Neuroinflammatory inhibitors from: Gardneria nutans Siebold & Zuccarini

Article abstract of DOI:10.1039/d1ra05204g

Two new monoterpene indole alkaloid glycosides nutanoside A-B (1-2), two new phenolic glycoside esters nutanester A-B (6-7), together with five known compounds (3-5, 8-9) were isolated from the ethanol extract of Gardneria nutans Siebold & Zuccarini. Their structures were established on the basis of extensive spectroscopic analysis and TDDFT/ECD calculations. Compounds 1 and 2 are two rare monoterpene indole alkaloids with the glucosyl moiety located at C-12 and represent the first two examples of enantiomer of ajmaline type monoterpene indole alkaloids. Compounds 3, 4 and 6 displayed significant inhibitory effects on NO production in over-activated BV2 microglial cells, with the IC50 values of 2.29, 6.36, and 8.78 μM, respectively. Compounds 1, 5, 7 could significantly inhibit the mRNA expression of inflammatory factors TNF-α and IL-6 induced by LPS in BV2 microglial cells at the effective concentration. Moreover, compound 3 exhibited stronger cytotoxicities against U87 and HCT116 cell lines than taxol with IC50 values of 10.58 and 14.60 μM, respectively.

Full text of DOI:10.1039/d1ra05204g

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Neuroinflammatory inhibitors from Gardneria
nutans Siebold & Zuccarini†
Cite this: RSC Adv., 2021, 11, 27085
ab
a
a
ab
Ying-Ying Si,
Wei-Wei Wang, Qing-Mei Feng, Zhen-Zhu Zhao,
ab
ab
ab
Jun-Im Young*c
Gui-Min Xue, Yan-Jun Sun, Wei-Sheng Feng,
and Xian-Shi Wang*
acd
Two new monoterpene indole alkaloid glycosides nutanoside A–B (1–2), two new phenolic glycoside esters
nutanester A–B (6–7), together with five known compounds (3–5, 8–9) were isolated from the ethanol
extract of Gardneria nutans Siebold & Zuccarini. Their structures were established on the basis of
extensive spectroscopic analysis and TDDFT/ECD calculations. Compounds 1 and 2 are two rare
monoterpene indole alkaloids with the glucosyl moiety located at C-12 and represent the first two
examples of enantiomer of ajmaline type monoterpene indole alkaloids. Compounds 3, 4 and 6
displayed significant inhibitory effects on NO production in over-activated BV2 microglial cells, with the
IC50 values of 2.29, 6.36, and 8.78 mM, respectively. Compounds 1, 5, 7 could significantly inhibit the
mRNA expression of inflammatory factors TNF-a and IL-6 induced by LPS in BV2 microglial cells at the
effective concentration. Moreover, compound 3 exhibited stronger cytotoxicities against U87 and
HCT116 cell lines than taxol with IC50 values of 10.58 and 14.60 mM, respectively.
Received 6th July 2021
Accepted 3rd August 2021
DOI: 10.1039/d1ra05204g
rsc.li/rsc-advances
a vicious cycle, and eventually lead to the gradual degenerative
changes or apoptosis of neurons and the occurrence of neuro-
Neuroinammation is considered to be one of the main causes degenerative diseases such as AD. Therefore, inhibition of
of neurodegenerative diseases like Alzheimer's disease (AD) and over-activation of microglia cells has become an effective way to
Parkinson's disease (PD), and microglia cells play an impor- inhibit neuroinammation. The lack of effective drugs to treat
tant role in the occurrence and development of neuro- neuroinammation has led researchers to turn their attention
inammation. Microglia cells are the permanent immune cells to natural products.
Introduction
6,7
1,2
3
of central nervous system, which have endogenous immune
In our screening of natural herbals for therapy of neuro-
4
defense function. Under normal physiological conditions, inammation, the ethanol extract of Gardneria nutans Siebold &
microglia cells are in a resting or dormant state and provide Zuccarini was selected as a good candidate. G. nutans is derived
nutritional support for neurons. In the brain of AD patients, from the genus Gardneria in the Loganiaceae family. The genus
microglia cells can be activated by Ab and then clear them, thus Gardneria is endemic to Asia and mainly distributed in the
5
playing a role in the repair and protection of nervous system.
southeast of Asia. The plants of this genus are usually used as
However, over-activation of microglia cells leads to the release folk medicines in China, such as G. multiora and G. angusti-
8,9
of large amounts of inammatory factors, including nitric oxide folia. Studies on the chemical constituents and pharmaco-
NO), tumor necrosis factor (TNF-a), interleukin (IL-6, IL-1b), logical activities of this genus have revealed that monoterpene
(
reactive oxygen species (ROS) and so on. The continuous and indole alkaloids were responsible for diverse bioactivities
a mass of release of these neurotoxic factors will directly exhibited by plants of the Gardneria genus, including anti-
damage neurons, or further activate microglia cells to form tumor, sympathetic block, central nervous excitation, dilate
10–14
blood vessels, reduce blood pressure and nerve conduction
activities and so on. G. nutans is a member of the genus, but
there are few reports on its chemical constituents and biological
activities. Thus, investigation on the ethanol extract of G. nutans
was conducted, which led to the isolation of ve monoterpene
indole alkaloids (1–5) and four phenolic glucosides (6–9)
a
College of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046,
China
b
The Engineering and Technology Center for Chinese Medicine Development of Henan
Province, Zhengzhou 450046, China
c
College of Pharmacy, Chonnam National University, Gwangju 500-757 61186, South
(
Fig. 1). Among the isolated compounds, four compounds (1–2,
Korea. E-mail: imyoungjun@jnu.ac.kr
d
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, 6–7) were previously undescribed and others were known. The
China. E-mail: xianshifree@126.com
Electronic supplementary information (ESI) available: The 1D & 2D NMR, and
HR-ESI-MS data of compounds 1–9. See DOI: 10.1039/d1ra05204g
anti-neuroinammation activities and cytotoxicities against
U87 and HCT116 cell lines were also investigated. Herein, the
†
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Table 1 The NMR data of compounds 1–2
Compound 1
Compound 2
a
H
b
C
a
H
b
No.
d
J (Hz)
d
d
J (Hz)
d
C
1
2
3
4
5
6
5.33 d, 3.2
3.51 d, 4.2
3.34 m
6.29 s
4.01 br. s
3.68 br. s
68.9
53.8
63.4
70.9
3.22 m
2.40 dd, 10.9, 4.8
61.0
36.0
3.72 d, 3.5
2.31 d, 11.9
2.24 m
74.7
31.2
1.47 d, 10.9
7
8
9
1
1
1
1
1
57.7
57.0
131.9
119.6
118.2
114.3
143.0
141.7
22.1
129.7
118.9
118.6
112.7
143.4
141.2
21.8
6.75 d, 7.6
6.56 t, 7.6
6.84 d, 7.6
6.66 d, 7.7
6.53 t, 7.7
6.77 d, 7.7
Fig. 1 Structures of compounds 1–9.
0
1
2
3
4
isolation, structural elucidation, anti-neuroinammation
activities, and cytotoxicities of these components are discussed.
2.46 dd, 13.8, 4.9
2.56 dd, 13.2, 4.1
1.89 t, 11.8
1.36 dd, 13.8, 9.9
1
1
1
1
1
5
6
7
8
9
3.35 m
30.0
59.9
73.2
12.5
114.2
139.0
54.8
3.49 d, 4.1
29.0
60.5
72.7
12.3
117.8
131.3
69.7
Results and discussion
4.08 d, 6.6
1.52 d, 6.6, 3H
5.14 q, 6.6
3.93 s
1.57 d, 6.2, 3H
5.33 q, 6.2
Nutanoside A (1) was obtained as a white acicular crystal. The
molecular formula was determined as C H N O on the basis
of the HR-ESI-MS peak at m/z 531.2359 [M + H] (C27
2
7
34
2
9
20
21
+
H
35
N
2
O
9
,
3.28 m
3.25 m
4.22 d, 15.0
3.65 m
calcd for 531.2343), corresponding to 12 indices of hydrogen
1
13
22
172.7
101.7
73.4
77.1
69.9
76.0
60.9
170.7
100.6
73.4
76.8
70.0
76.0
60.9
deciency. The H NMR and C NMR data (Table 1) displayed
0
1
4.67 d, 7.3
3.23 m
3.29 m
3.14 m
3.26 m
3.72 m
4.73 d, 7.3
3.23 m
3.32 m
3.15 m
3.30 m
3.71 m
3.45 m
3.60 s, 3H
signals characteristic of a 1,2,3-trisubstituted phenyl group
0
2
provided by signals at d
7
1
H
6.84 (1H, d, J ¼ 7.6 Hz), 6.75 (1H, t, J ¼
.6 Hz), 6.56 (1H, d, J ¼ 7.6 Hz), and d 143.0 (C-12), 141.7 (C-13),
31.9 (C-8), 119.6 (C-9), 118.2 (C-10), 114.3 (C-11); a methoxy at
0
3
0
0
C
4
5
0
6
d 3.53 (3H, s) and d 50.9 (22-OCH ); a methyl at d 1.52 (3H, d,
H
C
3
H
3.46 m
J ¼ 6.6 Hz) and d
C
12.5 (C-18); a carbonyl group at d
5.14 (1H, q, J ¼ 6.6 Hz)
4.67
C
172.7 (C-
–OCH
3
3.53 s, 3H
50.9
51.7
2
2); a trisubstituted double bond at d
H
a
b
Recorded at 500 MHz in DMSO-d
6
.
6
.
Recorded at 125 MHz in DMSO-
and d
C
114.2 (C-19), 139.0 (C-20); and a glucosyl group at d
H
d
0
0
0
(
(
1H, d, J ¼ 7.3 Hz) and d
C
101.7 (C-1 ), 73.4 (C-2 ), 77.1 (C-3 ), 69.9
0
0
0
C-4 ), 76.0 (C-5 ), 60.9 (C-6 ). Comparison of its 1D NMR data
15
with those of quebrachidine indicated that they have a similar
monoterpenoid indole alkaloid skeleton, except for the pres-
ence of a glucosyl group in compound 1. The attachment of the
Comparison of the calculated ECD spectra data with that of
experimental data of compound 1 indicated the absolute
conguration of compound 1 was assigned to be (2R, 3R, 5S, 7R,
15S, 16R, 17S).
Nutanoside B (2) was obtained as a yellowish oil. It had
a molecular formula of C H N O by the HR-ESI-MS peak at
glucosyl group to C-12 was conrmed by the correlations from
0
H-1 (d
H
4.67) to C-12 (d
C
143.0) in HMBC spectrum (Fig. 2), and
the conguration was conrmed to be b-D by the large coupling
constant (7.3 Hz) of the anomeric proton and acid hydrolysis as
described in the Experimental section. The NOESY correlations
of H-2 with H-5 (Fig. 3) suggested the same b-orientations of H-2
and H-5. The polycyclic rigid framework formed by the
connections of C15–C16(C5)–C17–C7 determined the same b-
orientations of H-15, H-3 and H-5. The relative conguration at
C-17 can be deduced from the observed NOESY correlation
signals between H-15 and H-17, which indicated the orientation
of H-17 is b. The reciprocal NOESY correlations observed for H-
2
7
34 2 10
+
m/z 547.2296 [M + H] (C H N O , calcd for 547.2292), which
2
7
35 2 10
was 16 mass units higher than that of compound 1. The NMR
spectra data (Table 1) were similar to those of 1, except for the
characteristic downeld shis of the carbon resonances for C-3,
C-5, and C-21. Accordingly, the planar structure of compound 2
was identied as the N(4)-oxide of compound 1. The relative
conguration was secured by 2D NMR data, including HSQC,
1
1
HMBC, H– H COSY, and NOESY. The NOESY correlations
Fig. 3) of H-21a with H-2, H-3 and H-5; H-15 with H-17 indi-
(
15/H-18 and H-19/H-21 established the geometry conguration
cated the same relative conguration at these chiral centers
compared with compound 1. Similarly, the NOESY correlations
observed for H-15/H-18 and H-19/H-21 established the geometry
conguration of the 19,20-double bond as E. In addition, HMBC
correlations (Fig. 2) from H-1 (d
of the 19,20-double bond as E. Then, theoretical calculations
were carried out to determine the absolute conguration of 1.
The ECD spectra of (2R, 3R, 5S, 7R, 15S, 16R, 17S)-1a and its
enantiomer (2S, 3S, 5R, 7S, 15R, 16S, 17R)-1b were calculated at
B3LYP/6-31+G** level on the base of TDDFT method (Fig. 4).
0
H C
4.73) to C-12 (d 143.4)
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1
1
Fig. 2 Key HMBC and H– H COSY correlations of 1, 2, 6, and 7.
assigned the C-12 attachment of the glucosyl moiety, and the by two magnetically equivalent aromatic proton signals at [d
coupling constants (7.3 Hz) of anomeric protons and acid 7.14 (2H, s) and d 124.7 (C-1), 106.8 (C-2, 6), 152.6 (C-3, 5), 137.9
hydrolysis established the sugar units as b-D-glucose. The (C-4)], with two magnetically equivalent methoxy by signals at
absolute conguration of 2 was established as (2R, 3R, 5S, 7R, 3.73 (6H, s) and d 56.2; a 1,4-disubstituted benzene ring at
5S, 16R, 17S), which was same as 1, according to their highly [d 120.2 (C-
7.55 (2H, d, J ¼ 8.5 Hz), 6.76 (2H, d, J ¼ 8.5 Hz); d
similarity on ECD spectrum (Fig. 4). Generally, the glucosyl 1 ), 131.3 (C-2 , 6 ), 115.1 (C-3 , 5 ), 162.0 (C-4 )]; a glucosyl group
H
C
d
H
C
1
H
C
0
0
0
0
0
0
0
00
0
00
00
moiety of monoterpene indole alkaloids are located at mono- at [dH 5.10 (1H, d, J ¼ 6.5 Hz, H-1 ); d 101.6 (C-1 ), 73.9 (C-2 ),
C
0
0
00
00
terpene moiety and occasionally at C-10 or C-11 of the indole 76.5 (C-3 ), 70.4 (C-4 ), 74.1 (C-5 ), 63.5 (C-6 )]; two carbonyl
0
skeleton. Compounds 1 and 2 are two rare monoterpene indole groups at d
alkaloids with the glucosyl moiety located at C-12. Moreover, 3.85 (3H, s) and d
compounds 1 and 2 represent the rst two examples of enan- 4 was conrmed by the HMBC correlation from H-1 to C-4
tiomer of ajmaline type monoterpene indole alkaloids. (Fig. 2). The p-hydroxy benzoyl was shown to be involved in an
Nutanester A (6) was obtained as a yellowish amorphous ester linkage at the C-6 position by HMBC correlations
C
165.8 (C-7) and 165.2 (C-7 ); and a methoxy at d
H
C
52.2. The connection of the glucose unit at C-
0
0
0
0
0
0
0
powder. The molecular formula of 6 was determined as observed from H-6 to C-7 . Then, the large coupling constants
C H O on the basis of the quasi-molecular ion peak at m/z (6.5 Hz) of anomeric protons, acid hydrolysis and alkaline
17.1334 [M + Na] (C23H O12Na, calcd for 517.1322) in its HR- methanolysis established the sugar units as b-D-glucose.
26
2
3
26 12
+
5
1
13
ESI-MS spectrum. The H NMR and C NMR spectrum (Table 2) Accordingly, compound 6 was identied as 4-[6-O-(p-hydroxy
showed the presence of a 1,3,4,5-tetrasubstituted benzene ring
Fig. 3 Key NOESY correlations of 1–2.
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1
13
compound 6. The H NMR and C NMR spectra data (Table 2)
were similar to those of 6, except for the presence of an addi-
tional methoxy at [dH 3.71 (3H, s); dC 55.5]. Meanwhile, the
aromatic proton signals for 1,4-disubstituted benzene ring in
compound 6 were substituted by a set of ABX coupling proton
signals at [d
H
7.22 (1H, br. s), 6.71 (1H, d, J ¼ 8.1 Hz), and 7.17
(
1H, br. d, J ¼ 8.1 Hz)] in compound 7. Thus, compound 7 was
0
identied as the 3 -methoxy substituted analogue of compound
, which was further conrmed by 2D NMR spectrum (Fig. 2).
6
16
The ve known compounds, isovallesiachotamine (3), vin-
cosamide (4), strictosamide (5), methyl syringate 4-O-a-L-
rhamnoside (8), methyl salicylate 2-O-b-D-xylosyl (1/6) b-D-
glucopyranoside (9), were identied by analysis of physico-
chemical properties and comparison of spectral data with those
reported in literatures.
17
17
18
19
Fig. 4 Calculated ECD spectrum of compound 1 and experimental Biological activities
ECD curves of 1–2.
The anti-inammatory activities of the isolated compounds 1–8
were assayed in BV2 microglial cells by monitoring LPS-induced
NO production. Beforehand, cell viabilities of compounds 1–8
on BV2 microglial cells were tested by MTT assay in order to avoid
the possible effect of reduced viability on NO release. Results
benzoyl)-b-D-glucopyranoside]-3,5-dimethoxy methyl benzoate,
named as nutanester A.
Nutanester B (7) was obtained as a yellowish amorphous
(Fig. 5) showed that compounds 3, 4, 8 exhibited cytotoxicity at 10
powder. The HR-ESI-MS spectrum exhibited a quasi-molecular
+
ion peak at m/z 547.1429 [M + Na] (C24
H
28
O
13Na, calcd for
547.1428), allowing the molecular formula of 7 to be assigned as
C H O , which was 30 mass units higher than that of
2
4
28 13
Table 2 The NMR data of compounds 6–7
Compound 6
Compound 7
a
H
b
C
a
H
b
No.
d
J (Hz)
d
d
J (Hz)
d
C
1
2
3
4
5
6
7
124.7
106.8
152.6
137.9
152.6
106.8
165.8
120.2
131.3
115.1
162.0
115.1
131.3
165.2
101.6
73.9
124.7
106.8
152.5
138.0
152.5
106.8
165.7
119.6
112.4
147.5
142.3
115.1
123.5
165.4
101.7
74.3
7.14 s
7.14 s
7.13 s
7.13 s
0
1
0
2
7.55 d, 8.5
6.76 d, 8.5
7.22 br. s
0
3
0
4
0
5
6.76 d, 8.5
7.55 d, 8.5
6.71 d, 8.1
7.17 br. d, 8.1
0
6
0
7
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
5.10 d, 6.5
3.27 m
3.26 m
3.22 m
3.38 m
5.11 d, 6.0
3.28 m
3.27 m
3.21 m
3.40 m
76.5
70.4
74.1
63.5
76.5
70.5
74.1
63.6
4.43 d, 12.1
4.43 m
4.09 dd, 12.1, 7.3
4.10 m
3
7
3
,5-OCH
3
3.73 s, 6H
3.85 s, 3H
56.2
52.2
3.73 s, 6H
3.84 s, 3H
3.71 s, 3H
56.2
52.2
55.5
-OCH
3
0
-OCH
3
a
b
Recorded at 500 MHz in DMSO-d
6
.
Recorded at 125 MHz in DMSO-
d .
6
Fig. 5 Cell viability assay of compounds 1–8 in BV2 microglial cells.
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Table 3 Inhibitory effects of compounds 1–8 on NO production in
LPS-activated microglia cells (mean ꢀ SD)
Compounds
IC50 (mM)
Compounds
IC50 (mM)
1
2
3
4
74.43 ꢀ 1.34
47.30 ꢀ 0.69
2.29 ꢀ 0.08
6.36 ꢀ 0.72
5
6
7
8
15.18 ꢀ 0.81
8.78 ꢀ 1.57
>100
14.41 ꢀ 0.03
mM, and compounds 2, 7 were toxic to the cells at 100 mM, which
may affect their inhibitory effect on LPS-induced NO release. That
is to say, such effect of these compounds may mainly due to cell
death and not to their inhibitory effect on LPS-induced NO
release. Taking this into consideration, concentrations that did
not affect the cell viability were selected to test for anti-
inammatory activities of compounds 1–8 in BV2 microglial
cells. Among them, 1 mM was the maximum test concentration for
compounds 3, 4, 8 and 10 mM for compounds 1–2, 5–7. As shown
in Table 3, compounds 3, 4 and 6 displayed signicant inhibitory
effects on NO production in over-activated BV2 microglial cells,
with the IC50 values of 2.29, 6.36, and 8.78 mM, respectively. In
addition, compounds 2, 5 and 8 also showed moderate inhibitory
effects with IC50 values ranging from 14.41 to 47.30 mM.
Then, quantitative real-time PCR (qRT-PCR) was also used to
further investigate the anti-neuroinammatory activities of
compounds 1–8. The results (Fig. 6) revealed that compounds 1, 7
(0.1, 1 and 10 mM), and compound 5 (10 mM) could signicantly
inhibit the mRNA expression of inammatory factors TNF-
a induced by LPS in BV2 microglial cells. Compounds 5 (1 and 10
mM) and 7 (10 mM) could also greatly inhibit the mRNA expression
of inammatory factors IL-6. In addition, compounds 1, 3, 6 and 8 0.05, **p < 0.01, ***p < 0.001, compared to LPS group;
Fig. 6 Effect of compounds 1–8 on LPS-induced mRNA expressions
of TNF-a, and IL-6 in BV-2 microglial cells. Each bar represents the
means ꢀ SD of three independent experiments. Significance: *P <
###
P < 0.001,
compared to control group.
also exhibited a moderate inhibitory effect on the inammatory
factors TNF-a or IL-6 at 0.3–10 mM.
Considering the anti-neuroinammatory activities and struc-
b
Table 4 Cytotoxicity data of isolated compounds 3, 4 and 8
tural characteristics of the isolated compounds, a brief structure–
activity relationship (SAR) could be concluded as follows: by
comparing compound 4 with 5, we found that the 3R congura-
IC50 (mM)
a
a
tion could cause an increase of NO inhibitory activity but atten- Compounds
U87
HCT116
uated the inhibitory effect on inammatory factors TNF-a and IL-
3
4
8
10.58 ꢀ 0.23
>100
>100
16.30 ꢀ 0.06
14.60 ꢀ 0.17
49.21 ꢀ 0.03
>100
23.57 ꢀ 0.11
6
in over-activated BV2 microglial cells. A similar situation
0
occurred between compounds 6 and 7, the presence of 3 -OCH
3
signicantly reduced the NO inhibitory activity, while greatly
enhanced the inhibitory activities on inammatory factors TNF-
a and IL-6. Moreover, the presence of coordination bond at N-4 in
c
Taxol
a
U87, human glioma cell line; HCT116, human colon cancer cell line.
Data are expressed as the mean
b
ꢀ
SD of three parallel
c
compound 2 (compared with 1) could weaken the inhibitory effect measurements. Positive control.
on inammatory factors TNF-a and IL-6 in BV2 microglial cells.
Furthermore, considering the effect of some compounds on
the survival rate of microglia cells, the cytotoxic activities on two
human cancer cell lines (U87 and HCT116) of compounds 3, 4 and
Experimental method
General experimental procedures
CD spectrum was tested using Chirascanq CD spectrometer
Applied Photophysics) at room temperature. Optical rotation
was analyzed using the APIV (Rudolph Research Analytical).
NMR spectra were performed on Bruker Avance III 500 MHz
spectrometer (Bruker, Karlsruhe, Germany) using TMS as
8
were investigated by MTT method. As shown in Table 4,
compound 3 exhibited stronger cytotoxic activities against U87
and HCT116 cell lines than taxol, which was used as the positive
control, with IC50 values of 10.58 and 14.60 mM, respectively.
Compound 4 showed selective inhibitory effect on HCT116 cells
with IC50 values of 49.21 mM.
(
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25
a standard (500 MHz for H NMR and 125 MHz for C NMR).
Nutanoside A (1), white acicular crystal; [a]
D
-68.28 (c 0.07,
ꢂ1
Bruker maXis HD mass spectrometer was used to collect HR MeOH); IR n
ESI-TOF MS data in m/z (rel.%). MPLC was performed on Agela
ODS ash column (C-18, 120 g, 40–60 mm, Tianjin, China), and d ) see Table 1; HR-ESI-MS m/z 531.2359 [M + H] (calcd for
3369, 1645, 1463, 1297, 1249, 1207, 1017 cm
H NMR (500 MHz, DMSO-d ) and C NMR (125 MHz, DMSO-
;
max
1
13
6
+
6
the FL-H050G MPLC system (Tianjin, China). HPLC separation
was carried out on a semi-preparative YMC-pack ODS-A column
C
27
H
35
N
2
O
9
, 531.2343).
Nutanoside B (2), yellowish oil; [a]
nmax 3314, 2923, 1731, 1593, 1482, 1458, 1230, 1244, 1206, 1175,
2
5
D
-13.10 (c 0.07, MeOH); IR
(
250 ꢁ 10 mm, YMC-pack, Kyoto, Japan) equipped with Sai-
ꢂ1
1
puruisi LC-52 instrument with a UV200 detector (Beijing, 1076, 1042, 879, 742 cm ; H NMR (500 MHz, DMSO-d
6
) and
) see Table 1; HR-ESI-MS m/z
for column chromatography were obtained from Qingdao 547.2296 [M + H] (calcd for C H N O , 547.2292).
1
3
China). Silica gel GF254 for TLC and silica gel (200–300 mesh)
C NMR (125 MHz, DMSO-d
6
+
2
7
35 2 10
Haiyang Chemical Co., Ltd. (Qingdao, China). D-Glucose and L-
Nutanester A (6), yellowish amorphous powder; IR nmax 3424,
glucose (SigmaAldrich, St. Louis, MO, USA) were used to 1698, 1640, 1608, 1502, 1462, 1341, 1277, 1126, 1066, 1018, 768,
ꢂ1
1
13
determine the absolute conguration of sugar moiety. Sephadex 699 cm ; H NMR (500 MHz, DMSO-d
LH-20 for column chromatography was obtained from Phar- MHz, DMSO-d
macia (Piscataway, NJ, USA).
6
) and C NMR (125
+
6
) see Table 2; HR-ESI-MS m/z 517.1334 [M + Na]
12Na, 517.1322).
Nutanester B (7), yellowish amorphous powder; IR nmax 3486,
26
(calcd for C23H O
ꢂ1
1
2
920, 1649, 1258, 775, 725, 701 cm
;
H NMR (500 MHz,
Plant material
1
3
DMSO-d ) and C NMR (125 MHz, DMSO-d ) see Table 2; HR-
6
6
The whole plant of Gardneria nutans Siebold & Zuccarini was
collected from Guizhou province, China, in October, 2018, and
identied by Prof. Cheng-Ming Dong (Henan University of
Chinese Medicine). A voucher specimen (no. 201810) was
deposited in the Research Department of Natural Product
Chemistry, School of Pharmacy, Henan University of Chinese
Medicine.
+
ESI-MS m/z 547.1429 [M + Na] (calcd for C H O Na,
47.1428).
2
4
28 13
5
Absolute congurations determination of sugar moieties
Compounds 1–2 (each 1.0 mg) were separately treated with 2 M
HCl (2 mL) for 3 h at 85 C. Each reaction product was dissolved
ꢃ
in H
2 3
O aer evaporation and then extracted with CHCl for
Extraction and isolation
three times. Aer being concentrated to dryness, the aqueous
The dried whole plant of G. nutans (17.5 kg) were extracted with layer was subjected to HPLC on a chiral-phase CHIRALPAK AD-
9
5% ethanol under reux for three times (2 h/time). Aer H column (250 ꢁ 4.6 mm) eluting with n-hexane: EtOH : TFA
ꢂ1
concentration and combination, the crude extract was (750 : 250 : 0.25, v/v/v, 0.5 mL min ), and equipped with an
dispersed in 0.5% HCl and extracted with isopyknic EtOAc for evaporative light scattering detector (ELSD). D-Glucose was
three times. The acidic water layer was basied with 10% detected in the acid hydrolysates of compounds 1–2 according
ammonia solution to pH 9–10 and extracted with EtOAc to give to their same retention times with those of known D-glucose (t
an alkaloidal extract (17 g). The alkaloidal extract was subjected ¼ 18.3 min) in the same condition.
R
to MPLC on ODS column, eluting gradually with MeOH–H
2
O
Compounds 6–7 (each 1.0 mg) were treated with 6% NaOMe
(
20 : 80, 40 : 60, 60 : 40, 80 : 20, and 100 : 0, v/v) to afford ve in MeOH (2 mL) at room temperature for 2 h. The reaction
fractions Fr. A–E. Fr. B was applied to a silica gel column and mixture was dissolved in H O aer evaporation and then
eluted with a CH Cl –MeOH gradient (from 100 : 0 to 0 : 100) to extracted with CHCl for three times. The aqueous layer was
afford 11 fractions Fr. B1–B11. Fr. B9 was further puried by dried and then D-glucose was determined by using acid hydro-
semi-preparative HPLC eluted with 32% MeOH–H O to yield lysis method described above.
compound 1 (20 mg, t
¼ 64 min). Fr. B10 was chromato-
graphed over pre-HPLC (MeOH–H O, 22 : 78, v/v) to yield
compound 2 (12.5 mg, t
¼ 32 min). Fr. B4 was further puried
by pre-HPLC (MeOH–H O, 40 : 60, v/v) to afford compounds 6 Cell viability was detected by MTT method. Cells were seeded in
2.1 mg, t
¼ 57 min), 7 (1.5 mg, t
38 min). Fr. B7 was applied to Sephadex LH-20 with MeOH Aer treatment with tested compounds for 24 h, cells were
2
2
2
3
2
R
2
Anti-inammatory assay in BV2 cells
R
2
5
(
R
R
¼ 50 min), and 8 (4.6 mg, t
R
96-well plates (3 ꢁ 10 cells per mL) and incubated for 24 h.
¼
ꢂ1
ꢃ
eluent to obtain ten fractions Fr. B7-1–B7-10, and then incubated with MTT (0.25 mg mL ) for 4 h at 37 C. The culture
compound 9 (13.4 mg, t
by pre-HPLC (MeOH–H
R
¼ 21 min) was obtained from Fr. B7-4 medium was removed, and then the crystals were dissolved in
2
O, 35 : 65, v/v). Fr. C was subjected to DMSO. The cell viability rate was measured by optical densities
Sephadex LH-20 (MeOH) to give four fractions Fr. C1–C4. Fr. C3 at 570 nm with a microplate reader.
was chromatographed on a silica gel column eluting with The nitrite concentration was measured in the supernatant
CH Cl –MeOH (20 : 1, 13 : 1, 1 : 0) to give 16 subfractions Fr. of cells by the Griess reagent system according to protocol. Cells
C3-1–C3-16. Compound 4 (3.6 mg, t
2
2
¼ 42 min) was obtained were seeded into 96-well plates and treated with tested
R
ꢂ1
from Fr. C3-5 by pre-HPLC (48% MeOH), and compound 5 compounds in presence of LPS (100 ng mL ) for 24 h. 50 mL of
(
(
2.8 mg, t ¼ 23 min) was yielded from Fr. C3-12 by pre-HPLC culture supernatant uids were mixed with 50 mL Griess reagent
R
57% MeOH). Compound 3 (41 mg) was puried by Sephadex at room temperature, and then the absorbance at 540 nm was
LH-20 (MeOH) and recrystallization from Fr. D.
determined aer 15 min.
27090 | RSC Adv., 2021, 11, 27085–27091
© 2021 The Author(s). Published by the Royal Society of Chemistry

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Quantitative real-time PCR
Conflicts of interest
5
BV-2 microglial cells were seeded in 96-well plates (4 ꢁ 10 cells
There are no conicts to declare.
per mL) and incubated for 24 h. Aer treatment with tested
ꢂ1
compounds for 1 h, cells were incubated with LPS (100 ng mL
)
for 24 h. Total RNA was extracted with Trizol (Invitrogen)
according to the manufacturer's protocol, and qRT-PCR assays Acknowledgements
were performed with the CFX Connect™ real-time PCR detec-
tion system (Bio-Rad) using total RNA and the GoTaq one-step
real-time PCR kit with SYBR green.
This research project was nancially supported by the 2018
doctoral research start-up foundation of Henan University of
Chinese Medicine (BSJJ2018-02), and innovation training
program for college students of Henan University of Chinese
Medicine (No. 202110471034).
Cytotoxicity assay
A MTT assay was used to determine the cytotoxicity of each
compound against two cultured human cancer cell lines. The cell
lines used were U87 (human glioma cell line) and HCT116
Notes and references
(
human colon cancer cell line). In brief, cells suspensions were
4
plated in 96-well microtiter plates (2.5 ꢁ 10 cells per mL) and
1
F. D. Zhu, Y. J. Hu, L. Yu, X. G. Zhou, J. M. Wu, Y. Tang,
ꢃ
incubated for 24 h at 37 C under 5% CO
2
. Aer treatment with
D. L. Qin, Q. Z. Fan and A. G. Wu, Front. Pharmacol., 2021,
tested compounds for 72 h, cells were incubated with MTT (50 mL,
1
2, 683935.
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0
.4%) for 4 h at 37 C. Then, the MTT was removed from the wells
2
3
4
5
and the formazan crystals were dissolved in DMSO (200 mL) for
0 min with shaking. The plate was read immediately on
8
1
a microtiter plate reader at a wavelength of 570 nm to record the
optical density. The IC50 value was dened as the concentration of
the control in the MTT assay. Taxol was used as a positive control.
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ECD computation method
2
Conformational search of compound 1 was carried out by
Spartan soware using the MMFF94S force eld. Then, the
structural optimization of target conformations was performed
by DFT theory at the B3LYP/6-31G* level. TDDFT ECD calcula-
tions for these optimized conformers were carried out at the
6 Z. Fan, D. J. Brooks, A. Okello and P. Edison, Brain, 2017, 140,
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B3LYP/6-31+G* level with a CPCM model in MeOH solvent.
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two new monoterpene indole alkaloid glycosides nutanoside X. D. Luo, Bioorg. Med. Chem. Lett., 2013, 23, 5563–5565.
A–B (1–2), two new phenolic glycoside esters nutanester A–B (6– 12 W. X. Yang, T. Huang, J. X. Zhang, J. H. Liu, X. J. Hao and
) and ve known compounds (3–5, 8–9). Compounds 1 and 2 Y. H. Zhang, Chin. Pharm. J., 2016, 51, 1113–1115.
are two rare monoterpene indole alkaloids with the glucosyl 13 G. H. Xie, L. Ma, Z. P. Zhang and L. H. Hu, Chin. J. Nat. Med.,
moiety located at C-12. Moreover, compounds 1 and 2 represent 2007, 5, 255–258.
7
the rst two examples of enantiomer of ajmaline type mono- 14 M. Harada and Y. Ozaki, Chem. Pharm. Bull., 1978, 26, 48–52.
terpene indole alkaloids. The results of anti-neuroinammatory 15 M. R. Yagudaev, Chem. Nat. Compd., 1982, 18, 693–696.
activities showed that compounds 3, 4 and 6 displayed signi- 16 F. Bahadori, G. Topçu, M. Bo ˘g a, A. T u¨ rkekul, U. Kolak and
cant inhibitory effects on NO production in over-activated BV2
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microglial cells, with the IC50 values of 2.29, 6.36, and 8.78 mM, 17 Z. Z. Zhang, H. N. ElSohly, M. R. Jacob, D. S. Pasco,
respectively, and compounds 1, 5, 7 could signicantly inhibit
the mRNA expression of inammatory factors TNF-a and IL-6
L. A. Walker and A. M. Clark, J. Nat. Prod., 2001, 64, 1001–
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induced by LPS in BV2 microglial cells at the effective concen- 18 Y. L. Wang, Y. J. Li, A. M. Wang, X. He, S. G. Liao and
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©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 27085–27091 | 27091