Stilbenes from the tubers of Bletilla striata with potential anti-neuroinflammatory activity

Article abstract of DOI:10.1016/j.bioorg.2020.103715

Neuroinflammation are involved in the pathogenesis of many neurodegenerative disorders. In our screening of natural effective neuroinflammatory inhibitors from natural products, stilbenes, such as resveratrol and its analogues, have received considerable attention over the last several decades as anti-neuroinflammatory agents. Then, Bletilla striata attracted our attention due to its abundant stilbenes portion, PE fraction. So, three new stilbenes: dusuanlansin E1 (23a), dusuanlansin E2 (23b), 3-hydroxy-5-methoxybibenzyl-3′-O-β-D-glucopyranoside (27), and 30 known stilbene compounds were isolated from B. striata. These structures of the compounds were established on the basis of extensive spectroscopic analysis including 1D and 2D NMR and circular dichroism (CD) data. Furthermore, all the isolated components were tested in vitro for their inhibitory effects on the nitric oxide generation in LPS-stimulated BV2 cells. As a result, compounds 2, 5, 6, 16, 17 can greatly inhibit the NO production without cytotoxicity. In addition, SARs between stilbenes and anti-neuroinflammation effects were discussed briefly. In conclusion, stilbenes were characteristic constituents of the tubers of B. striata with potential anti- neuroinflammatory effects.

Full text of DOI:10.1016/j.bioorg.2020.103715

Bioorganic Chemistry 97 (2020) 103715
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Stilbenes from the tubers of Bletilla striata with potential anti-
neuroinflammatory activity
Di Zhou , Wenhui Chang , Bo Liu , Gang Chen , Yanqiu Yang , Yingtu Hao , Yue Houb,⁎,
a
a
a
a
b
b
a,⁎
Ning Li
a
School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug, Shenyang Pharmaceutical University,
Wenhua Road 103, Shenyang 110016, PR China
b
College of Life and Health Sciences, Northeastern University, Shenyang 110819, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Neuroinflammation are involved in the pathogenesis of many neurodegenerative disorders. In our screening of
natural effective neuroinflammatory inhibitors from natural products, stilbenes, such as resveratrol and its
analogues, have received considerable attention over the last several decades as anti-neuroinflammatory agents.
Then, Bletilla striata attracted our attention due to its abundant stilbenes portion, PE fraction. So, three new
stilbenes: dusuanlansin E1 (23a), dusuanlansin E2 (23b), 3-hydroxy-5-methoxybibenzyl-3′-O-β-D-glucopyrano-
side (27), and 30 known stilbene compounds were isolated from B. striata. These structures of the compounds
were established on the basis of extensive spectroscopic analysis including 1D and 2D NMR and circular di-
chroism (CD) data. Furthermore, all the isolated components were tested in vitro for their inhibitory effects on
the nitric oxide generation in LPS-stimulated BV2 cells. As a result, compounds 2, 5, 6, 16, 17 can greatly inhibit
the NO production without cytotoxicity. In addition, SARs between stilbenes and anti-neuroinflammation effects
were discussed briefly. In conclusion, stilbenes were characteristic constituents of the tubers of B. striata with
potential anti- neuroinflammatory effects.
Bletilla striata
Stilbene
BV2 microglial cell
NO production inhibition
1
. Introduction
Neurodegenerative diseases are a group of chronic, progressive
effects. Recently, resveratrol’s analogues, pterostilbene, have gained a
significant amount of attention due to their potent antioxidant, anti-
inflammatory properties [7,8]. Furthermore, the stilbene scaffold is a
basic element for synthetic compounds, and it is considered as a pri-
vileged structure [8]. Therefore, stilbenes are of significant interest for
drug research and synthetic fields.
disorders characterized by the gradual loss of neurons in discrete areas
of the central nervous system (CNS) [1]. Neuroinflammatory processes
are involved in the pathogenesis of many neurodegenerative disorders,
such as Alzheimer’s disease (AD) [2]. To overcome the limitations of
current therapeutics for neurodegenerative diseases, extensive research
is underway to identify new targets, together with new drugs that are
effective and free of undesirable side effects. Some stilbenes with an-
tioxidant and anti-inflammatory properties have received considerable
attention as alternative candidates for neurodegenerative diseases
prevention or therapy [3-5].
Traditional Chinese medicines not only hold economic value of
healthcare system, but also possess unique and great potential for new
drug development [9]. Bletilla striata, is a perennial herb which is
widely distributed in China, North Korea, Japan and Burma, belonging
to Orchidaceae family. As a well-known Chinese folk herb medicine, its
tubers have been employed to treat hemostasia, detumescence, healing
and enhancement of bodily function [10,11]. Previous phytochemical
studies on Bletilla species have led to the isolation of many stilbene
compounds. Based on the traditional applications and structures types,
stilbenes may be the material basis of anti-neuroinflammatory effects.
As part of our search for new and bioactive stilbenes constituents, the
petroleum ether (PE)-soluble fraction of 95% EtOH extract of was
phytochemically studied, which furnished three new stilbenes and
thirty known components. The structures of these compounds were
Stilbenes are widely found in nature, have been paid extensive at-
tention for their various functions about healthy in human diet and
medical treatments, such as antioxidative, anticancer activities.
They represent a class of compounds with a common 1, 2-dipheny-
lethylene backbone that have shown extraordinary potential in the
biomedical fields, especially in the treatment of neuroinflammation [6].
As the most well-known example, resveratrol proved to have anti-aging
⁎
Corresponding authors at: School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China.
E-mail addresses: houyue@mail.neu.edu.cn (Y. Hou), liningsypharm@163.com (N. Li).
https://doi.org/10.1016/j.bioorg.2020.103715
Received 10 October 2019; Received in revised form 2 February 2020; Accepted 28 February 2020
Available online 28 February 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

D. Zhou, et al.
Bioorganic Chemistry 97 (2020) 103715
elucidated mainly by NMR spectroscopic and mass spectroscopic
methods. Furthermore, the anti-neuroinflammatory effects of the ex-
tract and purified constituents were performed by NO assays in LPS-
stimulated BV2 cells [12].
Table 1
1
13
H (600 MHz) and C (150 MHz) spectral data of compounds 23 and 27.
a
b
No.
23
No.
27
δ
H
, J (Hz)
δ
C
δ
H
, J (Hz)
δ
C
2
. Experimental
1
2
3
4
5
6
α
143.3
120.0
161.4
99.2
1
143.4
108.2
160.3
98.9
2
6.21, m
6.13, m
2.1. General experimental procedures
3
6.33, d, 2.4
4
159.0
109.7
36.7
5
160.3
104.3
37.2
NMR spectra were recorded on Bruker ARX-400 and 600 M AVIII
6.23, d, 2.4
2.74, m
6
6.21, m
spectrometers, using TMS as an internal standard. Silica gel (200–300
mesh) for chromatography was produced by Qingdao Ocean Chemical
Group Co. of China. Optical rotations were measured using MCP 200
polarimeter from Anton Paar GmbH (Graz, Austria). Sephadex LH-20
was purchased from Pharmacia Company (Uppsala, Switzerland). ODS
α
2.81–2.88, m
2.81–2.88, m
α′
1′
2.89, m
39.7
α′
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
36.7
144.3
120.9
158.5
114.0
130.4
116.4
181.5
32.2
143.1
113.5
157.5
116.3
129.0
121.8
100.4
73.3
2
3
4
5
′
′
′
′
6.62, m
6.83, m
6.60, m
6.89, m
(
50 µm) for column chromatography was afforded by YMC Co. (Ltd) in
7.06, t, 7.8
6.58, m
7.17, t, 7.8
6.84, m
Japan. HPLC separations were performed on a YMC-pack Prep-ODS
column (250 × 20 mm) equipped with a shimadzu RID-20A UV de-
tector and a shimadzu LC-6AD series pumping system (Tokyo, Japan).
Dimethyl sulfoxide (DMSO), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe-
6′
2″
4.80, d, 7.2
3.21, t, 7.2
3.26, t, 7.2
3.15, t, 7.2
3.30, m
3
4
5
3
″
2.50, m; 2.39, m
2.32, m; 2.06, m
5.03, dd, 9.0, 5.0
3.75, s
″
27.4
76.7
″
53.4
69.7
nyltetrazolium bromide (MTT), CDCl
3
, DMSO‑d
6
and CD OD were ob-
3
- OCH
3
55.9
77.0
tained from Sigma-Aldrich Company (St. Louis, MO, USA). All the
chromatographic and analytical grade reagents were obtained from
Tianjin DaMao Chemical Company (Tianjin, China).
3.67, m; 3.46, m
3.65, s
60.7
5
-OCH
3
54.7
a
b
Measured in CD
3
OD.
Measured in DMSO‑d
6
.
2.2. Plant material
(
73.2 mg), 13 (120.2 mg), 20 (35.2 mg) and 23 (16.6 mg). Using the
The tubers of B. striata were obtained from Shaanxi Tasly Plant
same method, 45% part and 55% part were separated by HPLC to ob-
Medicine Limited Liability Company, China, in November 2016. The
plant material was identified by Professor Yingni Pan (School of
Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University). A voucher specimen (No. 20161123) is deposited in School
of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University.
tain compounds 24 (22.8 mg) and 25 (37.7 mg), compounds 26
(
12.3 mg), 27 (17.4 mg), 28 (45.9 mg), 29 (25.2 mg) and 30 (17.0 mg),
respectively.
2
.3.1. Dusuanlansin E1 (23a)
Brownish powder; [α] − 30 (c 0.1, MeOH); UV (MeOH) λmax (logε)
1
13
2
10 (4.60) nm; H NMR (600 MHz, CD
3
OD) and C NMR (150 MHz,
2
.3. Extraction and isolation
−
CD
3
OD) data see Table 1; HRESIMS m/z: 328.1539 [M+H] , calcd for
C
19
H
22NO , 328.1543.
4
The dried and powdered tubers of B. striata (16 kg) were refluxed
with 95% EtOH (3 times, 3 h for each). The extract was evaporated to
dryness under vacuum to afford a crude residue (1.82 kg) (11.38%),
which was partitioned successively with petroleum ether (PE), ethyl
acetate (EtOAc) and n-BuOH.
2
.3.2. Dusuanlansin E2 (23b)
Brownish powder; [α] + 30 (c 0.1, MeOH); UV (MeOH) λmax (logε)
1
13
2
10 (−4.60) nm; H NMR (600 MHz, CD
3
OD) and C NMR (150 MHz,
The PE portion was evaporated under reduced pressure to afford a
crude extract. The effective PE extract (200.0 g) was subjected to silica
−
CD
3
OD) data see Table 1; HRESIMS m/z: 328.1539 [M+H] , calcd for
C
19
H
22NO , 328.1543.
4
gel column (CH
2
Cl : MeOH, 100:0–0:100). Then similar fractions were
2
collected together on the basis of similarity in R
f
values and afforded 10
2
.3.3. 3-Hydroxy-5-methoxybibenzyl-3′-O-β-D-glucopyranoside (27)
fractions (Fr. 1-Fr. 10). Fr. 1 was chromatographed on open ODS
Brownish powder; [α] − 19 (c 0.1, MeOH); UV (MeOH) λmax (logε)
column (100 × 3.5 cm) and eluted with MeOH-H O (0: 100–100:0) to
2
1
13
2
10 (- 3.20) nm; H NMR (600 MHz, DMSO‑d
6
) and
C NMR
yield sub-fractions Fr. 3.1. This fraction was purified by HPLC with ODS
(
150 MHz, DMSO‑d
6
) data see Table 1; HRESIMS m/z: 405.1554
column (250 mm × 10 mm, 3 mL/min) to yield compounds 1
−
[
M−H] , calcd for C21
H
25
O , 405.1555.
8
(
25.5 mg), 8 (17.8 mg), 15 (22.3 mg) and 19 (29.4 mg) using MeOH/
H O (68: 32) as mobile phase. Similarly, Fr.2 was separated by HPLC by
2
ODS column with elution phase MeOH/H
2
O (68: 32) to provide com-
2.4. Acid hydrolysis of compound 27 and HPLC analysis for sugar residues
Compound 27 (1.0 mg) was heated in 4 M HCl (2 mL) for 3 h in a
pound 21 (37.1 mg). Fr.3 was firstly isolated by silica gel column
chromatography (50 × 3 cm) with gradient elution of PE/EtOAc
(
0:100–100:0) to get Fr. 3–1 and 3–2, and then these subfractions were
H O bath at 90 °C. After cooling, these mixture were extracted by
2
further eluted by MeOH/H
2
O with ODS column to gain compounds 3
CH
2
Cl
2
and get CH
2
2
Cl extract. Then aqueous layer was evaporated to
(
(
(
30.2 mg), 4 (12.0 mg), 14 (18.8 mg), 16 (22.6 mg), 17 (27.4 mg), 18
dryness. The water layer and D-glucose (1.0 mg) were added in 3.0 mg
L-cysteine methyl ester and were dissolved in pyridine (1 mL) and he-
ated at 60 °C for 1 h and then o-tolyl isothiocyanate (5 μL) was added to
the mixture and heated further for 1 h. The reaction mixture was
20.2 mg), 22 (20.1 mg). And Fr. 4 was loaded on a ODS column
75 × 3.5 cm), eluting with MeOH and further purified by HPLC with
ODS column (250 mm × 10 mm, 3 mL/min) to yield compounds 6
(
32.0 mg), 12 (123.2 mg) and 7 (10.2 mg) using MeOH/H
eluting solvent. Fr. 6 was subjected to ODS column (MeOH-H
:100–100:0) and similar fractions were pooled together followed by
TLC analysis. Among them, 30% part was chromatographed over HPLC
and eluted with MeOH-H O (55:45) to yield compounds 2 (20.2 mg), 5
2
O (64:36) as
analyzed by HPLC and detected at 250 nm, t (min): D-glucose (18.73).
R
2
O,
The HPLC analysis was carried out on a shimadzu SPD-20A (UV/visible
detector) using shimpack ODS (H) KIT (5 μm particle size,
4.6 × 250 mm) at 35 °C with a flow rate of 1 mL/min. Mobile phase
was 25% CH CN/H O (0.1% formic acid of water).
0
a
2
3
2
2

D. Zhou, et al.
Bioorganic Chemistry 97 (2020) 103715
2.5. Nitrite assays of compounds 1–30 in LPS-induced BV2 cells
−
Using the griess method, accumulation of nitrite (NO
2
), an in-
dicator of NO synthase activity, in culture medium was measured. BV2
5
cells (3 × 10 cells/well) were plated in 96-well microtiter plates and
treated with extract (0, 1, 10, 30, 100 μg/mL) and each compounds (0,
1
2
5
, 10, 30, 100 μM) in presence of lipopolysacchride (LPS; 1 μg/mL) for
4 h. Fifty microliter culture medium supernatants were mixed with
0 μL griess reagent at room temperature for 15 min. The optical
density of each well was measured at 540 nm.
2.6. Cytotoxicities assay of compounds 1–30
Fig. 2. The key HMBC (
) correlations of compounds 23 and 27.
Cell viability was evaluated by MTT assay. Briefly, BV2 microglial
5
cells were seeded at 3 × 10 cells/well in 96-well microtiter plates.
After overnight incubation, the cells were treated with LPS (1 μg/mL) in
the absence or presence of the test extracts (0, 1, 10, 30, 100 μg/mL)
and each compounds (0, 1, 10, 30, 100 μM) for 24 h, the medium was
removed and the cells were incubated with MTT (0.25 mg/mL) for 4 h
at 37 °C. The formazan crystals in the cells were dissolved in DMSO. The
absorbance was measured at 490 nm by a microplate reader.
(
5''R)-23a
3
. Results and discussion
3.1. Isolation of compounds from the PE extract of the tubers of B. striata
(
5''S)-23b
Bio-guided fractionation and isolation of PE-soluble fraction of the
9
5% ethanol extract of the tubers of B. striata afforded 33 compounds
(
Fig. 1) by means of chromatographic methods and recrystallization
[
13]. Finally, their structures were determined as follow: (E)-3,3′,5-
trimethoxystilbene (1) [15], dihydropinosylvi (2) [16], gigantol (3)
[
17], 3,3′,5-trimethoxybibenzyl (4) [18], batatasin III (5) [19], 5-[2-(3-
methoxyphenyl)ethyl]-1,3-benzenediol (6) [20], 3,3′,4-trihydrox-
ybibenzyl (7) [21], 3- hydroxy-5-methoxybibenzyl (8) [22], 3′-O-me-
thylbatatasin III (9) [22], 3-hydroxy-5- methoxybibenzyl (10) [22],
Fig. 3. Comparison of experimental ECD spectra of compounds 23a and 23b (in
MeOH).
shancigusin
D (11) [22], 3,3′-dihydroxy-2-(4-hydroxyl benzyl)-5-
methoxy-bibenzyl (12) [23], 3′,5-dihydroxy-2-(4-hydroxybenzyl)-3-
methoxy-bibenzyl (13) [24], bulbocol (14) [25], 3′-hydroxy-2-(4-hy-
droxybenzyl)- 3,5-dimethoxy-bibenzyl (15) [26], gymconopin D (16)
species for the first time (Fig. 1).
[
27], 5-hydroxy-2-(p- hydroxybenzyl)-3-methoxybibenzyl (17) [28], 4-
3.2. Structural elucidation of new compounds
hydroxy-5-(p-hydroxybenzyl)-2- methoxybibenzyl (18) [26], arudinan
(
19) [26], 3,3′-dihydroxy-4-(4-hydroxybenzyl)-5-methoxybibenzyl (20)
The molecular formula of compound 23, C19
H
21NO , with 10 de-
4
+
[
[
[
29], 5-hydroxy-4-(p-hydroxybenzyl)-3′,3-dimethoxy bibenzyl (21)
30], 3′-dihydroxy-4-(4-hydroxybenzyl)-3,5-dimethoxybibenzyl (22)
26], dusuanlansin E1 (23a), dusuanlansin E2 (23b), dusuanlansin A
grees of unsaturation, was evidenced by the [M+H] ion at m/z
328.1539 (calcd. 328.1543 for C19
H
22NO ) in HRESI-MS. Analysis of
4
the NMR data (Table 1) revealed the presence of a disubstituted [δ
H
(
24a) [31], dusuanlansin B (24b) [31], dusuanlansin C (25a) [31],
7.06 (1H, t, J = 7.8 Hz, H-5′), 6.60 (1H, m, H-4′), 6.58 (1H, m, H-6′),
6.62 (1H, m, H-2′)] and a tetrasubstituted [6.33 (1H, d, J = 2.4 Hz, H-
4), 6.23 (1H, d, J = 2.4 Hz, H-6)] phenyl groups, four methylenes, a
dusuanlansin D (25b) [31], 3,3′-dihydroxy-2′,6′-bis(p-hydroxybenzyl)-
5
-methoxybibenzyl (26) [32], 3-hydroxy- 5-methoxybibenzyl-3′-O-β-D-
glucopyranoside (27), 3′,5-dimethoxybibenzyl-3-O- β-D-glucopyrano-
side (28) [33], batatsin III-3-O-glucoside (29) [33], 5-methoxy- bi-
benzyl-3,3′-di-O-β-D-glucopyranoside (30) [34]. Among them, com-
pounds 1, 7, 15, 17–18, 22, 24–25, and 30 were isolated from this
methine, a methoxy and a carboxylic carbon (δ
C
181.5). The signals of
2.74–2.89
(4H, m, H-α, α′)] suggest the existence of a bibenzyl skeleton, which is
similar to batatasin III. Additionally, the methoxy group [δ 3.75 (3H,
two phenyl groups combined with a pair of methylenes [δ
H
H
Fig. 1. The structures of new compounds.
3

D. Zhou, et al.
Bioorganic Chemistry 97 (2020) 103715
A 140
(
-)LPS
+)LPS
140
(
(-)LPS
(
+)LPS
1
20
1
20
00
80
#
##
100
1
8
6
4
2
0
0
0
0
0
*
**
***
6
0
0
4
*
**
20
0
*
**
0
0
1
10
50
100
0
0
1
100
10
50
EXT (μg/mL)
EXT (μg/mL)
1
1
1
40
B
(
-)LPS
+)LPS
(
140
120
(
-)LPS
+)LPS
20
00
(
###
**
100
80
60
40
20
0
80
60
40
20
0
*
**
*
**
0
0
1
10
50
100
0
0
1
10
50
100
EXT (μg/mL)
EXT (μg/mL)
C
Con
LPS
LPS+1μM
LPS+10μM
LPS+30μM
LPS+100μM
1
1
50
00
#
#
*
#
#
*
#
#
#
#
**
#
#
#
*
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
5
0
0
*
*
*
*
*
2
5
6
12
14
16
17
21
23
25 MINO
Con
LPS
LPS+1μM
LPS+10μM
LPS+30μM
LPS+100μM
1
1
50
00
*
*
*
*
5
0
0
*
*
*
*
2
5
6
12
14
16
17
21
23
25 MINO
Fig. 4. Anti-neuroinflammatory activities and cytotoxicities of ethanol extract, PE extract and isolated compounds on LPS-induced NO production in BV2 microglial
cells. A: Anti-neuroinflammatory effect and cytotoxicity of ethanol extract; B: Anti-neuroinflammatory effect and cytotoxicity of PE extract; C: Anti-neuroin-
flammatory effect and cytotoxicities of compounds 2, 5, 6, 12, 14, 16, 17, 21, 23, 25. (Each bar represents the means ± SE of three independent experiments.
#
Significance: *P < 0.001 compared to LPS groups. P < 0.001 compared to control groups; Ext-1: ethanol extract; Ext-2: PE extract; Con: control, NO: nitric oxide,
LPS: lipopolysaccharide, MINO: minocycline).
4

D. Zhou, et al.
Bioorganic Chemistry 97 (2020) 103715
Table 2
3.3. Neuroinflammatory activities of compounds 1–30 in LPS-induced BV2
Effects of extracts and identified compounds from B. striata on NO production
cells
by LPS-activated BV2 microglia cells (Mean ± SEM).d
IC50a
IC50a
To investigate the anti-inflammatory effects of the extracts and
isolated components (1–30), the inhibitory activities were evaluated by
NO assay in LPS-induced BV2 cells. In order to avoid that the inhibitory
activities exhibited by tested samples were due to their cytotoxicities,
the viabilities of BV2 cells were measured using MTT methods before
NO assays. Concentration higher than 10 µM used in our present study
is mainly based on other reports in which the highest concentration of
compounds was set to 100 µM or higher to investigate their anti-in-
flammation effect in BV2 cells [35,36]. Moreover, we also want to in-
vestigate compounds cytotoxic activities comprehensively in a wider
range, thus we set the highest dose to 100 µM. As you mentioned the
compounds are toxic at 100 µM which should not be used for anti-
inflammation evaluation.
Sample name
Ext-1b
Sample name
4.3 ± 1.6
75.3 ± 2.1
72.7 ± 1.1
> 100
5
31.8 ± 2.1
66.1 ± 2.4
61.1 ± 2.6
58.8 ± 2.6
18.1 ± 2.8
b
Ext-2
6
b
Ext-3
16
17
b
Ext-4
c
2
96.0 ± 2.6
Minocycline
a
b
IC50 (µg/mL for extracts and µM for compounds).
Ext-1: total ethanol extract of B. striata; Ext-2: PE extract of ethanol extract
of B. striata; Ext-3: EtOAc extract of ethanol extract of B. striata; Ext-4: n-BuOH
extract of ethanol extract of B. striata.
c
d
Minocycline was used as a positive control.
Compounds 1, 3, 4, 7, 8, 9, 10, 11, 13, 15, 18, 19, 20, 22, 24, 26, 27, 28,
2
9 and 30 showed no inhibitory activity at tested concentrations (1, 10, 30,
As shown in Fig. 4A, B and Table 2, the ethanol extract displayed
remarkable inhibitory effects against NO production, with IC50 value of
1
00 µM), compounds 12, 14, 21, 23, 25 showed toxicities at 30 or 100 µM.
4
.3 µg/mL and cytotoxicity was not observed at the all tested con-
s), δ
C
55.9] was deduced to be linked at C-3 from the HMBC correlation
3.75 (3-OCH ) and 161.4 (C-3). A pyrrolidone moiety was es-
centrations (1, 3, 10, 30, and 100 μg/mL). Of note, stilbenes-rich
fraction, PE extract could decrease the production of NO with IC50
value at 75.3 µg/mL without cytotoxicity observed in the experiment
of δ
H
3
tablished by 5.03 (1H, dd, J = 9.0, 5.0 Hz, H-5″), 2.39 (1H, m, H-3″a),
2
.50 (1H, m, H-3″b), 2.06 (1H, m, H-4″a) and 2.32 (1H, m, H-4″b), as
well as the HMBC correlations of H-3″/C-5″, H-4″/C-2″ (C]O), H-5″/C-
″, and C-2″ (see Fig. 2). The HMBC correlations from δ 5.03 (H-5″) to
[
the tested results of other portions, EtOAc (IC50, 72.7 µg/mL) and n-
BuOH extract (IC50 > 100 µg/mL)]. Therefore, PE and ethanol extracts
may have potential anti-inflammation activity, however PE is less ac-
tive compared to ethanol extract (Fig. 4).
3
H
δ 143.3 (C-1) and 161.4 (C-3), and from H-4″ to C-2 allowed the at-
C
tachment of the 2-oxopyrrolidin-5-yl group at C-2 (See Fig. 1 and
As shown in Fig. 4C, compounds 12, 14, 21, 23, 25 were toxic to
cells at a concentration of 30 µM or 100 µM, which may affect their
inhibitory effects on LPS-induced NO release. Taking the anti-in-
flammatory and cytotoxic activities into consideration, we found that
compounds 2, 5, 6, 16, 17 can greatly inhibit the production of NO
without showing cytotoxicity, with IC50 values at 96.0, 31.8, 66.1, 61.1,
Table 1). Therefore, structure 23 was determined as dusuanlansin E
(
Fig. 2).
Due to negligible or weak optical rotations and Cotton effects in
circular dichroism (CD) spectrum of compound 23, chiral analysis and
optical resolution of 23 were achieved by HPLC with a Chiral pack IF
chiral column (n-hexane/EtOH, 85:15) at 1.0 mL/min, which afforded
compounds 23a and 23b. The relative peak area ratio of 23a to 23b
was approximately 1:1, the absolute configuration of 23a was con-
firmed to be 5′R, and the absolute configuration of 23b was confirmed
as 5′S by combining the CD spectrum of 23a (Fig. 3) [31]. Thus, the
structures of 23a and 23b were assigned and named dusuanlansin E1
and dusuanlansin E2 (Fig. 3).
5
8.8 μM, respectively. Previous studies also reported that the effect of
compound on NO release can reflect its anti-inflammation activity.
However, this single assay may not be sufficient to indicate anti-in-
flammation activity of a compound. In future, we will further in-
vestigate the level of other pro-inflammatory mediators such as TNF-α,
IL-6 and IL-1β, and explore the effect of the compound on signaling
pathways related with inflammatory response to clarify its anti-in-
flammatory activity comprehensively.
Compound 27 was obtained as a brownish powder. High resolution
ESI-MS analysis of 27 yielded a quasi-molecular ion peak at m/z
−
On the corresponding references data (only some compounds):
compounds 5 (50.2 µM), 14 (80.2 µM), and 25 (> 100 µM) [31,37].
And these results are also very close to our measured values. But there
are few reports in the literature about SAR. Combining the activities
with structures of the isolated components, brief structure-activity re-
lationships could be suggested as follows. Firstly, the glycosidations of
stilbenes were considered to be the negative factor for their anti-in-
flammatory activities, such as compounds 5 vs 27, 29, 30 (31.8 μM
vs > 100 μM). Secondly, the presence of pyrrolidone moiety was det-
rimental to the inhibitory effects and increased the cytotoxicities, such
as 5 vs 23 (31.8 μM vs 67.7 μM). In addition, as to compounds 2 and 6,
the presence of 3-methoxy was beneficial to the activity of dihy-
dropinosylvi (2) with IC50 values of 96.0 and 66.1 μM, respectively
4
05.1554 [M−H] (calcd. 405.1555 for C21
H
25
O
8
), in accordance with
1
13
the molecular formula, C21
H
26
O
8
. Its H NMR and C NMR spectral
characteristics were very similar to those of analogous compound ba-
1
tatasin III. However, signals of 4.80 (H-1″) in H NMR spectrum in-
dicated the presence of a glucose substituent. This conclusion was also
supported by signals of 100.4 (C-1″), 73.3 (C-2″), 76.7 (C-3″), 69.7 (C-
4
″), 77.0 (C-5″), 60.7 (C-6″). And glycosyl unit was shown to be located
at C-3′ according to the long range correlations between 4.80 (1H, d,
J = 7.2 Hz, H-1″) and 157.5 (C-3′). According to the large coupling
constant, the configuration was confirmed to be β. Acid hydrolysis of 27
produced glucose as the sole sugar identified on the basis of derivati-
zation by comparing with an authentic sugar sample [14]. The structure
of 27 was elucidated as 3-hydroxy-5-methoxybibenzyl-3′-O-β-D-gluco-
pyranoside.
(
Fig. 5).
Fig. 5. The relationships of.
5

D. Zhou, et al.
Bioorganic Chemistry 97 (2020) 103715
4
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Declaration of Competing Interest
The authors declare no competing financial interests.
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