Pregnane glycosides from Dregea volubilis and their α-glucosidase inhibitory activity

Article abstract of DOI:10.1016/j.phytol.2020.04.013

Three new pregnane glycosides namely volubilosides D-F (1–3) along with three known, dregeoside Da1 (4), volubiloside A (5), and drevoluoside N (6) were isolated from the methanol extract of the leaves of Dregea volubilis using combined chromatographic methods. Their structures were elucidated by 1D-, 2D-NMR, and HR-ESI-MS spectra and comparing with those reported in the literature. Compounds 5 and 6 showed the most significant α-glucosidase inhibitory activity at the concentration of 40 μM with inhibition of 51.3 ± 3.2% and 50.4 ± 3.1%, respectively, compared to acarbose (59.8 ± 1.6%).

Full text of DOI:10.1016/j.phytol.2020.04.013

Phytochemistry Letters 37 (2020) 90–94
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Pregnane glycosides from Dregea volubilis and their α-glucosidase inhibitory
activity
a
e
f
g
b
Nguyen Thi Kim Thuy , Phan Tuan Phuong , Nguyen Thi Mai , Truong Thi Thu Hien ,
d
d,e
d,e
d
Nguyen Thi Cuc , Bui Huu Tai , Phan Van Kiem , Chau Van Minh , Ki Sung Kang ,
c
d,e,
Seung Huyn Kim , Nguyen Xuan Nhiem
*
a
Center for High Technology Development, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam
College of Korean Medicine, Gachon University, Seongnam 13120, South Korea
b
c
d
e
f
College of Pharmacy, Yonsei Institute of Pharmaceutical Science, Yonsei University, Incheon 21983, South Korea
Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam
Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam
University of Transport and Communications, 3 Cau Giay, Dongda, Hanoi, Viet Nam
g
Vietnam Military Medical University, 160 Phung Hung, Hadong, Hanoi, Viet Nam
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new pregnane glycosides namely volubilosides D-F (1–3) along with three known, dregeoside Da1 (4),
volubiloside A (5), and drevoluoside N (6) were isolated from the methanol extract of the leaves of Dregea
volubilis using combined chromatographic methods. Their structures were elucidated by 1D-, 2D-NMR, and HR-
ESI-MS spectra and comparing with those reported in the literature. Compounds 5 and 6 showed the most
significant α-glucosidase inhibitory activity at the concentration of 40 μM with inhibition of 51.3 ± 3.2% and
Dregea volubilis
Apocynaceae
Pregnane glycoside
Volubiloside
α-Glucosidase
5
0.4 ± 3.1%, respectively, compared to acarbose (59.8 ± 1.6%).
1
. Introduction
5.49 (br d, J = 5.5 Hz), one secondary methyl group at δ
Hz), and two tertiary methyl groups at δ 1.12 (s) and 1.20 (s), suggested
the presence of a pregnane aglycone. In addition, one anomeric proton at
4.86 (br d, J = 7.0 Hz), one secondary methyl group at δ 1.24 (d,
H
1.22 (d, J = 6.5
H
Dregea volubilis (L.f.) Benth. ex Hook. f. (Apocynaceae) has been
used in oriental medicine for the treatment of inflammation, rheumatic
pain, stomach ache, fever, cough, severe cold, and snake bite (Chi,
δ
H
H
J = 6.0 Hz), and one methoxy group at δ 3.45 (s) suggested the ap-
H
13
2
012). The phytochemical investigation of this plant showed preg-
pearance of one sugar unit. The C-NMR and HSQC spectra (Table 1.)
revealed the signals of 28 carbons, including 4 non-protonated carbons, 12
nanes, pregnane glycosides (Sahu et al., 2002; Yoshimura et al., 1985),
and flavonoids as main components. The extract and compounds from
this plant have exhibited anti-diabetic, antioxidant (Das et al., 2017),
and anti-inflammatory activities (Hossain et al., 2010). As a part of our
ongoing investigation on anti-diabetic compounds from various Viet-
namese medicinal plants (Nhiem et al., 2010), we report herein the
isolation and structural elucidation of pregnane-type glycosides and
their α-glucosidase inhibitory activity Fig. 1.
1
methines, 7 methylenes, and 5 methyl carbons. The analysis of H- and
1
3
C-NMR data indicated the structure of 1 was similar to those of dre-
geoside Da1 (4) (Yoshimura et al., 1985) with the difference of a mono-
saccharide moiety at C-3 of aglycone. The position of the double bond at
C-5/C-6 was confirmed by HMBC (Fig. 2) correlations between H-19 (δ
H
1.20) and C-1 (δ
C
40.2)/C-5 (δ
C
141.3)/C-9 (δ
1.25) to C-11 (δ
80.9)/C-13 (δ
C
50.5)/C-10 (δ
C
40.2). The
HMBC correlations from H-9 (δ
H
C
72.1)/C-12 (δ 80.9);
C
from H-18 (δ
H
1.12) to C-12 (δ
C
H
C
54.4)/C-14 (δ 85.7)/C-17
C
2
. Results and discussion
(δ
C
54.7); and from H-21 (δ
1.22) to C-17 (δ
C
54.7)/C-20 (δ 71.4)
C
confirmed the positions of hydroxyl groups at C-11, C-12, C-14, and C-20.
Compound 1 was isolated as a white amorphous powder and its mo-
The large coupling constants of H-9 and H-11, J = 10.5 Hz; H-11 and H-
lecular formula was determined to be C28
H
46
O
8
by HR-ESI-MS at m/z
12, J = 10.0 Hz and NOESY correlations of H-11 (δ
H
3.67) and H-19 (δ
H
―
―
1
5
45.2887 [M + Cl] (calcd for [C28
H O
46 8
Cl] , 545.2887). The H-NMR
1.20); H-12 (δ
H
3.04) and H-17 (δ 2.18) suggested the configurations of
H
spectrum of 1 (CD
3
OD) showed the signals of one olefinic proton at δ
H
hydroxyl groups at C-11 and C-12 to be α and β, respectively. The
⁎
Corresponding author at: Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam.
E-mail address: nxnhiem@yahoo.com (N.X. Nhiem).
https://doi.org/10.1016/j.phytol.2020.04.013
Received 8 January 2020; Received in revised form 26 March 2020; Accepted 20 April 2020
1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

N.T.K. Thuy, et al.
Phytochemistry Letters 37 (2020) 90–94
Fig. 1. Chemical structures of compounds 1–6.
Table 1
1
H- (500 MHz) and 13C-NMR (125 MHz) spectroscopic data for compounds 1–3 in CD
3
OD.
C
1
2
3
δ
C
δ
H
(mult, J in Hz)
δ
C
δ
H
(mult, J in Hz)
δ
C
δ
H
(mult, J in Hz)
Aglycon
1
40.2
1.15 (m)
41.2
1.09 (ddd, 3.5, 10.0, 13.5)
2.67 (br d, 13.5)
1.65 (m)/1.80 (m)
3.53 (m)
40.4
1.15 (ddd, 3.5, 10.5, 13.5)
2.68 (br d, 13.5)
1.57 (m)/1.83 (m)
3.50 (m)
2
.68 (dt, 3.5, 14.0)
2
3
4
30.8
79.2
40.3
1.58 (m)/1.84 (m)
3.51 (m)
30.4
79.6
40.2
30.8
79.2
40.3
2.22 (m)
2.31 (br d, 13.5)
2.37 (ddd, 1.5, 5.0, 13.5)
–
2.20 (m)
a
2
–
.35 (dd, 2.5, 12.0)
2.35
5
6
7
141.3
122.9
28.7
142.0
118.8
36.1
141.2
122.9
28.7
–
5.49 (br d, 5.5)
5.34 (br d, 5.5)
1.65 (m)
5.48 (br d, 5.5)
1.80 (m)/2.27 (m)
1.83 (m)/2.25 (m)
2
–
.17 (dd, 2.0, 12.5)
8
9
38.5
50.5
40.2
72.1
80.9
54.4
85.7
33.9
27.0
54.7
11.0
19.2
71.4
23.0
1.76 (m)
76.9
51.6
40.2
70.8
82.6
54.0
86.0
36.0
27.3
56.9
11.5
18.0
70.5
22.6
37.8
50.4
40.0
72.3
78.8
56.0
85.9
35.4
24.9
59.0
10.4
19.2
218.9
32.6
1.69 (ddd, 4.5, 7.0, 11.5)
a
a
1.25
1.44 (d, 10.5)
–
1.27
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
–
–
3.67 (dd, 10.0, 10.5)
3.04 (d, 10.0)
–
4.02 (dd, 10.0, 10.5)
3.17 (d, 10.0)
–
3.65 (dd, 10.0, 10.5)
3.08 (d, 10.0)
–
–
–
–
1.63 (m)/1.75 (m)
1.62 (m) /1.92 (m)
2.18 (m)
1.75 (m)/2.15 (m)
1.61 (m)/1.84 (m)
2.08 (m)
1.80 (m)/2.00 (m)
1.96 (m)/2.03 (m)
3.57 (dd, 4.0, 9.0)
0.93 (s)
1.12 (s)
1.28 (s)
1.20 (s)
1.40 (s)
1.18 (s)
3.79 (dq, 6.0, 7.5)
1.22 (d, 6.5)
3.75 (dq, 6.0, 7.5)
1.20 (d, 6.0)
–
2.27 (s)
Cym I
1
2
3
4
5
6
97.1
35.9
79.1
74.4
71.4
18.7
58.1
4.86 (br d, 7.0)
1.54 (m)/2.16 (m)
3.61 (br dd, 3.0, 3.0)
3.19 (dd, 3.5, 9.5)
3.75 (m)
97.1
36.6
78.6
83.9
70.0
18.5
58.5
4.88 (dd, 2.0, 9.5)
1.57 (m)/2.09 (m)
97.1
36.7
78.6
83.8
69.9
18.5
58.5
4.87 (dd, 2.0, 9.5)
1.56 (m)/2.07 (m)
a
a
3.86
3.85
a
3.24 (dd, 2.0, 6.0)
3.83 (dq, 6.5, 9.5)
1.21 (d, 6.5)
3.25
3.82 (dq, 6.5, 9.5)
1.19 (d, 6.5)
3.45 (s)
1.24 (d, 6.0)
3
-OMe
3.45 (s)
3.45 (s)
Cym II
1
2
3
4
5
6
101.1
36.4
78.7
84.0
70.1
18.8
58.5
4.82 (dd, 2.0, 11.0)
1.61 (m)/2.16 (m)
101.1
36.3
78.7
84.0
70.1
18.8
58.5
4.81 (dd, 2.0, 11.0)
1.62 (m)/2.15 (m)
a
a
3.86
3.85
a
3.25 (dd, 2.0, 6.0)
3.89 (dq, 6.5, 9.5)
1.31 (d, 6.5)
3.25
3.88 (dq, 6.5, 9.5)
1.31 (d, 6.5)
3.46 (s)
3
-OMe
3.46 (s)
All
1
104.0
73.3
83.9
74.9
70.9
18.3
62.5
4.60 (d, 8.0)
104.0
73.3
83.8
74.9
70.9
18.3
62.6
4.60 (d, 8.0)
2
3.38 (dd, 3.0, 8.0)
3.65 (t, 3.0)
3.38 (dd, 3.0, 8.0)
3.64 (t, 3.0)
3
4
3.20 (dd, 3.0, 9.5)
3.69 (dq, 6.5, 9.5)
1.24 (d, 6.5)
3.20 (dd, 3.0, 9.5)
3.68 (dq, 6.5, 9.5)
1.24 (d, 6.5)
5
6
3
-OMe
3.62 (s)
3.62 (s)
Assignments were done by HSQC, HMBC, and COSY experiments.
a
Overlapped signal; Cym, β-D-cymaropyranosyl; All, 6-deoxy-3-O-methyl-β-D-allopyranosyl.
91

N.T.K. Thuy, et al.
Phytochemistry Letters 37 (2020) 90–94
Fig. 2. The key HMBC and COSY correlations of compounds 1–2.
1
3
configuration at C-20 was proved as R by comparing their C-NMR
chemical shifts at C-17 (δ 54.7), C-20 (δ 71.4), and C-21 (δ 23.0) to
those of (20R)-3β,12β-diacetoxy-8β,14β,20-trihydroxypregn-5-ene [C-17
between All H-1 (δ
4.82) and Cym I C-4 (δ
3 (δ
H
4.60) and Cym II C-4 (δ
83.9); and between Cym I H-1 (δ
79.6) determined the sugar linkages as 6-deoxy-3-O-methyl-β-D-
C
84.0); Cym II H-1 (δ
H
C
C
C
C
H
4.88) and C-
C
(δ
C
53.1), C-20 (δ
C
70.6), and C-21 (δ
C
23.4)] and (20S)-3β,12β-diacetoxy-
51.9), C-20 (δ 65.3), and C-
allopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyr-
anosyl and at C-3 of aglycone. These sugar linkages previously reported
by Yoshimura S et al. from Dregea genus (Yoshimura et al., 1985). Thus,
the structure of 2 was determined as (20R)-3β,8β,11α,12β,14β,20-hex-
ahydroxy-pregn-5-ene 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→
4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside and named as
volubiloside E.
8
2
β,14β,20-trihydroxypregn-5-ene [C-17 (δ
C
C
1 (δ 22.4)] (Kimura et al., 1982). The acid hydrolysis of 1 gave D-
C
cymarose which identified by comparing its specific rotation with those
reported (Warashina and Noro, 2000). The HMBC correlations between
Cym I H-1 (δ
H
4.86) and C-3 (δ 79.2) determined the position of O-β-D-
C
cymaropyranosyl at C-3 of aglycone. Thus, the structure of 1 was eluci-
dated as (20R)-3β,11α,12β,14β,20-pentahydroxy-pregn-5-ene 3-O-β-D-
cymaropyranoside and named volubiloside D.
Compound 3 was obtained as a white amorphous powder and its
molecular formula was determined to be C42
H
68
O
15 by HR-ESI-MS at
+
+
1
Compound 2 was isolated as a white amorphous powder. The mo-
m/z 813.4614 [M + H] (calcd for [C42
H
69
O
15
]
, 813.4631). The H-
lecular formula was determined to be C42
H
70
O
16 by HR-ESI-MS at m/z
NMR spectrum of 3 exhibited one pregnane aglycone and three sugar
+
+
1
13
8
53.4561 [M + Na] (calcd for [C42
H
70
O
16Na] , 853.4556). The H-
units. The C-NMR and HSQC spectra indicated that 3 contained one
NMR spectrum of 2 (CD
3
OD) showed the specific signals of one preg-
carbonyl, four non-protonated carbons, twenty methines, eight me-
1
13
nane aglycone: one olefinic proton, one secondary methyl group, and
two tertiary methyl groups; three sugar units [three anomeric protons
thylenes, and nine methyl carbons. The analysis of H- and C-NMR
data indicated the structure of 3 was similar to those of dregeoside Da1
(4) (Yoshimura et al., 1985) with the appearance of a ketone group at
C-20. The result of hydroxymethine oxidative process at C-20 to form
the ketone group. This was confirmed by HMBC correlations from H-21
at δ 4.60 (d, J = 8.0 Hz), 4.82 (dd, J = 2.0, 11.0 Hz), and 4.88 (dd, J
H
=
2.0, 9.5 Hz), three secondary methyl groups at δ
H
1.21 (d, J = 6.5
Hz), 1.24 (d, J = 6.5 Hz), and 1.31 (d, J = 6.5 Hz), three methoxy
1
13
groups at δ
of 2 (Table 1) were found to be similar to those of drevoluoside N
Zhang et al., 2013) except for the absence of one sugar moiety. The
HMBC correlations from H-6 (δ 5.34)/H-9 (δ 1.44) to C-8 (δ 76.9);
70.8)/C-12 (δ 82.6); H-18 (δ 1.28) to C-12
54.0)/C-14 (δ 86.0)/C-17 (δ 56.9); and from H-21
1.20) to C-17 (δ 56.9)/C-20 (δ 70.5) confirmed the positions of
H
3.45 (s), 3.46 (s), and 3.62 (s)]. The H- and C-NMR data
(δ
H
2.27) to C-17 (δ
C
59.0)/C-20 (δ 218.9). The configurations of
C
functional groups of aglycone 3 were determined by the analysis of
coupling constants and the observation on NOESY spectrum. Acid hy-
drolysis of 3 confirmed the existence of D-cymarose and 6-deoxy-3-O-
(
H
H
C
H-9 (δ
H
1.44) to C-11 (δ
C
C
H
methyl-D-allose. In addition, the HMBC correlations from All H-1 (δ
H
(
δ
δ
C
82.6)/C-13 (δ
C
C
C
4.60) to Cym II C-4 (δ
C
84.0); Cym II H-1 (δ
H
4.81) to Cym I C-4 (δ
C
(
H
C
C
83.8); Cym I H-1 (δ 4.87) to C-3 (δ
H
C
79.2) proved the sugar linkages as
1
3
hydroxyl groups at C-8, C-11, C-12, C-14, and C-20. The C-NMR
3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-cymaropyr-
anosyl-(1→4)-β-D-cymaropyranosyl. Based on the above evidence,
the structure of 3 was elucidated as 3β,11α,12β,14β-tetrahydroxy-
pregn-5-ene-20-one 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-
β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside and named as
volubiloside F.
chemical shifts at C-17 (δ
C
56.9), C-20 (δ
C
70.5), and C-21 (δ 22.6)
C
were identical to 2 confirmed the configuration at C-20 as R. The acid
hydrolysis of 2 gave monosaccharides, which were recognized as D-
cymarose, and 6-deoxy-3-O-methyl-D-allose based on specific rotations
(
Abe et al., 1999; Warashina and Noro, 2000). The HMBC correlations
92

N.T.K. Thuy, et al.
Phytochemistry Letters 37 (2020) 90–94
Table 2
were obtained from DV2D1B on HPLC column eluting with ACN in H O
2
α-Glucosidase activity of compounds 1–6 at the concentration of
(35%, v/v). DV2F was chromatographed on a RP-18 column eluting
with acetone/water (1/1.8, v/v) to give three smaller fractions, DV2F1-
DV2F3. DV2F1 fraction was chromatographed on a RP-18 column
eluting with methanol/water (1/1, v/v) to give two smaller fractions,
DV2F1A- DV2F1B. The DV2F1A was purified on HPLC column eluting
with 20% acetonitrile to yield 1 (40.0 mg). Compounds 6 (7.3 mg) and
4
0 μM.
Compounds
α-glucosidase inhibition (%)
1
11.8 ± 0.5
16.9 ± 0.7
< 10
2
3
5
(151.0 mg) were obtained from DV2F1B chromatographed on HPLC
4
14.7 ± 0.3
51.3 ± 3.2
50.4 ± 3.1
59.8 ± 1.6
5
column eluting with ACN in H
2
O (24%).
6
Acarbosea
3
.3.1. Volubiloside D (1)
a
25
Acarbose was used as positive control at the concentration of
White amorphous powder; [ ] ― 14.1 (c 0.1, MeOH); C28
H
O ,
D
46
Cl] ,
8
―
―
4
0 μM.
HR ESI MS m/z: 545.2887 [M + Cl] (calcd for [C28
H
O
46
8
1
13
5
45.2887); H- (CD
3
OD, 500 MHz) and C-NMR (CD
3
OD, 125 MHz)
The known compounds were identified as dregeoside Da1 (4)
Yoshimura et al., 1985), volubiloside A (5) (Sahu et al., 2002), and
data, see Table 1.
(
drevoluoside N (6) (Zhang et al., 2013) by analysis of 1D and 2D NMR
spectra and by comparison with those reported in the literature.
All compounds were screened for the α-glucosidase inhibitory ac-
tivity at the concentration of 40 μM. Acarbose, an α-glucosidase in-
hbitor was used as a positive control. As shown in Table 2, compounds
3
.3.2. Volubiloside E (2)
2
5
White amorphous powder; [ ] ― 27.5 (c 0.1, MeOH); C42
H
70
16Na]
O
16
,
,
D
+
+
HR ESI MS m/z: 853.4561 [M + Na] (calcd for [C42
H
70
O
1
13
8
53.4556); H (CD
3
OD, 500 MHz) and C NMR (CD
3
OD, 125 MHz)
data, see Table 1.
5
and 6 showed the most significant α-glucosidase inhibitory activity
with inhibition of 51.3 ± 3.2% and 50.4 ± 3.1%, compared to those of
acarbose (59.8 ± 1.6%). This is the first report of α-glucosidase in-
hibitory activity of compounds from D. volubilis.
3.3.3. Volubiloside F (3)
2
5
White amorphous powder; [ ] ― 34.9 (c 0.1, MeOH); C42
H
68
15
O
15
]
,
,
D
+
+
HR ESI MS m/z: 813.4614 [M + H] (calcd for [C42
H
69
O
1
13
8
13.4631); H (CD
3
OD, 500 MHz) and C NMR (CD
3
OD, 125 MHz)
3
. Experimental
data, see Table 1.
3
.1. General
3.4. Acid hydrolysis
All NMR spectra were recorded on a Bruker 500 MHz AVANCE III
HD NMR spectrometer. HR-ESI-MS spectra were obtained using
AGILENT Q-TOF system (Agilent HPLC 1290 infinity, Agilent Q-TOF
Each compound (1–3, 10.0 mg) was separately dissolved in 1.0 N
HCl (dioxane―H O, 1:1, v/v, 1.0 mL) and heated to 80 °C in a water
bath for 3 h. The acidic solution was neutralized with silver carbonate
and the solvent thoroughly driven out under N overnight. After ex-
traction with CHCl , the aqueous layer was concentrated to dryness
using N to give aqueous residue (A). The aqueous residue (A) was
separated by silica gel CC eluting with CH Cl –MeOH (10:1, v/v) and
then further fractionated by RP-18 CC using a stepwise gradient of
2
6
550 iFunnel, dual AJS ESI source). HPLC was carried out using an
AGILENT 1200 HPLC system (J’sphere ODS M-80 column, 150 mm
length × 20 mm ID with the flow rate of 3 mL/min, detector DAD).
Column chromatography (CC) was performed on silica-gel (Kieselgel
2
3
2
6
0, 230–400 mesh, Merck) or RP-18 resins (30–50 μm, Fuji Silysia
2
2
Chemical Ltd.). For thin layer chromatography (TLC), pre-coated silica-
gel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm, Merck) plates
were used.
MeOH–H
2
O (6:4, 7:3, and 8:2, v/v) to give the saccharides. The specific
25
rotation of these sugars was determined. The specific rotation ([ ] ) of
D
sugars was determined after dissolving in H O for 24 h and compared to
2
3
.2. Plant material
the literature (lit): D-cymarose: found +50.0 (c 0.4, H
2
O), lit +51.8
(
Warashina and Noro, 2000); 6-deoxy-3-O-methyl-D-allose: found +
The Dregea volubilis leaves were collected at Lang Son, Vietnam in
11.0 (c 0.4, H
2
O); lit + 10.0 (Abe et al., 1999). Based on the above
September 2017 and identified by Dr. Nguyen The Cuong, Institute of
Ecology and Biological Resources. A voucher specimen (NCCT-P75) was
deposited at the Institute of Marine Biochemistry, VAST.
evidence and experiments, sugar components were found in compound
1: D-cymarose; compounds 2 and 3: D-cymarose and 6-deoxy-3-O-me-
thyl-D-allose.
3.3. Extraction and isolation
3.5. α-Glucosidase inhibitory assay
The dried powder of D. volubilis leaves (5.0 kg) was sonicated three
The α-glucosidase (Sigma–Aldrich, St. Louis, MO) enzyme inhibition
times with hot methanol and then removed solvent to yield dark solid
extract (630 g). The extract was suspended in water and successively
partitioned with n-hexane, dichloromethane, ethyl acetate giving n-
hexane (DV1, 90 g), dichloromethane (DV2, 200 g), ethyl acetate ex-
tracts (DV3, 23 g) and residual water layer (DV4). The DV2 fraction was
chromatographed on a silica gel column eluting with n-hexane/acetone
assay was performed as the previously described method (Hanh et al.,
2
014). The sample solution (2.0 mL) dissolved in dimethyl sulfoxide)
and 0.5 U/mL α-glucosidase (40 μL) were mixed in 0.1 M phosphate
buffer (pH 7.0, 120 μL). After 5 min pre-incubation, p-nitrophenyl-α-D-
−3
glucopyranoside solution (5.10
M, 40 μL) was added, and the solu-
tion was incubated at 37 °C for 30 min. The absorbance of released 4-
nitrophenol was measured at 405 nm by using a microplate reader
(
100:0 → 0:100, v/v) to give six sub-fractions, DV2A-DV2F. DV2D was
chromatographed on a RP-18 column eluting with methanol/water (2/
(
Molecular Devices, Sunnyvale, CA).
1
, v/v) to give six sub-fractions, DV2D1-DV2D6. DV2D1 was chroma-
tographed on a RP-18 column eluting with acetone/water (1.2/1, v/v)
to give two smaller fractions, DV2D1A-DV2D1B. DV2D1A was further
Declaration of Competing Interest
chromatographed on HPLC column eluting with ACN in H O (35%) to
2
yield compound 3 (40.6 mg). Compounds 2 (19.1 mg) and 4 (88.4 mg)
The authors declared no conflict of interest.
93

N.T.K. Thuy, et al.
Phytochemistry Letters 37 (2020) 90–94
Acknowledgment
Dat, N.T., 2014. Inhibitors of alpha-glucosidase and alpha-amylase from Cyperus ro-
tundus. Pharm. Biol. 52, 74–77.
Hossain, E., Sarkar, D., Maiti, A., Chatterjee, M., Mandal, S.C., Gupta, J.K., 2010. Anti-
inflammatory effect of a methanolic extract of leaves of Dregea volubilis. J.
Ethnopharmacol. 132, 525–528.
This research is funded by Vietnam National Foundation for Science
and Technology Development (NAFOSTED) under Grant number
Kimura, M., Hayashi, K., Narita, H., Mitsuhashi, H., 1982. Studies on the constituents of
Asclepiadaceae plants. LI. Oxidation at the 18-methyl group of C/D-cis-pregnane type
steroids and carbon-13-nuclear magnetic resonance spectra of 18-oxygenated preg-
nanes and related compounds. Chem. Pharm. Bull. 30, 3932–3941.
1
04.99-2017.340.
Appendix A. Supplementary data
Nhiem, N.X., Kiem, P.V., Minh, C.V., Ban, N.K., Cuong, N.X., Tung, N.H., Minh, H.l., Ha,
D.T., Tai, B.H., Quang, T.H., Ngoc, T.M., Kwon, Y.I., Jang, H.D., Kim, Y.H., 2010. α-
Glucosidase inhibition properties of cucurbitane-type triterpene glycosides from the
fruits of Momordica charantia. Chem. Pharm. Bull. 58, 720–724.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2020.04.013.
Sahu, N.P., Panda, N., Mandal, N.B., Banerjee, S., Koike, K., Nikaido, T., 2002.
Polyoxypregnane glycosides from the flowers of Dregea volubilis. Phytochemistry 61,
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