Aspidsaponins E-H, Four new steroidal saponins from the rhizomes of Aspidistra elatior Blume and their anti-inflammatory activity

Article abstract of DOI:10.1016/j.phytol.2019.09.014

Four new furostanol saponins, named aspidsaponins E-H (1–4) were isolated from the rhizomes of Aspidistra elatior Blume. Their structures were determined based on chemical methods and spectral data. Compounds 1 and 2 were a pair of diastereoisomeric furostanol saponins and possessed the highly oxidized structural feature of 1β, 2β, 3β, 4β, 5β, 26-hexanol-steroids, and compounds 3-4 were steroidal glycosides with a tetra-saccharide chain at C3. The isolated compounds (1–4) were tested in vitro for inhibitory activities against LPS-induced nitric oxide production in RAW264.7 macrophages. Among them, compounds 3 and 4 showed excellent anti-inflammatory activities with IC₅₀ values 82.1 and 65.9 μM, respectively.

Full text of DOI:10.1016/j.phytol.2019.09.014

Phytochemistry Letters 34 (2019) 68–73
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Aspidsaponins E-H, Four new steroidal saponins from the rhizomes of
Aspidistra elatior Blume and their anti-inflammatory activity
⁎
Zhi-Ying Sun, Sheng-Qi Zuo, Xia Yang, Jia-Hui Lan, Cheng-Xiong Liu , Zhi-Yong Guo, Fan Cheng,
⁎
Kun Zou
Hubei Key Laboratory of Natural Products Research and Development, College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang 443002,
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Four new furostanol saponins, named aspidsaponins E-H (1–4) were isolated from the rhizomes of Aspidistra
elatior Blume. Their structures were determined based on chemical methods and spectral data. Compounds 1 and
Aspidistra elatior blume
Steroidal glycosides
NMR
2
were a pair of diastereoisomeric furostanol saponins and possessed the highly oxidized structural feature of 1β,
2
β, 3β, 4β, 5β, 26-hexanol-steroids, and compounds 3-4 were steroidal glycosides with a tetra-saccharide chain
Structural elucidation
Anti-inflammatory activity
at C3. The isolated compounds (1–4) were tested in vitro for inhibitory activities against LPS-induced nitric oxide
production in RAW264.7 macrophages. Among them, compounds 3 and 4 showed excellent anti-inflammatory
activities with IC50 values 82.1 and 65.9 μM, respectively.
1
. Introduction
Aspidistra elatior Blume belongs to the family Liliaceae
were steroidal glycosides with a tetra-saccharide chain at C3. In this
paper, we reported the isolation and structural elucidations of com-
pounds 1–4, and their the inhibitory activities against lipopoly-
saccharide (LPS)-induced nitric oxide (NO) production in the
RAW264.7 macrophage were also described here.
(
Convallarieae), and it’s folk name is “Zhi Zhu Bao Dan” in China. The
rhizomes of A. elatior Blume as a traditional Chinese medicine are used
to treat fractures, congestion, and snake-bite, and mainly distributed in
southwestern China, Japan and northern Vietnam (Cui et al., 2013). In
recent years, research into its biochemical constituents had mainly fo-
cused on the steroidal compounds (Cui et al., 2016). Steroidal saponins
are characterized by a steroid type skeleton glycosidically linked to
carbohydrate moieties, which possessed a wide range of biological ac-
tivities including antiinflammatory (Adão et al., 2011), antiviral (Gosse
et al., 2002), antifungal (Sautour et al., 2005), platelet aggregation
inhibition (Cai et al., 2001), antihypertensive (Oh et al., 2003), and
cytotoxic (Mosad et al., 2017). In our previous studies, there were more
than 40 new steroidal saponins had been reported the isolation and
identification from the rhizomes of Convallarieae species Tupistra chi-
nensis and Reineckia carnea (Liu et al., 2012; Zheng et al., 2016), and
some of them showed strong antitumor activities, such as RCE-4 (Wang
et al., 2013).
2. Results and discussion
The structures of new compounds 1-4 (Fig. 1) were elucidated by
analysis using HR-ESI-MS, GC, 1D and 2D NMR spectroscopy, including
1
1
DEPT, H- H COSY, HMBC, HSQC, HSQC-TOCSY, and NOESY experi-
ments.
Compound 1 was obtained as a white amorphous powder, which
showed positive reactions in both the Molish and Liebermann–Buchard
tests. Its high resolution mass spectrum HR-ESI-MS showed quasi-mo-
+
lecular ion peak m/z 827.4029 [M + Na] (calcd. for C39
H
64NaO17,
827.4041). On acid hydrolysis, compound 1 liberated D-glucose only,
1
identified by GC analysis of the chiral derivative. H-NMR (in C
5
D N,
5
400 MHz) of 1 showed 4 methyl proton signals at δ 0.71 (s, H-18), 1.74
(s, H-19), 1.62 (s, H-21) and 1.03 (d, J = 6.5 Hz, H-27), two exo-me-
thylene protons at δ 4.31 (br.s, H-1), 4.42 (br.s, H-2), and also showed
two anomeric proton signals at δ 5.33 (d, J = 6.9 Hz, H-1'), 4.85 (d, J =
7.7 Hz, H-1”) (Tables 1 and 2), which indicated that 1 contained two
sugar moieties. The DEPT135 spectra showed 34 carbon signals,
In this paper, four new steroidal saponins(1-4) were obtained from
the rhizomes of Aspidistra elatior Blume. Among them, compounds 1
and 2 were rarely steroidal glycosides of the C5-linked sugar and pos-
sessed highly oxidized structural feature at C1∼C5, and compounds 3-4
⁎
Corresponding author at: Hubei Key Laboratory of Natural Products Research and Development, College of Biological and Pharmaceutical Sciences, China Three
Gorges University, Yichang, 443002, China.
E-mail addresses: liuchengxiong666@126.com (C.-X. Liu), kzou@ctgu.edu.cn (K. Zou).
https://doi.org/10.1016/j.phytol.2019.09.014
Received 6 July 2019; Received in revised form 19 September 2019; Accepted 27 September 2019
1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

Z.-Y. Sun, et al.
Phytochemistry Letters 34 (2019) 68–73
Fig. 1. The structures of 1-4.
Table 1
1
H (400 MHz) and 13C NMR (100 MHz) data of the aglycone moiety of compounds 1–4 in Pyridine-d
5
.
Position
1
2
3
4
δ
H
J (Hz)
δ
C
δ
H
J (Hz)
δ
C
δ
H
J (Hz)
δ
C
δ
H
J (Hz)
δ
C
1
2
4.31 (br. s)
4.42 (br. s)
77.8
68.1
4.32 (br. s)
4.45 (br. s)
77.8
68.1
0.96 (m)
37.4
30.0
0.95 (m)
1.67 (m)
1.69 (m)
2.08 (m)
3.87 (m)
2.43 (d, 10.2)
37.3
30.0
1
.72 (m)
1.86 (m)
2
.08 (m)
3
4
4.05 (overlapped)
4.13 (m)
75.2
67.6
4.09 (overlapped)
4.15 (m)
75.2
67.6
3.86 (m)
2.74 (m)
78.0
38.8
78.0
39.1
2
–
.65 (dd, 13.2, 2.5)
5
6
–
87.4
24.9
–
87.4
24.9
–
140.7
121.7
140.8
121.5
1.97 (m)
1.99 (m)
2.85 (d, 11.0)
1.15 (m)
1.56 (m)
1.63 (overlapped)
1.26 (m)
–
5.29 (d, 3.9)
5.29 (d, 3.9)
2
.84 (d, 11.0)
7
1.12 (m)
28.6
28.5
1.49 (m)
1.87 (m)
1.47 (m)
0.88 (m)
–
32.3
1.48 (m)
1.85 (m)
1.46 (m)
0.87 (m)
–
32.2
1
.52 (m)
8
9
1.61 (overlapped)
1.24 (m)
–
34.4
46.7
46.3
21.9
39.7
34.4
46.6
46.2
21.8
39.6
31.3
50.2
37.0
21.1
39.5
31.2
50.1
36.8
21.1
39.5
1
1
1
0
1
2
1.47 (m)
1.07 (m)
1.47 (m)
1.11 (m)
1.68 (m)
–
1.44 (overlapped)
1.14 (m)
1.72 (m)
–
1.42 (overlapped)
1.14 (m)
1.73 (m)
–
1
–
.64 (m)
1
1
1
3
4
5
43.4
54.3
31.4
43.4
54.2
31.3
43.3
54.8
34.4
43.3
54.8
34.3
0.80 (m)
1.47 (m)
0.80 (m)
1.48 (m)
1.84 (m)
4.82 (m)
2.47 (d, 10.2)
0.71 (s)
1.77 (s)
–
0.85 (m)
1.49 (m)
2.11 (m)
4.80 (m)
2.44 (d, 10.2)
0.71 (s)
1.06 (s)
–
0.85 (m)
1.46 (m)
2.10 (m)
4.78 (m)
2.43 (d, 10.2)
0.71 (s)
0.87 (s)
–
1
.84 (m)
1
1
1
1
2
2
2
2
6
7
8
9
0
1
2
3
4.80 (m)
2.46 (d, 10.2)
0.71 (s)
1.74 (s)
–
84.4
64.5
14.3
13.7
103.5
11.7
152.5
34.3
84.4
64.4
14.3
13.7
103.4
11.7
152.4
34.3
84.4
64.4
14.0
19.3
103.5
11.7
152.3
23.5
84.3
64.3
14.0
19.3
103.5
11.7
152.2
23.5
1.62 (s)
–
1.62 (s)
–
1.63 (s)
–
1.63 (s)
–
1.48 (m)
1.49 (m)
2.09 (m)
2.23 (m)
2.20 (m)
2.21 (m)
2
.07 (m)
2
4
2.21 (m)
23.7
23.6
1.46 (m)
31.3
1.48 (m)
31.3
1
.84 (m)
1.84 (m)
2
2
5
6
1.95 (m)
33.5
75.0
1.97 (m)
33.7
75.1
1.94 (m)
33.6
75.1
1.95 (m)
33.3
74.9
3.63 (dd, 9.2, 5.5)
3.50 (dd, 9.3, 6.9)
4.09 (m)
3.48 (dd, 9.3, 6.8) 4.06 (m)
3.63 (dd, 9.2, 5.5)
3.96 (m)
3
.97 (m)
2
7
1.03 (d, 6.5)
17.3
1.06 (d, 6.5)
17.1
1.03 (d, 6.6)
17.1
1.02 (d, 6.6)
17.2
Assignments were based on DEPT, HSQC, COSY, HMBC, and NOESY experiments.
69

Z.-Y. Sun, et al.
Phytochemistry Letters 34 (2019) 68–73
including 4 methyls, 8 methylenes, 22 methines and 5 quaternary
correlations of H-19 and H-8, H-1 and H-9 supported the B/C trans ring
junction pattern; the NOE correlations of H-9 and H-14, H-8 and H-18/
H-19, which indicated that the H-14 was α configuration, supported the
C/D trans ring junction pattern; the NOE correlations of H-8 and H-16/
H-17 supported the D/E cis ring junction pattern (Fig. 3). In summary,
the structure of 1 was identified as (25R)-26-O-β-D-glucopyranoside-
furost-20(22)-en-1β, 2β, 3β, 4β, 5β, 26-hexahydroxy-5-O-β-D-gluco-
pyranoside (Fig. 1), named aspidsaponins E.
1
3
carbons. The C-NMR (in C
5
5
D N, 100 MHz) of 1 showed a total of 39
carbon signals. In addition to the 12 carbon signals of 2 glucoses (δc
7.4, 76.2, 78.6, 71.8,78.8, 62.9, 104.9, 75.8, 78.5, 71.8, 78.6, 62.8),
there were 27 carbon signals belonged to the aglycone carbons. Among
9
carbon signals of the aglycone, δ
C
14.3, 13.7 11.7 and 17.3 were due to
84.4, 68.1, 75.2, 67.6, 87.4, 84.4, 64.5 and
5.2 were due to 8 oxygenated carbon groups, which indicated that the
4
7
methyl groups, and δ
C
compound 1 was a steroidal saponin with multiple hydroxyl groups.
Compound 2 was obtained as a white amorphous powder, and it was
found by HPLC analysis that the peaks of 2 and 1 were next to each other.
1
3
Comparing the C-NMR data of 1 with the 1β, 2β, 3β, 4β, 5β, 26-
hexahydroxyfurost-20 (22), 25 (27)-dien-5, 26-O-β-D-glucopyranoside
data (Li et al., 2015), it was found that the two carbon signals of the C-
13
Furthermore, the C-NMR data of the two compounds were completely
1
1
identical. However, the H-NMR data were slightly different. H NMR
2
5 (δc 146.2) and C-27 (δc 111.6) double bonds disappeared, and a
spectrum of 2 showed two signals of H-26 at δ 4.09 and 3.50 (Table 1),
ab
methyl carbon signal (δc 17.3) and a methine carbon signal appeared. It
was speculated that the C-27 position became a methyl group, and the
correctness of this speculation was verified by the HMBC spectrum
△
(δH26a-δH26b) = 0.59 > 0.57, which indicated 25S configuration for
2 (Agrawal, 2004, 2005). Obviously, 1 and 2 were a pair of diastereoi-
somers. Therefore, the structure of 2 had been identified as (25S)-26-O-β-
D-glucopyranoside-furost-20(22)-en-1β, 2β, 3β, 4β, 5β, 26-hexahydroxy-5-
O-β-D-glucopyranoside (Fig. 1), named aspidsaponins F.
(
Fig. 2). In addition, the HMBC correlation signals of H-Glc-1′/C-5 and
H-Glc-1′′/C-26, indicated that a glucosyl was attached to each of the C-
3
5
and C-26 (Fig. 2). The large coupling constants ( J1,2 > 7 Hz) showed
Compound 3 was isolated as a white amorphous powder, which
showed positive reactions in the Liebermann-Burchard and Molish tests.
that the configurations of two sugars were all the beta configuration
(
Agrawal, 1992). The 25R configuration was deduced based on the
Its high resolution mass spectrum HR-ESI-MS showed quasi-molecular ion
ab
+
difference of the chemical shifts for the geminal protons at H-26, △
peak m/z 1215.5758 [M + Na] (calcd. for C57
H
92NaO26, 1215.5775).
(
δ
H26a-δH26b) = 0.34 < 0.48, since the difference is usually < 0.48 for
On acid hydrolysis, compound 3 liberated L-rhamnose and D-glucose,
2
5R saponins and > 0.57 for 25S ones (Agrawal, 2004, 2005). In the
identified by GC analysis of their chiral derivatives, and the integral ratio
1
NOESY spectrum of 1, the NOE correlations of Me-19 and H-8, H-4 and
H-2, and H-1 and H-3, indicated α-axial configurations of H-1, H-2, H-3,
and H-4, and β-orientation of Me-19, 1-OH, 2-OH, 3-OH, 4-OH and 5-
OH, which supported the A/B cis ring junction pattern; the NOE
was 2:3. The H-NMR spectrum of 3 showed six methyl groups at δ 0.71 (s,
H-18), 1.06 (s, H-19), 1.63 (s, H-21), 1.03 (d, J = 6.6 Hz, H-27), 1.71 (d,
6.2, H-6”')and 1.76 (d, 6.2, H-6””), and five anomeric proton signals at δ
4.93 (d, J = 7.6 Hz, H-1'), 5.07 (d, J = 7.6 Hz, H-1”), 6.20 (br.s, H-1”'),
Table 2
1
13
H (400 MHz) and C NMR (100 MHz) data of the aglycone moiety of compounds 1-4 in Pyridine-d .
5
a
a
a,b
4a
Position
1
2
3
δ
H
J (Hz)
δ
C
δ
H
J (Hz)
δ
C
δ
H
J (Hz)
δ
C
δ
H
J (Hz)
δ
C
Glc 1'
5.33 (d, 6.9)
4.76 (overlapped)
4.06 (m)
97.4
76.2
78.6
71.8
78.8
62.9
5.35 (d, 7.7)
4.77 (overlapped)
4.09 (m)
97.4
76.2
78.6
71.8
78.8
62.8
Glc 1'
2'
4.93 (d, 7.6)
4.20 (m)
4.46 (m)
4.18 (m)
3.82 (m)
4.52 (m),
4.44 (m)
5.07 (d, 7.6)
4.00 (m)
3.74 (m)
4.15 (m)
4.45 (m)
4.17 (m)
4.06 (m)
6.20 (br.s)
4.72 (m)
4.56(m)
99.8
77.6
77.5
81.6
76.1
61.5
Gal 1'
4.90 (d, 7.6)
4.42(overlapped)
4.04 (m)
102.6
73.0
75.2
79.7
75.0
60.4
2
3
4
5
6
'
'
'
'
'
2'
3'
4'
5'
6'
3'
4.08 (m)
4.10 (m)
4'
4.61 (d, 2.2)
3.96 (m)
4.25 (m)
4.28 (m)
5'
4.26 (m)
4.29 (m)
6'
4.18 (m)
4
.57 (d, 11.1)
4.60 (d, 11.1)
4.87 (d, 7.7)
4.03 (m)
4.67 (t, 9.0)
5.21 (d, 7.7)
4.39(overlapped)
4.17 (m)
Glc 1”
4.85 (d, 7.7)
3.99 (m)
4.26 (m)
4.25 (m)
3.98 (m)
4.25 (m)
104.9
75.8
78.5
71.8
78.6
62.8
105.2
75.8
78.5
71.7
78.6
62.7
Glc 1”
2”
104.9
75.1
77.0
76.3
77.5
60.8
Glc 1”
2”
105.0
81.2
86.5
70.8
77.4
62.8
2
3
4
5
6
”
”
”
”
”
4.28 (m)
3”
3”
4.27 (m)
4”
4”
3.82 (m)
3.99 (m)
5”
5”
3.87 (m)
4.28 (m)
6”
6”
4.05 (m)
4
.57 (m)
4.59 (m)
4.56 (m)
Rha 1”'
101.6
72.4
72.6
73.8
69.4
18.4
Glc 1”'
2”'
5.57 (d, 7.3)
4.09(overlapped)
4.11 (m)
104.7
76.1
77.5
71.5
78.4
62.3
2
3
4
5
6
”'
”'
”'
”'
”'
3”'
4.34 (m)
4.92 (m)
1.71 (d, 6.2)
4”'
4.22 (m)
5”'
3.96 (m)
6”'
4.39 (m)
4
.56 (m)
Rha 1””
5.87 (br.s)
4.66 (m)
4.56 (m)
4.34 (m)
4.98 (m)
102.5
72.5
72.6
74.0
70.3
Xyl 1””
2””
5.23 (d, 7.8)
3.96(overlapped)
4.08 (m)
104.8
75.0
78.5
70.6
78.5
2
3
4
5
””
””
””
””
3””
4””
4.10 (m)
5””
3.97 (m)
4
.25 (overlapped)
6
””
1.76 (d, 6.2)
4.84 (d, 7.6)
4.04 (m)
18.5
105.1
75.1
78.4
71.6
78.5
62.7
Glc 1””'
Glc 1””'
2””'
4.84 (d, 7.8)
4.04(overlapped)
4.26 (m)
104.7
75.0
78.5
71.5
78.5
62.7
2
3
4
5
6
””'
””'
””'
””'
””'
4.25 (m)
3””'
4.24 (m)
4””'
4.25 (m)
3.97 (m)
5””'
4.09 (m)
4.39 (m)
6””'
4.39 (m)
4
.56 (m)
4.56 (m)
a, assignments were based on DEPT, HSQC, COSY, HMBC, and NOESY experiments.
b, assignments were based on HSQC-TOCSY experiment.
70

Z.-Y. Sun, et al.
Phytochemistry Letters 34 (2019) 68–73
Fig. 2. The key HMBC and 1H- H COSY correlations of 1–4.
1
5
.87 (br.s, H-1””), and 4.84 (d, J = 7.6 Hz, H-1””') (Tables 1 and 2), which
aspidsaponins A (Zuo et al., 2018), it was found to be basically the same.
13
13
indicated that 3 contained five sugar moieties. C-NMR (in C
5
D
5
N,
However, the C-NMR data of the sugar fraction showed that two distinct
4
00 MHz) of 3 showed a total of 57 carbon signals. In addition to the 30
methyl groups (δ
C
18.4, 18.5) belonged to 6-methyl groups of two
carbon signals of five sugars, there were 27 carbon signals belonged to the
rhamnoses, which were consistent with GC results. The HMBC correlation
signals of H-Glc-1′/C-3, H-Glc-1′′/C-Glc-4′, H-Rha-1′′′/C-Glc-2′, H-Rha-
aglycone carbons. Comparing the 13C-NMR data of aglycone of 3 with
Fig. 3. The key NOESY correlations of 1 and 3.
71

Z.-Y. Sun, et al.
Phytochemistry Letters 34 (2019) 68–73
1
′′′′/C-Glc-4′′, indicated that a tetra-saccharide chain was attached to C-3
hydroxyl groups could enhance the NO production inhibitory activities.
Meanwhile, comparison of the NO production inhibitory activities and
structures of compounds 3 and 4 suggested that the type of sugar and
led to slight changes in activity.
of aglycone (Fig. 2). In addition, the HMBC correlation signal of H-Glc-
1
′′′′′/C-26, indicated that a glucosyl was attached to C-26 (Fig. 2). The
3
large coupling constants ( J1,2 > 7 Hz) showed that the configurations of
three glucose were all the β configuration, and the single wide peak at H-
Rha-1′′′ and H-Rha-1′′′′ indicated that the configurations of two rhamnoses
were all the α configuration (Agrawal, 1992). Furthermore, NOESY cor-
relations between H-1a and H-3/H-9, H-8 and H-18/H-19, H-19 and H-1b,
H-18 and H-21, and H-14 and H-16/H-17 indicated that the B/C trans, C/D
Through literatures review, we found that the common anti-in-
flammatory pathways were NF-κB (Hwang et al., 2019), NOS/NO (Fan
et al., 2009), JAK2/STAT3 (Tao et al., 2015), PI3K/Akt (Villegas et al.,
2018), AMPK/mTOR (Li et al., 2018) and so on. The anti-inflammatory
activity of steroidal saponins may be related to PI3K/Akt, MARK and
Nrf2/HO-1 pathways (Yan et al., 2016). This paper mainly focused on
chemical constituents of n-butanol fraction and their preliminary anti-in-
flammatory activity. However, the number of the isolated compounds
(1–4) was insufficient for the analysis of the structure-activity relation of
steroidal saponins, and the further studies on more steroidal saponins and
their mechanism of NO production inhibitory activities are ongoing.
1
trans, and D/E cis ring junctions (Fig. 3). The H NMR spectrum of 3
ab
showed two signals of H-26 at δ 4.06 and 3.48 (Table 1), △ (δH26a
-
δ
H26b) = 0.58 > 0.57, which indicated 25S configuration for 3 (Agrawal,
2
004, 2005). Therefore, the structure of the compound 3 was identified as
(
25S)-26-O-β-D-glucopyranosyl-furost-5,20-dien-3β,
26-diol-3-O-α-L-
rhamnopyranosyl (1→2)-[α-L-rhamnopyranosyl (1→4)-β-D-glucopyranosyl
(1→4)]-β-D-glucopyranoside (Fig. 1), named aspidsaponins G.
Compound 4 was obtained as a white amorphous powder, and
3. Experimental
showed positive reactions in the Liebermann-Burchard and Molish tests.
The molecular formula was determined as C56
H
90NaO27 from [M
3.1. General experimental procedures
+
+
Na] ion at m/z 1217.5557 (calcd. for 1217.5567) in the HR-ESI-MS.
On acid hydrolysis, compound 4 liberated D-glucose, D-xylose and D-
galactose, identified by GC analysis of their chiral derivatives, and the
The optical rotations were measured using a JASCO P-1020 digital
polarimeter (Maryland, USA). The melting points were measured on an X-4
digital melting point apparatus without correction. The IR spectra were
measured on a Nicolet FT360 instrument (San Francisco, USA) as samples in
1
3
integral ratio was 3:1:1. The C-NMR data of this compound was
completely identical to 26-O-β-D-glucopyranosyl- (25S)-furost-5, 20
1
13
(
22)-dien-3β, 16β, 26-triol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylo-
pressed KBr disks. The H, C and 2D NMR spectra were recorded on the
Bruker AVANCE 400 MHz with Me4Si as the intestinal standard. The mass
spectra (HR-ESI-MS) were measured in the electron spray mode using the
Finnigan-MAT LCQ DECA XP plus mass spectrometer, and ions were given
in m/z. The HPLC was performed using the Dionex Ultimate 3000 system
for analytical (COSMOSIL-Pack C18-MS-Ⅱcolumn: 250 × 4.6 mm I.D.), and
Waters 1525 EF preparative HPLC (COSMOSIL-Pack 5C18-MS-Ⅱcolumn:
250 × 10 mm I.D.), respectively. The macroporous resins (AB-8, Nankai,
China), and RP Silica Gel (SP-120-40/60-ODS-B, DAISO CO, Japan) was
used as packing materials for column chromatography, respectively. The
optical density (OD) values in the inhibitory Activities against LPS-Induced
NO Production by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) assays were read on automated microplate reader (TECAN
infinite M200PRO, Switzerland).
pyranosyl-(1→3)]-β-D-
glucopyranosyl-(1→4)-β-D-galactopyranoside
1
(
Jiang et al., 2016). However, the H-NMR data were slightly different
1
at H-26. The H NMR spectrum of 4 showed two signals of H-26 at δ
ab
3
.96 and 3.63 (Table 1), △ (δH26a-δH26b) = 0.33 < 0.48, which in-
dicated 25S configuration for 4 (Agrawal, 2004, 2005). Apparently, 4
and the reported compound (Jiang et al., 2016) were a pair of dia-
stereoisomeric saponins, and the structure of 4 was assigned as (25R)-
2
6-O-β-D-glucopyranosyl-furost-5, 20-dien-3β, 26-diol-3-O-β-D-gluco-
pyranosyl (1→2)-[β-D-xylopyranosyl (1→3)]-β- D-glucopyranosyl (1→
4
)-β-D-galactopyranoside, named aspidsaponins H.
In this paper, all isolated compounds (1-4) were evaluated the in-
hibitory activities against lipopolysaccharide (LPS)-induced nitric oxide
(
NO) production in the RAW264.7 macrophage using the MTT assay
method. The results of their NO production inhibitory activities were
shown in Table 3. Among them, compounds 3 and 4 exhibited the
strongest NO production inhibitory activities with IC50 values 82.1 and
3.2. Plant material
6
5.9 μM, respectively, and the inhibitory activity of compound 4 was
The rhizomes of A. elatior Blume were collected from Changyang,
Hubei province of China in October, 2016 and identified by Dr. Yu-Bing
Wang. A voucher specimen (Herbarium No. 2016ZW11008) was de-
posited at Hubei Key Laboratory of Natural Products Research and
Development, China Three Gorges University, Yichang, China.
equal with positive drugs dexamethasone. The structure-activity re-
lationship of steroidal saponins for the NO production inhibitory ac-
tivities associated with the type of the aglycones, the site of the hy-
droxyl group, the number of the hydroxyl group in aglycone, and the
type, number, sequence and binding sites of the sugar moieties (Liu
et al., 2016; Man et al., 2010).
3.3. Extraction and isolation
Compared with compounds 1-2, compounds 3–4 possessed more
sugars and fewer hydroxyl groups and exhibited a stronger inhibitory
effect, it seemed that more number of sugars and fewer number of
Air-dried powdered rhizomes (7.0 kg) were extracted with methanol
(50 L × 3) under reflux. After the removal of the solvent in vacuo and
freeze-drying, the methanol extract (1200 g) was obtained. The extract
(
1000 g)was suspended in water (3.0 L), and then extracted with pet-
Table 3
roleum ether, EtOAc and n-BuOH, respectively. The n-BuOH-soluble
extract (185 g) was dissolved in water (2.0 L), and then was subjected to
macroporous resin column chromatography (10 cm × 120 cm) with
gradient elution (EtOH/water, V/V: 0%, 30%, 60%, 90%, respectively).
The 60% EtOH eluate (20 g out of 60 g) was separated by Rp-C18 silica
gel column chromatography (5.5 cm × 50 cm) in elution with gradient
solvent system (30%→100% methanol/water) to give 160 fractions.
The fractions 160 (600 mg) were further separated by repeated pre-
parative HPLC eluted with 50% acetonitrile (within 90 min, 3.0 mL/
min, 203 nm), giving compound 1 (15 mg) and compound 2 (5 mg). The
fractions 79 (200 mg) were further separated by repeated preparative
HPLC eluted with 35% acetonitrile (within 70 min, 3.0 mL/min,
203 nm), to afford compound 3 (10 mg). The fractions 103 (500 mg)
Inhibitory effects of compounds 1-4 against LPS-Induced
NO production in RAW264.7 macrophages (n = 4,
mean ± SD).
Compounds
IC50(μM)
Dexamethasoneb
69.3 ± 1.03
1
2
3
4
> 100
> 100
82.1 ± 1.18
65.9 ± 0.65
a
IC50 values were average value from three independent
experiments.
b
The positive control.
72

Z.-Y. Sun, et al.
Phytochemistry Letters 34 (2019) 68–73
were further separated by repeated preparative HPLC eluted with gra-
dient solvent (35% acetonitrile, within 70 min, 3.0 mL/min, 203 nm),
giving compound 4 (8 mg).
Research Project of Education Department of Hubei Province (No.
Q20181201), and the Master's Degree Dissertation Foundation of Three
Gorges University in 2019 (No. 2019SSPY109). We are grateful to Dr.
Hai-Bo He for anti-inflammatory activity assay.
2
0
Compound 1, white amorphous powder, m.p. 224-226℃; [α]
D
−
1
1
-
39.2° (c = 0.56, CH OH); IR (KBr) cm : 3428, 2935, 1631, 1563; H
3
1
3
and C NMR data shown in Table 1 and 2; HR-ESI-MS (positive ion
Appendix A. Supplementary data
+
mode) m/z: 827.4029 [M + Na] (calcd. for C39
H
64NaO17, 827.441).
2
1
0
Compound 2, white amorphous powder, m.p. 223-225℃; [α]
D
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2019.09.014.
−
1
-
38.4° (c = 0.52, CH OH); IR (KBr) cm : 3431, 2932, 1627, 1567; H
3
1
3
and C NMR data shown in Table 1 and 2; HR-ESI-MS (positive ion
+
mode) m/z: 827.4031 [M + Na] (calcd. for C39
H
64NaO17, 827.441).
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Compound 3, white amorphous powder, m.p. 209-211℃; [α]
D
-52.5°
−1
1
13
(
c = 0.46, CH
3
OH); IR (KBr) cm : 3436, 2950, 1627, 1561; H and
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Declaration of Competing Interest
2
2, 2541–2549.
Wang, G.P., Huang, W.F., He, H.B., Fu, X.J., Wang, J.Z., Zou, K., Chen, J.F., 2013. Growth
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The authors declared that they have no conflicts of interest to this
work. We declare that we do not have any commercial or associative
interest that represents a conflict of interest in connection with the
work submitted.
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trilliumoside from Trillium tschonoskii inhibits lipopolysaccharide-induced inflammation
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Acknowledgments
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This research was financially supported by grants from the National
Natural Science Foundations of China (No. 81803383), the Scientific
73