Triterpenoids and their glycosides from Glinus oppositifolius with antifungal activities against Microsporum gypseum and Trichophyton rubrum

Article abstract of DOI:10.3390/molecules24122206

Four new triterpenoids, 3β,12β,16β,21β,22-pentahydroxyhopane (1), 12β,16β,21β,22-tetrahydroxyhopan-3-one (2), 3-oxo-olean-12-ene-28,30-dioic acid (3), and 3β-hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl ester (4); 21 new triterpenoid saponins, glinusopposides A–U (5–25); and 12 known compounds (26–37) were isolated from the whole plants of Glinus oppositifolius. The structures of the new compounds were elucidated based on the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. All compounds from the plants were measured for antifungal activities against Microsporum gypseum and Trichophyton rubrum. Glinusopposide B (6), glinusopposide Q (21), glinusopposide T (24), and glinusopposide U (25) showed strong inhibitory activities against M. gypseum (MIC₅₀ 7.1, 6.7, 6.8, and 11.1 μM, respectively) and T. rubrum (MIC₅₀ 14.3, 13.4, 11.9, and 13.0 μM, respectively). For those active compounds with an oleanane skeleton, glycosylation (21–26) or oxidation (3) of 3-OH was helpful in increasing the activity; replacement of the 30-methyl group (29) by a carboxymethyl group (26) enhanced the activity; the presence of 11,13(18) double bonds (20) decreased the activity.

Full text of DOI:10.3390/molecules24122206

molecules
Article
Triterpenoids and Their Glycosides from Glinus
Oppositifolius with Antifungal Activities against
Microsporum Gypseum and Trichophyton Rubrum
Dongdong Zhang 1,2 , Yao Fu 1,2, Jun Yang 1,2, Xiao-Nian Li 2, Myint Myint San 3,
Thaung Naing Oo 3, Yuehu Wang 1,2,* and Xuefei Yang 1,2,
*
1
Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw 05282,
Myanmar; zhangdongdong@mail.kib.ac.cn (D.Z.); fuyao@mail.kib.ac.cn (Y.F.); yangjuna@mail.kib.ac.cn (J.Y.)
Key Laboratory of Economic Plants and Biotechnology and Yunnan Key Laboratory for Wild Plant
Resources, State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201, China; lixiaonian@mail.kib.ac.cn
Forest Research Institute, Yezin, Nay Pyi Taw 05282, Myanmar;
2
3
dawmyintmyintsanyezin@gmail.com (M.M.S.); tnoo71@gmail.com (T.N.O.)
*
Correspondence: wangyuehu@mail.kib.ac.cn (Y.W.); xuefei@mail.kib.ac.cn (X.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 May 2019; Accepted: 7 June 2019; Published: 12 June 2019
Abstract: Four new triterpenoids, 3
tetrahydroxyhopan-3-one ( ), 3-oxo-olean-12-ene-28,30-dioic acid (
11,13(18)-diene-28,30-dioic acid 30-methyl ester ( ); 21 new triterpenoid saponins, glinusopposides
A–U ( 25); and 12 known compounds (26 37) were isolated from the whole plants of Glinus
β,12
β,16β,21β,22-pentahydroxyhopane (
1
), 12
β,16β,21β,22-
2
3
), and 3β-hydroxyoleana-
4
5
–
–
oppositifolius. The structures of the new compounds were elucidated based on the analysis of
one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and mass
spectrometry (MS) data. All compounds from the plants were measured for antifungal activities
against Microsporum gypseum and Trichophyton rubrum. Glinusopposide B (6), glinusopposide Q
(
21), glinusopposide T (24), and glinusopposide U (25) showed strong inhibitory activities against
M. gypseum (MIC50 7.1, 6.7, 6.8, and 11.1 µM, respectively) and T. rubrum (MIC50 14.3, 13.4, 11.9,
and 13.0
µM, respectively). For those active compounds with an oleanane skeleton, glycosylation
(
21 26) or oxidation (3
–
) of 3-OH was helpful in increasing the activity; replacement of the 30-methyl
group (29) by a carboxymethyl group (26) enhanced the activity; the presence of 11,13(18) double
bonds (20) decreased the activity.
Keywords: Glinus oppositifolius; triterpenoids and triterpenoid saponins; antifungal activity
1. Introduction
Dermatophytosis is one of the most common skin diseases in animals and humans, which is mainly
caused by Epidermophyton, Microsporum and Trichophyton [1,2]. As a chronic disease, dermatophytosis
is difficult to treat due to the drug resistance developed by the related fungus [2]. Therefore, it is
important to search for novel agents to treat dermatophytosis.
Glinus oppositifolius (L.) Aug. DC. (Syn: Mollugo spergula L. and Mollugo oppositifolia L; family:
Molluginaceae) is a small herb widely distributed in tropical Asia, tropical Africa, and Australia [ ].
Traditionally it has been used for treating skin and various infectious diseases in Bangladesh, China,
India, Mali and Myanmar [ ]. As a Chinese folk medicine, the whole plants of G. oppositifolius are
used to treat diarrhea, coughs, hyperthermia, heat rashes, pinkeye, furuncles, snakebites, and burns [ ].
The plant is reputed in Indian medicine due to its antiseptic and antidermatitic properties [ ]. It is
used to treat leprosy, leukoderma, heart, and skin diseases in the traditional medicine of Myanmar [ ].
3
4–6
6
7
5
Molecules 2019, 24, 2206; doi:10.3390/molecules24122206
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2206
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The major secondary metabolites from G. oppositifolius are triterpenoids and their glycosides, which
exhibit -glucosidase inhibitory [ ], cytotoxic [ ], and antiprotozoal activities [10]. There is little research
α
8
9
reported the anti-fungal activities of G. oppositifolius. In this study, we isolated 25 new triterpenoids and
triterpenoid saponins (Figure 1), along with 12 known compounds in the whole plants of G. oppositifolius.
Their antifungal properties against Microsporum gypseum and Trichophyton rubrum were analyzed.
Figure 1. Chemical structures of compounds 1–26 from Glinus oppositifolius.
2. Results and Discussion
2.1. Structure Elucidation of the Compounds
Compound
positive ion at m/z 515.3718 [M + Na]+ (calcd. for C30H52NaO5, 515.3712) in the high resolution
1
had the molecular formula C30H52O5 based on 13C-NMR data (Table 1) and the

Molecules 2019, 24, 2206
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electrospray ionization mass spectroscopy (HRESIMS). The 1H-NMR spectrum showed resonances
for eight methyl groups at δH 1.61 (s), 1.56 (s), 1.21 (s), 1.19 (s), 1.13 (s), 1.01 (s), 0.97 (s), and 0.82 (s),
as well as three oxymethines at δH 4.48 (m), 4.23 (m), and 3.44 (br t, J = 8.3 Hz) (Table 1). The 13C-NMR
spectrum showed resonances for thirty carbon atoms as expected from high resolution mass spectrum,
which were sorted by DEPT into eight methyls, eight methylenes, seven methines (three oxymethines,
δC 78.0, 69.1, and 66.8), and seven quaternary carbons group, including two oxygenated quaternary
carbons. These NMR data were very similar to those of a known hopane triterpenoid saponin from
this plant, glinoside C, except for the lack of signals for glucopyranose [8]. The full NMR assignments
and connections were determined by 1H-detected heteronuclear single quantum coherence (HSQC),
1H-detected heteronuclear multiple bond correlation (HMBC), and 1H-1H correlation spectroscopy
(COSY) analyses.
According to the 1H–1H COSY correlations in the 2D spectra of
1
(Figure 2), five connections,
C-1-C-2-C-3, C-5-C-6-C-7, C-9-C-11-C-12-C-13, C-15-C-16-C-17, and C-19-C-20, were confirmed.
The planar structure of was further deduced as 3,12,16,21,22-pentahydroxyhopane by the HMBC
1
correlations from H3-23 and H3-24 to C-3, C-4, and C-5; from H3-25 to C-1, C-5, C-9, and C-10;
from H3-26 to C-7, C-9, and C-14; from H3-27 to C-8, C-13, and C-15; from H3-28 to C-13, C-17,
C-18, and C-19; from H3-29 and H3-30 to C-21 and C-22; and from H-17 to C-19 and C-22.
The configurations of 3-OH, 12-OH, 16-OH, and 21-OH were deduced as 3
key nuclear overhauser effect spectroscopy (ROESY) correlations of H-3/H-5, H-5/H-9, H-9/H-12,
H-16/H3-27, H-16/H3-28, H-16/H3-30, and H3-30/H3-28. Thus, the structure of was determined to be
,12 ,16 ,21 ,22-pentahydroxyhopane. The absolute configuration was assigned by Cu K X-ray
crystallographic analysis (Figure 3).
β,12β,16β,21β by the
1
3β
β
β
β
α
Table 1. 1H (500 MHz) and 13C (126 MHz) NMR data of 1 and 2 in Pyridine-d5 (δ in ppm, J in Hz).
1
2
No.
1
δH
δC
δH
δC
1.68 m
0.97 m
1.79 m
1.31 m
2.48 m
2.42 m
39.1
28.3
39.4
34.4
2
1.81 m
3
4
5
3.44 br t (8.3)
78.0
39.5
55.7
216.4
47.4
54.8
0.78 dd (12.0, 1.7)
1.51 m
1.28 m
1.37 m
1.31 m
1.38 m
1.19 m
6
18.9
20.0
1.33 m
1.43 m
1.21 m
7
33.6
32.7
8
9
10
45.1
49.3
37.3
45.2
48.4
36.8
1.39 m
1.39 m
2.11 m
1.64 m
4.23 m
2.04 m
1.65 m
4.20 m
11
33.2
33.4
12
13
14
69.1
56.4
41.8
69.0
56.5
41.7
1.84 d (10.7)
1.84 d (10.8)
1.93 dd (12.7, 4.2)
1.71 m
4.48 m
2.41 d (11.7)
1.92 dd (12.6, 4.3)
1.70 m
4.48 m
2.41 d (11.6)
15
46.0
45.9
16
17
18
66.8
73.8
47.5
66.7
73.8
47.5
2.60 m
2.14 m
2.05 m
1.95 m
2.60 m
2.13 m
2.07 m
1.96 m
19
43.1
43.1
20
21
37.8
85.7
37.8
85.7

Molecules 2019, 24, 2206
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Table 1. Cont.
1
2
No.
δH
δC
δH
δC
22
23
24
25
26
27
28
29
30
75.5
28.7
16.3
16.1
17.0
19.5
17.3
26.6
27.3
75.5
26.6
21.3
15.6
16.6
19.4
17.3
26.7
27.4
1.21 s
1.01 s
0.82 s
0.97 s
1.13 s
1.19 s
1.56 s
1.61 s
1.11 s
1.00 s
0.81 s
0.95 s
1.10 s
1.19 s
1.56 s
1.61 s
3-OH
12-OH
5.78 br s
5.32 d (6.0)
5.38 d (6.9)
30
29
COOH
20
21
20
17
19
29
OH
OH
19
H
OH18
12
H
12
13
18
22
11
13
OH
11
9
25
25
12
26
OH
25
24
17
16
10
9
22
COOH
16
30
15
OH
30
28
1
4
9
16
3
OH
HO
1
4
10
5
2
8
29
H
8
27
10
2
3
15
5
3
H
H
5
OH
7
H
H
HO
7
6
O
6
23
24
1
23
3
24
1
30
29
COOMe
20
21
19
30
COOMe
H
12
8
22
18
13
H19
11
9
COOH
19
29
26
H
25
25
17
16
25
18
COOH
26
24
4
10
12
28
14
27
9
9
1
4
29
10
5
3
2
3
15
8
16
14
HO
5
27
5
H
7
H
HO
24
H
H
6
23
24
4
4
3
23
O
20
22
19
15
OH
30
12
13
11
H
OH18
H
O 13
30
22
H
25
26
25
5'
29
25
12
12
24
17
16
24
4'
10
HO
O
9
17
9
1
9
O
2
3
10
5
8
3
O
28
29
HO
16
O
H
H
3'
27
27
5
4
3
5
H
7
1'
H
H
HO
O
2'
H
H
H
H
6
5
OH
5
12
23
24
HO
6'
5'
HO
4'
30
29
O
1'
COOMe
HO 3'
20
21
20
19
19
15
2'
30
O
12
8
30
22
22
18
19
HO
29
12
13
13
11
6' COOMe
18
25
11
9
17
16
25
26
COOH
17
9
29
5'
HO
28
27
9
1
4
O
16
3
O
10
5
15
8
1
4
4'
2
3
5
10
5
2
8
3'
3
1'
7
HO
O
24
7
2'
6
HO
6
OH
23
23
24
26
1H-1H COSY
12
26
23
24
HMBC
ROESY
Figure 2. Key 2D-NMR correlations of 1, 3–5, 12, and 26.

Molecules 2019, 24, 2206
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Figure 3. X-ray crystallographic structure of 1.
Compound
the [M + Na]+ ion at m/z 513.3551 (calcd. for C30H50NaO5, 513.3556) in the HRESIMS. The NMR data
(Table 1) of were analogous to those of except that the signal (δC 78.0) for an oxygenated methine in
the 13C-NMR spectrum of was replaced by the signal (δC 216.4) for a carbonyl group in the 13C-NMR
spectrum of . The structure of was easily established as 12 , 16 , 21 , 22-tetrahydroxyhopan-3-one
by the COSY, HMBC, and ROESY spectra of 2 (Supplementary Materials).
Compound
was assigned the molecular formula C30H44O5 based on 13C-NMR data (Table 2) and
positive ion mode HRESIMS, which showed a pseudomolecular ion peak at m/z 507.3084 [M + Na]+
(calcd. for C30H44NaO5, 507.3086). The 1H-NMR data of
(Table 2) indicated the presence of six methyl
groups at δH 1.43 (s), 1.30 (s), 1.14 (s), 1.00 (s), 0.99 (s), and 0.86 (s) and one olefinic group at δH 5.72.
The 13C-NMR data of
(Table 2) indicated the presence of six methyl groups, two carboxylic carbons
2
showed a molecular formula of C30H50O5 based on 13C-NMR data (Table 1) and
2
1
1
2
2
β
β
β
3
3
3
at δC 180.1 and 179.5, one carbonyl carbon at δC 216.3, and two olefinic carbons (one quaternary at δC
144.8 and one methane at δC 122.8, suggesting the presence of a double bond), 10 sp3 methylenes, three
sp3 methines, and seven sp3 quaternary carbon atoms. The NMR data of
3 were very similar to those
of 3-oxo-olean-12-en-28,29-dioic acid [11], implying that 3 was also an oleanane triterpenoid.
Six fragments, C-1-C-2, C-5-C-6-C-7, C-9-C-11-C-12, C-15-C-16, C-18-C-19, and C-21-C-22,
were deduced from the 1H–1H COSY correlations in the 2D-NMR spectra of
3
(Figure 2). The structure
of
3 was deduced as 3-oxo-olean-12-ene-28,30-dioic acid by the HMBC correlations from H3-23 and
H3-24 to C-3, C-4, and C-5; from H2-1 and H2-2 to C-3; from H3-25 to C-1, C-5, C-9, and C-10; from H3-26
to C-7, C-9, and C-14; from H3-27 to C-8, C-13, and C-15; from H-18 to C-12, from H2-19 to C-17;
from H2-16 and H2-22 to C-28; and from H3-29 to C-19, C-20, C-21 and C-30; as well as the key ROESY
correlations of H-19α/H3-27 and H-19α/H3-29 (Figure 2).
The molecular formula of compound
4, C31H46O5, with nine degrees of unsaturation,
was determined by the 13C-NMR data in methanol-d4 (Table 2) and positive ion mode HRESIMS, which
showed a pseudomolecular ion peak at m/z 521.3234 [M + Na]+ (calcd. for C31H46NaO5, 521.3237).
The 1H-NMR data in methanol-d4 (Table 2) showed signals for six methyl groups at δH 1.11 (s), 1.00 (s),
0.98 (s), 0.94 (s), 0.81 (s), and 0.78 (s); a methoxy group at δH 3.67 (s); and a disubstituted double bond at
δH 6.33 (dd, J = 11.2, 2.8 Hz) and 5.72 (br d, J = 11.2 Hz). The NMR data (Table 2) were very similar to those
of 30-O-methyl spergulagenate (27) [12]. However, compound
than 30-O-methyl spergulagenate, which was supported by four olefinic carbons at δC 139.8, 130.8,
129.0, and 126.2 for two double bonds in the 13C-NMR spectrum of
measured in methanol-d4. Finally,
the structure of was elucidated to be 3 -hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl
ester by the key HMBC correlations from H-11 to C-10, from H-12 to C-8, and from H3-27 to C-13,
4 had one more degree of unsaturation
4
4
β

Molecules 2019, 24, 2206
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as well as the key ROESY correlations of H-3/H-5, H-5/H-9, H-9/H3-27, H3-29/H-19
α, H3-29/H-16α,
and H3-29/H3-27 (Figure 2).
Table 2. 1H and 13C-NMR data of 3 and 4 (δ in ppm, J in Hz).
3 (Pyridine-d5)
δH (500 MHz)
4 (Methanol-d4)
δH (600 MHz)
4 (Pyridine-d5)
δH (500 MHz)
No.
1
δC (126 MHz)
39.1
δC (151 MHz)
39.4
δC (126 MHz)
38.5
1.65 m
1.30 m
2.51 m
2.37 m
1.92 m
1.05 m
1.69 m
1.88 m
1.07 m
1.94 m
2
34.4
27.9
28.1
1.64 m
1.89 m
3
4
5
216.3
47.5
55.4
3.18 dd (11.7, 4.9)
79.8
40.1
56.4
3.49 dd (10.6, 5.0)
78.1
39.6
55.3
1.32 m
1.35 m
0.84 br d (12.1)
1.63 m
0.91 dd (12.2, 1.8)
1.61 m
6
19.8
19.6
18.8
1.46 m
1.44 m
1.46 m
1.31 m
7
32.7
1.35 m
33.7
1.31 m
32.9
8
9
39.7
47.2
42.2
56.0
41.2
54.9
1.70 m
1.99 br s
2.06 br s
10
11
12
13
14
36.9
23.8
122.8
144.8
42.2
38.0
37.1
1.88 m
5.72 br t (3.3)
5.72 br d (11.2)
6.33 dd (11.2, 2.8)
129.0
126.2
139.8
43.6
5.78 br d (10.5)
6.63 dd (10.5, 2.6)
127.8
125.7
138.3 a
42.7
1.72 m
1.08 m
1.71 m
1.95 m
1.97 m
1.08 m
1.78 m
2.25 m
15
2.18 m
28.5
26.2
25.6
16α
16β
17
2.19 m
2.08 m
24.0
33.8
49.3 a
130.8
33.1
46.4
43.5
48.8
18
3.63 dd (13.7, 4.0)
1.92 dd (13.7, 13.7)
2.50 m
131.2 a
19α
19β
20
2.85 dd (14.5, 1.7)
2.17 d (14.5)
3.18 d (15.2)
2.78 d (15.2)
43.1
44.1
31.2
35.9
44.8
33.0
35.6
43.9
32.5
2.41 m
1.47 m
2.41 m
2.08 m
1.14 s
0.99 s
0.86 s
1.00 s
1.30 s
1.83 m
1.57 m
2.29 m
1.41 m
0.98 s
0.78 s
0.94 s
0.81 s
1.00 s
2.28 m
1.78 m
2.67 ddd (13.8, 3.5, 3.5)
21
22
34.8
35.4
35.0
1.52 m
1.24 s
1.03 s
0.97 s
1.07 s
1.09 s
23
24
25
26
27
28
29
30
30-OMe
26.6
21.6
14.9
17.3
26.1
180.1
29.1
179.5
28.6
15.9
18.8
17.3
20.3
179.9
20.4
180.4
52.6
28.6
16.1
18.4
17.0
20.1
DAS b
20.2
1.43 s
1.11 s
3.67 s
1.28 s
3.60 s
178.5
51.8
a
Detected by HMBC. b Disappeared signal.
The HRESIMS of glinusopposide A (
positive ion at m/z 613.4068 [M + Na]+ (calcd. for C35H58NaO7, 613.4080). By comparing its NMR
data (Table 3) with those of spergulagenin A 3-O- -d-xylopyranoside (31) [13], compound might
be combined by a modified hopane and a -xylopyranose [δH 4.87 d (J = 7.6 Hz)]. The configuration
5) indicated a molecular formula of C35H58O7, with the
β
5
β
of xylopyranose in the plant was determined as the d-configuration by acidic hydrolysis of 31
followed by acetylation to yield 1,2,3,4-tetra-O-acetyl-d-xylopyranose. The genin was deduced as
16-deoxyspergulagenin A by 1H–1H COSY, HMBC, and ROESY experiments. The ROESY correlations
(Figure 2) of H-3/H-5, H-5/H-9, H-9/H-12, H-12/H3-28, and H3-28/H3-29 indicated that 3-OH, 12-OH,
and Me-29 were β-, β-, and α-oriented, respectively. The xylose was located at 3-OH based on the
HMBC correlations from H-3 to C-10 and from H-10 to C-3 (Figure 2). Finally, the structure of
5 was
elucidated to be 16-deoxyspergulagenin A 3-O-β-d-xylopyranoside (glinusopposide A).
Both glinusopposides B (
13C-NMR data (Table 3) and HRESIMS. The NMR data of
of the same genin in the two saponins as in compound
are two sugars, -d-xylopyranose and -l-rhamnopyranose, in the structures of
6
) and C (
7
) have the same molecular formula, C41H68O11, based on
and (Table 3) indicated the presence
, with differences in the sugars. There
and . Base
6
5
7
β
α
6
7

Molecules 2019, 24, 2206
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on the key HMBC correlations from H-100 to C-20 and from H-10 to C-3 in
6
, along with the
correlations from H-100 to C-30 and from H-10 to C-3 in
7
(Supplementary Materials), the linkages
2)-Xyl-O-C-3 and Rha-(1 3)-Xyl-O-C-3
, respectively. Therefore, the structures of and were determined to be
16-deoxyspergulagenin A 3-O-[ -l-rhamnopyranosyl-(1 2)]- -d-xylopyranoside (glinusopposide B)
-l-rhamnopyranosyl-(1 3)]- -d-xylopyranoside (glinusopposide
between the two sugars were easily established to be Rha-(1
for saponins and
→
→
6
7
6
7
α
→
β
and 16-deoxyspergulagenin A 3-O-[
α
→
β
C), respectively.
Table 3. 1H and 13C-NMR data of 5–7 in Pyridine-d5 (δ in ppm, J in Hz).
5
6
7
No.
1
δH (600 MHz)
δC (151 MHz)
39.3
δH (500 MHz)
δC (126 MHz)
39.1
δH (600 MHz)
δC (151 MHz)
39.3
1.70 m
0.97 m
1.63 m
0.90 m
1.67 m
0.94 m
2.21 m
1.91 m
2.13 m
1.88 m
2.14 m
1.87 m
2
27.4
27.0
27.3
3
4
5
3.38 dd (11.7, 4.4)
89.1
40.1
56.2
3.29 dd (11.8, 4.2)
88.6
39.7
56.0
3.31 dd (11.9, 4.4)
89.3
40.1
56.2
0.79 br d (11.9)
1.51 m
0.71 br d (9.3)
1.46 m
0.76 br d (12.0)
1.50 m
6
19.1
18.6
19.0
1.33 m
1.33 m
1.32 m
1.39 m
1.19 m
1.33 m
1.15 m
1.40 m
1.19 m
7
34.0
33.5
34.0
8
9
10
43.8
50.0
37.4
43.4
49.6
37.0
43.8
50.0
37.4
1.40 m
1.35 m
1.40 m
2.12 m
1.64 m
4.20 m
2.07 m
1.60 m
4.17 m
2.10 m
1.63 m
4.20 m
11
33.5
33.0
33.5
12
13
14
69.2
56.9
42.1
68.7
56.5
41.6
69.1
57.0
42.1
1.72 d (10.7)
1.68 d (11.1)
1.72 overlapped
1.51 m
1.20 m
1.44 m
1.48 m
1.17 m
1.42 m
1.52 m
1.21 m
1.46 m
15
34.8
34.4
34.8
16
17
18
20.2
56.3
45.8
19.8
55.9
45.4
20.2
56.3
45.8
1.82 dd (12.1, 2.9)
1.78 dd (11.9, 2.9)
1.82 dd (12.0, 2.8)
2.58 m
1.68 m
2.17 m
1.68 m
2.55 m
1.65 m
2.14 m
1.65 m
2.58 m
1.68 m
2.17 m
1.68 m
19
45.2
44.8
45.2
20
36.2
35.7
36.2
21
22
23
24
25
26
27
28
29
30
10
54.5
213.0
28.6
17.2
16.5
17.5
18.4
17.2
21.5
25.9
108.2
76.0
79.1
71.7
54.0
212.6
28.0
16.9
16.2
17.0
18.0
16.8
21.0
25.4
106.2
78.0
79.7
71.6
54.5
213.0
28.5
17.2
16.5
17.5
18.4
17.2
21.5
25.9
107.8
75.9
83.5
70.2
1.33 s
1.01 s
0.82 s
0.99 s
1.05 s
1.12 s
1.22 s
2.19 s
1.25 s
1.17 s
0.79 s
0.95 s
1.01 s
1.09 s
1.19 s
2.16 s
1.27 s
0.97 s
0.81 s
0.99 s
1.05 s
1.12 s
1.22 s
2.19 s
4.87 d (7.6)
4.05 dd (8.6, 7.6)
4.20 m
4.83 d (7.3)
4.24 dd (8.2, 7.3)
4.18 m
4.77 d (7.5)
4.04 dd (8.6, 7.5)
4.32 dd (8.8, 8.8)
4.17 m
20
30
40
4.26 m
4.15 m
4.41 dd (11.3, 5.2)
3.80 dd (11.3, 10.5)
4.33 m
4.36 m
3.74 dd (11.2, 10.3)
6.30 br s
4.82 dd (3.4, 1.3)
4.62 dd (9.3, 3.4)
4.37 dd (9.3, 9.3)
5.01 m
50
67.6
67.0
67.4
3.71 dd (10.5, 9.9)
6.54 d (0.9)
4.88 m
4.69 dd (9.5, 3.1)
4.35 m
100
200
102.0
72.5
72.6
74.2
69.8
18.8
103.2
73.1
73.2
74.6
70.4
19.1
300
400
500
4.77 m
600
1.70 d (6.2)
5.23 d (6.2)
6.69 br s
6.72 br s
1.71 d (6.2)
1020-OH
2 -OH
400-OH

Molecules 2019, 24, 2206
8 of 23
Based on 13C-NMR data (Table 4) and HRESIMS, the molecular formulae of glinusopposides
D–G ( 11) were deduced to be C43H70O13, C45H72O13, C47H74O14, and C44H70O13, respectively.
By comparing their NMR data (Table 4) with those of spergulagenin A 3-O- -d-xylopyranoside
31) [13], saponins 8–11 were deduced to be disaccharide glycosides of spergulagenin A. The presence
8
–
β
(
of trans-2-butenoyl (crotonyl) group in
9
was confirmed by the 1H-NMR signals at δH 7.06
(m), 6.02 (dq, J = 15.6, 1.6 Hz), and 1.66 (3H, d, J = 6.8 Hz), along with COSY correlations
of H-2000/H-3000 and H-3000/H-4000 (Supplementary Materials). The closely similar data and
correlations can also be found in 10 and 11, herein the trans-2-butenoyl group was assigned in
10 and 11 as same way. The trans-2-butenoyl moiety of
9
–
11 was located at C-40 by the key
1
HMBC correlation from H-4’ to C-1"’. According to the correlations in the H–1H COSY, HMBC,
and ROESY spectra (Supplementary Materials), the structures of 8–11 were easily elucidated to be
3-O-[
D), 3-O-[
(glinusopposide E), 3-O-[
12-O-acetylspergulageninA(glinusopposideF), and3-O-[
β-d-xylopyranosyl] spergulagenin A (glinusopposide G), respectively.
α
-l-rhamnopyranosyl-(1
-l-rhamnopyranosyl-(1
-l-rhamnopyranosyl-(1
→
3)-4-O-acetyl-
3)-4-O-trans-2-butenoyl-
3)-4-O-trans-2-butenoyl-
-d-xylopyranosyl-(1 3)-4-O-trans-2-butenoyl-
β
-d-xylopyranosyl] spergulagenin A (glinusopposide
-d-xylopyranosyl] spergulagenin A
-d-xylopyranosyl]
α
→
β
α
→
β
β
→
Table 4. 1H and 13C-NMR data of 8–11 in Pyridine-d5 (δ in ppm, J in Hz).
8
9
10
11
δH
(500 MHz)
δC
δH
(500 MHz)
δC
δH
(800 MHz)
δC
δH
(800 MHz)
δC
No.
(126 MHz)
(126 MHz)
(201 MHz)
(201 MHz)
1.62 m
0.85 m
2.03 m
1.63 m
0.86 m
2.04 m
1.41 m
0.66 m
1.98 m
1.64 m
0.87 m
2.05 m
1
2
38.8
38.8
38.6
38.8
26.8
26.8
26.6
26.8
1.80 m
1.81 m
1.76 m
1.82 m
3
4
5
3.25 dd (11.8, 4.4)
88.8
39.6
55.7
3.26 dd (11.8, 4.4)
88.8
39.6
55.7
3.22 dd (11.9, 4.6)
88.8
39.5
55.5
3.29 dd (11.9, 4.0)
88.8
39.6
55.7
0.69 d (11.7)
1.43 m
1.27 m
1.40 m
1.21 m
0.69 br d (11.8)
1.44 m
0.64 m
1.41 m
1.23 m
1.35 m
1.17 m
0.70 br d (12.5)
1.41 m
6
18.6
18.6
18.4
18.6
1.27 m
1.41 m
1.22 m
1.31 m
1.42 m
1.23 m
7
33.6
33.6
33.3
33.6
8
9
10
45.7
49.1
36.9
45.7
49.1
36.9
45.8
48.5
36.8
45.7
49.1
36.9
1.33 m
1.34 m
1.28 m
1.35 m
2.07 m
1.62 m
4.19 m
2.09 m
1.63 m
4.21 m
1.98 m
1.37 m
5.47 m
2.09 m
1.63 m
4.20 m
11
33.0
33.0
28.1
33.0
12
13
14
68.6
55.8
41.8
68.6
55.8
41.8
72.2
52.4
41.6
68.6
55.8
41.7
1.79 d (10.7)
1.79 d (10.5)
1.90 d (11.5)
1.79 d (10.6)
1.87 m
1.69 m
4.12 m
1.88 m
1.70 m
4.11 m
1.83 m
1.64 m
4.06 m
1.88 m
1.70 m
4.12 m
15
45.7
45.7
45.2
45.6
16
17
18
65.5
63.7
47.1
65.5
63.7
47.1
65.0
63.3
46.4
65.5
63.7
47.1
2.28 d (11.3)
2.28 d (11.4)
2.23 d (11.5)
2.28 d (11.7)
2.61 m
1.87 m
2.05 m
1.74 m
2.63 m
1.90 m
2.06 m
1.74 m
1.83 m
1.68 m
2.04 m
1.66 m
2.62 m
1.89 m
2.06 m
1.73 m
19
45.8
45.8
44.8
45.7
20
37.6
37.6
37.5
37.6
21
22
23
24
25
26
27
28
29
30
10
53.6
214.9
28.0
16.6
16.0
17.0
19.2
17.8
21.0
26.3
107.2
75.9
78.3
53.6
215.0
28.0
16.6
16.0
17.0
19.2
17.8
21.0
26.3
107.2
75.8
79.0
53.4
214.6
27.9
16.6
15.9
16.8
18.9
17.7
20.9
26.3
107.2
75.8
79.0
53.6
214.9
28.0
16.7
16.0
17.0
19.1
17.8
21.0
26.3
106.9
75.0
83.9
1.22 s
0.89 s
0.76 s
0.98 s
1.11 s
1.21 s
1.66 s
2.34 s
4.74 d (7.7)
4.00 m
1.22 s
0.90 s
0.76 s
0.99 s
1.11 s
1.21 s
1.66 s
2.34 s
1.21 s
0.88 s
0.72 s
0.93 s
1.08 s
1.03 s
1.61 s
2.37 s
1.25 s
0.94 s
0.77 s
0.99 s
1.12 s
1.21 s
1.66 s
2.35 s
4.76 d (7.5)
4.00 dd (8.3, 7.5)
4.43 m
4.75 d (7.5)
4.01 dd (8.4, 7.5)
4.43 m
4.83 d (7.3)
4.08 m
4.35 m
20
30
4.40 m
5.28 ddd
(9.7, 9.7, 5.5)
4.30 m
3.58 dd
(11.3, 11.0)
40
71.3
5.38 overlapped
71.0
5.39 m
70.9
5.44 m
70.7
4.35 m
3.63 dd (11.3 10.0)
4.38 m
3.65 m
4.35 m
3.68 m
50
63.1
63.2
63.2
63.3

Molecules 2019, 24, 2206
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Table 4. Cont.
8
9
10
11
δH
(500 MHz)
δC
δH
(500 MHz)
δC
δH
(800 MHz)
δC
δH
(800 MHz)
δC
No.
(126 MHz)
(126 MHz)
(201 MHz)
(201 MHz)
100
200
300
400
6.26 d (1.2)
4.69 br s
4.45 m
102.6
72.4
72.7
73.9
6.24 br s
4.74 dd (3.2, 1.4)
4.45 m
102.8
72.5
72.6
73.9
6.25 br s
4.72 m
4.44 m
102.9
72.5
72.6
74.0
5.25 d (7.8)
3.99 t (7.8)
4.14 m
106.9
75.8
78.4
71.0
4.30 t (9.3)
4.30 t (9.4)
4.29 dd (9.5, 9.5)
4.15 m
4.31 m
3.70 m
500
4.43 m
70.0
4.40 m
70.0
4.40 m
70.1
67.6
600
1.70 d (6.2)
18.8
170.4
21.1
1.68 d (6.3)
18.9
165.9
122.7
146.0
17.8
1.69 d (6.2)
18.9
165.9
122.8
145.9
17.8
1000
165.9
123.1
145.4
17.8
2000
2.15 s
6.02 dq (15.6, 1.6)
7.06 m
1.66 d (6.8)
6.04 br d (14.6)
7.07 m
1.61 dd (7.0, 1.5)
5.99 br d (15.5)
7.09 m
1.61 br d (6.9)
3000
4000
10000
170.4
21.9
20000
2.15 s
12-OH
106-OH
2 -OH
200-OH
400-OH
5.34 d (6.1)
5.49 d (5.0)
7.68 d (6.1)
6.80 br s
6.80 br s
Glinusopposide H (12) was assigned the molecular formula C36H58O8, with eight degrees of
unsaturation as determined by 13C-NMR data (Table 5) and the positive ion at m/z 641.4021 [M +
Na]+ (calcd. for C36H58NaO8, 641.4024) in the HRESIMS. The 1H and 13C-NMR data indicated the
compound might be a hopane triterpenoid saponin with eight methyl groups [δH 1.67 (s), 1.20 (s), 1.44
(s), 1.44 (s), 0.99 (s), 0.94 (s), 0.88 (s), and 0.79 (s)], one tetrasubstituted double bond (δC 152.5 and 146.8),
and one β-glucopyranose [δH 5.19 (d, J = 7.6 Hz); δC 100.1, 78.8, 78.4, 75.9, 72.4, and 63.4]. In addition
to the signals for the sugar, signals (δC 84.2, 78.1, 76.0, and 74.5) for four oxygenated carbon atoms were
observed. The sugar was attached to C-12 based on the HMBC correlations from H-12 to C-10 and from
H-10 to C-12, and the 17(21)-double bond was confirmed by the correlations from H3-28 to C-17 and
from H3-29 and H3-30 to C-21 (Figure 2). The other three oxygenated carbon atoms were C-3, C-16,
and C-22 based on the correlations from H3-23 and H3-24 to C-3, from H-16 to C-21, and from H3-29
and H3-30 to C-22. According to the deduced molecular formula and the degrees of unsaturation, a
dihydrofuran ring containing the C-16-C-17-C-21-C-22-O fragment must be formed in the structure of
12, which was further confirmed because of the shift in the 13C-NMR signals for C-16 (δC 76.0) and C-22
(
δC 84.2) to downfield compared with the analogues
The 3 ,12 ,16 configurations were determined by the key ROESY correlations of H-3/H3-23, H-3/H-5,
H-5/H-9, H-9/H-12, H-12/H3-27, H-12/H3-28, H-16/H3-27, and H-16/H3-28 (Figure 2). Thus, the structure
1, 2, and 22,24,28-trihydroxy-hop-17(21)-ene [14].
β
β
β
of 12 was elucidated to be 3β,12β-dihydroxy-16β,22-epoxyhop-17(21)-ene 12-O-β-d-glucopyranoside
(glinusopposide H).
Based on 13C-NMR data (Tables 5 and 6) and HRESIMS, the molecular formulae of glinusopposides
I–K (13
the NMR data of 13
suggested these compounds with the same genin, 3
The position of connectivity of sugars to sapogenin were established according to the
correlations in the 2D-NMR spectra (Supplementary Materials). Therefore, the structures
–
15) were deduced to be C35H56O7, C41H66O11, and C41H66O11, respectively. Comparison of
15 (Tables 5 and 6) with those of 12 (Table 5) which are closely similar that were
,12 -dihydroxy-16 ,22-epoxyhop-17(21)-ene.
–
β
β
β
of saponins 13
3-O- -d-xylopyranoside (glinusopposide I),
3-O-[ -l-rhamnopyranosyl-(1 2)]-
16 ,22-epoxyhop-17(21)-ene 3-O-[ -l-rhamnopyranosyl-(1
K), respectively.
–
15 were determined to be 3
β
,12
β
-dihydroxy-16
-dihydroxy-16
β
,22-epoxyhop-17(21)-ene
,22-epoxyhop-17(21)-ene
β
3β
,12
β
β
α
→
β-d-xylopyranoside (glinusopposide J), and 3
β,12β-dihydroxy-
β
α
→
3)]- -d-xylopyranoside (glinusopposide
β

Molecules 2019, 24, 2206
10 of 23
Table 5. 1H and 13C-NMR data of 12–14 in Pyridine-d5 (δ in ppm, J in Hz).
12
13
14
No.
1
δH (500 MHz)
δC (126 MHz)
39.0
δH (600 MHz)
δC (151 MHz)
39.5
δH (600 MHz)
δC (151 MHz)
1.61 m
0.84 m
1.72 m
0.98 m
1.68 m
0.95 m
39.7
2.21 m
1.90 m
3.37 dd (11.8, 4.5)
2.17 m
1.92 m
3.31 dd (11.9, 4.2)
2
1.78 m
3.41 m
28.3
27.4
27.5
3
4
5
78.1
39.5
56.0
89.0
40.1
56.3
88.9
40.1
56.6
0.69 m
1.49 m
1.28 m
1.27 m
0.75 br d (10.8)
1.51 m
0.71 br d (11.5)
1.47 m
6
18.8
19.0
19.0
1.33 m
1.32 m
1.31 m
1.29 m
7
8
9
34.2
46.8
48.4
37.6
34.6
47.2
49.5
37.6
34.6
47.2
49.5
37.5
1.22 m
1.40 m
1.37 m
10
2.37 m
1.48 m
4.37 m
2.09 m
1.66 m
4.04 m
2.07 m
1.65 m
4.04 m
11
27.2
33.5
33.5
12
13
14
74.5
54.0
41.5
69.7
56.2
41.9
69.7
56.2
41.9
1.87 d (11.2)
1.92 d (11.1)
1.91 d (11.1)
1.92 m
1.29 m
4.84 m
1.99 dd (11.6. 6.2)
1.38 m
1.97 dd (11.7, 6.1)
1.36 m
15
43.0
43.7
43.7
16
17
18
76.0
152.5
46.0
4.93 m
76.6
153.0
46.4
4.92 m
76.6
153.0
46.4
3.15 m
2.70 ddd
(13.6, 8.2, 2.6)
2.50 m
2.88 m
2.54 m
3.15 m
2.70 m
19
53.7
54.6
54.5
2.37 m
2.04 m
2.50 m
2.20 m
20
25.6
26.1
26.1
2.21 m
21
22
23
24
25
26
27
28
29
30
10
146.8
84.2
28.7
16.4
16.6
16.5
15.9
22.2
29.2
28.6
100.1
75.9
78.8
72.4
147.5
84.5
28.5
17.2
17.0
16.9
16.2
23.0
29.2
29.5
108.2
76.0
79.1
71.7
147.5
84.5
28.3
17.4
17.1
16.9
16.2
23.0
29.2
29.5
106.6
78.4
80.1
72.0
1.20 s
0.99 s
0.79 s
0.88 s
0.94 s
1.67 s
1.44 s
1.44 s
5.19 d (7.5)
4.07 m
1.31 s
0.99 s
0.85 s
1.02 s
1.07 s
1.59 s
1.49 s
1.47 s
4.86 d (7.5)
4.05 m
1.25 s
1.19 s
0.85 s
1.01 s
1.06 s
1.59 s
1.49 s
1.47 s
4.85 d (7.4)
4.27 dd (8.3, 7.4)
4.20 dd (8.7, 8.3)
4.17 m
20
30
4.37 m
4.26 dd (9.5, 9.5)
4.20 dd (8.7, 8.7)
4.26 m
40
4.39 dd (11.3, 5.2)
3.79 dd (11.3,
10.7)
4.34 dd (11.4, 4.8)
3.73 dd (11.4, 9.9)
50
4.05 m
78.4
63.4
67.6
67.4
4.57 dd (11.7, 2.2)
4.36 m
60
100
200
6.58 br s
4.90 dd (3.4, 1.4)
4.71 dd (9.4, 3.4)
4.38 dd (9.3, 9.3)
4.80 m
102.4
72.9
73.0
74.6
70.2
19.2
300
400
500
600
1.73 d (6.2)
12-OH
5.46 br s
The HRESIMS of glinusopposide L (16) exhibited an ion peak at m/z 731.4353 [M + Na]+ (calcd.
for C39H64NaO11, 731.4346), implying a molecular formula of C39H64O11. The NMR data of 16 (Table 6)
were highly similar to those of spergulin B (35) [13], indicating that the compound might also be a
bisnor hopane saponin with the same genin, spergulatriol, and the same sugars, xylose and rhamnose.
The difference between the two saponins was the linkage mode of the two sugars. The rhamnose was
linked to 3-OH of the inner sugar, xylose, based on the HMBC correlations from H-100 to C-30 and from

Molecules 2019, 24, 2206
11 of 23
H-30 to C-100 (Supplementary Materials). Finally, the structure of 16 was elucidated to be spergulatriol
3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide L).
According to 13C-NMR data (Table 6) and the positive ion HRESIMS at m/z 731.4347 [M + Na]+
(calcd. for C39H64NaO11, 731.4346), glinusopposide M (17) had the same molecular formula, C39H64O11,
as saponin 16. The 1D and 2D-NMR spectra (Supplementary Materials) indicated that 17 had a
tetrasubstituted double bond rather than the terminal double bond of 16. The 17(21) double bond
was identified based on the HMBC correlations from H3-28 to C-17 and from H3-22 to C-17 and C-21.
Therefore, the structure of 17 was determined to be 29,30-bisnor-3β,12β,16β-trihydroxyhop-17(21)-ene
3-O-[α-l-rhamnopyranosyl-(1 → 3)]-β-d-xylopyranoside (glinusopposide M).
Table 6. 1H and 13C-NMR data of 15–17 in Pyridine-d5 (δ in ppm, J in Hz).
15
16
17
No.
1
δH (600 MHz)
δC (151 MHz)
39.5
δH (500 MHz)
δC (126 MHz)
38.8
δH (800 MHz)
δC (201 MHz)
39.0
1.69 m
0.95 m
1.64 m
0.90 m
1.63 m
0.91 m
2.14 m
1.87 m
2.11 m
1.83 m
2.10 m
1.83 m
2
27.3
26.8
26.9
3
4
5
3.30 dd (11.9, 4.3)
89.2
40.1
56.3
3.27 dd (11.8, 4.4)
88.8
39.6
55.7
3.26 dd (12.0, 4.3)
88.8
39.6
55.9
0.73 br d (11.3)
1.48 m
0.72 br d (11.9)
1.47 m
0.73 br d (11.7)
1.44 m
6
19.0
18.6
18.6
1.31 m
1.30 m
1.30 m
1.45 m
1.27 m
1.40 m
1.31 m
7
1.31 m
1.39 m
34.6
33.5
33.6
8
9
10
47.2
49.5
37.5
45.4
49.2
36.9
44.8
49.6
37.0
1.38 m
1.47 m
2.09 m
1.65 m
4.04 m
2.07 m
1.61 m
4.19 m
2.08 m
1.62 m
4.08 m
1.91 m
11
33.5
33.1
33.4
12
13
14
69.7
56.2
47.2
69.3
53.9
41.8
69.6
55.1
41.7
1.92 d (11.1)
1.77 d (10.9)
2.00 m
1.38 m
4.93 m
1.87 dd (12.5, 4.2)
1.73 m
4.32 m
2.14 d (10.8)
1.92 m
1.87 m
4.99 overlapped
15
43.7
45.1
44.1
16
17
18
76.6
153.0
46.4
67.2
62.2
45.4
68.1
143.9
52.6
3.15 m
2.70 m
2.50 m
2.21 m
2.53 m
1.78 m
2.58 m
2.20 m
2.54 m
2.18 m
19
54.6
43.2
46.5
20
21
22
26.1
147.5
84.5
2.42 m
29.9
152.2
106.1
38.1
128.5
16.0
6.06 t (2.0)
5.14 t (2.0)
1.23 s
0.93 s
0.78 s
1.01 s
1.16 s
1.06 s
2.33 s
23
24
25
26
27
28
29
30
10
1.25 s
0.95 s
0.83 s
1.02 s
1.07 s
1.59 s
1.49 s
1.47 s
4.76 d (7.5)
4.03 m
28.4
17.2
17.0
16.9
16.2
23.0
29.2
29.5
107.8
75.9
83.5
70.2
28.0
16.7
16.0
17.0
19.1
16.0
1.21 s
0.92 s
0.79 s
0.98 s
1.25 s
1.39 s
28.0
16.7
16.4
16.5
17.5
20.6
4.74 d (7.4)
4.01 m
4.30 t (8.9)
4.14 m
107.4
75.4
83.0
69.7
4.73 d (7.5)
4.00 m
4.29 t (8.8)
4.13 m
107.4
75.4
83.0
69.7
20
30
4.32 dd (8.9, 8.9)
4.16 m
40
4.32 m
3.72 dd (11.3,
10.2)
4.35 m
3.73 dd (11.2, 10.4)
4.33 m
3.71 dd (11.2, 10.3)
50
67.4
67.0
66.9

Molecules 2019, 24, 2206
12 of 23
Table 6. Cont.
15
16
17
No.
δH (600 MHz)
δC (151 MHz)
δH (500 MHz)
δC (126 MHz)
δH (800 MHz)
δC (201 MHz)
100
200
6.30 br s
4.82 dd (3.3, 1.5)
4.62 dd (9.4, 3.3)
4.37 dd (9.4, 9.4)
5.00 m
103.3
73.1
73.2
74.6
70.4
19.2
6.26 d (1.2)
4.78 br s
4.60 br d (9.1)
4.34 m
4.98 overlapped
1.68 d (6.2)
5.27 d (6.5)
5.72 d (5.9)
7.29 d (6.0)
6.79 d (5.9)
6.74 br s
102.9
72.6
72.8
74.2
70.0
18.7
6.26 br s
4.78 br s
4.59 br d (8.7)
4.33 m
4.97 overlapped
1.68 d (6.1)
5.20 d (6.2)
6.03 d (5.5)
7.26 d (6.0)
6.76 d (5.6)
6.71 br s
102.8
72.6
72.8
74.2
70.0
18.7
300
400
500
600
1.71 d (6.2)
12-OH
16-OH
20-OH
40-OH
200-OH
300-OH
400-OH
6.47 br s
6.74 br s
6.43 br s
6.71 br s
According to 13C-NMR data (Table 7) and HRESIMS, the molecular formulae of glinusopposides
N (18) and O (19) were deduced to be C33H52O6 and C39H62O10, respectively. Comparison of their
NMR data (Table 7) with those of 17 indicated that saponins 18 and 19 were 29,30-bisnor hopane
saponins with two double bonds and two hydroxy substitutions in the structure of the genin. 3
β-OH
and 12 -OH were determined based on the key HMBC correlations from H3-23 and H3-24 to C-3 and
β
from H-9 to C-12, as well as the key ROESY correlations of H-3/H-5, H-5/H-9, H-9/H-12, H-12/H3-27,
and H-12/H3-28 (Supplementary Materials). The 15,17(21) double bonds were identified by the
HMBC correlations from H3-22 to C-17 and C-21, from H3-27 to C-15, from H3-28 to C-17, and from
H-16 to C-14 and C-18. Finally, based on other correlations in the 2D-NMR spectra (Supplementary
Materials), 18 and 19 were elucidated to be 29,30-bisnor-3
β,12
β-dihydroxyhopa-15,17(21)-diene
3-O- -d-xylopyranoside (glinusopposide N) and 29,30-bisnor-3
β
β,12
β-dihydroxyhopa-15,17(21)-diene
3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide O), respectively.
The molecular formula of glinusopposide P (20) was determined to be C44H66O15 based
on 13C-NMR data (Table 7) and the positive ion at m/z 857.4300 [M + Na]+ (calcd. for
C44H66NaO15, 857.4299) in the HRESIMS. The NMR data (Table 7) indicated a moiety of
3β-hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl ester (4), an α-rhamnopyranosyl
group [δH 6.33 (br s), 5.08 (m), 4.76 (dd, J = 3.3, 1.4 Hz), 4.57 (dd, J = 9.3, 3.3 Hz), 4.35 (dd,
J = 9.3, 9.3 Hz), and 1.71 (d, J = 6.1 Hz); δC 103.4, 74.6, 73.2, 73.0, 70.3, and 19.1], and a
6-O-methyl-β-glucuronopyranosyl group [δH 4.92 (d, J = 7.9 Hz), 4.58 (d, J = 9.3 Hz), 4.45 (dd, J = 9.3,
8.7 Hz), 4.41 (dd J = 9.3, 9.3 Hz), 4.07 (dd, J = 8.7, 7.9 Hz), and 3.79 (s); δC 171.3, 107.6, 82.3, 77.6, 76.2, 71.9,
and 52.7]. The linkage of the sugar chain was determined to be Rha-(1
→
3)-[6-O-methyl-GlcA]-O-C-3
based on the key HMBC correlations from H-100 to C-30, from H-30 to C-100, from H-10 to C-3,
and from H-3 to C-10 (Supplementary Materials). Thus, the structure of 20 was elucidated to be
3-O-[
α
-l-rhamnopyranosyl-(1
→
3)-6-O-methyl-β-d-glucuronopyranosyl]-3β-hydroxyoleana-11,13(18)-
diene-28,30-dioic acid 30-methyl ester (glinusopposide P).
Table 7. 1H and 13C-NMR data of 18–20 in Pyridine-d5 (δ in ppm, J in Hz).
18
19
20
δH (J in Hz)
(600 MHz)
No.
δH (600 MHz)
δC (151 MHz)
δH (500 MHz)
δC (126 MHz)
δC (151 MHz)
1.69 m
0.98 m
2.21 m
1.61 m
0.90 m
2.10 m
1.70 m
0.89 m
2.14 m
1
2
39.2
27.4
38.8
26.9
38.5
27.0
1.91 m
1.82 m
1.87 m
3
4
5
3.39 dd (11.9, 4.3)
89.1
40.2
56.5
3.27 dd (11.9, 4.4)
88.8
39.7
56.0
3.32 dd (11.9, 4.5)
89.8
40.1
55.6
0.84 m
0.77 overlapped
0.79 br d (12.1)

Molecules 2019, 24, 2206
13 of 23
Table 7. Cont.
19
18
20
δH (J in Hz)
(600 MHz)
No.
δH (600 MHz)
δC (151 MHz)
δH (500 MHz)
δC (126 MHz)
δC (151 MHz)
1.57 m
1.42 m
1.52 m
1.54 m
1.37 m
1.48 m
1.52 m
1.33 m
1.28 m
6
19.1
18.6
18.8
7
8
9
33.9
41.8
50.2
37.7
33.5
41.3
49.7
37.3
33.2
41.6
55.2
37.1
1.50 m
1.45 m
1.98 br s
10
2.18 m
1.66 m
4.33 m
2.14 m
1.61 m
4.28 m
11
34.3
33.9
5.69 br d (10.1)
128.2
12
13
14
69.4
53.3
47.5
69.0
52.8
47.0
6.60 dd (10.1, 2.9)
126.2
138.7
43.1
2.17 d (11.1)
2.13 d (11.1)
1.96 m
1.09 m
2.26 m
1.78 m
15
5.76 d (10.3)
6.42 d (10.3)
135.5
5.72 d (10.3)
6.39 d (10.3)
135.1
26.0
16
120.4
120.0
33.5
17
18
141.9
48.6
141.5
48.2
49.2
131.4
2.59 m
2.24 m
2.60 m
2.14 m
2.55 m
2.21 m
2.55 m
2.09 m
3.17 d (14.5)
2.77 d (14.5)
19
20
21
22
45.3
36.8
44.9
36.3
36.0
44.3
32.9
35.3
2.31 m
1.79 m
2.66 m
1.52 m
1.25 s
0.89 s
0.84 s
1.02 s
1.11 s
131.8
14.4
131.4
14.0
1.73 s
1.70 s
23
24
25
26
27
28
29
30
1.35 s
1.02 s
0.81 s
1.03 s
1.34 s
1.32 s
28.6
17.2
16.3
19.7
19.5
20.4
1.25 s
0.94 s
0.76 s
0.99 s
1.30 s
1.29 s
28.0
16.7
15.9
19.3
19.0
20.0
28.2
16.8
18.7
17.4
20.5
179.1
20.7
178.9
52.2
107.6
76.2
82.3
71.9
1.27 s
30-Me
3.60 s
4.92 d (7.9)
4.07 dd (8.7, 7.9)
4.45 dd (9.3, 8.7)
4.41 dd (9.3, 9.3)
10
4.88 d (7.7)
4.05 dd (8.4, 7.7)
4.20 dd (8.8, 8.4)
4.26 m
108.2
76.0
79.1
71.7
4.74 d (7.5)
4.00 dd (8.4, 7.5)
4.28 m
107.4
75.4
83.0
69.7
20
30
40
4.12 m
4.32 m
3.70 dd (11.0,
10.5)
4.40 dd (11.0, 5.1)
3.80 dd (11.0, 10.4)
50
67.6
67.0
4.58 d (9.3)
77.6
60
60-OMe
100
171.3
52.7
103.4
73.0
73.2
74.6
70.3
19.1
3.79 s
6.33 br s
4.76 dd (3.3, 1.4)
4.57 dd (9.3, 3.3)
4.35 dd (9.3, 9.3)
5.08 m
6.27 br s
4.79 br s
4.59 dd (9.3, 3.0)
4.34 dd (9.3, 9.3)
4.97 m
102.8
72.6
72.8
74.2
69.9
18.7
200
300
400
500
600
1.67 d (6.1)
1.71 d (6.1)
Based on 13C-NMR data (Tables 8 and 9) and HRESIMS, the molecular formulae of glinusopposides
Q–U (21 25) were deduced to be C39H61NO10, C44H68O15, C45H70O15, C36H56O9, and C42H66O13,
–
respectively. By comparing their NMR data with those of 30-O-methyl spergulagenate (27) [12],
these saponins were determined to have the same genin, 30-O-methyl spergulagenate. The NMR
signals of 21 at δH 8.94 (d, J = 9.0 Hz) and 2.15 (s), along with δC 170.3 and 23.8 manifested the
presence of an acetylamino unit which was further confirmed by the HMBC correlations from
δH 2.15 (H-200) to δC 170.3 (C-100) and from δH 8.94 (NH) to δC 170.3 (C-100). The position of the
acetylamino moiety of 21 was determined by the HMBC correlation from H-2’ to C-100. The location
of the sugar in 21 was also confirmed by the HMBC correlations from H-3 to C-1’ and H-1’ to
C-3. Two anomeric carbons at δC 107.1 and 102.9 of 22 suggested that the presence of two sugars,

Molecules 2019, 24, 2206
14 of 23
of which the positions were assigned by the key HMBC from H-3’ to C-100, from H-100 to C-3’,
from H-10 to C-3, and from H-3 to C-1’. The NMR date of 23 were almost identical to those of
22 except for the replacement of the methoxy group in 22 by ethoxy group (δC 61.4 and 14.3).
Comparison of NMR data of 30-O-methyl spergulagenate (27), signals for an additional sugar in
compound 24 and for two additional sugars in compound 25 were observed. According to these
correlations in the 2D-NMR spectra (Supplementary Materials), saponins 21
3-O-(2-acetylamino-2-deoxy- -d-glucopyranosyl)-30-O-methyl spergulagenate (glinusopposide Q),
3-O-[ -l-rhamnopyranosyl-(1 3)-6-O-methyl- -d-glucuronopyranosyl]-30-O-methyl spergulagenate
(glinusopposide R), 3-O-[ -l-rhamnopyranosyl-(1 3)-6-O-ethyl- -d-glucuronopyranosyl]-30-O-methyl
spergulagenate (glinusopposide S), 30-O-methyl spergulagenate 3-O- -d-xylopyranoside
-l-rhamnopyranosyl-(1 3)]- -d-
–25 were determined as
β
α
→
β
α
→
β
β
(glinusopposide T), and 30-O-methyl spergulagenate 3-O-[
xylopyranoside (glinusopposide U), respectively.
α
→
β
Table 8. 1H and 13C-NMR data of 21–23 in Pyridine-d5 (δ in ppm, J in Hz).
21
22
23
No.
1
δH (500 MHz)
δC (126 MHz)
38.6
δH (500 MHz)
δC (126 MHz)
38.5
δH (500 MHz)
δC (126 MHz)
38.5
1.35 m
0.82 m
1.36 m
0.82 m
1.37 m
0.83 m
2.19 m
1.76 m
2.04 m
1.77 m
2.07 m
1.78 m
2
26.4
26.6
26.6
3
4
5
3.25 dd (11.9, 4.4)
89.2
39.3
55.8
3.28 overlapped
89.4
39.5
55.7
3.27 overlapped
89.3
39.5
55.7
0.75 overlapped
1.49 m
0.75 overlapped
1.46 m
0.74 overlapped
1.46 m
6
18.6
18.4
18.4
1.28 m
1.25 m
1.26 m
1.46 m
1.27 m
1.44 m
1.26 m
1.45 m
1.26 m
7
33.3
33.2
33.2
8
9
39.7
48.1
42.1
48.0
42.1
48.0
1.60 m
1.61 m
1.62 dd (8.6, 8.6)
10
11
12
13
14
37.0
23.8
122.5
145.5
42.2
36.9
23.7
123.1
144.5
39.7
36.9
23.7
123.1
144.5
39.7
1.85 m
5.58 br s
1.85 m
5.59 br s
1.85 m
5.59 dd (3.4, 3.4)
2.12 m
1.18 m
2.12 m
2.02 m
2.13 m
1.27 m
2.13 m
2.03 m
15
2.21 m
2.08 m
28.6
28.4
28.4
16
24.2
23.9
23.9
17
18
46.4
43.6
46.2
43.4
46.2
43.4
3.36 dd (13.5, 3.6)
2.26 m
3.29 overlapped
2.25 br d (13.2)
1.81 m
3.29 overlapped
2.25 br d (12.0)
1.82 m
19
20
21
43.2
44.3
31.2
42.7
44.2
30.9
42.7
44.2
30.9
1.82 m
2.16 m
1.45 m
2.06 m
1.96 m
1.19 s
0.98 s
0.75 s
2.19 m
1.46 m
2.08 m
1.97 m
1.24 s
0.90 s
0.75 s
0.95 s
1.31 s
2.19 br d (13.5)
1.47 m
2.09 m
1.98, m
1.23 s
22
34.9
34.6
34.6
23
24
25
26
27
28
29
30
28.2
17.0
15.4
28.1
16.9
15.4
17.3
26.2
179.9
28.5
177.2
51.8
107.1
74.1
81.9
71.4
28.1
16.9
15.4
17.3
26.2
179.9
28.5
177.2
51.7
107.2
75.8
81.9
71.4
0.90 s
0.76 s
0.95 s
1.31 s
0.89 br s
1.31 s
17.6
26.2
DAP a
28.7
1.22 s
1.23 s
1.23 s
177.4
51.7
105.0
58.1
76.3
72.7
30-OMe
3.63 s
5.05 d (8.2)
4.58 m
4.41 dd (9.3, 8.7)
4.18 dd (9.3, 9.3)
3.65 s
4.88 d (7.8)
4.34 m
4.42 dd (9.0, 9.0)
4.37 m
3.65 s
4.88 d (7.9)
4.05 dd (8.4, 7.9)
4.44 dd (8.9, 8.4)
4.41 dd (9.4, 8.9)
10
20
30
40

Molecules 2019, 24, 2206
15 of 23
Table 8. Cont.
21
22
23
No.
50
δH (500 MHz)
δC (126 MHz)
78.4
δH (500 MHz)
4.56 d (9.4)
δC (126 MHz)
77.2
δH (500 MHz)
4.55 d (9.4)
δC (126 MHz)
3.97 m
4.57 m
4.37 m
77.2
60
63.0
170.8
170.3
60-OMe,
OEt
100
3.77 s
52.2
4.29 m
1.19 t (7.1)
6.33 d (1.2)
4.77 dd (3.4, 1.2)
4.56 dd (9.2, 3.4)
4.36 dd (9.2, 9.2)
5.08 m
61.4
14.3
102.8
72.6
72.7
74.1
69.8
18.7
170.3
23.8
6.30 br s
4.75 m
4.55 m
4.34 m
5.05 m
102.9
72.5
72.7
74.1
69.9
18.6
200
2.15 s
300
400
500
600
1.69 d (6.1)
1.69 d (6.2)
NH
8.94 d (9.0)
a
Disappeared.
Table 9. 1H- and 13C-NMR data of 24–26 in Pyridine-d5 (δ in ppm, J in Hz).
24
25
26
No.
1
δH (500 MHz)
δC (126 MHz)
38.8
δH (500 MHz)
δC (126 MHz)
38.7
δH (500 MHz)
δC (126 MHz)
38.6
1.51 m
0.97 m
1.47 m
0.93 m
1.39 m
0.85 m
2.16 m
1.87 m
2.09 m
1.81 m
2.13 m
1.83 m
2
26.8
26.7
26.6
3
4
5
3.35 dd (11.7, 4.4)
88.7
39.6
55.9
3.28 overlapped
88.8
39.5
55.8
3.36 dd (11.6, 4.3)
89.2
39.6
55.8
0.82 overlapped
1.49 m
0.79 overlapped
1.47 m
0.79 overlapped
1.47 m
6
18.5
18.5
18.5
1.28 m
1.27 m
1.26 m
1.47 m
1.28 m
1.46 m
1.27 m
1.45 m
1.27 m
7
33.2
33.2
33.2
8
9
39.7
48.1
42.0
48.0
39.7
48.0
1.67 dd (9.0, 8.7)
1.65 m
1.63 dd (9.0, 8.6)
10
11
12
13
14
37.1
23.8
123.2
144.5
42.1
37.0
23.7
123.1
144.4
39.7
37.0
23.7
123.1
144.5
42.1
1.89 m
5.60 br t (3.1)
1.88 m
5.60 dd (3.4, 3.4)
1.87 m
5.60 br t (3.3)
2.13 m
1.19 m
2.13 m
2.03 m
2.13 m
1.27 m
2.12 m
2.02 m
2.13 m
1.20 m
2.12 m
2.03 m
15
28.4
28.4
28.4
16
23.9
23.8
23.9
17
18
46.2
43.4
46.2
43.4
46.2
43.4
3.30 dd (13.6, 3.6)
3.29 overlapped
2.26 br d (13.5)
1.82 dd (13.5,
13.5)
3.29 dd (13.6, 3.9)
2.26 m
1.82 dd (13.6, 13.6)
2.26 m
1.82 m
19
42.7
42.7
42.7
20
21
44.2
30.9
44.2
30.8
44.2
30.9
2.19 m
1.46 m
2.10 m
1.98 m
1.30 s
0.97 s
0.82 s
0.97 s
1.30 s
2.19 br d (13.1)
1.46 m
2.09 m
1.98 m
1.24 s
2.20 m
1.46 m
2.09 m
1.98 m
1.30 s
0.95 s
0.78 s
0.95 s
1.30 s
22
34.6
34.5
34.6
23
24
25
26
27
28
29
30
30-OMe
28.2
17.0
15.5
17.4
26.2
179.9
28.5
177.2
51.8
28.1
16.9
15.5
17.3
26.1
179.9
28.5
177.2
51.7
28.2
17.0
15.5
17.4
26.2
179.9
28.5
177.2
51.8
0.92 s
0.80 s
0.96 s
1.30 s
1.23 s
3.65 s
1.23 s
3.64 s
1.23 s
3.65 s

Molecules 2019, 24, 2206
16 of 23
Table 9. Cont.
24
25
26
No.
δH (500 MHz)
δC (126 MHz)
δH (500 MHz)
δC (126 MHz)
δH (500 MHz)
δC (126 MHz)
10
20
30
40
4.83 d (7.6)
4.01 dd (8.7, 7.6)
4.17 dd (8.7, 8.7)
4.23 m
107.8
75.6
78.7
71.3
4.73 d (7.5)
4.00 dd (8.8, 7.5)
4.29 dd (8.8, 8.8)
4.13 m
107.4
75.4
82.9
69.7
5.00 d (7.8)
107.3
75.4
77.9
73.3
4.09 dd (9.0, 7.8)
4.28 dd (9.0, 9.0)
4.48 dd (9.0, 9.7)
4.34 m
3.73 dd (10.6,
10.2)
4.38 dd (11.2, 5.1)
3.78 dd (11.2, 10.2)
50
67.2
66.9
4.61 d (9.7)
3.73 s
77.3
60
60-OMe
100
170.9
52.1
6.27 d (1.2)
4.79 dd (3.3, 1.2)
4.60 dd (9.3, 3.3)
4.34 dd (9.3, 9.3)
4.98 m
102.7
72.6
72.7
74.1
69.9
18.7
200
300
400
500
600
1.67 d (6.2)
The NMR data of compound 26 in methanol-d4 (Supplementary Materials) were the same
as those of coryternic acid 3-O- -O-(6-O-methyl-
-d-glucuronopyranoside-60-O-methyl ester [3
-d-glucuronopyranosyl)-olean-12-ene-28,29-dioic acid 29-methyl ester] [15]. Based on the 1D and
2D-NMR spectra of 26 both in methanol-d4 and pyridine-d5 (Table 9, Figure 2, and Supplementary
Materials), especially on the ROSEY correlations of H3-29/H-19 and H-19 /H3-27, the structure of
26 was determined to be 3 -O-(6-O-methyl- -d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid
30-methyl ester. Therefore, the structure of coryternic acid 3-O-
-d-glucuronopyranoside-60-O-methyl
-O-(6-O-methyl- -d-glucuronopyranosyl)-
β
β
β
α
α
β
β
β
ester reported in the literature is suggested to be revised to 3
olean-12-ene-28,30-dioic acid 30-methyl ester.
β
β
Other known compounds, 30-O-methyl spergulagenate (27) [12], 28-
-hydroxyolean-12-en-28,30-dioate (28) [16], oleanolic acid 3-O-60-O-methyl-
29) [17], oppositifolone (30) [18], spergulagenin A 3-O- -d-xylopyranoside (31) [13], spergulin A
32) [13], spergulacin A (33) [13], spergulacin (34) [13], spergulin B (35) [13], grasshopper ketone (36) [19],
and -carboline (37) [20], were determined by comparing their NMR data (for all compounds) and
β-d-glucopyranosyl-30-methyl
3β
β-d-glucuronopyranoside
(
β
(
β
optical rotation values (for all compounds except 37) with those reported in the literature.
2.2. Biological Evaluation
We proposed antifungal activities of G. oppositifolius according to traditional healthcare use.
The 70% ethanol extract of the whole plants of G. oppositifolius showed inhibitory activity against
M. gypseum with an inhibition of 23.0
compounds ( 37) were measured for antifungal activities against M. gypseum and T. rubrum, and the
results are presented in Table 10. Glinusopposide B ( ), glinusopposide Q (21), glinusopposide T
24), and glinusopposide U (25) showed the most notable inhibitory activities against M. gypseum
±
1.9% at a concentration of 128 µg/mL. All the isolated
1
–
6
(
(MIC50 7.1, 6.7, 6.8, and 11.1
µ
M, respectively) and T. rubrum (MIC50 14.3, 13.4, 11.9, and 13.0
M against
M against T. rubrum). Glinusopposide K (15), glinusopposide N (18),
glinusopposide O (19), glinusopposide R (22), glinusopposide S (23), glinusopposide U (25),
and 3 -O-(6-O-methyl- -d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid 30-methyl ester (26
showed moderate inhibitory activities against M. gypseum, with MIC50 values ranging from 22.0 to
46.8 M. Additionally, 3 ,12 ,16 ,21 ,22-pentahydroxyhopane ( ), 3-oxo-olean-12-ene-28,30-dioic
acid ( ), glinusopposide H (12), and glinusopposide L (16) showed weak activities against M. gypseum,
µM,
respectively) compared with the positive control terbinafine hydrochloride (MIC50 0.008
M. gypseum and 1.647
µ
µ
β
β
)
µ
β
β
β
β
1
3
with MIC50 values ranging from 105.0 to 260.1 µM. Other compounds did not display activity against
M. gypseum or T. rubrum (MIC50 > 300 µM).
The active compounds of G. oppositifolius against M. gypseum and T. rubrum have two types of
carbon skeletons, hopane and oleanane. For those oleanane-type compounds, glycosylation (21 26) or
–

Molecules 2019, 24, 2206
17 of 23
oxidation (
3) of 3-OH was helpful in increasing the activity based on a comparison of the MIC50 values
of and 21
3
–
26 with those of 27 and 28. Replacement of the 30-methyl group (29) with a carboxymethyl
group (26) enhanced the activity. The presence of 11,13(18) double bonds (20) decreased the activity.
The structure-activity relationships (SARs) of the hopane-type compounds against the two fungi were
not clear.
Table 10. Antifungal Effects of Compounds from G. oppositifolius against Microsporum gypseum and
Trichophyton rubrum a.
MIC50 (µM) ± SD
Compound
M. gypseum
T. rubrum
1
3
6
12
15
16
18
19
21
22
23
24
25
26
105.0 ± 0.6
128.1 ± 1.4
7.1 ± 1.2
>300
>300
14.3 ± 2.1
260.1 ± 2.3
46.8 ± 0.1
120.7 ± 1.4
29.3 ± 3.4
34.9 ± 1.2
6.7 ± 2.1
>300
>300
>300
>300
>300
13.4 ± 1.1
40.3 ± 0.5
39.9 ± 1.2
6.8 ± 3.2
>300
>300
11.9 ± 0.3
13.0 ± 1.3
>300
1.647 ± 0.101
11.1 ± 2.4
22.0 ± 0.9
0.008 ± 0.373
terbinafine hydrochloride (positive control)
a
Compounds 2, 4–7, 9–11, 13, 14, 17, 20, and 27–37 were inactive (MIC50 > 300 µM).
3. Experimental Section
3.1. General Experimental Procedures
This part can be found in the Supplementary Materials.
3.2. Plant Material
Whole plants of G. oppositifolius were bought from Zay cho market of Mandalay in Myanmar,
in December 2015. The plants were identified by author, Jun Yang. A voucher specimen (No.
MD1612078) was deposited at the Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute
of Botany, Chinese Academy of Sciences.
3.3. Extraction and Isolation
Powdered whole plants of G. oppositifolius (3.0 kg) were extracted with 70% EtOH at 60 ◦C for
six times (each for 4 h) to obtain a crude extract (650.1 g), which was suspended in H2O and then
extracted with petroleum ether. The water-soluble phase was adjusted to pH 1
then partitioned with EtOAc to afford the EtOAc-soluble extract (B, 130.0 g). The aqueous phase was
basified with 5% NaOH solution to pH 9 10 and then extracted with CHCl3 to yield the CHCl3-soluble
−
2 with 1% HCl and
−
extract (A, 25.2 g). The aqueous phase was extracted with n-butanol to yield the n-butanol-soluble
extract (C, 142.0 g).
The CHCl3 extract (A, 25.2 g) was subjected to silica gel column chromatography (CC,
CH2Cl2-MeOH, 50:1
subjected to reversed phase (RP-C18) silica gel CC eluted with MeOH-H2O (30%
MeOH-eluted part (86.3 mg) was separated on a Sephadex LH-20 CC (MeOH) and purified by
→
0:1, v/v) to yield four main fractions A1–A4. Fraction A1 (931.1 mg) was
→100%). The 30%
semipreparative HPLC (Welch Ultimate AQ-C18, MeOH-H2O, 18:82, 0.8 mL/min) to yield 36 (6.0 mg,

Molecules 2019, 24, 2206
18 of 23
tR = 56.431 min). The 60% MeOH-eluted part (399.8 mg) was separated on a Sephadex LH-20 CC
(MeOH) to yield (4.9 mg), (4.7 mg), and 31 (24.6 mg) recrystallized from MeOH, as well as 37 (0.4 mg)
1
2
recrystallized from CH2Cl2. The 80% MeOH-eluted part (127.3 mg) was purified by Sephadex LH-20
CC (CH2Cl2-MeOH, 1:1) and semipreparative HPLC (Agilent Zorbax SB-C18, MeOH-H2O (containing
0.05% TFA), 67:33, 2 mL/min) to obtain 11 (1.1 mg, tR = 36.803 min) and
Fraction A2 (2.1 g) was separated on an RP-18 silica gel CC eluted with MeOH-H2O (30%
The 70% MeOH-eluted part (96.8 mg) was purified by silica gel CC (CH2Cl2-MeOH-H2O, 300:10:1) to
yield (24.3 mg) recrystallized from MeOH. Fraction A3 (2.5 g) was separated on an RP-18 silica gel CC
eluted with MeOH-H2O (20% 100%). The 60% MeOH-eluted part (965.5 mg) was purified by silica
9
(5.7 mg, tR = 43.850 min).
→100%).
8
→
gel CC (EtOAc-MeOH, 20:1) to yield two main subfractions (A3-1 and A3-2). The subfraction A3-1
(91.8 mg) was purified by silica gel CC (CH2Cl2-MeOH, 10:1) and semipreparative HPLC (Agilent
Zorbax SB-C18, CH3CN-H2O, 35:65, 2 mL/min) to yield 17 (1.0 mg, tR = 28.240 min). The subfraction
A3-2 (192.0 mg) was purified by silica gel CC (CH2Cl2-MeOH-H2O, 80:10:1) to yield two further
subfractions (A3-2-1 and A3-2-2). The subfraction A3-2-1 (33.1 mg) was purified by semipreparative
HPLC (Agilent Zorbax SB-C18, MeCN-H2O, 30:70, 2 mL/min) to yield 34 (16.6 mg, tR = 31.175 min)
and 16 (7.6 mg, tR = 45.162 min). The subfraction A3-2-2 (7.0 mg) was purified by semipreparative
HPLC (Welch Ultimate AQ-C18, MeCN-H2O, 30:70, 1 mL/min) to yield 35 (1.4 mg, tR = 7.176 min).
Fraction A4 (3.0 g) was separated on an RP-C18 silica gel CC eluted with MeOH-H2O (20% 100%)
→
to yield two further subfractions. The 40% MeOH-eluted part (217.9 mg) was purified by silica gel
CC (CH2Cl2-MeOH-H2O, 70:10:1) to yield 32 (38.4 mg). The 50% MeOH-eluted part (494.4 mg) was
recrystallized from MeOH to yield 33 (290.3 mg).
The part of EtOAc extract (B, 27.0 g) was separated on an RP-18 silica gel CC eluted with
MeOH-H2O (5%
was purified by silica gel CC (CH2Cl2-MeOH, 50:1
part (B2, 545.7 mg) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) to yield 12 (5.5 mg). The 70%
MeOH-eluted part (B3, 2.5 g) was purified by silica gel CC (CH2Cl2-MeOH, 30:1 20:1) to afford
→
100%) to yield five main fractions (B1–B5). The 50% MeOH-eluted part (B1, 2.7 g)
→30:1) to afford 30 (26.8 mg). The 60% MeOH-eluted
→
28 (11.1 mg) and three main subfractions (B3-1–B3-3). Subfraction B3-1 (29.0 mg) was purified by
semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 47:53, 2 mL/min)
and further by semipreparative HPLC (Agilent Zorbax SB-C18, MeOH-H2O (containing 0.05% TFA),
75:25, 2 mL/min) to yield 13 (3.9 mg, tR = 30.891 min) and
(91.7 mg) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) and semipreparative HPLC (Agilent Zobrax
SB-C18, MeCN-H2O (containing 0.05% TFA), 50:50, 2 mL/min) to yield (5.3 mg, tR = 14.414 min) and
5 (2.1 mg, tR = 41.804 min). Subfraction B3-2
7
15 (5.3 mg, tR = 16.844 min). Subfraction B3-3 (459.4 mg) was purified by silica gel CC (CH2Cl2-MeOH,
30:1) and semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O, 45:55, 2 mL/min) to yield 21
(5.7 mg, tR = 12.996 min),
6 (5.9 mg, tR = 16.490 min), and 14 (3.6 mg, tR = 17.736 min). The 80%
MeOH-eluted part (B4, 1.1 g) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) to yield two main
subfractions (B4-1 and B4-2). Subfraction B4-1 (97.3 mg) was purified by semipreparative HPLC
(Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 45:55, 2 mL/min) to afford 24 (18.3 mg,
tR = 42.714 min), 26 (29.0 mg, tR = 51.174 min), and 10 (1.6 mg, tR = 62.511 min). Subfraction B4-2
(153.1 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing
0.05% TFA), 43:57, 2 mL/min) to yield 25 (12.4 mg, tR = 37.244 min), 22 (42.6 mg, tR = 45.406 min),
23 (16.2 mg, tR = 66.231 min), and a mixture. The mixture was purified by semipreparative HPLC
(Agilent Eclipse XDB-C18, MeCN-H2O (containing 0.05% TFA), 40:60, 1 mL/min) to yield 20 (3.5 mg,
tR = 18.741 min). The 90% MeOH-eluted part (B5, 1.1 g) was separated on a Sephadex LH-20 CC
(MeOH) to yield two main subfractions (B5-1 and B5-2). Subfraction B5-1 (535.3 mg) was purified by
silica gel CC (CH2Cl2-MeOH, 30:1) and semipreparative HPLC (Agilent Zorbax SB-C18, MeCN/H2O
(containing 0.05% TFA), 49:51, 2 mL/min) to yield 19 (3.9 mg, tR = 43.926 min). Subfraction B5-2
(244.5 mg) was purified by silica gel CC (petroleum ether-EtOAc, 2:1
subfractions (B5-2-1 and B5-2-2). Subfraction B5-2-1 (52.9 mg) was purified by semipreparative HPLC
(Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 60:40, 2 mL/min) to yield (7.6 mg,
→
1:1) to yield two further
3

Molecules 2019, 24, 2206
19 of 23
tR = 35.867 min),
4
(3.5 mg, tR = 47.842 min), and 27 (21.0 mg, tR = 53.439 min). Subfraction B5-2-2
(20.2 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing
0.05% TFA), 70:30, 2 mL/min) to yield 29 (0.7 mg, tR = 15.194 min) and 18 (3.1 mg, tR =16.328 min).
3.4. Spectroscopic and Physical Data
3
β
,12
β
,16
β
,21
β
,22-Pentahydroxyhopane (
1
). Colorless blocks (Me-H2O, 10:1); [α]D25
∆ε) 240 (+0.35), 226 (
−
18 (c 0.05,
0.36),
MeOH); UV (MeOH) λmax (log
198 (+1.15) nm; 1H and 13C-NMR data, see Table 1; ESIMS m/z 515 [M + Na]+; HRESIMS m/z 515.3718
[M + Na]+ (calcd for C30H52NaO5, 515.3712).
ε
) 203 (3.34) nm; ECD (c 0.05, MeOH) λmax
(
−
Crystal data for
1
: C30H52O5, M = 524.75, a = 7.8358(3) Å, b = 17.7389(6) Å, c = 20.4841(7) Å, α = 90◦
,
,
β = 90◦ = 90◦, V = 2847.26(17) Å3, T = 100.(2) K, space group P212121, Z = 4, µ(Cu Kα) = 0.653 mm−1
,
γ
51,068 reflections measured, 5642 independent reflections (Rint = 0.0514). The final R1 values were
0.0363 (I > 2 (I)). The final R1 values were 0.0371
(I)). The final wR(F2) values were 0.1081 (I > 2
σ
σ
(all data). The final wR(F2) values were 0.1102 (all data). The goodness of fit on F2 was 0.998.
Flack parameter = 0.02(5). The supplementary crystallographic data can be obtained free of charge
from the Cambridge Crystallographic Data Centre (CCDC) (deposition number CCDC 1917520) via
http://www.ccdc.cam.ac.uk.
12
β,16
β,21
β,22-Tetrahydroxyhopan-3-one (2
). White powder; [α]2D5 +2 (c 0.12, MeOH); UV (MeOH)
λmax (log
ε
) 280 (1.98), 219 (2.73), 203 (2.91) nm; ECD (c 0.12, MeOH) λmax
(
∆ε) 289 nm (+0.17), 232
(+0.28), 208 (–0.29) nm; IR νmax (KBr) 3443, 3427, 1690, 1452, 1385, 1084, 1047, 879 cm−1; H and
13C-NMR data, see Table 1; ESIMS m/z 513 [M + Na]+; HRESIMS m/z 513.3551 [M + Na]+ (calcd for
C30H50NaO5, 513.3556).
1
The 3-Oxo-olean-12-ene-28,30-dioic acid (
3
). White powder; [α]2D5 +59 (c 0.26, MeOH); UV (MeOH)
1
λmax (log
ε
) 371 (1.73), 252 (2.75), 239 (2.72) nm; H and 13C-NMR data, see Table 2; ESIMS m/z 507
[M + Na]+; HRESIMS m/z 507.3084 [M + Na]+ (calcd for C30H44NaO5, 507.3086).
The 3 -Hydroxyoleana-11,13(18)-diene-28,29-dioic acid 29-methyl ester (
). White powder; [α]2D6–7
∆ε) 250
β
4
(c 0.15, MeOH); UV (MeOH) λmax (log
ε
) 250 (2.65), 242 (2.59) nm; ECD (c 0.09, MeOH) λmax
(
(–3.92) nm; H and 13C-NMR data, see Table 2; ESIMS m/z 521 [M + Na]+; HRESIMS m/z 521.3234
[M + Na]+ (calcd for C31H46NaO5, 521.3237).
1
Glinusopposide A (
ESIMS m/z 613 [M + Na]+; HRESIMS: m/z 613.4068 [M + Na]+ (calcd for C35H58NaO7, 613.4080).
5
). White powder; [α]2D6 –8 (c 0.05, MeOH); 1H and 13C-NMR data, see Table 3;
Glinusopposide B (
). White powder; [α]2D5 –20 (c 0.1, MeOH); 1H and 13C-NMR data, see Table 3;
ESIMS m/z 759 [M + Na]+; HRESIMS m/z 759.4650 [M + Na]+ (calcd for C41H68NaO11, 759.4659).
Glinusopposide C (
). White powder; [α]2D6 –6 (c 0.35, MeOH); 1H and 13C-NMR data, see Table 3;
ESIMS m/z 759 [M + Na]+; HRESIMS m/z 759.4656 [M + Na]+ (calcd for C41H68NaO11, 759.4659).
Glinusopposide D ( ) 275 (2.77),
). White powder; [α]2D1 –36 (c 0.16, pyridine); UV (MeOH) λmax (log
245 (3.02), 204 (3.59) nm; ECD (c 0.099, MeOH) λmax ∆ε) 284 (+1.00), 249 (–0.17), 218 (+1.03), 197
6
7
8
ε
(
(–1.11) nm; 1H and 13C-NMR data, see Table 4; ESIMS m/z 833 [M + K]+, 817 [M + Na]+; HRESIMS m/z
817.4709 [M + Na]+ (calcd for C43H70NaO13, 817.4714).
Glinusopposide E (9 ε) 415 (2.39), 206
). White powder; [α]D20 –32 (c 0.09, MeOH); UV (MeOH) λmax (log
(4.09) nm; ECD (c 0.065, MeOH) λmax (∆ε) 284 (+0.48), 206 (+0.73), 200 (–1.80) nm; IR νmax (KBr) 3442,
3428, 1677, 1644, 1449, 1431, 1385, 1202, 1144, 1086, 1047, 879 cm−1; 1H and 13C-NMR data, see Table 4;
ESIMS m/z 843 [M + Na]+; HRESIMS m/z 843.4867 [M + Na]+ (calcd for C45H72NaO13, 843.4871).
Glinusopposide F (10). White solid; [α]D25 –31 (c 0.04, MeOH); H and 13C-NMR data, see Table 4;
1
ESIMS m/z 885 [M + Na]+; HRESIMS m/z 885.4930 [M + Na]+ (calcd for C47H74NaO14, 885.4976).

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Glinusopposide G (11). White powder; [α]2D0 –7 (c 0.15, MeOH); UV (MeOH) λmax (log
ε) 252 (3.21),
206 (3.81) nm; ECD (c 0.078, MeOH) λmax (∆ε) 201 (–1.59), 197 (+1.29) nm; IR νmax (KBr) 3448, 3427,
1639, 1447, 1383, 1084, 1046, 879 cm−1; 1H and 13C-NMR data, see Table 4; ESIMS m/z 829 [M + Na]+;
HRESIMS m/z 829.4711 [M + Na]+ (calcd for C44H70NaO13, 829.4714).
Glinusopposide H (12). White powder; [α]2D5 +7 (c 0.28, MeOH); 1H and 13C-NMR data, see Table 5;
ESIMS m/z 641 [M + Na]+; HRESIMS m/z 641.4021 [M + Na]+ (calcd for C36H58NaO8, 641.4024).
Glinusopposide I (13). White powder; [α]2D5 –10 (c 0.07, MeOH); 1H and 13C-NMR data, see Table 5;
ESIMS m/z 611 [M + Na]+; HRESIMS m/z 611.3898 [M + Na]+ (calcd for C35H56NaO7, 611.3924).
Glinusopposide J (14). White powder; [α]2D5 –22 (c 0.11, MeOH); 1H and 13C-NMR data, see Table 5;
ESIMS: m/z 757 [M + Na]+; HRESIMS m/z 757.4486 [M + Na]+ (calcd for C41H66NaO11, 757.4503).
Glinusopposide K (15). White powder; [α]2D5 –16 (c 0.13, MeOH); 1H and 13C-NMR data, see Table 6;
ESIMS m/z 757 [M + Na]+; HRESIMS m/z 757.4510 [M+ Na]+ (calcd for C41H66NaO11, 757.4503).
Glinusopposide L (16). White powder; [α]2D5 –25 (c 0.1, MeOH); UV (MeOH) λmax (log
ε) 203 (3.82)
nm; ECD (c 0.076, MeOH) λmax (∆ε) 196 (–6.89) nm; IR νmax (KBr) 3425, 1632, 1454, 1385, 1129, 1094,
1047, 974 cm−1; 1H and 13C-NMR data, see Table 6; ESIMS m/z 731 [M + Na]+; HRESIMS m/z 731.4353
[M + Na]+ (calcd for C39H64NaO11, 731.4346).
Glinusopposide M (17). White powder; [α]2D0 –25 (c 0.05, MeOH); UV (MeOH) λmax (log
ε) 203 (3.54) nm;
ECD (c 0.05, MeOH) λmax (∆ε) 206 (–4.38), 196 (+4.32) nm; IR νmax (KBr) 3443, 3426, 1639, 1453, 1421,
1384, 1084, 1047, 879 cm−1; 1H and 13C-NMR data, see Table 6; ESIMS m/z 731 [M + Na]+; HRESIMS
m/z 731.4347 [M + Na]+ (calcd for C39H64NaO11, 731.4346).
Glinusopposide N (18). White powder; [α]2D6 –7 (c 0.18, MeOH); UV (MeOH) λmax (log
ε) 250 (3.76),
213 (3.38) nm; 1H and 13C-NMR data, see Table 7; ESIMS m/z 567 [M + Na]+; HRESIMS m/z 567.3655
[M + Na]+ (calcd for C33H52NaO6, 567.3656).
Glinusopposide O (19). White powder; [α]2D5 –14 (c 0.13, MeOH); UV (MeOH) λmax (log
ε) 250 (3.76),
218 (3.43) nm; 1H and 13C-NMR data, see Table 7; ESIMS m/z 729 [M + K]+, 713 [M + Na]+; HRESIMS
m/z 713.4200 [M + Na]+ (calcd for C39H62NaO10, 713.4241).
Glinusopposide P (20). White powder; [α]2D5 –39 (c 0.1, MeOH); UV (MeOH) λmax (log
ε) 250 (4.07),
217 (3.68) nm; 1H and 13C-NMR data, see Table 7; ESIMS m/z 857 [M + Na]+; HRESIMS m/z 857.4300
[M + Na]+ (calcd for C44H66NaO15, 857.4299).
Glinusopposide Q (21). White powder; [α]2D5 +40 (c 0.13, MeOH); 1H and 13C-NMR data, see Table 8;
ESIMS m/z 726 [M + Na]+; HRESIMS m/z 726.4201 [M + Na]+ (calcd for C39H61NNaO10, 726.4193).
Glinusopposide R (22). White powder; [α]2D5 +10 (c 0.15, MeOH); ECD (c 0.078, MeOH) λmax (∆ε) 221
1
(–1.44) nm; H and 13C-NMR data, see Table 8; ESIMS m/z 859 [M + Na]+; HRESIMS m/z 859.4452
[M + Na]+ (calcd for C44H68NaO15, 859.4450).
Glinusopposide S (23). White powder; [α]2D5 +8 (c 0.1, MeOH); 1H and 13C-NMR data, see Table 8;
ESIMS m/z 873 [M + Na]+; HRESIMS m/z 873.4606 [M + Na]+ (calcd for C45H70NaO15, 873.4607).
Glinusopposide T (24). White powder; [α]2D4 +71 (c 0.22, MeOH); 1H and 13C-NMR data, see Table 9;
ESIMS m/z 655 [M + Na]+; HRESIMS m/z 655.3820 [M + Na]+ (calcd for C36H56NaO9, 655.3822).
Glinusopposide U (25). White powder; [α]2D5 +11 (c 0.1, MeOH); 1H and 13C-NMR data, see Table 9;
ESIMS m/z 801 [M + Na]+; HRESIMS m/z 801.4393 [M + Na]+ (calcd for C42H66NaO13, 801.4396).
The 3
β
-O-(6-O-Methyl-
β
-d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid 30-methyl ester
26). White powder; [α]2D5 +66 (c 0.12, MeOH); H and 13C-NMR data, see Table 9; ESIMS m/z 713
1
(
[M + Na]+; HRESIMS m/z 713.3871 [M + Na]+ (calcd for C38H58NaO11, 713.3877).

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3.5. Acid Hydrolysis and Sugar Analysis
3.5.1. Acid Hydrolysis of 31 and Acetylation of Xylose
◦
Compound 31 (15.5 mg) was dissolved in 2 M HCl (1 mL) and stirred at 90 C for 4 h. After
cooling, the solution was evaporated to dryness under reduced pressure. The reaction mixture was
purified by silica gel column chromatography (CH2Cl2-MeOH-H2O, 500:10:1, 300:10:1, 200:10:1) to
afford xylose (1.9 mg). The sugar was dissolved in pyridine (0.1 mL) and acetic anhydride (0.1 mL)
and stirred for 21 h at room temperature. Then, water (5 mL) was added to the reaction mixture,
followed by extraction with EtOAc (5 mL). The organic layer was dried under reduced pressure to yield
1,2,3,4-tetra-O-acetyl-d-xylopyranose (0.9 mg), which was identified based on its 1H-NMR spectrum
and optical rotation value: [α]2D1 −31 (c 0.08, CHCl3) [21].
3.5.2. Acid Hydrolysis of the Saponin Mixture and Acetylation of Rhamnose
The n-butanol-soluble part (20.0 g) was subjected to D101 resin column chromatography, eluted
using water (discarded) and 60% EtOH to yield the saponin mixture (4.0 g). The latter (1.0 g) was
dissolved in 2 M HCl (3 mL) and stirred at 90 ◦C for 5 h. The reaction mixture was dried and purified by
silica gel column chromatography (CH2Cl2-MeOH-H2O, 500:10:1, 200:10:1, 100:10:1) to yield rhamnose
(97.9 mg) and glucose (9.4 mg). The glucose was identified as d-glucose based on its 1H-NMR spectrum
and optical rotation value: [α]1D9 +40 (c 0.22, H2O) [22]. The rhamnose (97.9 mg) was dissolved in
pyridine (0.1 mL) and acetic anhydride (0.1 mL) and stirred for 21 h at room temperature. Then, water
(5 mL) was added to the reaction mixture, followed by extraction with EtOAc (5 mL). The organic
layer was dried under reduced pressure and purified by silica gel column chromatography (petroleum
ether-EtOAc, 50:1) to yield 1,2,3,4-tetra-O-acetyl-α-l-rhamnopyranose (1.4 mg), which was identified
based on its 1H-NMR spectrum and optical rotation value: [α]D21 −27 (c 0.14, CHCl3) [23].
3.6. Antimicrobial Assays
The fungi strains T. rubrum ATCC 4438 and M. gypseum CBS118893) were purchased from the
Institute of Dermatology and Hospital for Skin Diseases, Chinese Academy of Medical Sciences.
An antifungal assay was performed according to modified versions of the clinical and laboratory
standards institute (CLSI), formerly national committee for clinical laboratory standards (NCCLS)
methods, as described previously [24,25]. Terbinafine hydrochloride was used as a positive control.
The 50% minimum inhibitory concentration (MIC50) was calculated by the Reed-Muench method [26].
4. Conclusions
In this study, four new triterpenoids (1−4), 21 new triterpenoids glycosides (5−25), and 12
known compounds were isolated from G. oppositifolius. However, we cannot exclude the possibility
that some of the isolated compounds might be artifacts resulted from the extraction treatment, for
example compound 23 might be artifacts of ethanol extraction. The triterpenoids and their glycosides
were hopane-type and oleanane-type which have been proofed to be existing in this plant [
9,13].
Four compounds including glinusopposide B ( ), glinusopposide Q (21), glinusopposide T (24),
6
and glinusopposide U (25) showed considerable inhibitory activities against M. gypseum and T. rubrum.
According to the study of SARs, sugars at 3-hydroxy, 30-carboxymethyl group, and the double bond
at C-12 play a key role in oleanane type compounds for antifungal activities. The SARs of hopane
type compounds for antifungal activities remain for further research. This study provides a scientific
evidence of traditional practice on applying G. oppositifolius to treat dermatophytosis.
Supplementary Materials: The following are available online. General experimental procedures; Figures S1–S198:
1D and 2D-NMR and HRESIMS spectra of compounds 1–26, structures of known compounds (26–37), and key
2D-NMR correlations of 2, 6–11, and 13–25.

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Author Contributions: Y.W. and X.Y. designed the research; D.Z. performed the research; J.Y. identified the plant
of study; M.M.S. and T.N.O. provided the material and traditional knowledge; D.Z. and Y.W. analyzed the data
and wrote the paper. Y.W., X.Y., and Y.F. revised the manuscript. X.-N.L. provided the data of single-crystal X-ray
diffraction. All authors have read and approved the final manuscript.
Funding: This study was supported by the Southeast Asia Biodiversity Research Institute, Chinese
Academy of Sciences (no. 2015CASEABRIRG001 and Y4ZK111B01), the National Natural Science Foundation
of China (no. 31670338), and the International Partnership Program of Chinese Academy of Sciences
(no. 153631KYSB20160004).
Acknowledgments: We thank to Xue Bai, at Kunming Institute of Botany, Chinese Academy of Sciences, for the
antifungal assay.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8, 31, 33, and 34 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).