, 4.0 Hz, H-2a) and 4.19 (dd,
J = 11.5, 6.5 Hz, H-2b), and δH 3.13 (dd, J = 14.0, 9.0 Hz, H-9a) and 2.72 (dd, J = 14.0
,
7.0 Hz, H-9b), a methine proton at δH 2.96 (m, H-3), 1,4-disubstituted aromatic ring at
0
0
0
0
δH 7.16 (d, J = 8.5 Hz, H-2 and H-6 ) and 6.86 (d, J = 8.5 Hz, H-3 and H-5 ), and an aromatic
proton at δH 6.31 (s, H-8), and two methoxy groups at δH 3.77 (s) and 3.75 (s) (Table 3).
Furthermore, signals for two anomeric protons and carbons were observed at δH 4.96 (d,
J = 7.5 Hz); δC 101.7 and δH 4.68 (br s); δC 102.2. The enzymatic hydrolysis of 12 gave
5,7-dihydroxy-6-methoxy-3-(40-methoxybenzyl)-chroman-4-one (12a) [19] and D-glucose

Int. J. Mol. Sci. 2021, 22, 1262
6 of 12
(Glc) and L-rhamnose (Rha) as the carbohydrate moieties. The absolute configuration at
C-3 was confirmed to be R because of the CD spectrum [(MeOH, ∆ε): 284 ( 1.98) nm], and
−
the specific rotation of 12a almost agreed with that of 14. In the HMBC spectrum of 12
,
long-range correlations were observed between0t0he anomeric proton (H-1000) of Rha at δH
00
4.68 and C-6 of Glc at δC 67.6, and between H-1 of Glc at δH 4.96 and C-7 of the aglycone
at δC 160.8. Thus, 12 was identified as (3R)-5-hydroxy-6-methoxy-3-(40-methoxybenzyl)-7-
[(O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl)oxy]-chroman-4-one.
1
Table 3. H and 13C NMR (500 and 125 MHz, CD3OD) spectroscopic assignments of 12 and 13.
12
13
C
δ
J (Hz)
δC
δ
J (Hz)
δC
H
H
2
4.36
4.19
2.96
—
—
—
—
—
6.31
—
3.13
2.72
—
dd
dd
m
11.5, 4.0
11.5, 6.5
70.8
4.35
4.20
2.96
—
—
—
—
—
6.31
—
3.09
2.68
—
7.06
6.73
—
6.73
7.06
3.75
—
4.95
3.50
3.46
3.63
3.59
4.01
3.60
dd
dd
m
11.5, 4.5
11.5, 7.0
71.4
3
4
4
5
6
7
8
8
9
48.2
200.0
101.8
155.7
131.2
160.8
97.4
49.4
201.7
105.2
156.4
132.0
160.8
98.1
a
a
s
s
160.1
33.0
162.2
33.9
dd
dd
14.0, 9.0
14.0, 7.0
dd
dd
14.0, 9.5
14.0, 7.3
0
1
2
3
4
5
6
131.2
131.2
115.2
160.0
115.1
131.2
61.4
55.7
101.7
74.8
78.1
71.4
77.3
67.6
132.0
132.0
117.2
158.3
117.2
132.0
62.7
0
0
0
0
0
7.16
6.86
—
d
d
8.5
8.5
d
d
8.5
8.5
6.86
7.16
3.75
3.77
4.96
3.49
3.46
3.36
3.59
4.02
3.59
d
d
s
s
d
dd
t
dd
m
dd
br d
8.5
8.5
d
d
s
8.5
8.5
6-OCH3
4 -OCH3
0
00
Glc 1
7.5
8.5, 7.5
8.5
d
dd
t
t
m
br d
br d
7.5
8.7, 7.5
8.7
102.5
75.5
78.8
72.2
78.1
68.3
00
2
3
4
5
00
00
9.2, 8.5
8.7
00
00
6
12.5, 5.0
12.5
11.2
11.0
000
Rha 1
4.68
3.88
3.69
3.32
3.62
1.18
br s
br s
dd
t
m
d
102.2
72.1
72.5
74.1
69.8
18.0
4.68
3.88
3.69
3.33
3.62
1.18
br s
br dd
dd
t
m
103.0
72.9
73.2
74.9
70.6
15.2
000
2
3
4
5
3.4, 1.4
9.8, 3.5
9.8
000
9.6, 3.5
9.6
000
000
000
6
6.3
br d
6.2
The 1H and 13C NMR spectra of 13 (C29H36O15) showed a close similarity to those of 12
,
including the signals attributed to the sugar moiety attached to C-7. However, 13 differed
from 12 in the lack of the 1H and 13C signals for the methoxy group at C-40 [δH 3.77 (s)
δC 55.7] (Table 3). Furthermore, the C-40 signal of 13 was shifted upfield by 1.7 ppm
when compared with that of 12. These data implied that the aglycone of 13 corresponded
;
to 15 and that 13 had an O-
C-7. Thus, 13 was identified as (3R)-5-hydroxy-6-methoxy-3-(4 -hydroxybenzyl)-7-[(O-
α
-L-rhamnopyranosyl-(1
→
6)-
β
-D-glucopyranosyl group at
0
α-L-
rhamnopyranosyl-(1→6)-β-D-glucopyranosyl)oxy]-chroman-4-one.
Structures of known compounds 14–17, including their absolute configurations, were
determined based on a spectroscopic analysis [19–22].

Int. J. Mol. Sci. 2021, 22, 1262
7 of 12
2.2. Lipase Inhibitory Activity
Various types of triterpenes and triterpene glycosides have been reported as pancreatic
lipase inhibitors and expected to be promising treatments for obesity [12 14 16 23]. The pan-
creatic lipase inhibitory activity of the isolated lanosterol-type triterpene glycosides ( 11
was measured using a commercially available kit (Figure 3 and Table 4). Cetilistat was used
as a positive control (IC50 1.43 0.052 M). As a result, , and showed the inhibitory ac-
tivity in a dose-dependent manner (IC50 0.84 0.029, 0.59
–
,
,
1–
)
±
µ
2
,
3
6
±
± 0.020, and 0.81 ± 0.029 mM).
Interestingly, 11, a tetranorlanosterol glycoside, inhibited the pancreatic lipase activity
in an inverted dose-dependent manner. Of the compounds with the peruvianoside A
derivatives (
1–
5
), the modification of the hemiacetal group to the corresponding methyl
and ) enhanced the lipase inhibitory activity. In the lanosterol derivatives
), had the most potent lipase inhibitory activity. Next,
on TG levels in ddY mice fed a single-dose administration of a high-fat
diet (intralipid) were examined (Figure 4). Compound at 150 or 300 mg/kg was orally
acetal group (
2
3
with a spiro-lactone group (
6–8 6
the effects of
6
6
administrated to mice, respectively, 15 min before intralipid loading. Serum TG levels were
determined at 120 min after intralipid oral treatment. In the control group, serum TG levels
increased from 48.3
compared to those of the untreated group. When the mice were treated with cetilistat as a
positive control, serum TG levels were held at 130.2 23.4 mg/dL. Although inhibition
potency of cetilistat in vivo was weaker than that of the lipase inhibitory activity in vitro
(IC50 1.43 M), the same range was reported in previous works [24 25]. The single oral
administration of at 300 mg/kg significantly suppressed the elevation in serum TG levels,
with values held at 90.9 ± 16.2 mg/dL in mice.
±
8.2 to 264.9
±
32.2 mg/dL following administration of intralipid
±
µ
,
6
Figure 3. The pancreatic lipase activity of
9–11 (0.45, 0.90, and 4.5 mM), and cetilistat (c) (0.1 [ ], 1.0 [ ], and 10 [ ] µM); Data are represented as
the mean S.E.M. of three experiments performed in triplicate; *** p < 0.0001 vs. control, ** p < 0.001
vs. control, ˙ p < 0.05 vs. control.
1–11. Compounds 1 and 8 (0.45 and 0.90 mM), 2–7 and
±
3β-Hydroxylanosta 9,24-dien-21-oic acid, a lanostane-type triterpene, isolated from
Protorhus longifolia, was reported to inhibit the pancreatic lipase activity [26]. However, no
previous reports have shown the effect of purified lanostane-type triterpene glycoside on
both lipase inhibition in vitro and TG reduction in vivo [27]. This is the first report of the
lipase inhibitory activity of lanosterol glycosides. The inhibition of lipase activity might be
only one of the possible mechanisms inhibiting triglycerides absorption. In conclusion, five
rearranged pentacyclic-lanosterol glycosides (
with a modified spirocyclic side chain ( ), two pentacyclic-norlanosterol glycosides
and 10), a pentacyclic-tetranorlanosterol glycoside (11), a homoisoflavanone glycosides
12 and 13), and four homoisoflavanones (14 17) were isolated from the MeOH extract of
1–5), three hexacyclic-lanosterol glycosides
6–8
(9
(
–
S. peruviana bulbs. Their structures were determined by extensive spectroscopic analysis
and the results of hydrolytic cleavage. Compounds 12, and 13 are undescribed
glycosides, and 10 11, and 14 17 were isolated from S. peruviana for the first time.
1–3,
8
,
,
–

Int. J. Mol. Sci. 2021, 22, 1262
8 of 12
The pancreatic lipase inhibitory activity of the isolated triterpene glycosides (
evaluated. Compounds , and
dependent manner (IC50 0.84
oral dose of at 300 mg/kg reduced serum TG levels in mice loaded with intralipid. It is
concluded that suppress elevated serum TG levels partially via lipase inhibition. Further
1
–
11) was
2,
3
6
showed significant lipase inhibitory activity in a dose-
±
0.029, 0.59 0.020, and 0.81 0.029 mM) and a single
±
±
6
6
studies are needed to reveal the mechanism of the effects of 6 on the serum TG reduction.
Table 4. The pancreatic lipase inhibitory activity of 1–11.
Compounds
IC50 (mM)
Cetilistat
1.43 ± 0.052 µM
1
–
2
3
4
5
6
7
8
0.84 ± 0.029
0.59 ± 0.020
1.42 ± 0.040
1.40 ± 0.035
0.81 ± 0.029
1.10 ± 0.015
-
9
10
11
0.91 ± 0.023
1.13 ± 0.062
>4.50
Data are represented as the mean ± S.E.M. of three experiments performed in triplicate.
Figure 4. Effects of 6 on the serum TG levels in mice fed a single-dose administration of a high-fat
diet. Compound 6 (150 or 300 mg/kg) and cetilistat (180 mg/kg); Data are represented as the
mean ± S.E.M. (n = 5); *** p < 0.0001 vs. control, ** p < 0.001 vs. control.
3. Materials and Methods
3.1. General Experimental Procedures
The instruments and experimental conditions were the same as those described in a
previous paper [28]. Optical rotations were measured on a JASCO P-1030 automatic digital
polarimeter (Jasco). IR spectra were obtained using a JASCO FT-IR 620 spectrophotometer
(Jasco). NMR spectra were recorded on a Bruker DRX-500 or AV-600 spectrometer (Bruker)
using standard Bruker pulse programs. Chemical shifts (δ) were given with reference to
tetramethylsilane (TMS) as an internal standard. HR-ESI-TOF-MS data were obtained
using a Water-Micromass LCT mass spectrometer (Waters-Micromass). CC was conducted
by Diaion HP-20 (Mitsubishi-chemical), silica gel Chromatorex BW-300 (Fuji-Silysia Chem-
ical), and ODS silica gel COSMOSIL 75C18-OPN (Nacalai Tesque). Analytical TLC was
performed on precoated silica gel 60 F254 or RP18 F254S plates (0.25 mm thick) (Merck). The
spots were detected by spraying the plates with 10% H2SO4 aqueous solution and then
heating. HPLC was conducted using a system consisting of a CCPM pump (Shimadzu), an
RI-8021 (Tosoh) or a Shodex OR-2 (Showa-Denko) detector, and a Rheodyne injection port
(Rohnert Park). A TSK gel ODS-100Z column (10 mm i.d.
×
250 mm, 5 µm) (Tosoh) was

Int. J. Mol. Sci. 2021, 22, 1262
9 of 12
used for the preparative HPLC. Enzymatic hydrolysis was carried out using naringinase
(EC 232-962-4, Sigma), acetic acid (AcOH) and potassium acetate (AcOK) (Wako).
3.2. Plant Material
The bulbs of Scilla peruviana. (11.2 kg dry weight) were obtained from Takii (Kyoto,
Japan) in 2014. The bulbs were cultivated, and the morphological characteristics of the
flowering plants allowed us to identify the plant material. A voucher specimen was
deposited at the Herbarium of the Tokyo University of Pharmacy and Life Sciences (KS-
2014-017).
3.3. Extraction and Isolation
The bulbs of S. peruviana (11.2 kg) were treated with MeOH (50 L) at 50 ◦C for 2 h.
After evaporating the solvent in vacuo, the MeOH extract (981 g) was subjected to a Diaion
HP-20 column (2200 g, 85 mm i.d.
×
600 mm) and successively partitioned by eluting
with MeOH–H2O (3:7; 1:1), MeOH, EtOH, and EtOAc (each 10 L) in order of decreasing
polarity. The MeOH-eluted fraction (221 g) was passed through a silica gel CC (2400 g,
42 mm i.d. × 430 mm) eluted with gradient mixtures of CHCl3–MeOH–H2O (20:10:1; 7:4:1)
and MeOH, to give 8 fractions (Frs. A-H). Fr. A was separated by ODS silica gel CC
(930 g, 32 mm i.d.
22 mm i.d. × 380 mm) eluted with CHCl3–MeOH–H2O (40:10:1) to give 13 (13.1 mg). Fr.
C was separated by silica gel CC (1800 g, 80 mm i.d. 400 mm) eluted with EtOAc–MeOH
(2:1; 1:1) and sequentially ODS silica gel CC (700 g, 30 mm i.d. 250 mm) eluted with
MeOH–H2O (2:1; 3:1) and MeCN–H2O (1:2; 2:3) to yield (22.1 mg). Fr. E was fractionated
by ODS silica gel CC (930 g, 35 mm i.d. 300 mm) using MeOH–H2O (2:1; 3:1; 4:1) and
MeCN–H2O (1:2; 2:3; 1:1), and preparative TLC using CHCl3–MeOH–H2O (20:10:1) to
give (15.5 mg), (16.0 mg), and 10 (43.8 mg). Fr. E-1 was separated by ODS silica gel
CC (930 g, 35 mm i.d. 300 mm) eluted with MeOH–H2O (2:1; 3:1; 4:1) and silica gel CC
(430 g, 22 mm i.d. 280 mm) eluted with CHCl3–MeOH–H2O (70:40:1; 10:10:1) to give
12 (21.8 mg). Fr. F was purified by ODS silica gel CC (930 g, 32 mm i.d. 320 mm) using
MeOH–H2O (2:1; 3:1; 4:1) and MeCN–H2O (1:2; 2:3; 1:1), and preparative HPLC using
MeCN–H2O (2:3) to yield (16.8 mg), (59.0 mg) and (15.6 mg). Fr. G was fractionated
by ODS silica gel CC (500 g, 22 mm i.d. 330 mm) using MeCN–H2O (1:2; 2:3) and silica
gel CC (450 g, 22 mm i.d. 280 mm) eluted with CHCl3–MeOH–H2O (10:10:1) to give
(69.3 mg), (56.1 mg), (25.0 mg) and 11 (50.8 mg). The EtOH-eluted fraction (5.4 g)
passed through a silica gel CC (1000 g, 32 mm i.d. 320 mm) eluted with gradient mixtures
of hexane–EtOAc (3:1; 1:1) and MeOH, to give 7 fractions (Frs. a–g). Fr. d was fractionated
by ODS silica gel CC (280 g, 17 mm i.d. 300 mm) using MeOH–H2O (5:2; 7:1) and
×
300 mm) eluted with MeOH–H2O (2:1) and silica gel CC (580 g,
×
×
7
×
1
4
×
×
×
5
6
8
×
×
2
3
9
×
×
preparative HPLC using MeOH–H2O (4:1) to give 14 (7.4 mg), 15 (42.3 mg), 16 (74.2 mg),
and 17 (9.7 mg).
3.4. Structural Characterization
10.1 (c 0.27, MeOH); IR νmax (film) cm−1
:
25
Compound
1
: An amorphous solid; [
α
]
D
−
3398 (OH), 2963 (CH), 1699 (C=O ester), 1670 (C=O ketone and C=C), 1587 (C=O ke-
1
tone); UV
λ
(MeOH) nm (log
ε
): 264 (3.98) (C=O ketone), 204 (4.26) (C=O ester). H
max
and 13C NMR (500 and 125 MHz, C5D5N), see Tables 1 and 2; HR-ESI-TOF-MS (m/z:
1037.4563 [M + Na]+, calculated for C49H74NaO22: 1037.4569).
Enzymatic hydrolysis of
in AcOH/AcOK buffer (pH 4.3, 4.0 mL) for 24 h. The crude hydrolysate was subjected to
Diaion HP-20 CC (6 mm i.d. 200 mm) eluted with MeOH–H2O (3:7), Me2CO–EtOH (1:1),
and MeOH to yield the sugar fraction (0.31 mg). The aglycone moiety of was decomposed.
The sugar fraction of was analyzed by HPLC under the following conditions: Capcell
Pak NH2 UG80 column (4.6 mm i.d. 250 mm, 5 m, Shiseido), mobile phase of MeCN–
1: Compound 1 (4.9 mg) was treated with naringinase (80 mg)
×
1
1
×
µ
H2O (7:3), detection by refractive index and optical rotation, and flow rate of 1.0 mL/min.
D-Glucose and L-rhamnose were identified by comparing their retention times and optical

Int. J. Mol. Sci. 2021, 22, 1262
10 of 12
rotation with those of authentic samples. tR (min): 16.85 (D-glucose, positive optical
rotation) and 7.97 (L-rhamnose, negative optical rotation).
25
Compound
2
: An amorphous solid; [
α
]
D
+10.4 (c 0.17, MeOH); IR ν
(film) cm−1
:
max
3390 (OH), 2935 (CH), 1699 (C=O ester), 1648 (C=C); UV λmax (MeOH) nm (log ε): 219 (4.37)
(C=C), 205 (4.28)(C=O ester); 1H and 13C NMR (500 and 125 MHz, C5D5N), see
Tables 1 and 2; HR-ESI-TOF-MS (m/z: 1023.5141 [M + Na]+, calculated for C50H80NaO20:
1023.5141).
25
Compound
3
: An amorphous solid; [
α
]
−
31.1 (c 0.36, MeOH): IR
ν
(film)
max
D
cm−1: 3382 (OH), 2927 (CH), 1715 (C=C), 1643 (C=O ester); UV
λmax (MeOH) nm (log ε):
220 (4.42) (C=C), 206 (4.42) (C=O ester); 1H and 13C NMR (500 and 125 MHz, C5D5N), see
Tables 1 and 2; HR-ESI-TOF-MS (m/z: 1023.5140 [M + Na]+, calculated for C50H80NaO20:
1023.5141).
13.1 (c 0.05, MeOH): IR νmax (film) cm−1
max (MeOH) nm (log ε):
:
25
Compound 12: An amorphous solid; [
3413 (OH), 2922 (CH), 1730(C=O), 1590, 1456 (aromatic rings); UV
α
]
D
−
λ
1
284 (3.72), 205 (C=O ester); CD
λ
max
(MeOH) nm (∆ε): 282 (
1.80); H and 13C NMR
−
(500 and 125 MHz, CD3OD), see Table 3; HR-ESI-TOF-MS (m/z: 661.2119 [M + Na]+,
calculated for C30H38NaO15: 661.2108).
Enzymatic hydrolysis of 12: Compound 12 (4.0 mg) was subjected to enzymatic
hydrolysis with naringinase (80 mg) as described for
fraction (1.2 mg). The HPLC analysis of the sugar fraction under the same conditions as in
the case of showed the presence of D-glucose and L-rhamnose. tR (min): 12.71 (D-glucose,
1 to give 12a (2.0 mg) and a sugar
1
positive optical rotation) and 7.83 (L-rhamnose, negative optical rotation).
25
Compound 12a: An amorphous solid; [
α
]
D
−
32.8 (c 0.05, MeOH): CD
λ
max
(MeOH)
nm (∆ε): 284 (
−
1.98); 1H NMR (500 MHz, CD3OD) δH 7.15 and 6.86 (each 2H, d, J = 8.2 Hz),
5.92 (s), 4.30 (dd, J = 11.4, 4.2 Hz), 4.15 (dd, J = 11.4, 7.0 Hz), 3.76 (s), 3.71 (s), 3.12 (dd, J = 13.8,
4.3 Hz), 2.83 (m), 2.70 (dd, J = 13.8, 10.5 Hz); HR-ESI-TOF-MS (m/z: 353.1005 [M + Na]+
,
calculated for C18H18NaO6: 353.1001).
25
Compound 13: An amorphous solid; [
α
]
−
1.2 (c 0.20, MeOH): IR
ν
(film)
max
D
cm−1: 3411 (OH), 2924 (CH), 1740(C=O),1626, 1539 (aromatic rings); UV
λ
(MeOH) nm
max
1
(
log ε): 285 (3.64), 205 (C=O ester); CD
λ
max
(MeOH) nm (∆ε): 271 (
−
0.01); H and 13
C
NMR (500 and 125 MHz, CD3OD), see Table 3; HR-ESI-TOF-MS (m/z: 647.1954 [M + Na]+,
calculated for C29H36NaO15: 647.1952).
3.5. Lipase-Inhibitory Activity
The pancreatic lipase activity of each test sample was measured by a modified method
using a commercially available kit (Lipase Kit S, Dainippon Pharmaceutical). The purities
of test samples were confirmed by HPLC analysis (Figure S6 in Supplementary Materials).
Cetilistat was used as a positive control at 0.1, 1.0, and 10 µM. Test sample was dissolved
in EtOH–H2O (1:1) and diluted to concentrations of 0.45, 0.90, and 4.5 mM, respectively.
Lipase (lipase from porcine pancreas, TYPE II, 100–500 units/mg) was purchased from
Sigma-Aldrich. Briefly, test sample was mixed with enzyme buffer, and incubated for 5 min
at 30 ◦C. After incubation, 2,3-dimercapto-1-propanol tributyrate (BALB) as substrate was
added and the enzyme reaction was proceeded for 30 min at 30 ◦C. Lipase activity was
determined by measuring the absorbance of the resulting 5-thio-2-nitrobenzoate anion
at 412 nm using a microplate reader. The inhibition rate was calculated by the following
formula: Inhibition rate (%) = [1
inhibitory concentration (IC50) was calculated by log-Probit analysis.
−
(
∆
Asample − ∆
A
blank)/(
∆
Acontrol − ∆
Ablank)]
×
100. The
3.6. Animals
Male ddY mice, 5 weeks old, were purchased from Japan Laboratory Animals and
maintained in the animal facility of the Tokyo University of Pharmacy and Life Sciences.
The animals were housed in cages under controlled temperature (23
light-dark cycle. The mice were allowed free access to breeding food and water. All
±
2 ◦C) and a 12 h

Int. J. Mol. Sci. 2021, 22, 1262
11 of 12
experiments were approved by the Tokyo University of Pharmacy and Life Sciences Animal
Use Committee (approval number: P17-84, date of approval: 24 April 2017).
3.7. Measurement of Triglyceride in Animals
After 5 weeks, mice were fasted overnight and randomly divided into five groups
(
n = 5). The body weight of each mouse was measured (24.0–30.7 g/body). 0.5% Car-
boxymethyl cellulose (CMC; Wako Pure Chemical Industries), cetilistat (Tokyo Kasei,
purity > 98.0%; 180 mg/kg), or compound (150 or 300 mg/kg) were prepared for test
samples and the intralipid (soybean oil 20% emulsion, 20 mL/kg, Sigma-Aldrich) was
used for the high-fat diet. Single dose of 0.5% CMC, cetilistat, and compound (150 or
6
6
300 mg/kg) were orally administered to mice in each group (n = 5) 15 min before the
intralipid loading. Blood samples were collected from the abdominal vena cava 120 min
after the intralipid-loaded treatment and centrifuged (700
samples. The TG levels in serum were measured using a commercially available kit (Wako
Triglyceride E-Test kit, Wako Pure Chemical Industries).
×
g for 10 min) to obtain serum
3.8. Statistical Analysis
Data are represented as the mean
±
standard error of the mean (S.E.M.) of three
experiments performed in triplicate. Dunnett’s test was used for statistical analysis, and
the level of significance is indicated by p values. All statistical analyses were performed
with R (R version 3.2.4).
Supplementary Materials: The supplementary materials can be found online at https://www.mdpi.
com/1422-0067/22/3/1262/s1.
Author Contributions: Conceptualization, Y.M. (Yukiko Matsuo), N.I., and Y.M. (Yoshihiro Mimaki);
methodology, Y.M. (Yukiko Matsuo). N.I. and Y.M. (Yoshihiro Mimaki); software, Y.M. (Yukiko
Matsuo); formal analysis, N.I.; investigation, A.Y., K.O., M.N., and H.T.; resources, Y.M. (Yoshihiro
Mimaki); data curation, Y.M. (Yukiko Matsuo). and N.I.; writing—original draft preparation, Y.M.
(Yukiko Matsuo) and N.I.; writing—review and editing, Y.M. (Yukiko Matsuo), N.I., and Y.M.;
visualization, N.I.; supervision, N.I.; project administration, N.I. and Y.M. (Yoshihiro Mimaki);
funding acquisition, Y.M. (Yukiko Matsuo). All authors have read and agreed to the published
version of the manuscript.
Funding: This work was financially supported in part by the Japan Society for the Promotion of
Sciences (JSPS) KAKENHI Grant Numbers JP16K18901.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or supplementary material.
Conflicts of Interest: The authors declare no conflict of interest.
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