Quinic acid derivatives and coumarin glycoside from the roots and stems of Erycibe obtusifolia

Article abstract of DOI:10.1016/j.phytol.2015.10.002

A new quinic acid derivative (1) and a new coumarin glycoside (8), together with six known compounds (2-7) were isolated from the roots and stems of Erycibe obtusifolia. The structures of the new compounds were elucidated by spectroscopic and chemical analyses. The in vitro antiviral activity against the respiratory syncytial virus (RSV) of seven quinic acid derivatives was evaluated by cytopathic effect (CPE) reduction assay. Among them, the dicaffeoylquinic acids (6 and 7) displayed potent in vitro anti-RSV activity.

Full text of DOI:10.1016/j.phytol.2015.10.002

Phytochemistry Letters 14 (2015) 185–189
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Quinic acid derivatives and coumarin glycoside from the roots and
stems of Erycibe obtusifolia
Long Fana,b,c,1, Ying Wanga,b,1, Ning Lianga, Xiao-Jun Huanga,b, Chun-Lin Fana,
a,b,
a,**
*
Zhen-Long Wua,b, Zhen-Dan Hec, Yao-Lan Li , Wen-Cai Ye
a
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China
JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, Jinan University, Guangzhou 510632, PR China
Department of Pharmacy, School of Medicine, Shenzhen University, Shenzhen 518060, PR China
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 16 May 2015
Received in revised form 2 September 2015
Accepted 5 October 2015
Available online xxx
A new quinic acid derivative (1) and a new coumarin glycoside (8), together with six known compounds
(2–7) were isolated from the roots and stems of Erycibe obtusifolia. The structures of the new compounds
were elucidated by spectroscopic and chemical analyses. The in vitro antiviral activity against the
respiratory syncytial virus (RSV) of seven quinic acid derivatives was evaluated by cytopathic effect (CPE)
reduction assay. Among them, the dicaffeoylquinic acids (6 and 7) displayed potent in vitro anti-RSV
activity.
Keywords:
ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Erycibe obtusifolia
Convolvulaceae
Quinic acid derivative
Coumarin glycoside
Antivirus activity
RSV
1. Introduction
obtusifolia, together with their in vitro antiviral effect against the
respiratory syncytial virus (RSV) (Fan et al., 2013). In order to
The genus Erycibe (Convolvulaceae) comprises more than 60
species, most of which are distributed in tropical Asia (Fang and
Huang, 1979), and some species are used as traditional medicines
in China and Thailand (Hsu et al., 1998; Matsuda et al., 2004;
Tanawan et al., 2007). Previous phytochemical investigations have
revealed that the main constituents of this genus plants are
coumarins, quinic acid derivatives, alkaloids, flavonoids, and
diglycosides (Liu et al., 2007, 2014; Lu et al., 1986; Morikawa
et al., 2006; Matsuda et al., 2004, 2007; Song et al., 2010; Yao and
Chen,1979; Pan et al., 2009; Fan et al., 2013). The roots and stems of
Erycibe obtusifolia are commonly used as a traditional Chinese
medicine to treat various rheumatoid diseases (Hsu et al., 1998). In
a previous study, we reported the isolation of several quinic acid
derivatives and coumarin glycosides from the roots and stems of E.
search for more anti-RSV constituents from this plant, our
continuing phytochemical studies have led to identification of a
new quinic acid derivative (1) and a new coumarin glycoside (8),
together with six known compounds (2–7) (Fig. 1). In addition,
seven quinic acid derivatives were tested for their in vitro anti-RSV
activity. Here, we describe the isolation and structural elucidation
of two new compounds, as well as the in vitro anti-RSV activity of
the quinic acid derivatives.
2. Results and discussion
Compound 1 was obtained as yellow oil. The molecular formula
of 1 was determined to be C27H30O13 based on HR–ESI–MS at m/z
563.1759 [M + H]+ (calculated for C27H31O13, 563.1759). The UV
spectrum of 1 showed absorption maxima at 216, 288 and 327 nm.
The IR spectrum implied the presence of hydroxyl (3414 cmꢀ1) and
carbonyl (1716 cmꢀ1) groups, as well as aromatic ring (1603 and
1517 cmꢀ1). The 1H NMR spectrum of 1 showed signals for three
oxygenated methine protons at dH 5.54 (1H, m), 5.01 (1H, dd, J = 7.2,
3.2 Hz) and 4.13 (1H, m), as well as four methylene protons at dH
2.30 (1H, dd, J = 13.6, 3.9 Hz), 2.04 (2H, m) and 2.01 (1H, m),
suggesting the presence of a quinic acid moiety. In addition, the 1H
NMR spectrum displayed signals for two aromatic protons at dH
* Corresponding author at: Institute of Traditional Chinese Medicine & Natural
Products, College of Pharmacy, Jinan University, No. 601 Huangpu Road West,
510632 Guangzhou, PR China.
** Corresponding author at: Institute of Traditional Chinese Medicine & Natural
Products, College of Pharmacy, Jinan University, No. 601 Huangpu Road West,
510632 Guangzhou, PR China. Fax: +86 20 85221559.
E-mail address: chyewc@gmail.com (W.-C. Ye).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.phytol.2015.10.002
1874-3900/ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

186
L. Fan et al. / Phytochemistry Letters 14 (2015) 185–189
Fig. 1. Chemical structures of compounds 1–8.
7.21 (2H, s) and two methoxy protons at dH 3.72 (6H, s), which
indicated the presence of a syringoyl moiety in 1. Furthermore, the
Table 1
1H and 13C NMR spectral data of compound 1 (DMSO-d6,
d
in ppm, J in Hz).
1H NMR spectrum showed proton signals due to
a 1,3,4-
Position
dH
dC
trisubstituted benzene ring at dH 7.24 (1H, d, J = 1.7 Hz), 7.03 (1H,
dd, J = 8.2, 1.7 Hz) and 6.78 (1H, d, J = 8.2 Hz), a trans-disubstituted
double bond at dH 7.49 (1H, d, J = 15.9 Hz) and 6.45 (1H, d,
J = 15.9 Hz), and one methoxy protons at dH 3.79 (3H, s), suggesting
the presence of a feruloyl moiety. The 13C NMR and DEPT spectra of
1 revealed the presence of 27 carbon signals, including character-
istic signals due to feruloyl, syringoyl and quinic acid moieties,
respectively. With the aid of 1D and 2D NMR experiments, all of the
1H and 13C NMR signals of 1 were assigned (Table 1).
1
2
–
73.0
35.6
a 2.30 dd (13.6, 3.9)
b 2.01 m
5.54 m
5.01 dd (7.2, 3.2)
4.13 m
2.04 m
3
4
5
6
68.5
72.8
64.4
39.8
7
–
174.2
119.6
107.1
147.4
140.7
164.7
125.4
111.0
148.0
149.5
115.5
123.2
145.3
114.3
166.0
51.8
10
–
20, 60
7.21 s
–
In the HMBC spectrum of 1, the correlations between H-3 (dH
5.54) of quinic acid moiety and C-70 (dC 164.7) of syringoyl moiety,
as well as between H-4 (dH 5.01) of quinic acid and C-900 (dC 166.0)
of feruloyl moiety were displayed, which suggested that the
syringoyl and feruloyl moieties were attached to C-3 and C-4
positions of quinic acid moiety, respectively. The additional
methoxy group (dH 3.65; dC 51.8) was located at C-7 of the quinic
acid based on the HMBC correlation between methoxy protons (dH
3.65) and C-7 (dC 174.2) of quinic acid (Fig. 2). In the ROESY
spectrum, the correlations between H-20, 60 (dH 7.21) and H-5 (dH
30, 50
40
–
70
–
100
–
200
7.24 d (1.7)
–
300
400
–
500
6.78 d (8.2)
7.03 dd (8.2, 1.7)
7.49 d (15.9)
6.45 d (15.9)
–
600
700
800
900
4.13)/ CH3O-300
CH3O-7 (dH 3.65) were observed, which indicated that H-3, H-4 and
the carbonyl group at C-7 were on the configuration, and H-5 was
on the configuration (Fig. 2). Hence, the structure of 1 was
(dH 3.79), as well as between H-4 (dH 5.01) and
7-OCH3
30, 50-OCH3
300-OCH3
3.65 s
3.72 s
3.79 s
55.9
55.7
b
a
determined to be 4-O-feruloyl-3-O-syringoyl quinic acid methyl
ester.
Erycibe are rich in this kind of quinic acid derivative. In addition,
during the isolation procedure, silica gel which was acidic property
and might lead to forming methyl ester if MeOH was used as the
eluent avoided being used, and the liquid chromatography peak of
compound 1 could be observed in the EtOH extract, fractions and
Although only one quinic acid derivative with methyl ester was
reported in this paper, actually there were some references which
have reported many other similar constituents with methyl ester,
even ethyl ester or butyl ester (Liu et al., 2007, 2014; Song et al.,
2010; Fan et al., 2013), indicating that the plants of the genus

L. Fan et al. / Phytochemistry Letters 14 (2015) 185–189
187
Table 2
1H and 13C NMR spectral data of compound 8 (DMSO-d6,
d
in ppm, J in Hz)
Position
dH
dC
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
–
160.6
113.3
144.3
109.9
145.9
149.7
102.8
148.9
112.4
99.0
6.32 d (9.5)
7.98 d (9.5)
7.31 s
–
–
7.26 s
–
–
5.23 d (7.1)
3.36 m
3.29 m
3.30 m
3.36 m
a 4.65 br d (12.0)
b 4.09 dd (12.0, 7.7)
5.05 d (7.4)
3.26 m
3.24 m
3.94 t (8.2)
3.30 m
72.8
70.0
76.8
76.4
64.5
100
99.2
73.0
69.2
73.7
76.7
200
300
400
Fig. 2. Selected HMBC and ROESY correlations of 1.
500
600
3.54 m
–
60.3
1000
122.6
112.5
148.5
150.5
113.9
122.6
165.3
56.0
subfractions in the HPLC experiments. Therefore, compound 1
might not be an artifact product.
2000
7.44 d (1.9)
–
3000
Compound 8 was obtained as yellow oil. The HR–ESI–MS
4000
–
spectrum of
8
exhibited an [M + Na]+ ion at m/z 689.1687
5000
7.19 d (8.8)
7.57 dd (8.8, 1.9)
–
(calculated for C30H34NaO17, 689.1688), corresponding to the
molecular formula C30H34O17. The UV spectrum of 8 showed
absorption maxima at 204, 218, 255, 291 and 342 nm. The IR
spectrum suggested the presence of hydroxyl (3395 cmꢀ1) and
carbonyl (1715 cmꢀ1) groups, as well as aromatic ring (1614, 1571
and 1509 cmꢀ1). The 1H NMR spectrum of 8 revealed the presence
of signals due to a cis-disubstituted double bond at dH 7.98 (1H, d,
J = 9.5 Hz) and 6.32 (1H, d, J = 9.5 Hz), and two aromatic proton
signals at dH 7.31 (1H, s) and 7.26 (1H, s), suggesting the existence
of a 6,7-disubstituted coumarin skeleton in 8 (Song et al., 2010). In
addition, the 1H NMR spectrum showed proton signals due to a
1,3,4-trisubstituted benzene ring at dH 7.57 (1H, dd, J = 8.8, 1.9 Hz),
7.44 (1H, d, J = 1.9 Hz) and 7.19 (1H, d, J = 8.8 Hz), two methoxy
protons at dH 3.82 (3H, s) and 3.77 (3H, s), as well as two anomeric
protons due to sugar moieties at dH 5.23 (1H, d, J = 7.1 Hz) and 5.05
(1H, d, J = 7.4 Hz).
6000
7000
6-OCH3
3000-OCH3
3.82 s
3.77 s
55.5
H-400 (dH 3.94) of another
D
-glucose and C-60 (dC 64.5) of
-glucose,
-glucose and C-4000
(dC 150.5)
D
as well as between H-100 (dH 5.05) of
D
of the vanillic acid moiety were observed, which led to the
elucidation of linkage sequence and positions of these moieties as
shown in Fig. 1. Hence, the structure of 8 was identified as 7-O-[6-
O-(1-O-vanillic acid 4-O-b-D-glucopyranosyl)-b-D-glucopyrano-
syl]-6-methoxycoumarin.
The six other compounds were identified as 4-O-caffeoyl-3-O-
sinapoylquinic acid (2) (Jaiswal et al., 2010), 5-O-caffeoyl-4-O-
sinapoylquinic acid (3) (Nishizawa et al., 1988), 4-O-caffeoyl-3-O-
syringoylquinic acid (4), 5-O-caffeoyl-4-O-syringoylquinic acid (5)
(Song et al., 2010), 3,4-dicaffeoylquinic acid (6) (Ono et al., 2000),
and 3,5-dicaffeoylquinic acid (7) (Wang and Liu, 2007) by
comparison of their spectroscopic data with those reported in
the literature.
Seven quinic acid derivatives were tested for their in vitro
antiviral activities against RSV using a CPE reduction assay. The
MTT method was applied to measure the cytotoxicity of the
compounds. The selectivity index (SI) value calculated from the
ratio of CC50/IC50 was used as an important parameter to evaluate
the antiviral activity of the active compounds. As shown in Table 3,
among these quinic acid derivatives, the two dicaffeoylquinic acids
(6 and 7) possessed strong anti-RSV activity with low IC50 values,
and showed lower cytotoxicity to HEp-2 cells, resulting in much
higher SI values than that of ribavirin. Based on our previous and
present studies (Fan et al., 2013; Geng et al., 2011), the caffeoyl
moiety may be important for antiviral activity. Moreover,
dicaffeoylquinic acids show weaker activity than that of dicaf-
feoylquinic acid methyl esters, suggesting that the methoxy group
at position C-7 provides an advantage in increasing antiviral
activity. These results will be useful to further structural
modifications of quinic acid derivatives.
Acid hydrolysis of 8 afforded D-glucose, which was identified by
high-performance liquid chromatography (HPLC) analysis using an
authentic sample as a reference. The relative anomeric config-
urations of the two sugar units were determined to be
b based on
3
their JH1,H2 coupling constants (J = 7.1 Hz and J = 7.4 Hz). The 13C
NMR and DEPT spectra of 8 revealed the presence of 30 carbon
signals, including signals due to a 6,7-disubstituted coumarin (dC
160.6, 149.7, 148.9, 145.9, 144.3, 113.3, 112.4, 109.9 and 102.8), a
1,3,4-trisubstituted benzene ring (dC 150.5, 148.5, 122.6, 113.9 and
112.5), and two
b-D-glucopyranosyl units (dC 99.2, 99.0, 76.8, 76.7,
76.4, 73.7, 73.0, 72.8, 70.0, 69.2, 64.5 and 60.3). In addition to the
above assigned signals, there were two methoxy (dC 56.0 and 55.5)
and one carbonyl (dC 165.3) groups in 8. With the aid of 1D and 2D
NMR experiments, all of the 1H and 13C NMR signals of 8 were
assigned (Table 2). In the HMBC spectrum of 8, the correlations
between H-2000
benzene ring and the carbonyl carbon (dC 165.3), as well as
between the methoxy protons (dH 3.77) and C-3000
dC 148.5) of
( (dH 7.57) of 1,3,4-trisubstituted
dH 7.44)/H-6000
(
1,3,4-trisubstituted benzene ring were observed, indicating the
presence of a vanillic acid moiety. The additional methoxy group
(
dH 3.82; dC 56.0) was located at position C-6 of coumarin based on
the HMBC correlation between methoxy protons (dH 3.82) and C-6
dC 145.9) of coumarin. In addition, the HMBC correlations between
H-10 (dH 5.23) of
-glucose and C-7 (dC 149.7) of coumarin, between
(
D

188
L. Fan et al. / Phytochemistry Letters 14 (2015) 185–189
Table 3
20:80; 25:75; 30:70, each 4.5 L) as the eluent to give six
In vitro anti-RSV activity of compounds 1–7.
subfractions (Fr. 2–1-Fr. 2–6). Fr. 2–3 (230 mg) was subsequently
purified by preparative HPLC [CH3CN–H2O (0.1% TFA), 25:75, 6 mL/
min] to yield compounds 1 (7 mg), 2 (17 mg), and 3 (11 mg). Fr. 2–2
(325 mg) was purified by preparative HPLC [CH3CN–H2O (0.1%
TFA), 20:80, 6 mL/min] to obtain compounds 4 (15 mg) and 5
(12 mg). Fr. 1 (6 g) was then subjected to an ODS column
(4 ꢁ 30 cm) eluted with gradient mixtures of MeOH–H2O (5:95;
10:90; 15:85; 20:80; 25:75; 30:70, each 4.5 L) to afford six
subfractions (Fr. 1–1-Fr. 1–6). Fr. 1–4 (563 mg) was purified by
preparative HPLC [MeOH–H2O (0.1% TFA), 45:55, 6 mL/min] to give
compounds 6 (21 mg), 7 (30 mg), and 8 (7 mg).
Compoundsa RSV long strain
RSV A2 strain
CC50 (m
g/mL)c
IC50
(
m
g/mL)b SId
IC50
(m
g/mL) SI
1
2
3
4
5
6
7
NAf
NA
NA
32.0
NA
2.5
2.5
2.5
–
–
–
NA
NA
NA
32.0
NA
–
–
–
>200.0
>200.0
>200.0
>200.0
>200.0
>6.3
–
>6.3
–
>80.0 3.0
>80.0 2.2
>66.7 >200.0
>90.9 >200.0
Ribavirine
20.0
2.5
20.0
50.0 ꢄ 2.3
“-” means no detection.
a
The purity of all tested compounds is greater than 98%.
IC50 is the concentration that reduced 50% of CPE in respect to virus control.
CC50 is the concentration of sample with half maximal inhibition on the growth
b
c
3.1.3. 4-O-feruloyl-3-O-syringoyl quinic acid methyl ester (1)
Yellow oil; ½a D
ꢂ
25: - 68.3 (c 0.13, MeOH); UV (MeOH) lmax (log
e):
and survival of HEp-2 cells.
216 (4.45), 288 (4.15), 327 (4.10) nm; IR (KBr): nmax 3414, 1716,
1603, 1517, 1458, 1424, 1334, 1274, 1210, 1119 cmꢀ1; HR–ESI–MS m/
z: 563.1759 [M + H]+ (calcd for C27H31O13, 563.1759); 1H NMR
(DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) data, see
Table 1.
d
SI is selective value = CC50/IC50.
e
Ribavirin (purity ꢅ98%) is the positive control in the test.
f
NA means no active.
3. Materials and methods
3.1. General experimental procedures
3.1.4. 7-O-[6-O-(1-O-vanillic acid 4-O-
glucopyranosyl]-6-methoxy coumarin (2)
25: - 51.0 (c 0.23, MeOH); UV (MeOH) lmax (log
e):
b-D-glucopyranosyl)-b-D-
Optical rotations were recorded by using a JASCO P-1020
polarimeter. UV spectra were measured on a JASCO V-550 UV/vis
spectrophotometer with a 1 cm length cell. IR spectra were
recorded on a JASCO FT/IR-480 plus FT-IR spectrometer. NMR
spectra were recorded on Bruker AV-400 and AV-300 spectrom-
eters. HR–ESI–MS data were obtained on an Agilent 6210 ESI/TOF
mass spectrometer. Macroporous resin Diaion HP-20 (Mitsubishi
Yellow oil; ½a D
ꢂ
204 (4.26), 218 (4.11), 255 (3.81), 291 (3.64), 342 (3.55) nm; IR
(KBr): nmax 3395, 1715, 1614, 1571, 1509, 1456, 1420, 1279, 1086,
1035 cmꢀ1
C
;
HR-ESI–MS m/z: 689.1687 [M + Na]+ (calcd for
30H34NaO17, 689.1688); 1H NMR (DMSO-d6, 400 MHz) and 13C
NMR (DMSO-d6, 100 MHz) data, see Table 2.
Chemical Corporation), MCI gel (75–150
mm; Mitsubishi), ODS
3.2. Acid hydrolysis and HPLC analysis of compound 8
(50 m; YMC) and Sephadex LH-20 (Pharmacia) were used for
m
column chromatography. TLC analyses were carried out using
precoated silica gel GF254 plates (Yantai Chemical Industry
Research Institute). Analytic high-performance liquid chromatog-
raphy (HPLC) was performed on an Agilent chromatography
equipped with a G1311C pump and a G1315D diode-array detector
Compound 8 (2 mg) was hydrolyzed with 2 mol/L HCl (5 mL) at
80 ꢃC for 2 h. The solution was then evaporated under reduced
pressure, after which 1 mL of pyridine and 2 mg of L-cysteine
methyl ester hydrochloride were added to the residue, and the
reaction mixture was heated at 60 ꢃC for 1 h. O-tolyl isothiocyanate
(5 m
L) was added to the mixture and heated at 60 ꢃC for an
(DAD) with a Cosmosil 5C18-MS-II column (4.6 ꢁ 250 mm, 5
mm).
Preparative HPLC was carried out on an Agilent instrument
equipped with a G1310B pump and a G1365D detector with a
additional 1 h. The reaction mixture was then analyzed by HPLC
and detected at 250 nm. Analytical HPLC was performed on a
Cosmosil 5C18-MS-II column (4.6 ꢁ 250 mm, 5
CH3CN-0.05% CH3COOH in H2O (25:75, 1.0 mL/min) as the mobile
phase. -Glucose (tR 16.36 min) was identified as the sugar moiety
of compound 8 based on comparison with authentic samples of
m
m) at 20 ꢃC using
Cosmosil 5C18-MS-II Waters column (20 ꢁ 250 mm, 5
mm).
3.1.1. Plant material
D
The roots and stems of E. obtusifolia were collected in Haikou
city, Hainan province of PR China, in June of 2010 and authenticated
by Prof. Guang-Xiong Zhou (Institute of Traditional Chinese
D
-
glucose (tR 16.36 min) and L-glucose (tR 14.99 min) (Tanaka et al.,
2007).
Medicine
& Natural Products, Jinan University). A voucher
specimen (No.100605) was deposited at the Institute of Traditional
Chinese Medicine & Natural Products, Jinan University, Guangzhou,
PR China.
3.3. Antiviral assay
3.3.1. Cells and viruses
Human larynx epidermoid carcinoma (HEp-2, ATCC CCL-23)
cells, as well as RSV A2 (ATCC-VR-1540) and Long (ATCC-VR-26)
strains were purchased from the Medicinal Virology Institute,
Wuhan University. HEp-2 cells were grown in Dulbecco Modified
Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine
serum (FBS, Gibco) and antimicrobials (growth medium, GM), then
cultured at 37 ꢃC in a humidified atmosphere supplied with 5% CO2.
For RSV propagation, RSV stock diluted using DMEM with 2% FBS
and antimicrobials (maintenance medium, MM) was added to the
confluent HEp-2 cells and continually cultured in the incubator
until the maximal RSV syncytia formation. Virus titers were
3.1.2. Extraction and isolation
The air-dried roots and stems of E. obtusifolia (20.0 kg) were
powdered and extracted with 95% (V/V) EtOH under reflux twice
(2 ꢁ 200 L, 2 h each). The EtOH extract was concentrated under
vacuum to afford a residue (963 g), which was suspended in H2O
and then partitioned successively with EtOAc and n-BuOH. After
removing the solvent, the n-BuOH soluble fraction (273 g) was
subjected to macroporous resin HP-20 column (15 ꢁ 60 cm) eluted
with EtOH–H2O mixtures (10:90; 30:70; 50:50; 70:30; 95:5, V/V,
each 30 L). The 30% EtOH eluate (77 g) was subjected to an MCI gel
column (7 ꢁ60 cm) using gradient mixtures of MeOH–H2O (0:100;
10:90; 15:85; 20:80; 25:75; 30:70; 40:60; 50:50; 100:0, each 15 L)
as the eluent to afford twelve fractions (Fr. 1-Fr. 12). Fr. 2 (7 g) was
separated by an ODS column (4 ꢁ 30 cm) with MeOH–H2O (15:85;
determined by the 50% tissue culture-infective dose (TCID50
)
method. The virus stock was stored at ꢀ80 ꢃC until use.

L. Fan et al. / Phytochemistry Letters 14 (2015) 185–189
189
3.3.2. Cytotoxicity assay
Liu, Z.Z., Feng, Z.M., Yang, Y.N., Jiang, J.S., Zhang, P.C., 2014. Acyl quinic acid
derivatives from the stems of Erycibe obtusifolia. Fitoterapia 99, 109–116.
Lu, Y., Yao, T.R., Chen, Z.N., 1986. Studies on the constituents of Erycibe elliptilimba.
The cytotoxicity of the compounds on HEp-2 cells was tested by
MTT assay as previously described (Wang et al., 2012). Due to the
quantitative limitation of the isolated compounds, the initial
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Matsuda, H., Morikawa, T., Xu, F.M., Ninomiya, K., Yoshikawa, M., 2004. New
isoflavones and pterocarpane with hepatoprotective activity from the stems of
Erycibe expansa. Planta Med. 70, 1201–1209.
concentrations of tested samples were set as 200 mg/mL.
Matsuda, H., Yoshida, K., Miyagawa, K., Asao, Y., Takayama, S., Nakashima, S., Xu, F.
M., Yoshikawa, M., 2007. Rotenoids and flavonoids with anti-invasion of
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3.3.3. CPE reduction assay
A cytopathic effect (CPE) reduction assay was adopted to
evaluate the antiviral activities of the compounds as previously
described (Wang et al., 2012). Ribavirin (Sigma) was used as a
positive control.
Appendix A. Supplementary data
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constituents from the roots and stems of Erycibe hainanesis. J. Nat. Prod. 73,177–
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Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2015.10.002.
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