Dipeptidyl peptidase-IV inhibitory activity of dimeric dihydrochalcone glycosides from flowers of Helichrysum arenarium

Article abstract of DOI:10.1007/s11418-015-0914-8

A methanol extract of everlasting flowers of Helichrysum arenarium L. Moench (Asteraceae) was found to inhibit the increase in blood glucose elevation in sucrose-loaded mice at 500 mg/kg p.o. The methanol extract also inhibited the enzymatic activity against dipeptidyl peptidase-IV (DPP-IV, IC₅₀ = 41.2 μg/ml), but did not show intestinal α-glucosidase inhibitory activities. From the extract, three new dimeric dihydrochalcone glycosides, arenariumosides V-VII (2-4), were isolated, and the stereostructures were elucidated based on their spectroscopic properties and chemical evidence. Of the constituents, several flavonoid constituents, including 2-4, were isolated, and these isolated constituents were investigated for their DPP-IV inhibitory effects. Among them, chalconaringenin 2′-O-β-D-glucopyranoside (16, IC₅₀ = 23.1 μM) and aureusidin 6-O-β-D-glucopyranoside (35, 24.3 μM) showed relatively strong inhibitory activities.

Full text of DOI:10.1007/s11418-015-0914-8

J Nat Med
DOI 10.1007/s11418-015-0914-8
ORIGINAL PAPER
Dipeptidyl peptidase-IV inhibitory activity of dimeric
dihydrochalcone glycosides from flowers of Helichrysum
arenarium
Toshio Morikawa1,2 Kiyofumi Ninomiya1,2 Junji Akaki1 Namiko Kakihara1
•
•
•
•
Hiroyuki Kuramoto1 Yurie Matsumoto1 Takao Hayakawa1 Osamu Muraoka1,2
Li-Bo Wang3,4 Li-Jun Wu4 Seikou Nakamura3 Masayuki Yoshikawa3
Hisashi Matsuda3
•
•
•
•
•
•
•
•
Received: 10 February 2015 / Accepted: 9 April 2015
Ó The Japanese Society of Pharmacognosy and Springer Japan 2015
Abstract A methanol extract of everlasting flowers of
Helichrysum arenarium L. Moench (Asteraceae) was found
to inhibit the increase in blood glucose elevation in sucrose-
loaded mice at 500 mg/kg p.o. The methanol extract also
inhibited the enzymatic activity against dipeptidyl pepti-
dase-IV (DPP-IV, IC50 = 41.2 lg/ml), but did not show
intestinal a-glucosidase inhibitory activities. From the ex-
tract, three new dimeric dihydrochalcone glycosides, are-
nariumosides V–VII (2–4), were isolated, and the
stereostructures were elucidated based on their spectro-
scopic properties and chemical evidence. Ofthe constituents,
several flavonoid constituents, including 2–4, were isolated,
and these isolated constituents were investigated for their
DPP-IV inhibitory effects. Among them, chalconaringenin
20-O-b-D-glucopyranoside (16, IC50 = 23.1 lM) and au-
reusidin 6-O-b-D-glucopyranoside (35, 24.3 lM) showed
relatively strong inhibitory activities.
Keywords Dwarf everlasting Á Dipeptidyl peptidase-IV
inhibitor Á Dimeric dihydrochalcone glycoside Á
Arenariumoside Á Helichrysum arenarium
Introduction
An Asteraceae plant, Helichrysum arenarium L. Moench
(common names ‘‘dwarf everlasting’’ or ‘‘immortelle’’), has
an extensive distribution throughout Europe. The choleretic,
hepatoprotective, and detoxifying activities of the inflores-
cence of this plant are known to European folk medicine [1].
The extract from the inflorescence of this plant has been
characterized as showing free-radical scavenging actions [1]
and antibacterial activities against lower respiratory tract
pathogens [2]. Several flavonoids, megastigmanes, a-py-
rones, and phthalides have been isolated from the flowers [3–
9], achenes [10], roots [11, 12], capitula, and leafy stems [13]
of H. arenarium. In the course of our studies on bioactive
constituents from the flowers of H. arenarium [4–6], we have
reported that the methanol extract and several flavonoids
showed inhibitory effects on tumor necrosis factor-a (TNF-
a)-induced cytotoxicity in L929 cells [4]. We further
evaluated the extract and its constituents and found that the
methanol extract inhibited blood glucose elevation in su-
crose-loaded mice. A serine protease dipeptidyl peptidase-
IV (DPP-IV) is widely expressed in the endothelial cells
throughout the body and is found in a circulating soluble
form [14]. It has been shown that the incretin hormone glu-
cagon-like peptide-1 (GLP-1) is released from the intestinal
L-cells into the circulation in response to the ingestion of
food and stimulates both insulin biosynthesis and secretion
[15, 16]. Apart from several other beneficial effects, GLP-1
regulates insulin in a strictly glucose-dependent manner, and
inhibition of the enzyme DPP-IV, which rapidly inactivates
& Toshio Morikawa
morikawa@kindai.ac.jp
& Hisashi Matsuda
matsuda@mb.kyoto-phu.ac.jp
1
Pharmaceutical Research and Technology Institute, Kinki
University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502,
Japan
2
Antiaging Center, Kinki University, 3-4-1 Kowakae,
Higashi-osaka, Osaka 577-8502, Japan
3
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku,
Kyoto 607-8412, Japan
4
Faculty of Natural Medicines, Shenyang Pharmaceutical
University, 103 Wenhua Rd., 110016 Shenyang,
People’s Republic of China
123

J Nat Med
Effects of the methanol extract and its fractions
on human recombinant DPP-IV activity
GLP-1, has been shownto increase the half-life ofGLP-1 and
to prolong the effects of this incretin hormone. The human
recombinant DPP-IV inhibitory activity was revealed to
regulate the antidiabetic effect of the extract. Further
separation of the active constituents in the extract allowed us
to isolate a known and novel new dimeric dihydrochalcone
glycosides, tomoroside A (1) [17] and arenarumosides V–
VII (2–4), respecitvely. Here, we describe the isolation and
elucidation of the structure of 2–4 as well as the DPP-IV
inhibitory activity of the flavonoid constituents (1–34).
To characterize the mode of action of the antihyper-
glycemic effect of the methanol extract, in vitro in-
hibitory activities on enzymes such as rat small intestinal
a-glucosidases, maltase, and sucrose, and human re-
combinant DPP-IV inhibitory activities were examined.
As shown in Table 2, the methanol extract was found to
show an inhibitory effect on DPP-IV (IC50 = 41.2 lg/
ml), but did not show a-glucosidase inhibitory activities
(both IC50 [300 lg/ml). Following this, the methanol
extract was partitioned with EtOAc–H2O (1:1, v/v) to
furnish an EtOAc-soluble fraction (0.58 %) and an
aqueous phase. The aqueous phase was subjected to
Diaion HP-20 column chromatography (H2O ? MeOH)
to yield H2O- and MeOH-eluted fractions (21.5 and
0.73 %, respectively). A bioassay-guided fractionation
revealed that the EtOAc-soluble and MeOH-eluted
Results and discussion
Effects of the methanol extract on plasma glucose
levels in sucrose-loaded mice
Dried flowers of H. arenarium were extracted with
methanol under reflux to yield a methanol extract (19.8 %
from the dried material). As shown in Table 1, the
methanol extract showed an inhibitory effect against the
increase in blood glucose levels in sucrose-loaded mice at a
dose of 500 mg/kg, p.o.
fractions
showed
DPP-IV
inhibitory
activities
(IC50 = 16.0 and 25.4 lg/ml, respectively), whereas the
H2O-eluted fraction showed no notable activity
(IC50 = 273 lg/ml).
Table 1 Inhibitory effect of the
Treatment
Dose (mg/kg, p.o.)
N
Blood glucose (mg/dl)a
methanol extract on plasma
glucose levels in sucrose-loaded
mice
0.5 h
1 h
2 h
99.3 11.7c
Normal
–
7
126.3 20.2c
279.5 10.0
236.8 10.7c
193.3 10.6c
179.7 3.6c
124.8 7.3c
218.7 4.0
115.3 10.6c
254.7 10.9
257.5 9.2
214.1 5.1c
199.5 4.4c
143.0 5.4c
208.9 6.8
183.8 3.8b
185.4 8.1b
Control
–
12
7
172.8 8.4
182.6 5.0
180.8 4.7
178.3 5.6
131.8 6.4c
163.7 3.7
151.5 6.3
152.8 3.8
MeOH extract
500
1000
2000
–
7
7
Normal
6
Control
–
9
Acarbose [32]
10
20
6
162.4 11.7c
153.8 10.2c
6
a
Values represent the mean SEM
b
c
Significantly different from the control group, p \ 0.05, p \ 0.01
Table 2 Inhibitory effects of
the methanol extract and its
fractions on rat small intestinal
a-glucosidases and human
recombinant DPP-IV activity
Rat a-glucosidase IC50 (lg/ml)a
DPP-IV
Maltase
Sucrase
[300 (32.0)b
[300 (46.6)b
[300 (43.3)b
[300 (7.1)b
IC50 (lg/ml)a
MeOH extract
[300 (28.2)b
271
[300 (46.2)b
[300 (10.2)b
41.2
16.0
25.4
273
EtOAc-soluble fraction
MeOH-eluted fraction
H2O-eluted fraction
a
Each value represents the mean of 2–4 experiments
b
Values in parentheses present inhibition % at 300 lM (for a-glucosidases)
123

J Nat Med
H-a), 6.06 (1H, dd, J = 5.8, 10.9 Hz H-b0), 6.11 (1H, dd,
J = 8.7, 10.3 Hz, H-b)], two pairs of ABX type 1,2,4,6-
tetrasubstituted aromatic protons [d 6.25, 6.70 (1H each,
both d, J = 2.2 Hz, H-5000,3000), 6.25, 6.74 (1H each, both d,
J = 2.2 Hz, H-50,30)], and two pairs of A2B2 type 1,4-
disubstituted aromatic protons [d 6.84, 7.90 (2H each, both
d, J = 8.7 Hz, H-300,500, 200,600), 6.93, 8.10 (2H each, both d,
J = 8.7 Hz, H-3,5, 2,6)] together with two glucopyranosyl
units [d 5.70 (1H, d, J = 8.0 Hz, 20-O-Glc-H-100000), 5.87
(1H, d, J = 8.0 Hz, 2000-O-Glc-H-100000)].
Isolation
In the present study, we additionally isolated four dimeric
dihydrochalcone glycosides, tomoriside A (1, 1.09 %) [17]
and arenariumosides V (2, 0.0030 %), VI (3, 0.010 %), and
VII (4, 0.018 %) from the MeOH-eluted fraction (Fig. 1)
and (2R)-helichrysin A (9, 0.12 %), (2S)-helichrysin A (10,
0.037 %), chalconeringenin 20-O-b-D-glucopyranoside
(16, 0.11 %), apigenin 7-O-b-D-glucopyranoside (18,
0.045 %), kaempferol 7-O-b-D-glucopyranoside (27,
0.39 %), and quercetin 3-O-b-D-glucopyranoside (32,
0.027 %) from the EtOAc-soluble fraction using normal-
phase silica gel and reversed-phase ODS column chro-
matography, and finally HPLC.
1
As shown in Fig. 2, the H-1H COSY experiment on 1
indicated the presence of partial structures written in bold
lines. In an HMBC experiment, long-range correlations
were observed between H-a and C-1 (dC 131.2), H-b and
C-70 (dC 203.8), H-a0 and C-100 (dC 131.6), H-b0 and C-7000
(dC 204.8), 20-O-Glc-H-10000 and C-20 (dC 161.4), and 2000-O-
Glc-H-100000 and C-2000 (dC 160.9). The stereochemistry of 1
was characterized by the rotating frame nuclear Overhauser
effect spectroscopy (ROESY) experiment, which showed
the rotating frame nuclear Overhauser effect (ROE) cor-
relations between H-b and H-a0/H2-2,6 as well as H-b0 and
H-a/H2-200,600 (Fig. 2). Thus, the configuration of the cy-
clobutane moiety in 1 was determined to be an a-truxillic
type structure [18–20]. Based on these findings, the struc-
ture of 1 was identified to be tomoroside A.
Structural identification and determination
of dimeric dihydrochalcone glycosides (1–4)
In the positive-ion fast atom bombardment (FAB) MS
spectrum of 1, a quasimolecular ion peak was observed at
m/z 891 [M?Na]?, and HRFABMS analysis revealed the
molecular formula to be C42H44O20. The 1H and 13C NMR
(pyridine-d5 at 40 °C with TMS as an internal standard)
spectroscopic properties of 1 were in accordance with those
of reported data [17] except for the deviations of the che-
mical shift, as shown in Table 3 (DdH: from 0 to 0.08 ppm;
Arenariumoside V (2) was obtained as a yellow powder
with positive optical rotation ([a]2D7 ?32.1 in MeOH). The
IR spectrum showed absorption bands at 1619 cm-1 (car-
bonyl), 1509 and 1458 cm-1 (aromatic ring) and broad
bands at 3568 and 1074 cm-1, suggestive of a glycosyl
moiety. In the positive ion FABMS, a quasimolecular ion
DdC: from 0.6 to 1.3 ppm). The H and 13C NMR spectra
1
(pyridine-d5) of 1, which were assigned with the aid of
1
DEPT, H-1H COSY, HMQC, and HMBC experiments,
showed signals assignable to four methines [d 5.56 (1H, dd,
J = 5.8, 10.3 Hz, H-a0), 5.97 (1H, dd, J = 8.7, 10.9 Hz,
Fig. 1 Dimeric dihydrochalcone
glycosides (1–4) from the flowers
of H. arenarium
123

J Nat Med
Table 3 1H (500 MHz) and 13C NMR (125 MHz) data of tomoroside A (1)
Position
1a
1b
DdH
DdC
dH (J, Hz)
dC
dH (J, Hz)
dC
dH1b–a
dC1b–a
1
131.2
129.8
115.2
156.5
132.1
130.9
116.0
157.5
0.9
1.1
0.8
1.0
2,6
3,5
4
8.10 (2H, d, 8.7)
6.93 (2H, d, 8.7)
8.15 (2H, d, 8.6)
6.97 (2H, d, 8.6)
0.05
0.04
4-OH
a
10.90 (br s)
5.97 (dd, 8.7, 10.9)
6.11 (dd, 8.7, 10.3)
41.6
53.4
6.02 (dd, 8.3, 11.1)
6.15 (dd, 8.3, 10.5)
42.6
54.3
0.05
0.04
1.0
0.9
0.9
1.0
1.1
1.2
b
10
20
30
106.0
161.4
94.5
106.9
162.4
95.6
6.74 (d, 2.2)
6.78 (d, 2.2)
0.04
40
165.5
166.7
40-OH
50
60
60-OH
70
100
200, 600
300, 500
400
12.68 (br s)
6.28 (d, 2.2)
6.25 (d, 2.2)
14.47 (br s)
97.3
98.3
0.03
0.08
1.0
1.0
167.1
168.1
14.55 (s)
203.8
131.6
129.8
115.4
156.5
204.9
132.6
130.9
116.2
157.4
1.1
1.0
1.1
0.8
0.9
7.90 (2H, d, 8.7)
6.84 (2H, d, 8.7)
7.92 (2H, d, 8.6)
6.84 (2H, d, 8.6)
0.02
0
400-OH
a0
b0
1000
2000
10.83 (br s)
5.56 (dd, 5.8, 10.3)
6.06 (dd, 5.8, 10.9)
41.6
54.6
5.60 (dd, 5.5, 10.5)
6.08 (dd, 5.5, 11.1)
42.5
55.8
0.04
0.02
0.9
1.2
0.9
1.0
0.9
1.3
106.8
160.9
94.8
107.7
161.9
95.7
3000
4000
6.70 (d, 2.2)
6.74 (d, 2.2)
0.04
165.6
166.9
4000-OH
5000
6000
6000-OH
7000
12.68 (br s)
6.27 (d, 2.2)
6.25 (d, 2.2)
14.47 (br s)
97.3
98.3
0.02
0.12
1.0
1.0
167.2
168.2
14.59 (s)
204.8
205.7
0.9
20-O-Glc
Glc-10000
Glc-20000
Glc-30000
Glc-40000
Glc-50000
Glc-60000
5.70 (d, 8.0)
5.10 (dd, 8.0, 9.0)
4.31 (m)
101.8
74.5
78.5
70.8
79.0
61.9
5.74 (d, 7.8)
5.16 (t, 8.2)
4.34 (m)
102.8
75.6
79.4
71.7
79.7
62.7
0.04
0.06
0.03
0.03
0.03
0.03
0.03
1.0
1.1
0.9
0.9
0.7
0.8
4.40 (m)
4.43 (m)
3.99 (m)
4.02 (ddd, 2.4, 5.4, 8.0)
4.36 (m)
4.33 (m)
4.40 (m)
4.43 (m)
2000-O-Glc
Glc-100000
Glc-200000
Glc-300000
Glc-400000
Glc-500000
5.87 (d, 8.0)
4.77 (dd, 8.0, 8.9)
4.34 (m)
100.7
74.2
78.6
70.5
79.0
5.92 (d, 7.8)
4.81 (t, 8.2)
101.5
75.2
79.8
71.4
79.6
0.05
0.04
0.04
0.04
0.04
0.8
1.0
1.2
0.9
0.6
4.38 (m)
4.32 (m)
4.36 (m)
3.91 (m)
3.95 (ddd, 2.3, 5.3, 7.8)
123

J Nat Med
Table 3 continued
Position
1a
1b
DdH
DdC
dH (J, Hz)
dC
61.7
dH (J, Hz)
dC
62.6
dH1b–a
dC1b–a
Glc-600000
4.23 (m)
4.32 (m)
4.27 (dd, 5.2, 12)
4.37 (m)
0.04
0.05
0.9
a
Measured in pyridine-d5 at 40 °C (tetramethylsilane as an internal standard)
Measured in pyridine-d5 [17]
b
peak was observed at m/z 891 [M?Na]?, and HRFABMS
hydroxyl and carbonyl moieties as well as the glucopyra-
nosyl parts in 2 were characterized on the basis of the
HMBC experiment, in which long-range correlations were
observed between H-a and C-1 (dC 136.2), H-b and C-70
(dC 204.5), H-a’ and C-100 (dC 130.2), H-b0 and C-7000 (dC
204.6), 20-O-Glc-H-10000 and C-20 (dC 162.6), and 2000-O-
Glc-H-100000 and C-2000 (dC 161.2). In the ROESY ex-
periment, the ROE correlations were observed between
H-b and H2-2,6/H2-200,600 and H-b0 and H2-2,6/H2-200,600
(Fig. 2). Consequently, the stereochemistry of the cy-
clobutane moiety in 2 was determined to be an e-truxillic
type structure.
analysis revealed the molecular formula to be C42H44O20,
which was the same as that of 1. Acid hydrolysis of 2 with
1 M hydrochloric acid (HCl) liberated D-glucose, which
1
was identified by HPLC analysis [21, 22]. The H and 13C
NMR spectra of 2 (pyridine-d5, Table 4) showed signals
assignable to a cyclobutane moiety [d 5.66 (1H, dd,
J = 9.6, 10.4 Hz, H-b), 5.95 (1H, dd, J = 9.6, 10.4 Hz,
H-a), 5.96 (1H, dd, J = 9.6, 9.6 Hz H-b0), 6.16 (1H, dd,
J = 9.6, 9.6 Hz, H-a0)], 12 aromatic protons {two pairs of
ABX type 1,2,4,6-tetrasubstituted aromatic protons [d 6.21,
6.68 (1H each, both d, J = 2.4 Hz, H-5000,3000), 6.24, 6.65
(1H each, both d, J = 2.4 Hz, H-50,30)] and two pairs of
A2B2 type 1,4-disubstituted aromatic protons [d 6.74, 7.82
(2H each, both br d, J = ca. 8 Hz, H-300,500, 200,600), 7.04,
7.86 (2H each, both br d, J = ca. 9 Hz, H-3,5, 2,6)], and
two glucopyranosyl units [d 5.55 (1H, d, J = 8.0 Hz, 20-O-
Glc-H-10000), 5.80 (1H, d, J = 8.0 Hz, 2000-O-Glc-H-100000)].
Arenariumosides VI (3) and VII (4), C42H44O20, were
also obtained as a yellow powders with negative and
positive optical rotations (3: [a]2D6 –16.7; 4: [a]D27 ?6.0 both
in MeOH). The acid hydrolysis of 3 and 4 liberated D-
glucose, respectively. The proton and carbon signals in the
1H and 13C NMR (pyridine-d5, Table 4) spectra of 3 and 4
were superimposable on those of 2 and indicated the
presence of the same functional groups: a cyclobutane
moiety [3: d 4.30, 5.96 (2H each, both br d, J = ca. 9 Hz,
H-a,a0, H-b,b0); 4: d 4.41 (1H, dd, J = 5.4, 9.6 Hz, H-a),
1
The H and 13C NMR spectroscopic properties of 2 were
1
quite similar to those of 1. The H-1H COSY experiment
on 2 indicated the presence of a partial structure shown in
bold lines as shown in Fig. 2. The connectivities of the
Fig. 2 1H-1H COSY, HMBC, and ROE correlations of 1–4
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J Nat Med
Fig. 3 Flavonoids (5–34) from the flowers of H. arenarium
5.04 (1H, dd, J = 9.2, 9.6 Hz, H-a0), 6.01 (1H, dd,
J = 9.2, 9.2 Hz H-b0), 6.12 (1H, dd, J = 5.4, 9.2 Hz,
H-b0)], two pairs of ABX type 1,2,4,6-tetrasubstituted
aromatic protons [3: d 6.38, 6.65 (2H each, both d,
J = 1.9 Hz, H-50,5000, 30,3000); 4: d 6.45, 6.65 (1H each,
both d, J = 2.3 Hz, H-50,30), 6.55, 6.85 (1H each, both d,
J = 2.3 Hz, H-5000,3000)], two pairs of A2B2 type 1,4-dis-
ubstituted aromatic protons [3: d 7.08, 7.59 (4H each, both
br d, J = ca. 8 Hz, H-3,5,300,500, 2,6,200,600); 4: d 6.94, 7.40
(2H each, both d, J = 8.4 Hz, H-300,500, 200,600), 7.08, 7.63
(2H each, both d, J = 8.4 Hz, H-3,5, 2,6)], and two glu-
copyranosyl units [3: d 5.50 (2H, d, J = 7.7 Hz, 20-O-Glc-
H-10000, 2000-O-Glc-H-100000); 4: d 5.35 (1H, d, J = 7.7 Hz,
2000-O-Glc-H-100000), 5.53 (1H, d, J = 8.0 Hz, 20-O-Glc-H-
10000)]. The head-to-head conjugated dimeric dihy-
drochalcone structures of 3 and 4 were determined on the
basis of the 1H-1H COSY and HMBC experiments
(Fig. 2). The ROE correlations in the ROESY ex-
periments of 3 and 4 were observed as shown in Fig. 2;
the stereochemistry of the cyclobutane moieties in 3 and 4
were elucidated to be d- and b-truxinic type structures,
respectively.
To our knowledge, dimeric dihydrocalcones isolated
from natural resources are rare, and the first report of a
dimeric dihydrochalcone, brackenin, which was isolated
from Brackenridgea zanguebarica, was established in 1983
[23]. A few other dimeric dihydrochalcones have been
reported [24–30]. Recently, a dimeric dihydrochalcone
glycoside, tomoroside A (1), was isolated from the same
genus Helichysum zivojinii [17]. This article is the second
report on the isolation and structural determination of
dimeric dihydrochalcone glycosides from the genus
Helichrysum.
Effects of the flavonoid constituents on human
recombinant DPP-IV activity
In order to specify the principal active constituents, the
inhibitory effects of the isolates from the active fractions
described above (the EtOAc-soluble and the MeOH-eluted
123

J Nat Med
Table 4 1H (500 MHz) and 13C NMR (125 MHz) data of arenariumosides V–VII (2–4)
Position
2a
2b
3a
4a
dH (J, Hz)
dC
dH (J, Hz)
dC
dH (J, Hz)
dC
dH (J, Hz)
dC
1
136.2
134.2
133.7
132.7
130.9
115.8
157.0
47.8
2, 6
3, 5
4
7.86 (2H, br d, ca. 9) 129.4 7.12 (2H, br d, ca. 8) 128.0
7.04 (2H, br d, ca. 9) 116.2 6.65 (2H, br d, ca. 8) 114.7
7.59 (2H, br d, ca. 8) 129.3
7.08 (2H, br d, ca. 8) 116.3
157.7
7.63 (2H, d, 8.4)
7.08 (2H, d, 8.4)
157.1
154.8
46.9
a
5.95 (dd, 9.6, 10.4)
5.66 (dd, 9.6, 10.4)
48.8 4.83 (dd, 9.4, 10.1)
56.2 4.70 (m)
106.8
4.30 (br d, ca. 9)
5.96 (br d, ca. 9)
48.1
52.5
4.41 (dd, 5.4, 9.6)
6.12 (dd, 5,4, 9.2)
b
10
20
30
40
50
60
60-OH
70
54.1
58.1
105.1
161.2
94.4
107.8
161.1
96.5
105.7
162.2
96.1
162.6
6.65 (d, 2,4)
6.24 (d, 2.4)
14.88 (s)
95.7 6.04 (br s)
167.5
6.65 (d, 1.9)
6.38 (d, 1.9)
13.73 (s)
6.65 (d, 2.3)
6.45 (d, 2.3)
14.10 (br s)
163.8
96.5
165.9
98.3
166.1
98.5
98.2 5.72 (br s)
167.9
165.6
166.1
167.6
204.5
130.2
203.2
128.5
204.8
133.7
204.8
132.1
130.0
115.6
156.6
43.4
100
200, 600
300, 500
400
a0
b0
1000
2000
3000
4000
5000
6000
6000-OH
7000
7.82 (2H, br d, ca. 8) 131.6 6.84 (2H, br d, ca. 8) 129.3
6.74 (2H, br d, ca. 8) 115.4 6.24 (2H, br d, ca. 8) 113.8
7.59 (2H, br d, ca. 8) 129.3
7.08 (2H, br d, ca. 8) 116.3
157.7
7.40 (2H, d, 8.4)
6.94 (2H, d, 8.4)
157.0
155.3
36.3
6.16 (dd, 9.6, 9.6)
5.96 (dd, 9.6, 9.6)
37.8 4.99 (m)
55.5 4.92 (dd, 9.4, 9.7)
107.9
4.30 (br d, ca. 9)
5.96 (br d, ca. 9)
48.1
52.5
5.04 (dd, 9.2, 9.6)
6.01 (dd, 9.2, 9.2)
53.8
52.8
106.5
159.7
95.9
107.8
161.1
96.5
107.1
161.8
96.1
161.2
6.68 (d, 2.4)
6.21 (d, 2.4)
14.42 (s)
96.7 6.04 (br s)
166.3
6.65 (d, 1.9)
6.38 (d, 1.9)
13.73 (s)
6.85 (d, 2.3)
6.55 (d, 2.3)
14.49 (br s)
164.4
96.9
165.9
98.3
166.9
98.6
98.5 5.70 (br s)
167.9
165.2
166.1
166.9
204.6
203.3
204.8
205.8
20-O-Glc
Glc-10000
Glc-20000
Glc-30000
Glc-40000
Glc-50000
Glc-60000
5.55 (d, 8.0)
5.19 (dd, 8.0, 8.8)
3.92 (m)
103.5 4.79 (d, 7.9)
74.5 3.42 (m)
79.2 3.92 (m)
71.2 3.27 (m)
79.8 3.31 (m)
62.4 3.59 (dd, 5.2, 12.2)
3.82 (m)
101.9
73.6
77.0
69.7
77.5
60.8
5.50 (d, 7.7)
4.38 (m)
3.86 (m)
4.22 (m)
3.83 (m)
4.22 (m)
4.33 (m)
102.5
74.5
79.0
71.3
79.0
62.6
5.53 (d, 8.0)
4.71 (dd, 8.0, 8.8)
3.73 (m)
104.1
74.0
78.9
70.9
79.3
62.1
4.16 (m)
4.47 (dd, 8.8, 9.0)
4.20 (m)
3.92 (m)
4.12 (m)
4.22 (m)
4.30 (m)
4.28 (m)
2000-O-Glc
Glc-100000
Glc-200000
Glc-300000
Glc-400000
Glc-500000
Glc-600000
5.80 (d, 8.0)
4.63 (dd, 8.0, 8.8)
4.26 (m)
102.4 5.05 (d, 7.6)
74.1 3.42 (m)
79.4 3.92 (m)
71.4 3.72 (m)
79.7 3.26 (m)
62.4 3.32 (dd, 5.2, 12.2)
3.67 (m)
100.9
73.5
77.0
69.7
77.4
60.7
5.50 (d, 7.7)
4.38 (m)
3.86 (m)
4.22 (m)
3.83 (m)
4.22 (m)
4.33 (m)
102.5
74.5
79.0
71.3
79.0
62.6
5.35 (d, 7.7)
3.63 (dd, 7.7, 8.5)
3.95 (m)
102.8
73.2
78.5
71.2
79.0
62.8
4.55 (m)
3.93 (m)
3.92 (m)
3.83 (m)
4.12 (m)
4.12 (m)
4.30 (m)
4.32 (m)
a
b
Measured in pyridine-d5 or DMSO-d6 at 40 °C (tetramethylsilane as an internal standard)
123

J Nat Med
fraction) on human recombinant DPP-IV activity were
tested.
Table 5 Inhibitory effects of the flavonoid constituents (1–35) on
human recombinant DPP-IV activity
IC50 (lM)a
In our previous study, we isolated 30 flavonoid con-
stituents from the active MeOH-eluted fraction, including
arenariumosides I (5, 0.0045 %), II (6, 0.0059 %), III (7,
0.0046 %), and IV (8, 0.0034 %), (2S)-helichrysin (9,
0.13 %), (2R)-helichrysin (10, 0.0016 %), neringenin 7-O-
b-D-glucopyranoside (11, 0.0053 %), 5,7-di-O-b-D-glu-
copyranosyl-(2S)-naringenin (12, 0.0045 %), 5,7-di-O-b-
D-glucopyranosyl-(2R)-naringenin (13, 0.0097 %), heli-
cioside A (14, 0.0015 %), (2R,3R)-dihydrokaempferol
7-O-b-D-glucopyranoside (15, 0.010 %), chalconeringenin
20-O-b-D-glucopyranoside (16, 0.013 %), chalconeringen-
in 20,40-di-O-b-D-glucopyranoside (17, 0.0060 %), api-
genin 7-O-b-D-glucopyranoside (18, 0.0025 %), apigenin
7-O-b-D-glucopyranosiduronic acid methyl ester (19,
0.0024 %), apigenin 7-O-gentiobioside (20, 0.0040 %),
apigenin 7,40-di-O-b-D-glucopyranoside (21, 0.019 %),
luteolin 7-O-b-D-glucopyranoside (22, 0.0025 %), luteolin
30-O-b-D-glucopyranoside (23, 0.0013 %), scutellarein
7-O-gentiobioside (24, 0.0017 %), 6-hydroxyluteolin 7-O-
b-D-glucopyranoside (25, 0.020 %), 6-hydroxy-30-O-
methylluteolin 7-O-b-D-glucopyranoside (26, 0.0033 %),
kaempferol 7-O-b-D-glucopyranoside (27, 0.58 %),
kaempferol 3-O-gentiobioside (28, 0.0070 %), kaempferol
3,7,-di-O-b-D-glucopyranoside (29, 0.0025 %), kaempfer-
ol 3,40-di-O-b-D-glucopyranoside (30, 0.0011 %), kaemp-
Tomoroside A (1)
[100 (46.8)b
83.0
Arenariumoside V (2)
Arenariumoside VI (3)
Arenariumoside VII (4)
Arenariumoside I (5)
52.0
83.0
[100 (31.4)b
[100 (41.6)b
54.1
Arenariumoside II (6)
Arenariumoside III (7)
Arenariumoside IV (8)
(2S)-Helichrysin (9)
[100 (34.2)b
[100 (39.4)b
[100 (25.6)b
[100 (4.6)b
[100 (48.7)b
[100 (30.0)b
[100 (33.3)b
[100 (1.3)b
23.1
(2R)-Helichrysin (10)
Naringenin 7-O-Glc (11)
5,7-Di-O-Glc-(2S)-naringenin (12)
5,7-Di-O-Glc-(2R)-naringenin (13)
Helicioside A (14)
(2R,3R)-Dihydrokaempferol 7-O-Glc (15)
Chalconaringenin 20-O-Glc (16)
Chalconaringenin 20,40-di-O-Glc (17)
Apigenin 7-O-Glc (18)
Apigenin 7-O-GlcA methyl ester (19)
Apigenin 7-O-Glc-(1 ? 6)-Glc (20)
Apigenin 7,40-di-O-Glc (21)
Luteolin 7-O-Glc (22)
Luteolin 30-O-Glc (23)
Scutellarein 7-O-Glc-(1 ? 6)-Glc (24)
6-Hydroxyluteolin 7-O-Glc (25)
26
70.0
62.7
50.5
32.0
[100 (39.6)b
37.5
[100 (49.1)b
[100 (43.7)b
60.3
ferol
3-O-b-D-glucopyranosyl-(1 ? 3)-b-D-glucopyra-
noside (31, 0.0040 %), quercetin 3-O-b-D-glucopyranoside
(32, 0.037 %), rutin (33, 0.0032 %), quercetin 3,30-di-O-
b-D-glucopyranoside (34, 0.0015 %), and aureusidin 6-O-
b-D-glucopyranoside (35, 0.0025 %), as shown in Fig. 3
[4–6].
73.2
[100 (44.2)b
58.0
Kaempferol 7-O-Glc (27)
Kaempferol 3-O-Glc-(1 ? 6)-Glc (28)
Kaempferol 3,7-di-O-Glc (29)
Kaempferol 3,40-di-O-Glc (30)
31
36.8
As shown in Table 5, we found that the following fla-
vonoids had DPP-IV inhibitory activities: arenariumosides
V (2, IC50 = 83.0 lM), VI (3, 52.0 lM), VII (4, 83.0 lM),
and III (7, 54.1 lM), chalconaringenin 20-O-b-D-glucopy-
ranoside (16, 23.1 lM), chalconeringenin 20,40-di-O-b-D-
glucopyranoside (17, 70.0 lM), apigenin 7-O-b-D-glu-
copyranoside (18, 62.7 lM), apigenin 7-O-b-D-glucopy-
ranosiduronic acid methyl ester (19, 50.5 lM), apigenin
7-O-gentiobioside (20, 32.0 lM), luteolin 7-O-b-D-glu-
copyranoside (22, 37.5 lM), 6-hydroxyluteolin 7-O-b-D-
glucopyranoside (25, 60.3 lM), 6-hydroxy-30-O-methyllu-
teolin 7-O-b-D-glucopyranoside (26, 73.2 lM), kaemp-
ferol 3-O-gentiobioside (28, 58.0 lM), kaempferol 3,
7,-di-O-b-D-glucopyranoside (29, 36.8 lM), kaempferol
3,40-di-O-b-D-glucopyranoside (30, 88.6 lM), kaempferol
3-O-b-D-glucopyranosyl-(1 ? 3)-b-D-glucopyranoside
(31, 85.2 lM), quercetin 3-O-b-D-glucopyranoside (32,
56.6 lM), quercetin 3,30-di-O-b-D-glucopyranoside (34,
73.9 lM), and aureusidin 6-O-b-D-glucopyranoside (35,
24.3 lM). Among them, 16 and 35 showed relatively
88.6
85.2
Quercetin 3-O-Glc (32)
56.6
[100 (17.2)b
73.9
Rutin (33)
Quercetin 3,30-di-O-Glc (34)
Aureusidin 6-O-Glc (35)
Alogliptin
24.3
0.0018
Diprotin A
2.3
a
Each value represents the mean of 2–4 experiments
b
Values in parentheses present inhibition % at 100 lM
strong inhibitory activity. In order to examine the inhibi-
tion type of DPP-IV by 16 and 35, the kinetic analysis was
examined. Analysis of the Lineweaver-Burk plots indicated
that the DPP-IV inhibition by 16 and 35 was of mixed type
(Fig. 4).
In conclusion, a methanol extract of the flowers of H.
arenarium was found to inhibit blood glucose elevation in
123

J Nat Med
Fig. 4 Lineweaver-Burk plots of the inhibition of human recombinant DPP-IV activities by 16 and 35
sucrose-loaded mice. The mode of action for the antihy-
perglycemic effect of the extract could be attributed to the
observed inhibition of DPP-IV. Three new dimeric dihy-
drochalcone glycosides arenariumoside V–VII (2–4) to-
gether with 32 flavonoids were isolated using bioassay-
guided fractionation. Compounds 2–4, 7, 16–20, 22, 25–32,
34, and 35 were able to inhibit DPP-IV enzymatic activity
at IC50 ranging from 23.1 to 88.6 lM. Among the active
constituents, chalconaringenin 20-O-b-D-glucopyranoside
(16, IC50 = 23.1 lM) and aureusidin 6-O-b-D-glucopyra-
noside (35, 24.3 lM) were found to be the most potent, and
analysis of Lineweaver-Burk plots showed that they act
through mixed competitive and non-competitive inhibition
of the enzymes. Thus, these flavonoid constituents from H.
arenarium may be useful agents for the prevention or
treatment of type 2 diabetes.
(250 9 4.6 mm i.d. and 250 9 20 mm i.d. for analytical
and preparative purposes, respectively).
The following experimental conditions were used for
column chromatography (CC): highly porous synthetic
resin, Diaion HP-20 (Mitsubishi Chemical Co., Tokyo,
Japan); ordinary-phase silica gel CC, silica gel 60 N
(Kanto Chemical Co., Tokyo, Japan; 63–210 mesh, sphe-
rical, neutral); reverse-phase ODS CC, Chromatorex ODS
DM1020T (Fuji Silysia Chemical Ltd., Aichi, Japan;
100–200 mesh); normal-phase TLC, pre-coated TLC plates
with silica gel 60F254 (Merck, Darmstadt, Germany;
0.25 mm); reversed-phase TLC, pre-coated TLC plates
with silica gel RP-18 F254S (Merck, 0.25 mm); reversed-
phase HPTLC, pre-coated TLC plates with silica gel RP-18
WF254S (Merck, 0.25 mm). Detection was achieved by
spraying with 1 % Ce(SO4)2–10 % aqueous H2SO4, fol-
lowed by heating.
Materials and methods
General
Plant material
This item was described in a previous report [4].
Extraction and isolation
The following instruments were used to obtain spectro-
scopic data: specific rotations, Horiba SEPA-300 digital
polarimeter (l = 5 cm); UV spectra, Shimadzu UV-1600
spectrometer; IR spectra, Shimadzu FTIR-8100 spec-
trometer; FAB-MS and high-resolution MS, JEOL JMS-SX
The dried flowers of H. arenarium (3.0 kg) were extracted
under reflux with methanol three times for 3 h. Evaporation
of the solvent under reduced pressure provided
a
1
102A mass spectrometer; H-NMR spectra, JEOL JNM-
methanolic extract (593.8 g, 19.8 %). This methanolic
extract (543.8 g) was partitioned with an EtOAc–H2O (1:1,
v/v) mixture, and removal of the solvents in vacuo yielded
an EtOAc-soluble fraction (210.0 g, 7.6 %) and an aqueous
phase. The aqueous phase was subjected to Diaion HP-20
CC (3.0 kg, H2O ? MeOH) to give H2O-eluted (237.2 g,
8.6 %) and MeOH-eluted (88.6 g, 3.2 %) fractions,
respectively.
LA500 (500 MHz) and EX-270 (270 MHz) spectrometers;
13C-NMR spectra, JEOL JNM-LA500 (125 MHz) and EX-
270 (68 MHz) spectrometers in CD3OD at room tem-
perature or pyridine-d5 at 40 °C with tetramethylsilane
(TMS) as an internal standard; HPLC detector, Shimadzu
RID-10A refractive index and SPD-10Avp UV–VIS de-
tectors; HPLC column, Cosmosil 5C18-MS-II (Nacalai
Tesque Inc., Kyoto, Japan) and Wakopak Navi C30-5
(Wako Pure Chemical Industries, Ltd., Osaka, Japan)
The MeOH-eluted fraction (68.6 g) was subjected to
normal-phase silica gel CC [2.5 kg, CHCl3–MeOH–H2O
123

J Nat Med
(20:3:1 ? 10:3:1 ? 7:3:1, lower layer ? 6:4:1, v/v/
v) ? MeOH] to give 12 fractions [Fr. 1 (0.85 g), Fr. 2
(1.20 g), Fr. 3 (0.90 g), Fr. 4 (1.80 g), Fr. 5 (6.40 g), Fr. 6
(11.00 g), Fr. 7 (5.40 g), Fr. 8 (4.00 g), Fr. 9 (7.10 g), Fr.
10 (5.80 g), Fr. 11 (6.10 g), and Fr. 12 (17.10 g)], as re-
ported previously [4–6]. Fraction 11 (5.0 g) was subjected
to reversed-phase ODS CC [300 g, MeOH–H2O
(10:90 ? 60:40, v/v) ? MeOH] to afford 13 fractions [Fr.
11-1 (140.0 mg), Fr. 11-2 (180.0 mg), Fr. 11-3 (200.0 mg),
Fr. 11-4 (130.0 mg), Fr. 11-5 (340.0 mg), Fr. 11-6
(180.0 mg), Fr. 11-7 (310.0 mg), Fr. 11-8 (310.0 mg), Fr.
11-9 (590.0 mg), Fr. 11-10 (255.0 mg), Fr. 11-11
(394.0 mg), Fr. 11-12 (470 0 mg), and Fr. 11-13
(498 mg)]. Fraction 11-9 (590.0 mg) was further purified
by HPLC [Cosmosil 5C18-MS-II, MeOH–H2O (30:70, v/v)
and Wakopak Navi C30-5, MeOH–H2O (45:55, v/v)] to
give arenariumoside VI (3, 41.0 mg, 0.010 %) together
with arenariumoside III (18.4 mg, 0.0046 %), 6-hy-
droxyluteolin 7-O-b-D-glucopyranoside (25, 4.0 mg,
0.0010 %), kaempferol 3,40-di-O-b-D-glucopyranoside
(30, 44.7 mg, 0.011 %), and quercetin 3,30-di-O-b-D-glu-
copyranoside (34, 6.0 mg, 0.0015 %), as reported previ-
ously [4]. Fraction 11-10 (255.0 mg) was purified by
HPLC [Wakopak Navi C30-5, MeOH–H2O (45:55, v/v)] to
give arenariumoside VIII (4, 70.0 mg, 0.018 %). Fraction
12 (17.10 g) was subjected to reversed-phase ODS CC
[500 g, MeOH–H2O (10:90 ? 40:60, v/v) ? MeOH] to
afford 11 fractions {Fr. 12-1 (1.10 g), Fr. 12-2 (1.50 g), Fr.
12-3 (1.30 g), Fr. 12-4 (0.67 g), Fr. 12-5 (0.46 g), Fr. 12-6
(0.40 g), Fr. 12-7 [= atomoroside A (1, 4055.0 mg,
1.014 %)] [17], Fr. 12-8 (576.0 mg), Fr. 12-9 (0.74 g), Fr.
12-10 (0.90 g), and Fr. 12-11 (2.97 g)}. Fraction 12-8
(576.0 mg) was separated by HPLC [Wakopak Navi C30-
5, MeOH–H2O (30:70, v/v)] to give 1 (250.0 mg,
0.062 %). Fraction 12-9 (740.0 mg) was separated by
HPLC [Wakopak Navi C30-5, MeOH–H2O (35:65, v/v)] to
give 1 (60.0 mg, 0.015 %). Fraction 12-10 (900.0 mg) was
separated by HPLC [Wakopak Navi C30-5, MeOH–H2O
(45:55, v/v)] to give arenariumoside V (2, 12.0 mg,
Fr. 11-8 (2.479 g), Fr. 11-9 (241.0 mg), Fr. 11-10
(182.0 mg), Fr. 11-11 (1.033 g), and Fr. 11-12
(813.0 mg)]. Fraction 11-5 (500.0 mg) was further purified
by HPLC [Cosmosil 5C18-MS-II, MeOH–1 % aqueous
AcOH (30:70, v/v)] to give (2R)-helichrysin A (9, 52.4 mg,
0.12 %) and (2S)-helichrysin A (10, 16.3 mg, 0.037 %).
Fraction 11-8 (500.0 mg) was further purified by HPLC
[Cosmosil 5C18-MS-II, MeOH–1 % aqueous AcOH
(40:60, v/v)] to give chalconeringenin 20-O-b-D-glucopy-
ranoside (16, 55.2 mg, 0.11 %), apigenin 7-O-b-D-glu-
copyranoside (18, 22.1 mg, 0.045 %), kaempferol 7-O-b-
D-glucopyranoside (27, 190.3 mg, 0.39 %), and quercetin
3-O-b-D-glucopyranoside (32, 13.2 mg, 0.027 %). These
isolates from the EtOAc-soluble fraction (9, 10, 16, 18, 27,
and 32) were unambiguously identified by comparison of
their physical and spectral data with those of authentic
samples [4].
Arenariumoside V (2)
Yellowish amorphous powder, [a]2D7 ?32.1 (c 0.65,
MeOH); UV [MeOH, nm (log e)]: 224 (3.61), 289 (3.43);
IR (KBr) vmax: 3568, 1619, 1509, 1458, 1074 cm-1; 1H and
13C NMR: given in Table 4; positive-ion FABMS m/z: 891
[M?Na]?; HRFABMS m/z: 891.2319 [M?Na]? (calcd for
C42H44O20Na, 891.2324).
Arenariumoside VI (3)
Yellowish amorphous powder, [a]2D6 -16.7 (c 0.20,
MeOH); UV [MeOH, nm (log e)]: 224 (3.62), 298 (3.42);
IR (KBr) vmax: 3590, 1620, 1509, 1458, 1075 cm-1; 1H and
13C NMR: given in Table 4; positive-ion FABMS m/z: 891
[M ? Na]?; HRFABMS m/z: 891.2332 [M?Na]? (calcd
for C42H44O20Na, 891.2324).
Arenariumoside VII (4)
Yellowish amorphous powder, [a]2D7 ?6.0 (c 0.28, MeOH);
UV [MeOH, nm (log e)]: 223 (3.70), 287 (3.53); IR (KBr)
0.0030 %) together with everlastoside
0.0018 %), as reported previously [4].
L
(6.9 mg,
v
max: 3568, 1638, 1509, 1458, 1073 cm-1 1H and 13C
;
The EtOAc-soluble fraction (18.3 g) was subjected to
normal-phase silica gel CC [550 g, hexane–EtOAc
(5:1 ? 2:1 ? 1:1, v/v) ? EtOAc ? MeOH] to give 11
fractions [Fr. 1 (0.27 g), Fr. 2 (9.56 g), Fr. 3 (0.43 g), Fr. 4
(0.22 g), Fr. 5 (0.31 g), Fr. 6 (0.56 g), Fr. 7 (0.93 g), Fr. 8
(1.05 g), Fr. 9 (0.72 g), Fr. 10 (0.65 g), and Fr. 11
(11.40 g)]. Fraction 11 (11.40 g) was subjected to re-
NMR: given in Table 4; positive-ion FABMS m/z: 891
[M ? Na]?; HRFABMS m/z: 891.2327 [M?Na]? (calcd
for C42H44O20Na, 891.2324).
Acid hydrolysis of 1–4
A solution of 1–4 (each 2.0 mg) in 1 M HCl (1.0 ml) was
stirred at 80 °C for 1 h. After being cooled, the reaction
mixture was neutralized with Amberlite IRA-400 (OH-
form), and the resin was removed by filtration. After removal
of the solvent from the filtrate under reduced pressure, the
residue was separated using a Sep-Pak C18 cartridge column
versed-phase
ODS
CC
[350 g,
MeOH–H2O
(30:70 ? 50:50 ? 70:30, v/v) ? MeOH ? acetone] to
afford 12 fractions [Fr. 11-1 (419.0 mg), Fr. 11-2
(265.0 mg), Fr. 11-3 (714.0 mg), Fr. 11-4 (435.0 mg), Fr.
11-5 (2.715 g), Fr. 11-6 (277.0 mg), Fr. 11-7 (116.0 mg),
123

J Nat Med
(H2O ? MeOH). The H2O-eluted fraction was subjected to
HPLC analysis under the following conditions: HPLC col-
umn, Kaseisorb LC NH2-60-5, 4.6 mm i.d. 9 250 mm
(Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical
rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo,
Japan); mobile phase, CH3CN-H2O (85:15, v/v); flow rate
0.8 ml/min]. Identification of D-glucose from 1–4 present in
the H2O-eluted fraction was carried out by comparing the
retention time and the optical rotation with the standard [tR:
13.9 min (positive optical rotation)] [21, 22].
activities of maltase and sucrase. A test sample was dis-
solved in dimethyl sulfoxide (DMSO), and the resulting
solution was diluted with 0.1 M maleate buffer to prepare
the test sample solution (concentration of DMSO: 10 %). A
substrate solution in the maleate buffer (maltose or sucrose:
74 mM, 50 ll), the test sample solution (25 ll), and the
enzyme solution (25 ll) were mixed at 37 °C for 30 min
and then immediately heated by boiling water for 2 min to
stop the reaction. The glucose concentrations were deter-
mined by a glucose-oxidase method. The final concentra-
tion of DMSO in the test solution was 2.5 %, and no
influence of DMSO on the inhibitory activity was detected.
The IC50 was determined graphically (N = 2–4). The in-
testinal a-glucosidase inhibitors acarbose (IC50 = 1.7 and
1.5 lM against maltase and sucrase, respectively) and
salacinol (IC50 = 6.0 and 1.3 lM) were used as reference
compounds [33–35].
In vivo assay
Animals
Male ddY mice (6 weeks old) were purchased from Kiwa
Laboratory Animal Co., Ltd., Wakayama, Japan. The ani-
mals were housed at a constant temperature of 23 2 °C
and were then fed a standard laboratory chow (MF, Oriental
Yeast Co., Ltd., Tokyo, Japan). The animals were fasted for
20–24 h prior to the beginning of the experiment, but were
allowed free access to tap water. All of the experiments were
performed on conscious mice unless otherwise noted. The
experimental protocol was approved by the Experimental
Animal Research Committee at Kinki University.
Inhibitory effect on human recombinant DPP-IV
The experiment was performed according to the method
described in our previous reports with a slight modification
[16]. Briefly, the assay was performed in 96-well half area
white microplates (flat bottom). Pre-incubation of 15 ll of
DPP-IV enzyme (from human recombinant; 17.3 mU/ml) or
a blank buffer (50 mM tris HCl buffer, pH 7.5) with 35 ll of
sample or control was incubated at room temperature
(25 °C) for 10 min. Fifty microliters of H-Gly-Pro-7-amino-
4-methylcoumarin (Gly-Pro-AMC) substrate (4 mg/ml) was
added, and the mixture was incubated at 25 °C for 30 min.
During the incubation, the fluorescence was measured using
a fluorescence microplate reader (SH-9000, CORONA) at an
excitation wavelength of 380 nm and an emission wave-
length of 460 nm. The final concentration of DMSO in the
test solution was 1.0 %, and no influence of DMSO on the
inhibitory activity was detected. The IC50 was determined
graphically (N = 2–4). The DPP-IV inhibitors alogliptin
(IC50 = 18 nM) and diprotin A (Ile-Pro-Ile, IC50 = 2.3 -
lM) were used as reference compounds.
For kinetic analysis of their inhibitory activity on human
recombinant DPP-IV by chalcogenin 20-O-b-D-glucopyra-
noside (16) and aureusidin 6-O-b-D-glucopyranoside (35),
the enzyme (final concentration: 0.42 mU/ml) and each test
compound [16 (23 lM), 35 (24 lM), or alogliptin (2 nM)]
were incubated with increasing concentrations of the sub-
strate (40–640 lM). A typical type of competitive inhibi-
tior, alogliptin (Ki = 2.3 nM), was used as a reference.
Effects on plasma glucose levels in sucrose-loaded mice
The experiments were performed according to the method
as described in our previous reports with a slight modifi-
cation [31, 32]. Thus, a mixture of each test sample and
sucrose (2 g/kg) suspended in 5 % (w/v) acacia solution
(20 ml/kg) was administered orally to fasted mice (body
weight 24–27 g). Blood samples (ca. 0.1 ml) were col-
lected from the infraorbital venous plexus under ether
anesthesia 0.5, 1, and 2 h after the oral administration. The
collected blood was immediately mixed with heparin
sodium (5 units/tube). After centrifugation of the blood
samples, the plasma glucose level was determined enzy-
matically by the Wako Glucose CII test (Wako Pure
Chemical Industries Ltd., Osaka, Japan) according to the
manufacturer’s instructions. The intestinal a-glucosidase
inhibitor acarbose was used as a reference compound [31].
In vitro assay
Inhibitory effects on rat intestinal a-glucosidases
The experiments were performed as described previously
[33–35]. Briefly, the assay was performed in 96-well mi-
croplates. Rat small intestinal brush border membrane
vesicles were prepared, and a suspension of these in 0.1 M
maleate buffer (pH 6.0) was used to determine the
Statistical analysis
Values were expressed as mean SEM. For statistical
analysis, one-way analysis of variance followed by Dun-
nett’s test was used.
123

J Nat Med
ˇ ´
17. Aljancic IS, Vuckovic I, Jadranin M, Pesic M, Ðordevic I,
Podolski-Renic A, Stojkovic S, Menkovic N, Vajs VE,
ˇ
´
ˇ ´
´
´
Acknowledgments This work was supported by the MEXT-Sup-
ported Program for the Strategic Research Foundation at Private
Universities, 2014–2018, and JSPS Kakenhi grant nos. 15K08008 and
15K08009. Thanks are also due to the Kobayashi International
Scholarship Foundation for financial support.
¯
´
´
´
Milosavljevic SM (2014) Two structurally distinct chalcone
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