Five new pregnane glycosides from Gymnema sylvestre and their α-glucosidase and &alphla;-amylase inhibitory activities

Article abstract of DOI:10.3390/molecules25112525

Gymnema sylvestre, a medicinal plant, has been used in Indian ayurvedic traditional medicine for the treatment of diabetes. Phytochemical investigation of Gymnema sylvestre led to the isolation of five new pregnane glycosides, gymsylosides A-E (1-5) and four known oleanane saponins, 3β-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (6), gymnemoside-W1 (7), 3β-O-β-D-xylopyranosyl-(1→6)-β-Dglucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (8), and alternoside XIX (9). Their structures were identified based on spectroscopic evidence and comparison with those reported in the literature. All compounds were evaluated for their α-glucosidase and α-amylase inhibitory activities. Compounds 2-4 showed significant α-amylase inhibitory activity, with IC₅₀ values ranging from 113.0 to 176.2 μM.

Full text of DOI:10.3390/molecules25112525

molecules
Article
Five New Pregnane Glycosides from
Gymnema sylvestre and Their α-Glucosidase
and α-Amylase Inhibitory Activities
Phan Van Kiem 1,2, Duong Thi Hai Yen 1,2, Nguyen Van Hung 1, Nguyen Xuan Nhiem 1,2
Bui Huu Tai 1,2, Do Thi Trang 1, Pham Hai Yen 1, Tran Minh Ngoc 3, Chau Van Minh 1,
,
SeonJu Park 4, Jae Hyuk Lee 5 , Sun Yeou Kim 6,7 and Seung Hyun Kim 6,
*
1
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet,
Cau Giay, Hanoi 100000, Vietnam; phankiem@yahoo.com (P.V.K.); haiyenk51a@gmail.com (D.T.H.Y.);
hungnvd8@yahoo.com (N.V.H.); nxnhiem@yahoo.com (N.X.N.); bhtaiich@gmail.com (B.H.T.);
trang2002.imbc@gmail.com (D.T.T.); yeninpc@yahoo.com (P.H.Y.); cvminh@vast.vn (C.V.M.)
Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay,
Hanoi 100000, Vietnam
Traditional Medicine Administration, Ministry of Health, 138A Giang Vo, Ba Dinh, Hanoi 100000, Vietnam;
tmngocvkn@gmail.com
Chuncheon Center, Korea Basic Science Institute (KBSI), Chuncheon 24341, Korea; sjp19@kbsi.re.kr
College of Pharmacy, Gachon University, 191, Hambakmoero, Yeonsu-gu, Incheon 21936, Korea;
wogur6378@naver.com
2
3
4
5
6
7
Yonsei Institute of Pharmaceutical Science, College of Pharmacy, Yonsei University, Incheon 21983, Korea;
sunnykim@gachon.ac.kr
Gachon Institute of Pharmaceutical Science, Gachon University, 191, Hambakmoero,
Yeonsu-gu, Incheon 21936, Korea
*
Correspondence: kimsh11@yonsei.ac.kr
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Natalizia Miceli
Received: 15 April 2020; Accepted: 26 May 2020; Published: 28 May 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Gymnema sylvestre, a medicinal plant, has been used in Indian ayurvedic traditional
medicine for the treatment of diabetes. Phytochemical investigation of Gymnema sylvestre
led to the isolation of five new pregnane glycosides, gymsylosides A–E (
known oleanane saponins, 3 -O- -D-glucopyranosyl (1 6)- -D-glucopyranosyl oleanolic acid
28-O- -D-glucopyranosyl ester ( ), gymnemoside-W1 ( ), 3 -O- -D-xylopyranosyl-(1 6)- -D-
glucopyranosyl-(1 6)- -D-glucopyranosyl oleanolic acid 28-O- -D-glucopyranosyl ester ( ), and
alternoside XIX ( ). Their structures were identified based on spectroscopic evidence and comparison
with those reported in the literature. All compounds were evaluated for their -glucosidase and
-amylase inhibitory activities. Compounds showed significant -amylase inhibitory activity,
with IC50 values ranging from 113.0 to 176.2 µM.
1–5) and four
β
β
→
β
β
6
7
β
β
→
β
→
β
β
8
9
α
α
2
–
4
α
Keywords: Gymnema sylvestre; Asclepiadacaea; pregnane; gymsyloside; α-glucosidase; α-amylase
1. Introduction
Gymnema sylvestre (Retz.) R.Br. ex Sm. (Apocynaceae) is a perennial woody climber native
to tropical and subtropical regions, such as India, Africa, and southeast Asia. In folk medicine,
G. sylvestre have been used to treat snake bites, arthritis, digestive, and enhancing laxative [
1,2].
Moreover, the plant has been explored for its benefits in blocking sugar craving and reducing sugar
consumption. The recent studies have indicated that G. sylvestre are potential anti-diabetic plants [
3,4
].
The bioactive components from this plant include pregnane glycosides [5], triterpene saponins [6,7
],
Molecules 2020, 25, 2525; doi:10.3390/molecules25112525
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2525
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and flavonoids [
their -glucosidase inhibitory activity [
compounds from Vietnamese plants [10], a methanol extract of the leaves of G. sylvestre was found to
inhibit -glucosidase and -amylase activities. Herein, we report the isolation, structural elucidation
8
]. Our previous study reported pregnane glycosides from Gymnema inodorum and
α
9]. As a part of our ongoing investigation on anti-diabetic
α
α
of pregnane-type saponins and oleanane saponins and the evaluation of
inhibitory activities of these compounds.
α-glucosidase and α-amylase
2. Results and Discussion
2.1. Isolation of Compounds
The methanol extract of the G. sylvestre leaves was suspended in water and then partitioned
with n-hexane, CH2Cl2 and EtOAc to obtain four layers. The CH2Cl2 and water extracts were
chromatographed using combined silica gel and RP-18 columns. The fractions were further
purified by HPLC to give five new pregnane glycosides and four known compounds (Figure 1
and Supplementary Materials).
Figure 1. Chemical structures of compounds of 1–5.
2.2. Compound Identification
Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined
as C47H76O17 by HRESIMS ion at m/z 935.4986 [M + Na]+ (calcd for [C47H76O17Na]+, 935.4975).
The 1H-NMR spectrum showed proton signals of three methyl groups at δH 1.13 (3H, s), 1.53 (3H, s),
and 1.05 (3H, d, J = 6.4 Hz), one olefinic proton at δH 5.32 (1H, br s), which represented a pregnane
aglycone. Two methyl groups at δH 1.80 (3H, d, J = 7.2 Hz) and 1.85 (3H, s) and one olefinic proton
at δH 6.98 (1H, q, J = 7.2 Hz), suggested the presence of a tigloyl moiety. Three anomeric protons
[
[
δH 4.84 (br d, J = 9.6 Hz), 4.57 (br d, J = 9.2 Hz), and 4.41 (br d, J = 8.0 Hz), three methoxy groups
δH 3.39, 3.42, and 3.60 (each 3H, s)], together with three secondary methyl groups [δH 1.19 (3H, d,
J = 6.0 Hz), 1.25 (d, J = 6.0 Hz), and 1.35 (d, J = 6.0 Hz)], confirmed the presence of three sugar units.
The 13C NMR and DEPT spectra indicated that
contained one carbonyl, seven non-protonated carbons
(three oxygenated), nineteen methines (sixteen oxygenateds), nine methylenes, and eleven methyl
carbons (three methoxys). The 1H and 13C-NMR spectroscopic data suggested that
was a pregnane
glycoside [11]. Of these, 21 carbons were assigned to the pregnane skeleton, 5 to one tigloyl moiety,
1
1

Molecules 2020, 25, 2525
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and 21 to a trisaccharide moiety. The HMBC correlations between H-19 (δH 1.13) and C-1 (δC 39.8)/C-5
δC 140.0)/C-9 (δC 44.7)/C-10 (δC 38.0) suggested the position of a double bond at C-5/C-6. The HMBC
correlations between H-18 (δH 1.53) and C-13 (δC 57.6)/C-14 (δC 89.3)/C-17 (δC 89.1); between H-21
δH 1.05) and C-17 (δC 89.1)/C-20 (δC 71.5); and between H-6 (δH 5.32)/H-7 (δH 2.11)/H-9 (δH 1.49) and
C-8 (δC 74.9) demonstrated the positions of hydroxyl groups at C-8, C-14, C-17, and C-20 (Figure 2).
(
(
The constitution of the aglycone of
similar reported-structures [11].
1 was demonstrated by the analysis of NOESY observations and
Figure 2. The key HMBC, COSY, and NOESY correlations of compounds 1–5.
The aglycone of
F and 12-O-(E)-cinnamoylgymnepregoside F from G. sylvestre [12], biogenetic derivatives of
C-3, C-8, C-12, C-14, C-17, and C-20. In addition, the alkaline hydrolysis of gave sarcostin,
((20S)-3 ,8 ,12 ,14 ,17 ,20-hexahydroxypregn-5-ene) [13]. The multiplicity of H-12 [δH 4.68 (dd,
J = 4.0, 11.6 Hz)] suggested that the configuration of H-12 was axial ( -configuration, Figure 2).
The NOESY correlations between H-3 (δH 3.50) and Hα-1 (δH 1.09)/ Hα-4 (δH 2.33), and between H-12
δH 4.68) and H-9 (δH 1.49)/Hα-15 (δH 1.88) suggested the configurations of the oxygenated groups at
C-3 and C-12, the hydroxy groups at C-8 and C-14 to be . The HMBC correlations between Tig H-5
δH 1.85) and Tig C-1 (δC 169.1)/Tig C-2 (δC 130.0)/Tig C-3 (δC 139.5) and between Tig H-4 (δH 1.80) and
1
was supposed to have the same configurations as those of gymnepregoside
1
at
1
β
β
β
β
β
α
(
β
(
Tig C-2/ Tig C-3 and NOESY correlations between Tig H-4 (δH 1.80) and Tig H-5 (δH 1.85) suggested the
presence of (E)-tigloyl moiety. In addition, the position of this moiety at C-12 was confirmed by HMBC
correlation from H-12 (δH 4.68) to Tig C-1 (δC 169.1). Acid hydrolysis of
1 gave three monosaccharides,
which were identified as D-cymarose [14], D-oleandrose [14], and D-thevetose [15], by comparing
their specific rotation with those reported [16]. The large coupling constants between H-1 and H-2
of monosaccharide moieties and also HMBC correlations between Thv H-1 (δH 4.41) and Ole C-4
(
δC 84.1), Ole H-1 (δH 4.57) and Cym C-4 (δC 83.8), and between Cym H-1 (δH 4.84) and aglycone
C-3 (δH 79.2) indicated the sugar linkages as -D-thevetopyranosyl-(1 4)- -D-oleandropyranosyl-
(1 4)- -D-cymaropyranoside and at C-3 of aglycone. Based on the above evidence, the
structure of was elucidated as (20S)-12 -tigloyloxy-3 ,8 ,14 ,17 ,20-pentahydroxypregn-5-ene
3-O- -D-thevetopyranosyl- (1 4)- -D-oleandropyranosyl-(1 4)- -D-cymaropyranoside, a new
β
→
β
→
β
1
β
β
β
β
β
β
→
β
→
β
compound named gymsyloside A.
1
The H and 13C-NMR spectra of
2
exhibited a pregnane aglycone, one tigloyl unit, and three
were similar to those of gymsyloside A ( ),
except for the difference of sugar unit at Ole C-4: D-thevetose replaced by 6-deoxy-3-O-methyl-D-allose.
Acid hydrolysis of confirmed the presence of D-cymarose, D-oleandrose, and 6-deoxy-3-O-methyl-D-allose
sugar units (Table 1). In addition, the NMR data of
2
1
2

Molecules 2020, 25, 2525
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1
as sugar components. Furthermore, the H and 13C-NMR data of
2
showed the sugar units as
β
-D-cymaropyranosyl, -D-oleandropyranosyl, and 6-deoxy-3-O-methyl-β-D-allopyranose. The HMBC
β
correlations between All H-1 (δH 4.70) and Ole C-4 (δC 84.0), Ole H-1 (δH 4.56) and Cym C-4 (δC 83.8), and
between Cym H-1 (δH 4.85) and aglycone C-3 (δC 79.3) confirmed the sugar linkages to be 3-O-6-deoxy-
3-O-methyl-
compound
3-O-6-deoxy-3-O-methyl-
β
-D-allopyranosyl-(1
was elucidated to be (20S)-12
-D-allopyranosyl-(1 4)-
new compound named gymsyloside B.
→
4)-
β
-D-oleandropyranosyl-(1
-tigloyloxy-3
-D-oleandropyranosyl-(1
→
4)-
,8β,14
β
-D-cymaropyranoside. Consequently,
,17 ,20-pentahydroxypregn-5-ene
4)- -D-cymaropyranoside, a
2
β
β
β
β
β
→
β
→
β
Table 1. NMR spectroscopic data for compounds 1–3 in CD3OD.
C
1
2
3
δC
δH (mult., J, in Hz)
δC
δH (mult., J, in Hz)
δC
δH (mult., J, in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
39.8
30.2
79.2
39.8
140.0
120.0
35.2
74.9
44.7
38.0
26.0
75.1
57.6
89.3
34.3
33.5
89.1
11.2
18.5
71.5
18.9
Tig
1.09, m, α/1.80, m, β
1.57, m, β/1.84, m, α
3.50, m
2.20, m, β/2.33, m, α
39.8
30.2
79.3
39.8
140.0
120.0
35.2
75.0
44.7
38.0
26.0
75.2
57.6
89.3
34.3
33.5
89.2
11.2
18.5
71.5
18.9
Tig
1.09, m, α/1.79, m, β
1.56, m, β/1.84, m, α
3.50, m
2.20, m, β/2.33, m, α
39.8
30.1
79.3
39.8
140.0
119.9
35.2
75.0
44.7
38.0
25.9
75.2
57.6
89.2
34.3
33.5
89.3
11.2
18.5
71.6
18.9
Tig
1.09, m, α/1.80, m
1.57, m, β/1.83, m, α
3.49, m
2.19, m, β/2.33, m, α
-
-
-
5.32, br s
2.11, m
5.32, br s
2.12, m
5.33, br s
2.12, m
-
-
-
1.49, m
1.48, m
1.49, m
-
-
-
1.63, m, α/2.00, m, β
4.68, dd, 4.0, 11.6)
1.62, m, α/2.00, m, β
1.62, m, α/2.00, m, β
4.70, dd, 4.0, 9.6)
4.68, m
-
-
-
-
1.83, m, β/1.88, m, α
1.74, m
-
1.82, m, β/1.91, m, α
1.75, m
-
1.81, m, β/1.89, m, α
1.74, m
-
-
-
1.53, s
1.13, s
3.45, m
1.05, d (6.4)
1.53, s
1.14, s
3.44, m
1.04, d (6.4)
1.52, s
1.14, s
3.46, m
1.06, d (6.4)
1
2
3
4
5
169.1
130.0
139.5
14.5
12.2
Cym
97.2
36.6
78.5
83.8
69.9
18.5
58.5
Ole
102.6
37.6
80.2
84.1
72.5
18.9
57.6
-
-
169.2
130.1
139.6
14.5
12.1
Cym
97.2
36.7
78.5
83.8
70.0
18.5
58.5
Ole
102.6
37.5
80.4
84.0
72.6
19.0
57.5
-
-
169.1
130.1
139.6
14.5
12.1
Cym I
97.2
36.6
78.5
83.8
70.1
18.5
58.5
Cym II
101.2
36.4
78.6
83.9
69.9
18.6
58.4
-
-
6.98, q (7.2)
1.80, d (7.2)
1.85, s
6.98, q (7.2)
1.80, d (7.2)
1.85, s
7.00, q (6.8)
1.81, d (6.8)
1.87, s
1
2
3
4
5
6
4.84, br d (9.6)
1.52, m, a/2.05, m, e
3.82, m
4.85, br d (9.6)
1.53, m, a/2.04, m, e
3.83, m
4.84, br d, 9.6)
1.55, m, a/2.05, m, e
3.82, m
3.24, m
3.79, m
1.19, d (6.0)
3.42, s
3.24, m
3.79, m
1.19, d (6.0)
3.42, s
3.23, m
3.79, m
1.18, d (6.4)
3.42, s
3-OMe
1
2
3
4
5
6
4.57, br d (9.2)
1.40, m, a/2.30, m, e
3.36, m
4.56, br d (6.8)
1.39, m, a/2.30, m, e
3.36, m
4.78, br d (9.6)
1.55, m, a/2.10, m, e
3.82, m
3.18, m
3.36, m
1.35, d (6.0)
3.39, s
3.17, m
3.35, m
1.34, d (6.0)
3.39, s
3.23, m
3.79, m
1.21, d (6.0)
3.42, s
3-OMe

Molecules 2020, 25, 2525
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Table 1. Cont.
C
1
2
3
δC
Thv
δH (mult., J, in Hz)
δC
δH (mult., J, in Hz)
δC
δH (mult., J, in Hz)
All
102.2
73.6
83.8
75.0
71.2
18.5
62.5
Ole
102.6
37.6
80.2
84.1
72.5
18.9
57.5
Thv
104.3
75.6
87.7
76.6
73.2
18.1
61.1
1
2
3
4
5
6
104.3
75.6
87.7
76.6
73.2
18.1
61.0
4.41, br d (8.0)
3.18, m
3.00, t (6.8)
3.00, t (6.8)
3.25, m
4.70, br d (7.6)
3.29, m
4.58, br d (8.4)
1.40, m, a)/2.30, m, e)
3.36, m
3.60, m
3.16, m
3.64, m
1.21, d (6.0)
3.58, s
3.18, m
3.35, m
1.36, d (5.6)
3.41, s
1.25, d (6.0)
3.60, s
3-OMe
1
2
3
4
5
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4.42, d (8.0)
3.18, m
3.00, m
3.00, m
3.25, m
1.27, d (6.0)
3.60, s
3-OMe
Assignments were done by Heteronuclear Single Quantum Coherence (HSQC), Heteronuclear Multiple Bond
Correlation (HMBC), Correlation Spectroscopy (COSY), and Rotating frame Overhauser Effect Spectroscopy (ROESY)
experiments. Tig, Tigloyl; Cym,
6-deoxy-3-O-methyl- -d-allopyranosyl; a, axial; e, equatorial;
β, atoms or groups laying above the plane of structure.
β
-d-cymaropyranosyl; Ole,
β
-d-oleandropyranosyl; Thv,
β-d-thevetopyranosyl; All,
β
α, atoms or groups laying below the plane of structure;
The HRESIMS of
to the molecular formula of C54H88O20. The 1H and 13C-NMR spectra of
pregnane aglycone, four sugar units, and one tigloyl unit (Table 1). The NMR data of
gymsyloside A ( ) and found the addition of one sugar unit in the sugar linkages. Acid hydrolysis of
gave three monosaccharides, which were identified as D-cymarose, D-oleandrose, and D-thevetose.
The tetrasaccharide was determined to be -D-thevetopyranosyl-(1 4)- -D-oleandropyranosyl-
(1 4)- -D-cymaropyranosyl-(1 4)- -D-cymaropyranoside, by the analysis of HMBC and COSY
correlations. The location of sugar linkages at C-3 was confirmed by the HMBC correlation
between Cym I H-1 (δH 4.84) and C-3 (δC 79.3). Consequently, the structure of was determined
to be (20S)-12 -tigloyloxy-3 ,8 ,14 ,17 , 20-pentahydroxypregn-5-ene 3-O- -D-thevetopyranosyl-
(1 4)- -D-oleandropyranosyl-(1 4)- -D-cymaropyranosyl-(1 4)- -D-cymaropyranoside, a new
compound named gymsyloside C.
The molecular formula of
was determined as C54H88O20 by the HRESIMS. The 1H and 13C-NMR
data (Table 2) indicated that the structure of was similar to those of , except for the difference of
monosaccharide at Ole C-4. The sugar components were found to be similar to those of (D-cymarose,
D-oleandrose, and 6-deoxy-3-O-methyl-D-allose) [17]. Moreover, the sugar linkages, 3-O-6-deoxy-3-
O-methyl- -D-allopyranosyl-(1 4)- -D-oleandropyranosyl-(1 4)- -D-cymaropyranosyl-(1 4)- -D-
3
gave a pseudo-molecular ion peak at m/z 1079.5769 [M + Na]+, corresponding
showed the presence of one
were compared to
3
3
1
3
β
→
β
→
β
→
β
3
β
β
β
β
β
β
→
β
→
β
→
β
4
4
3
2
β
→
β
→
β
→
β
cymaropyranoside was confirmed by the HMBC correlations from All H-1 (δH 4.70) to
Ole C-4 (δC 83.7), Ole H-1 (δH 4.56) to Cym II C-4 (δC 83.8), and from Cym II H-1
(
δH 4.77) to Cym I C-4 (δC 83.8). Similar to those of
(20S)-12 -tigloyloxy-3 ,8 , 14 ,17 , 20-pentahydroxypregn-5-ene. Consequently, the structure
of was determined as (20S)-12 -tigloyloxy-3 ,8 ,14 ,17 ,20-pentahydroxypregn-5-ene3-O-6-
deoxy-3-O-methyl- -D-allopyranosyl- (1 4)- -D-oleandropyranosyl-(1 4)- -D-cymaropyranosyl-
(1→4)-β-D-cymaropyranoside and named gymsyloside D.
1–3, the aglycone was found as
β
β
β
β
β
4
β
β
β
β
β
β
→
β
→
β

Molecules 2020, 25, 2525
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Table 2. NMR spectroscopic data for compounds 4 and 5 in CD3OD.
C
4
5
δC
δH (mult., J, in Hz)
δC
δH (mult., J, in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
39.8
30.2
79.3
39.8
140.0
120.0
35.2
74.9
44.7
38.0
26.0
75.1
57.6
89.3
34.3
33.5
89.1
11.2
18.6
71.5
19.0
Tig
1.07, m,
1.57, m,
3.50, m
2.18, m, /2.33, m,
α
/1.79, m,
β
α
39.8
30.2
79.3
39.8
140.0
120.0
35.2
74.9
44.7
38.0
25.9
75.1
57.6
89.3
34.3
33.5
89.1
11.2
18.6
71.5
18.9
Tig
1.08, m,
1.58, m,
3.50, m
2.20, m, /2.33, m,
α
/1.80, m,
β
α
β
/1.83, m,
β
/1.84, m,
β
α
β
α
-
-
5.32, br s
2.11, m
5.32, br s
2.11, m
-
-
1.49, m
-
1.63, m,
1.49, m
-
1.63, m,
α
/2.00, m,
β
α
/2.00, m,
β
4.67, m
4.68, m
-
-
-
-
1.80, m,
β
/1.88, m,
α
1.83, m,
β
/1.90, m,
α
1.74, m
-
1.53, s
1.13, s
3.46, m
1.05, d (6.0)
1.75, m
-
1.53, s
1.12, s
3.45, m
1.05, d (6.4)
1
2
3
4
5
169.1
130.1
139.5
14.6
12.2
Cym I
97.2
36.6
78.5
83.8
69.8
18.3
58.4
Cym II
101.2
36.4
78.4
83.8
69.9
18.6
58.5
Ole
-
-
169.1
130.1
139.5
14.5
12.2
Cym
97.2
36.7
78.5
83.8
69.9
18.4
58.5
Ole
102.6
37.6
80.3
84.1
72.6
18.8
57.6
Thv
104.2
75.2
86.3
82.9
72.5
18.5
61.2
Glc
-
-
6.98, q (7.2)
1.81, d (7.2)
1.85, s
6.98, q (7.2)
1.80, d (7.2)
1.85, s
1
2
3
4
5
6
4.83, br d (9.6)
1.54, m, a/2.04, m, e
3.82, m
4.84, br d (9.6)
1.53, m, a/2.05, m, e
3.81, m
3.24, m
3.78, m
1.17, d (6.4)
3.41, s
3.24, m
3.80, m
1.19, d (6.4)
3.42, s
3-OMe
1
2
3
4
5
6
4.77, br d (9.6)
1.54, m, a/2.04, m, e
3.82, m
4.57, br d (9.2)
1.40, m, a/2.30, m, e
3.35, m
3.24, m
3.78, m
1.19, d (5.6)
3.40, s
3.18, m
3.38, m
1.36, d (6.0)
3.39, s
3-OMe
1
2
3
4
5
6
102.6
37.5
80.3
83.7
72.5
18.9
57.4
All
4.56, br d (9.2)
1.40, m/2.30, m
3.35, m
3.18, m
3.35, m
1.35, d (6.0)
3.39, s
4.43 (d (7.6)
3.23, m
3.17, m
3.30, m
3.38, m
1.36, d (7.2)
3.60, s
3-OMe
1
2
3
4
5
6
102.2
73.5
83.9
75.0
71.2
18.6
62.5
4.70, d (8.0)
3.29, m
3.60, m
3.16, m
3.63, m
104.3
75.6
78.3
71.8
77.9
63.2
4.41, d (7.6)
3.15, m
3.23, m
3.20, m
3.31, m
1.21, d (6.0)
3.58, s
3.62, m/3.84, m
3-OMe
Tig, Tigloyl; Cym,
β
-d-cymaropyranosyl; Ole,
-d-allopyranosyl; a, axial; e, equatorial;
below the plane of structure; β, atoms or groups laying above the plane of structure.
β
-d-oleandropyranosyl; Thv,
β
-d-thevetopyranosyl; Glc,
β-d-glucopyranosyl; All, 6-deoxy-3-O-methyl-
β
α, atoms or groups laying

Molecules 2020, 25, 2525
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The molecular formula of 5, C53H86O22 was determined by the HRESIMS pseudo-ion at m/z
1097.5530 [M + Na]+. The 1H and 13C NMR data of
were similar to , except for an additional sugar
5
2
unit at Thv C-4 (Table 2). The sugar moieties were determined as D-cymarose [14], D-oleandrose [14],
D-thevetose [15], and D-glucose [17] by acid hydrolysis. The HMBC correlations between Glc H-1
(
δH 4.41) and Thv C-4 (δC 82.9), Thv H-1 (δH 4.43) and Ole C-4 (δC 84.1), Ole H-1 (δH 4.57) and Cym
C-4 (δC 83.8), and between Cym H-1 (δH 4.84) and aglycone C-3 (δC 79.3) confirmed the sequence of
sugar linkages, previously reproted from G. sylvestre [ ]. Thus, compound was characterized as
(20S)-12 -tigloyloxy-3 ,8 ,14 ,17 ,20-pentahydroxypregn-5-ene3-O- -D-glucopyranosyl-(1 4)-
4)- -D-oleandropyranosyl-(1 4)- -D-cymaropyranoside and named
5
5
β
β
β
β
β
β
→
β-
D-thevetopyranosyl-(1
gymsyloside E.
→
β
→
β
The known compounds were identified as 3
oleanolic acid 28-O- -D-glucopyranosyl ester (
xylopyranosyl-(1 6)- -D-glucopyranosyl-(1 6)-
glucopyranosyl ester ( ) [ ], and alternoside XIX (
from G. sylvestre. Thus, for oleanane saponins, the main components could be hightly species-specific
of G. sylvestre [ 18]. In addition, new pregnane glycosides were also found in G. alternifolium [19],
G. sylvestre [ ], and G. griffithii [15]. Five new pregnane glycosides in this report will contribute specific
β
-O-
) [
-D-glucopyranosyl oleanolic acid 28-O-
β
-D-glucopyranosyl (1
→
6)-β-D-glucopyranosyl
β
6
β
7
], gymnemoside-W1 (
7) [18], 3
β-O-
β
-D-
-D-
→
β
→
β
8
7
9) [18]. These compounds were already reported
7,
5
compounds in Gymnema genus.
2.3. α-Glucosidase and α-Amylase Inhibitory Activities
All compounds were evaluated for the
showed the weak -glucosidase inhibitory activity (16.4
compared tos the positive control, acarbose (inhibition percentage of 57.8
of 155 M) (Figure 3). Compounds showed -amylase inhibitory activity with inhibition
percent ranging from 57.9% to 66.8% at the concentration of 200 M (Figure 4). In the subsequent
concentration-dependent assay, compounds , and showed significant -amylase inhibitory activity,
2.7, and 113.0 0.7 M, respectively, compared to positive
M). This is the first report of -glucosidase and -amylase
α
-glucosidase and
α
-amylase inhibitory assays. Compound
2.3%) at the concentration of 200 M,
3.2% at the concentration
4
α
±
µ
±
µ
2
–
4
α
µ
2,
3
4
α
with IC50 values of 175.8
control, acarbose (IC50 value of 72.4
±
2.3, 162.2
±
±
µ
±
0.8
µ
α
α
inhibitory activities of compounds from G. sylvestre. Recent reports have shown insulin secretion
stimulation of G. sylvestre extract [ ], antihyperglycemic effects of gmynemic acids [20], -glucosidase
and -amylase inhibitory activities of pregnane glycosides from G. latifolium [11]. Previous studies
have indicated that pregnane glycosides from G. griffithii showed moderate -glucosidase inhibitory
4
α
α
α
activity [15]. Russelioside B, a pregnane glycoside, possessed antidiabetic and antihyperlipidemic
effect in streptozotocin induced diabetic rats [21]. Therefore, the results suggest that the discovery of
pregane glycosides may increase the possibility of finding antidiabetic agents.
100
80
60
40
20
0
200 M
500 g/ml
155 M
t
e
s
1
2
3
4
5
6
7
8
9
x
e
o
b
Compounds
H
r
a
O
c
e
A
M
Figure 3. α-Glucosidase inhibitory effects of the G. sylvestre extract and compounds 1–9.

Molecules 2020, 25, 2525
8 of 12
200 M
500 g/ml
155 M
100
80
60
40
20
0
t
e
s
1
2
3
4
5
6
7
8
9
x
a
e
o
b
Compounds
H
r
O
c
e
A
M
Figure 4. α-Amylase inhibitory effects of the G. sylvestre extract and compounds 1–9.
3. Materials and Methods
3.1. General
All NMR spectra were recorded on an Agilent 400-MR-NMR (Agilent technologies, Santa Clara, CA,
USA) spectrometer operated at 400 and 100 MHz for hydrogen and carbon, respectively. Data processing
was carried out with the MestReNova ver.6.0.2 program. HRESIMS spectra were obtained using
an AGILENT 6550 iFunnel Q-TOF LC/MS system (Agilent technologies, Santa Clara, CA, USA).
Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. Preparative HPLC
was carried out using an AGILENT 1200 HPLC system. Column chromatography was performed
on silica-gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) or YMC RP-18 resins (30–50 µm,
Fuji Silysia Chemical Ltd., Aichi, Japan). For thin layer chromatography (TLC), a pre-coated silica-gel
60 F254 (0.25 mm, Merck, Darmstadt, Germany) and RP-18 F254S plates (0.25 mm, Merck, Darmstadt,
Germany) were used.
3.2. Plant Material
The leaves of Gymnema sylvestre (Retz.) R.Br. ex Sm. were collected in Hai Loc, Hai Hau, Nam Dinh
in November, 2015, and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological
Resources. A voucher specimen (NCCT-P20) was deposited at the Herbarium Institute of Marine
Biochemistry, VAST.
3.3. Extraction and Isolation
The dried powders of G. sylvestre leaves (4.0 kg) were sonicated with hot methanol (3 times × 10 L
,
each 3 h) to give MeOH extract (450 g), after evaporation of the solvent. The MeOH extract was
suspended in water and successively partitioned with n-hexane, CH2Cl2 and EtOAc to obtain the
n-hexane (GS1, 47.0 g), CH2Cl2 (GS2, 60.0 g), EtOAc extracts (GS3, 27.0 g) and H2O layer (GS4).
GS2 was chromatographed on a silica gel column (180.0 g, silica gel) eluting with gradient solvent of
n-hexane:acetone (40:1, 20:1, 10:1, 5:1, 1:1, and 0:1, v/v, each 2 L), to give seven fractions, GS2A-GS2G.
The GS2F fraction was chromatographed on a silica gel column eluting with CHCl3:MeOH (11:1, v/v) to
give four fractions, GS2F1-GS2F4. GS2F1 was chromatographed on a RP-18 column using MeOH:H2O
(4:1, v/v) as a solvent, to give five fractions, GS2F1A-GS2F1E. Compounds
1 (17.0 mg, tR 38.5 min)
and (9.0 mg, tR 42.1 min) were yielded from GS2F1B fraction using HPLC system: J’sphere H-80
2
column (150
Compounds
J’sphere H-80 column (150
×
20 mm), flow rate of 3 mL/min, and solvent condition of 40% acetonitrile in water.
(5.0 mg, tR 44.7 min) and (5.0 mg, tR 49.4 min) were yielded from GS2F1D fraction on
20 mm), flow rate of 3 mL/min, and solvent condition of 40% acetonitrile
in water. GS2G was chromatographed on RP-18 column eluting with MeOH:H2O (4:1, v/v) to give
3
4
×

Molecules 2020, 25, 2525
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four smaller fractions, GS2G1-GS2G4. Finally, GS2G3 was chromatographed on J’sphere H-80 column
(150 20 mm), flow rate of 3 mL/min, and solvent condition of 35% acetonitrile in water to yield
compound (14.0 mg, tR 39.7 min). GS4 was chromatographed on a Diaion column and eluted with
×
5
H2O then increased concentrations of MeOH in H2O, to obtain sub-fractions, GS4A-GS4C. GS4C was
chromatographed on a silica gel column eluting with a gradient of CHCl3:MeOH (20:1, 10:1, 5:1, 1:1,
v/v) to give smaller fractions, GS4C1-GS4C4. GS4C4 was chromatographed on an RP-18 CC eluting
with MeOH:water (2:1, v/v) to give smaller fractions, GS4C4A-GS4C4E. GS4C4B was chromatographed
on an RP-18 column eluting with acetone:H2O (0.8:1, v/v) to yield
chromatographed on an RP-18 column eluting with acetone:H2O (1:1, v/v), to yield
7
(5.0 g) and
9
(40.0 mg). GS4C4E was
(100.0 mg) and
6
8
(5.0 mg).
3.3.1. Gymsyloside A (1)
White amorphous powder; [α]D25
−
20.0 (c 0.1, MeOH); C47H76O17, HRESIMS m/z: 935.4986
1
[M + Na]+ (calcd for [C47H76O17Na]+, 935.4975); H (CD3OD, 400 MHz) and 13C-NMR (CD3OD,
100 MHz) data, see Table 1.
3.3.2. Gymsyloside B (2)
White amorphous powder; [α]D25 + 35.0 (c 0.1, MeOH); C47H76O17, HRESIMS m/z: 935.4996
1
[M + Na]+ (calcd for [C47H76O17Na]+, 935.4975); H (CD3OD, 400 MHz) and 13C-NMR (CD3OD,
100 MHz) data, see Table 1.
3.3.3. Gymsyloside C (3)
White amorphous powder; [α]D25 + 80.0 (c 0.1, MeOH); C54H88O20, HRESIMS m/z: 1079.5769
1
[M + Na]+ (calcd for [C54H88O20Na]+, 1079.5769); H (CD3OD, 400 MHz) and 13C-NMR (CD3OD,
100 MHz) data, see Table 1.
3.3.4. Gymsyloside D (4)
White amorphous powder; [α]D25+ 58.7 (c 0.1, MeOH); C54H88O20, HRESIMS m/z: 1079.5778
1
[M + Na]+ (calcd for [C54H88O20Na]+, 1079.5769); H (CD3OD, 400 MHz) and 13C-NMR (CD3OD,
100 MHz) data, see Table 2.
3.3.5. Gymsyloside E (5)
White, amorphous powder; [α]D25+ 54.0 (c 0.1, MeOH); C53H86O22, HRESIMS m/z: 1097.5530
1
[M + Na]+ (calcd for [C53H86O22Na]+, 1097.5503); H (CD3OD, 400 MHz) and 13C NMR (CD3OD,
100 MHz) data, see Table 2.
3.4. Acid Hydrolysis
Each compound (1–5, 3.0 mg) was separately dissolved in 1.0 N HCl (dioxane—H2O, 1:1, v/v,
1.0 mL) and heated to 80 ◦C in a water bath for 3 h. The acidic solution was dried under N2 overnight.
After extraction with CHCl3, the aqueous layer was dried using N2 to give aqueous residue (A).
The aqueous residue (A) was separated by silica gel CC eluting with CH2Cl2–MeOH (10:1, v/v) and
then further fractionated by RP-18 CC using a solvent gradient of MeOH–H2O (6:4, 7:3, and 8:2, v/v),
to give the monosaccharides (50% yield). The specific rotations of these sugars were determined.
The specific rotations ([α]2D5) of sugars was determined after dissolving in H2O for 24 h and compared to
the literature (lit):D-cymarose: found +50.1 (c 0.4, H2O), lit +51.8 [14]; D-oleandrose: found 12.1 (c 0.4,
−
H2O), lit +11.7 [14]; D-thevetose: found +40.3 (c 0.4, H2O); lit +42.3 [15]; 6-deoxy-3-O-methyl-D-allose:
found +10.9 (c 0.4, H2O); lit +10.0 [17]; D-glucose: found + 49.2 (c 0.4, H2O); lit +48.0 [17]. Based on
the above evident, sugar components were found in: compounds
1 and 3: D-cymarose, D-oleandrose,

Molecules 2020, 25, 2525
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and D-thevetose; compounds
2
and
4
: D-cymarose, D-oleandrose, and 6-deoxy-3-O-methyl-D-allose;
compound 5: D-cymarose, D-oleandrose, D-thevetose, and D-glucose.
3.5. Alkaline Hydrolysis
A solution of compound
then neutralized with HCl 0.1 M. After that, the solution was partitioned with CHCl3 to give CHCl3
layer. CHCl3 layer was separated on HPLC system: J’sphere H-80 column (150 20 mm), solvent
1
(8 mg) in 1.0 mL of 5% KOH/MeOH was heated at 40 ◦C four 4 h and
×
condition of 55% acetonitrile, to give sarcostin (54% yield). In a similar way, sarcostin was found as
aglycone of compounds 2–5.
3.6. α-Glucosidase Inhibitory Assay
The
performed according to the previously described method [11]. The sample solution (2 mL dissolved
in dimethyl sulfoxide (DMSO)) and 0.5 U/mL -glucosidase (40 mL) were mixed in 120 mL of 0.1 M
phosphate buffer (pH 7.0). After 5 min pre-incubation, 5 mM p-nitrophenyl- -D-glucopyranoside
α-glucosidase (G0660-750UN, Sigma-Aldrich, St. Louis, MO) enzyme inhibition assay was
α
α
solution (40 mL) was added, and the solution was incubated at 37 ◦C for 30 min. The absorbance
of released 4-nitrophenol was measured at 405 nm by using a microplate reader (Molecular Devices,
Sunnyvale, CA, USA).
3.7. α-Amylase Inhibitory Assay
The α-amylase (A8220, Sigma-Aldrich, St. Louis, MO, USA) enzyme inhibitory activity was
measured using the reported method [11]. Substrate was prepared by boiling 100 mg potato starch
in 5 mL phosphate buffer (pH 7.0) for 5 min, then cooling to room temperature. The samples (2 mL
dissolved in DMSO) and substrate (50 mL) were mixed in 30 mL of 0.1 M phosphate buffer (pH 7.0).
After 5 min pre-incubation, 5 mg/mL α-amylase solution (20 mL) was added, and the solution was
incubated at 37 ◦C for 15 min. The reaction was stopped by adding 50 mL 1 M HCl and then 50 mL
iodine solution was added. The absorbances were measured at 650 nm by a microplate reader.
4. Conclusions
In summary, five new pregnane glycosides and four known oleanane saponins were isolated
and identified from G. sylvestre. Besides oleanane saponins-hightly species-specific of G. sylvestre,
new pregnane glycosides in this report will provide secondary metabolisms as specific compounds
in Gymnema genus. Compound
, and showed significant -amylase inhibitory activity. This is the first report of
α-glycosidase inhibitory activities of compounds from G. sylvestre.
4
showed the weak
α
-glucosidase inhibitory activity. Compounds
2,
3
4
α
α
-amylase and

Molecules 2020, 25, 2525
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Supplementary Materials: Figure S1: The chemical structures of compounds
6
–
9
; Figure S2: HR-ESI-MS of
compound
1
, Figure S3: 1H-NMR spectrum of compound
1
, Figure S4: 13C-NMR spectrum of compound
1
,
Figure S5: DEPT135 spectrum of compound
1
, Figure S6: HSQC spectrum of compound
1
, Figure S7: HMBC
spectrum of compound
1
, Figure S8: COSY spectrum of compound
1
, Figure S9: ROESY spectrum of compound
1
,
1
Figure S10: HR-ESI-MS of compound
spectrum of compound
compound , Figure S15: HMBC spectrum of compound
ROESY spectrum of compound , Figure S18: HR-ESI-MS of compound
compound
, Figure S20: 13C-NMR spectrum of compound
Figure S22: HMBC spectrum of compound
spectrum of compound , Figure S25: HR-ESI-MS of compound
Figure S27: 13C-NMR spectrum of compound
spectrum of compound , Figure S30: HMBC spectrum of compound
, Figure S32: ROESY spectrum of compound
spectrum of compound
, Figure S35: 13C-NMR spectrum of compound
compound , Figure S37: HSQC spectrum of compound ; Figure S38: HMBC spectrum of compound
COSY spectrum of compound
; Figure S40: 1H-NMR spectrum of compound ; Figure S41: 13C-NMR spectrum
of compound ; Figure S42: HSQC spectrum of compound
; Figure S43: 1H-NMR spectrum of compound
; Figure S44: 13C-NMR spectrum of compound
; Figure S45: HSQC spectrum of compound ; Figure S46:
1H-NMR spectrum of compound ; Figure S47: 13C-NMR spectrum of compound
; Figure S48: HSQC spectrum
of compound
; Figure S49: 1H-NMR spectrum of compound ; Figure S50: 13C-NMR spectrum of compound
Figure S51: HSQC spectrum of compound 9.
2
, Figure S11: H-NMR spectrum of compound
2
, Figure S12: 13C-NMR
2
, Figure S13: DEPT135 spectrum of compound
, Figure S16: COSY spectrum of compound
, Figure S19: 1H-NMR spectrum of
2, Figure S14: HSQC spectrum of
2
2
2
, Figure S17:
2
3
3
3
, Figure S21: HSQC spectrum of compound
3
,
3
, Figure S23: COSY spectrum of compound
3
, Figure S24: ROESY
3
4
, Figure S26: 1H-NMR spectrum of compound
4,
4
, Figure S28: DEPT135 spectrum of compound 4, Figure S29: HSQC
4
4
, Figure S31: COSY spectrum of compound
1
4
4, Figure S33: HR-ESI-MS of compound
5
, Figure S34: H-NMR
5
5, Figure S36: DEPT135 spectrum of
5
5
5; Figure S39:
5
6
6
6
7
7
7
8
8
8
9
9;
Author Contributions: Isolation, P.V.K., D.T.H.Y., and B.H.T.; Formal analysis, P.V.K., D.T.H.Y., N.X.N., B.H.T.,
D.T.T., P.H.Y., T.M.N., J.H.L., S.Y.K. and S.H.K.; Research idea, N.V.H., T.M.N., and C.V.M.; Structure elucidation,
P.V.K., D.T.H.Y., N.X.N., and B.H.T.; Biological assay, D.T.H.Y., S.P., J.H.L., S.Y.K., and S.H.K.; Writing, P.V.K.,
D.T.H.Y., N.X.N., N.V.H., and S.H.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research is funded by Vietnam National Foundation for Science and Technology Development
(NAFOSTED), under grant number 104.01-2017.25 and by the National Research Foundation of Korea (NRF) grant
[NRF-2016R1A2B4006742], funded by the Ministry of Education, Science and Technology, Republic of Korea.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–9 are available from the authors.
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