Iridoid glycosides from the fruits of Cornus officinalis

Article abstract of DOI:10.1080/10286020.2018.1497609

Four new iridoid glycosides named cornusphenosides A–D (1–4) were isolated from an ethanol extract of the fruits of Cornus officinalis (shan zhu yu). The structures of these compounds were elucidated on the basis of spectroscopic data (UV, IR, HRESIMS, and 1D and 2D NMR) and chemical evidence. The neuroprotective effects of compounds 1–4 were also assessed in vitro. (Figure presented.).

Full text of DOI:10.1080/10286020.2018.1497609

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Iridoid glycosides from the fruits of Cornus
officinalis
Xin Wang, Chong-Hui Liu, Jin-Jie Li, Bo Zhang, Lin-Lin Ji & Xiao-Ya Shang
To cite this article: Xin Wang, Chong-Hui Liu, Jin-Jie Li, Bo Zhang, Lin-Lin Ji & Xiao-Ya Shang
(2018): Iridoid glycosides from the fruits of Cornusꢀofficinalis, Journal of Asian Natural Products
Research, DOI: 10.1080/10286020.2018.1497609
To link to this article: https://doi.org/10.1080/10286020.2018.1497609
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1497609
Iridoid glycosides from the fruits of Cornus officinalis
†
†
Xin Wang , Chong-Hui Liu , Jin-Jie Li, Bo Zhang, Lin-Lin Ji and Xiao-Ya Shang
Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University,
Beijing 100191, China
ABSTRACT
ARTICLE HISTORY
Received 11 April 2018
Accepted 3 July 2018
Four new iridoid glycosides named cornusphenosides A–D (1–4)
were isolated from an ethanol extract of the fruits of Cornus offici-
nalis (shan zhu yu). The structures of these compounds were elu-
cidated on the basis of spectroscopic data (UV, IR, HRESIMS, and
KEYWORDS
Cornaceae; Cornus
officinalis; iridoid glycosides;
cornusphenosides A–D;
isolation; structural
elucidation
1
D and 2D NMR) and chemical evidence. The neuroprotective
effects of compounds 1–4 were also assessed in vitro.
GRAPHICAL ABSTRACT
O
H
O
H
OMe
O
H
OMe
O
O
O
O
O
O
O
O
O
H
O
H
O
O
O
O
O
HO
OH HO
HO
OH HO
OH
OH
1
2
7-β
7-α
3
4
7-β
7-α
Cornus officinalis
1. Introduction
The genus Cornus belongs to the family Cornaceae and consists of four species dis-
tributed mainly in Southern and Central Europe, Eastern Asia, and Eastern North
America [1]. Cornus officinalis is the most widely used specie in this genus, and its
fruits are known as “shan zhu yu” and used for treatment of various disorders in
traditional Chinese medicine [2]. Modern pharmacology studies revealed that the
extract and chemical constituents of C. officinalis are bioactive, and exhibit neuropro-
tective [3], anti-inflammatory [4], anti-diabetes [5], anti-oxidant [6], and cardiovascu-
lar system [7] activities. Previous phytochemical studies of this species have led to the
isolation and identification of iridoid glycosides, flavonoids, terpenoids, and several
other components [8–12]. Iridoid glycosides are an important type of constituent
contributing to bioactivity, and have been revealed to possess potential anti-
Alzheimer’s disease activity [13,14]. As a part of our ongoing search for bioactive nat-
ural products, we found that a 40% ethanol elution fraction of the ethanol extract of
CONTACT Xiao-Ya Shang
These authors contributed equally to this work.
shangxiaoya@buu.edu.cn
†
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2018.1497609.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
X. WANG ET AL.
O
5
O
6'''
6
'''
H
H
'''
O 11 OMe
5'''
O 11 OMe
O
O
7
1
2
12
H
H
6
6
4
'''
O
7
4
4'''
O
4
3
O
3
5
5
3
'''
1'''
3'''
1'''
9
9
O 8
O 8
10
O
1
O
1
H
6'
H
O
6'
O
O
'
O
O
1''
1''
1
0
3''
5''
3''
5''
O
2''
O
1
1'
2''
4
'' 9''
4''
9
''
HO 2'
OH
HO
HO 2'
OH
HO
OH
OH
2
7''
7
''
1
O
H
OMe
3
O
H
OMe
6
6
O
7
O
7
1'''
1
'''
3
5
8 9 5 1 O
9
O
O
8
1 O
O
1''
H
O
O
H
6
'
6
'
O
1'
O
O
'
1''
1
0
3''
1
0
3''
O
O
2''
2''
1
4'' 9''
4
'' 9''
HO 2'
OH
HO 2'
OH HO
HO
5''
5''
OH
OH
7''
7
''
4
3
Figure 1. Structures of compounds 1–4.
C. officinalis obtained over a macroporous resin showed potential anti-Alzheimer’s
disease activity in vitro and in vivo assays. Chemical and biological investigations of
this fraction were performed, and the subfraction without morroniside and loganin
still showed significant neuroprotective activity in vitro assay. In order to investigate
additional minor iridoid glycosides with novel structures and neuroprotective activity
from C. officinalis, bioassay-guided fractionation led to the isolation of four new
minor iridoid glycosides cornusphenosides A–D (1–4) (Figure 1). Herein, the isola-
tion and structure elucidation and biological assays of these iridoid glycosides have
been described.
2. Results and discussion
Compound 1 was isolated as a white amorphous powder with a specific rotation of
2
5
[
[
a]
–118. The HRESIMS data showed a molecular ion peak at m/z 685.2097
D
þ
M þ Na] , establishing the molecular formula as C H O . Its IR spectrum dis-
32
38 15
ꢀ
1
played absorption bands at 3431, 1741, 1704, and 1679 cm
attributed to the
1
hydroxyl and carbonyl functionalities. The H NMR spectrum of 1 exhibited one
methyl group at dH 1.23, one methoxy group at dH 3.64, two methines at dH
1
.75–1.77 and 2.96–3.01, two methylenes at dH 1.30–1.37, 1.76–1.80, 2.88, and 3.09,
two oxygen-bearing methines at dH 4.22 and 4.36, one oxygen-bearing methylene at
d 4.43 and 4.49, two acetal protons at d 5.79 and 4.70–4.72, seven olefinic protons
H
H
at dH 7.15–7.17, 7.21, 6.50 and 7.30, one oxygen-bearing olefinic proton at d 7.45,
H
1
and one aldehyde group at d 9.50. The H NMR data of 1 also showed the presence
H
of a b-glucopyranose moiety, the anomeric proton of which resonated at d 4.76
H
0
13
(
1H, d, J ¼ 7.4 Hz, H-1 ). Consistent with these observations, the C NMR spectrum

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
O
5
O
6
'''
6'''
H
H
1
1
11
'''
O
H
OMe
5'''
O
H
OMe
12
O
O
1
2
H
4
H
4
6
4
''
'''
O
6
8
4
'''
O
3
5
9
3
5
9
7
3
'''
1'''
7
3'''
1'''
O
O
O
O
1
8
0
H
H
1
1
O
10
O
6
'
6'
O
O
O
O
1'
1
''
1''
3
''
3''
5''
O
2''
HO
O
2''
1
'
4
9''
4''
9''
HO
OH
HO 2'
OH HO
2
'
OH
5''
OH
7
''
7''
1
2
1
1
O
H
OMe
O
H
11 OMe
12
1
2
H
H
6
6
8
O
O
4
1
4
5
9
3
3
7
O
7
O
5
1
'''
1
'''
O
9
1
O
8
0
H
H
1
O
10
6
'
6'
O
O
O
O
O
1
''
3''
1''
O
2''
O
3''
2''
1
'
1
'
4
''
9''
4''
9''
HO 2'
OH
HO
HO 2'
OH
HO
OH
5''
OH
5''
7
''
3
4
1
1
Figure 2. Key HMBC (
) and H- H COSY ( ) correlations of compounds 1–4.
O
O
5
6'''
6
'''
'''
H
H
'''
O 11 OMe
5'''
O 11 OMe
12
O
O
1
2
H
H
H
H
6
4
4
'''
O
6
4
4
O
3
3
O
5
7
H
5
7
H
3'''
1'''
3
'''
1'''
9
O
9
O
1
O
1
8
0
H
8
H
H
H
1
O
10
O
6
'
6'
O
O
O
O
1
''
1''
3
''
3''
2''
O
2''
O
1
'
1'
4
'' 9''
4'' 9''
HO
OH
HO 2'
OH
2
'
HO
HO
OH
5''
OH
2
5''
7
''
7''
1
O 11 OMe
O 11 OMe
12
1
2
H
H
H
H
6
4
6
O
O
4
3
O
3
O
5
5
7
H
7 H
1
'''
1'''
9
9
O
O
1
1
8
0
H
H
8
H
H
2'
1
O
10
O
6
'
6'
O
O
O
O
1
''
1''
3
''
3''
O
2''
HO
O
2''
HO
1
'
1'
4
'' 9''
4'' 9''
HO
OH
HO
OH
2
'
OH
5''
OH
5''
7
''
3
4
Figure 3. Key NOESY (
) correlations of compounds 1–4.

4
X. WANG ET AL.
showed 32 carbon resonances, including 1 methyl, 1 methoxyl, 2 methylenes, 2
methines, 1 oxygenated methylene, 2 oxygenated methines, 3 acetal carbons, 12 ole-
finic carbons (d 111.8, 154.5, 138.9, 130.3, 130.3, 129.4, 129.4, 127.8, 159.8, 112.7,
C
1
24.4, 154.1), three carbonyl carbons (d 168.6, 175.0, and 179.4), and resonances for
C
a glucopyranosyl group.
The HMBC correlations from H-1 to C-3, C-5, and C-8; from H-3 to C-11 and C-
0
5
; from H-6 to C-4 and C-7; from H-7 and C-5, C-8; from H-1 to C-1; together
with their chemical shifts, revealed the presence of an iridoid glycoside moiety
Figure 2). These correlations and chemical shifts were similar to those previously
(
reported for 7b-morroniside [15]. The evident differences were that 1 showed reso-
nances due to an additional 5-hydroxymethyl furfural group and a phenyllactic acid
0
00
000
000
group. In the HMBC spectrum, correlations from H-4 to C-2 and C-6 ; and
0
00
000
000
from H-3 to C-5 and C-1 revealed the presence of the 5-hydroxymethyl furfural
moiety in 1, and correlations from H-1 a/1 b to the carbon at d 97.9 indicated
0
00
000
C
that the 5-hydroxymethyl furfural moiety was presented at C-7 of the 7b-morroniside
0
0
00
00
00
moiety. In addition, the HMBC correlations from H-2 to C-1 , C-3 , and C-4 ;
00
00
00
00
from H-3 to C-1 , C-5 , and C-9 , together with the chemical shifts showed the
0
presence of a phenyllactic acid moiety which was connected to the Glc-6 . This was
0
0
further confirmed by the HMBC correlations of H-6 a/6 b with the ester carbonyl at
dC 175.0.
The relative stereochemistry of 1 was determined by the NOESY spectrum and
coupling constants (Figure 3). In the NOESY spectrum, correlations from H-1 to H-
10 and H-6a, from H-7 to H-6a indicated that H-1, H-6a, H-7 and H-10 were on the
same side in a orientation; correlations from H-5 to H-6b, H-8, and H-9 confirmed
that H-5, H-6b, H-8, and H-9, in turn, on the other side in b oriented. This was also
substantiated based on biogenetic grounds in that nearly all iridoids found in nature
have a configuration of 5b and 9b, and the coupling constant of H-1 and H-9
(
J ¼ 9.2 Hz) indicated that the iridoid moiety was cis-fused, thus H-1 should be a ori-
ented. In addition, the b configuration at C-7 was also determined by comparison of
the data to those of the known compound, 7b-morroniside [15]. Acid hydrolysis of 1
afforded D-glucose which was determined by GC-MS of methanolysate and silylated
derivatives, and the b-anomeric configuration of the glucosyl unit was judged from
its large coupling constant (J ¼ 7.4 Hz). Analysis of the HSQC, H- H COSY, HMBC,
and NOESY spectra led to the complete assignments of the proton and carbon signals
in compound 1 (Table 1). On the basis of the above data, cornusphenoside A was
characterized as shown.
1
1
Compound 2 was found to have the same molecular formula, C H O , as estab-
3
2
38 15
þ
lished by its HRESIMS (m/z 685.2096 [M þ Na] ). The IR spectrum of 2 showed
ꢀ
1
characteristic absorption bands for the hydroxyl (3424 cm ) and carbonyl (1739,
ꢀ
1
1
678 cm ) groups. The NMR spectroscopic data of 2 were also very similar to those
of 1 except for the downfield shifts of C-5 (d 31.7), C-6 (d 35.5), C-7 (d 102.5)
C
C
C
and C-8 (d 74.0). These changes suggested that the iridoid glycoside skeleton of 2
C
was 7a-morroniside [15] instead of 7b-morroniside, which was confirmed by the
NOESY correlations of H-7 with H-5 and H-8b. Thus, the spectroscopic data of 2

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5

6
X. WANG ET AL.
indicated that it is an epimer of 1 and the configuration of H-7 is b-oriented. Hence,
the structure of 2 (cornusphenoside B) was established as shown.
Compound 3 was isolated as a white amorphous powder, and its molecular for-
mula was established as C H O on the basis of HRESIMS (m/z 591.2045
2
7
36 13
þ
[
(
2
M þ Na] ). Its IR spectrum showed the absorption characteristic of hydroxyl
ꢀ1 ꢀ1 13
3441 cm ) and carboxyl (1739, 1708 cm ) groups. The C NMR spectrum showed
7 carbon signals including six for a glucopyranosyl unit, nine for a phenyllactic acid
group, and the remaining 12 for an iridoid skeleton. Comparison of the spectroscopic
data of 3 with those of 1 showed that they were almost superimposable on those of
the iridoid glycoside skeleton and phenyllactic acid group, but the 5-hydroxymethyl
furfural group at C-7 in 1 was replaced by a methoxy moiety in 3. In the HMBC
0
00
spectrum, the correlation from H-1 to C-7 confirmed the assignment of the
methoxy moiety to C-7 of the iridoid glycoside group. NOESY experiments indicated
that the relative stereochemistry of 3 was the same as that of 1. The structure of this
compound was confirmed by detailed analysis of the 2D NMR data including HSQC,
1
1
H- H COSY, HMBC, and NOESY spectra. Accordingly, cornusphenoside C was
characterized as 3.
Compound 4 shared the same molecular formula C H O as 3 with HRESIMS
2
7
36 13
þ
ꢁ
ion at m/z 591.2045 [M þ Na] and required 10 of unsaturation. It exhibited similar
UV, IR, and NMR data to 3, suggesting their structural resemblance. However, a
comparison of NMR data revealed that 7b-O-methyl-morroniside in 3 was replaced
by 7a-O-methyl-morroniside in 4. This discrepancy was confirmed by NOESY correl-
ation of H-7 with H-8b. With the exception of this difference, other 1D NMR data of
4
and 3 were matched. Thus compound 4 was named as cornusphenoside D.
Compounds 1–4 were tested for their neuroprotective effects against hydrogen per-
oxide (H O )-induced cell injury in SH-SY5Y cells. However, at 10 lM, all exhibited
2
2
no effect on H O -induced neurotoxicity.
2
2
In conclusion, four new iridoid glucosides, cornusphenosides A–D, were isolated
from the fruit of C. officinalis and evaluated for their neuroprotective activities. Four
new compounds all have a phenyllactic acid group in sugar moiety; additionally, cor-
nusphenosides A and B also have a rare furan ring in iridoid skeleton. Experiments
were taken to prove that 1–4 are genuine natural products.
3. Experimental
3.1. General experimental procedures
IR spectra were recorded on a Nicolet 5700 spectrometer by FT-IR microscope trans-
mission method (Nicolet Instrument, Inc., Madison, Wisconsin, USA). CD spectra
and UV data were recorded on a JASCO J-810 circular dichroism spectrometer
(JASCO Corporation, Tokyo, Japan). HRESIMS was performed using an Agilent 1100
series LC/MSD ion trap mass spectrometer (Agilent, Santa Clara, California, USA).
The 1D- and 2D-NMR spectra were acquired in CD OD with TMS as internal stand-
3
ard on Varian 500 MHz and Bruker AV500-III spetrometers (Bruker Corporation,
Billerica, Massachusetts, USA). Column chromatography was performed with silica
gel (160–200 mesh, Qingdao Marin Chemical, Inc., Qingdao, China) and Sephadex

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC experiments were performed
with an instrument consisting of a Waters 600 pump, and a Waters 2996 dual-wave-
length absorbance detector (Waters Corporation, Milford, Massachusetts, USA) with
a Sunfire (Waters, 250 ꢂ 10 mm i.d.) preparative column packed with C18 (5 lm)
(Alltech Associates, Inc., Bannockburn, Illinois, USA). TLC was carried out with glass
precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying
with 8% H SO in 95% EtOH followed by heating.
2
4
3
.2. Plant material
The fruits of Cornus officinalis were purchased from Tong-Ren-Tang Company in
Beijing, and authenticated by Professor Wen Wang (Xuanwu Hospital of Capital
Medical University, China). A voucher specimen (20090305) has been deposited at
the Beijing Union University, Beijing Key Laboratory of Bioactive Substances, and
Functional Foods, Beijing, China.
3
.3. Extraction and isolation
The dried fruits (10 kg) of C. officinalis were extracted with 50% EtOH (100 L ꢂ 3) at
room temperature. The combined extracts were concentrated under reduced pressure to
dryness. The residue was suspended in H O and applied to a Diaion HP-20 column that
2
was eluted using a stepwise gradient of EtOH-H O (0:100, 20:80, 40:60, 70:30, and 95:5,
2
v/v), to afford five fractions. After removing the solvent, the EtOH-H O (40:60) eluate
2
(500 g) was separated subsequently by silica gel chromatography eluting with
CHCl –MeOH (15:1 to 8:1, v/v) to afford four major fractions (F –F ) based on TLC
3
1
4
analysis. Fraction F was chromatographed on a reversed-phase C18 silica column,
2
eluted with an EtOH-H O (5:95–100:0) gradient, to give three subfractions F2-1–F2-3
.
2
Fraction F2-3 (128.3 g) was subjected to a column of silica gel, eluting with
CHCl –MeOH (20:1–3:1, v/v), to give six subfractions F2-3-1–F2-3-6. Fraction F2-3-2
3
(
(
(
10.1g) was chromatographed over Sephadex LH-20 and eluted with CHCl –MeOH
2:1) as the mobile phase to give four subfractions F2-3-1-1–F2-3-1-4. Fraction F2-3-1-1
2.7 g) was separated using a reversed-phase C18 silica gel column, by eluting with a
3
MeOH-H O (30:70–100:0) gradient, to yield subfractions F2-3-1-1-1–F2-3-1-1-7. Fraction F2-3-
2
(
76 mg) was purified by preparative reversed-phase HPLC, eluting with MeOH–H O
1-1-6
2
(
58:42, 2.5 ml/min) to afford 1 (9.3 mg, t ¼ 16 min) and 3 (5.1 mg, t ¼ 20 min). Further
R
R
separation of fractions F
(103mg) by Sephadex LH-20 gel, eluting with MeOH,
-3-1-1-7
2
which was purified by preparative reversed–phase HPLC, eluting with MeOH-H O (58:42,
2
2
.5 ml/min) to afford 2 (4.3 mg, t ¼ 40min), and 4 (7.8 mg, t ¼ 44 min).
R
R
3
.3.1. Cornusphenoside A
2
5
White amorphous powder, [a] ꢀ118 (c 0.08, CH OH); UV k
(MeOH, log e):
max
D
3
ꢀ
1
4
2
2
35 (4.75), 280 (4.20) nm; CD (c 3.7 ꢂ 10 , MeOH) De
–5.64; IR ꢀmax 3431,
24 nm
2
ꢀ
1
13
912, 1741, 1704, 1679, 1522, 1440 cm ; H NMR (methanol-d , 500 MHz) and
C
4
NMR (methanol-d , 125 MHz) spectral data, see Table 1; (þ)-HRESIMS m/z:
4
þ
6
85.2097 [M þ Na] (calcd for C H O Na, 685.2103).
3
2
38 15

8
X. WANG ET AL.
3
.3.2. Cornusphenoside B
2
5
White amorphous powder, [a] ꢀ28 (c 0.02, CH OH); UV k
(MeOH, log e): 235
D
3
max
ꢀ
4
(
1
4.10), 280 (3.92) nm; CD (c 3.1 ꢂ 10 , MeOH) De
–5.61; IR ꢀmax 3424, 2938,
28 nm
2
ꢀ
1
1
13
739, 1678, 1641, 1440 cm ; H NMR (methanol-d , 500 MHz) and C NMR
4
(
[
methanol-d , 125 MHz) spectral data, see Table 1; (þ)-HRESIMS m/z: 685.2096
4
þ
M þ Na] (calcd for C H O Na, 685.2103).
3
2
38 15
3
.3.3. Cornusphenoside C
2
5
White amorphous powder, [a] ꢀ97.6 (c 0.05, CH OH); UV k
(MeOH, log e):
3441, 2940, 1739,
D
3
max
ꢀ
4
2
1
38 (4.36) nm; CD (c 2.2 ꢂ 10 , MeOH) De
–3.03; IR ꢀ
2
25 nm
max
13
ꢀ
1 1
708, 1639, 1440 cm ; H NMR (methanol-d , 500 MHz) and C NMR (methanol-
4
þ
d , 125 MHz) spectral data, see Table 1; (þ)-HRESIMS m/z: 591.2045 [M þ Na]
4
(calcd for C H O Na, 591.2048).
27 36 13
3
.3.4. Cornusphenoside D
2
5
White amorphous powder, [a] ꢀ68 (c 0.03, CH OH); UV k
(MeOH, log e): 238
3418, 2938, 1704, 1702,
D
3
max
ꢀ
4
(
1
1
4.82) nm; CD (c 4.2 ꢂ 10 , MeOH) De
–9.81; IR ꢀ
2
27 nm
max
13
ꢀ1
1
640, 1441 cm ; H NMR (methanol-d , 500 MHz) and C NMR (methanol-d ,
4 4
þ
25MHz) spectral data, see Table 1; (þ)-HRESIMS m/z: 591.2045 [M þ Na] (calcd
for C H O Na, 591.2048).
2
7
36 13
3
.4. Acid hydrolysis of compounds 1–4
Each compound (1 mg) was individually refluxed in 2 M HCl (2.0 ml) at 80 C for
.5 h. The reaction mixture was extracted with EtOAc, and H O phase evaporated
ꢁ
1
2
under vacuum, diluted repeatedly with H O, and evaporated in vacuo to furnish a
2
neutral residue. The residue was then dissolved in anhydrous pyridine (1.0 ml), to
which 1 mg of L-cysteine methyl ester hydrochloride was added. The mixture was
ꢁ
stirred at 60 C for 2 h and after evaporation in vacuo to dryness, 0.1 ml of N-trime-
ꢁ
thylsilylimidazole was added. After stirring the mixture another 2 h at 60 C, it was
partitioned between n-hexane and H O (1.0 ml each), and then the n-hexane extract
2
was analyzed by GC under the following conditions: capillary column, HP-5 (30 m,
ꢁ
0
.25 mm, with a 0.25 lm film; Dikma); detection, FID; detector temperature, 280 C;
ꢁ
ꢁ
ꢁ
injection temperature, 250 C; initial temperature, 160 C, then raised to 280 C at
ꢁ
5
C/min, final temperature maintained for 10 min; carrier gas, N . From the acid
2
hydrolysate of each compound, D-glucose was confirmed by comparison of the reten-
tion time of its derivative with that of authentic sugar derivatized in a similar way,
which showed a retention time of 20.55 min.
3
.5. Neuroprotective activity assay
Human neuroblastoma SH-SY5Y cells were cultured in 100 mm dishes and grown in
DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100
ꢁ
lg/ml streptomycin at 37 C with 5% CO . Cells were cultured in 96-well plates at a
2
4
density of 1 ꢂ 10 cells/well in 200 ll for 24 h. Cells were incubated with test

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
compounds (10 lM) for 12 h. To induce an oxidative stress, 100 lM H O freshly
2
2
ꢁ
prepared was added to the cells and incubated at 37 C for 1.5 h. Cell viability was
determined colourimetrically using MTT assay. Then 20 ll of the MTT solution
ꢁ
(
5 mg/ml) was add into each well. After incubation for 4h at 37 C, the cells were
finally lysed with 150 ll of DMSO. The absorbance was read at 490 nm with a micro-
plate reader. The value for cell viability was expressed as the percentage of the control
value.
Disclosure statement
There are no potential conflicts of interest to declare.
Funding
The authors gratefully acknowledge financial support from the Key projects of the Beijing
Natural Sciences Foundation and Beijing Municipal Education Committee (Grant No.
KZ201811417049) and Scientific Research Common Program of Beijing Municipal
Commission of Education (Grant No. KM201811417008).
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