Polyacetylenes and anti-hepatitis B virus active constituents from Artemisia capillaris

Article abstract of DOI:10.1016/j.fitote.2014.03.017

Three new polyacetylenes, 8-(Z)-decene-4, 6-diyne-1, 3, 10-triol (1), 1, 3S, 8S-trihydroxydec-9-en-4, 6-yne (2), 3S, 8S-dihydroxydec-9-en-4, 6-yne 1-O-β -D-glucopyranoside (3), and one new glucosyl caffeoate, 1-O-ethyl-6-O-caffeoyl-β -D-glucopyranose (4), together with 34 known compounds were isolated from Artemisia capillaris. The structures of the new compounds were determined by extensive spectroscopic analyses including 1D and 2D NMR, HRESIMS, [α]_D and CD experiments. Among them, 19 compounds showed activity inhibiting HBsAg secretion; 20 compounds showed activity inhibiting HBeAg secretion; and 25 compounds possessed inhibitory activity against HBV DNA replication according to our anti-HBV assay on HepG 2.2.15 cell line in vitro. The most active compound 12 could inhibit not only the secretions of HBsAg and HBeAg, but also HBV DNA replication with IC ₅₀ values of 15.02 μM (SI = 111.3), 9.00 μM (SI = 185.9) and 12.01 μM (SI = 139.2).

Full text of DOI:10.1016/j.fitote.2014.03.017

Fitoterapia 95 (2014) 187–193
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Polyacetylenes and anti-hepatitis B virus active constituents
from Artemisia capillaris
Yong Zhao a,b, Chang-An Geng a, Chang-Li Sun a,b, Yun-Bao Ma a, Xiao-Yan Huang a, Tuan-Wu Cao a,
Kang He a, Hao Wang a,b, Xue-Mei Zhang a, Ji-Jun Chen a,
⁎
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China
b
University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new polyacetylenes, 8-(Z)-decene-4, 6-diyne-1, 3, 10-triol (1), 1, 3S, 8S-trihydroxydec-9-en-4,
6-yne (2), 3S, 8S-dihydroxydec-9-en-4, 6-yne 1-O-β -D-glucopyranoside (3), and one new glucosyl
caffeoate, 1-O-ethyl-6-O-caffeoyl-β -D-glucopyranose (4), together with 34 known compounds
were isolated from Artemisia capillaris. The structures of the new compounds were determined by
extensive spectroscopic analyses including 1D and 2D NMR, HRESIMS, [α]D and CD experiments.
Among them, 19 compounds showed activity inhibiting HBsAg secretion; 20 compounds showed
activity inhibiting HBeAg secretion; and 25 compounds possessed inhibitory activity against HBV
DNA replication according to our anti-HBV assay on HepG 2.2.15 cell line in vitro. The most active
compound 12 could inhibit not only the secretions of HBsAg and HBeAg, but also HBV DNA
replication with IC50 values of 15.02 μM (SI = 111.3), 9.00 μM (SI = 185.9) and 12.01 μM (SI =
139.2).
Received 29 January 2014
Accepted in revised form 14 March 2014
Available online 29 March 2014
Keywords:
Polyacetylenes
Anti-HBV activity
Artemisia capillaris
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
hepatoprotective, choleretic, anti-inflammatory and diuretic
purposes in the folk. Currently, many formulae containing
Hepatitis B virus (HBV) infection is a serious global health
problem; however, the current treatment strategies includ-
ing vaccines, α-interferon and nucleoside analogues are
still unsatisfied. Therefore, new anti-HBV drugs with unique
mechanism are further needed. Traditional Chinese medi-
cines (TCMs) with hepatoprotective and antiviral activity are
fascinating sources for anti-HBV drug discovery, from which
many anti-HBV leading compounds (silymarin, schisanthein A,
oxymatrine, etc.) have been obtained [1]. Artemisia capillaris
(Yin-Chen) belonging to the Artemisia genus of the family
Compositae is a famous TCM documented in every edition of
“Chinese Pharmacopoeia”. The earliest recording of Yin-Chen
was found in the first Chinese dispensary “Shennong Bencao
Jing”. This plant is mainly distributed in Shananxi, Shanxi,
Anhui provinces of China, and has been widely used for
Yin-Chen have been used for treating acute and chronic
hepatitis in clinical [2,3]. Previous investigation suggested
that coumarins, flavonoids, organic acids, polyacetylenes,
chromones, etc. were the main constituents in A. capillaris,
of which p-hydroxyacephenone, scoparone, cirsimaritin,
arcapillin, quercetin, chlorogenic acid, capillarisin and capillene
were revealed to be hepatoprotective and choleretic compo-
nents [4,5]. Meanwhile, a series of constituents with antiplate-
let aggregation and anti-HIV activity were also isolated from
this herb [2]. However, its anti-HBV active constituents are still
unclear.
According to our bioassay on HepG 2.2.15 cell line in vitro,
the 90% ethanol extract of A. capillaris exhibited activity against
the secretions of hepatitis B surface antigen (HBsAg), hepatitis
B e antigen (HBeAg), and HBV DNA replication with the IC50
value of 460.33 μg/mL (SI = 1.4), 295.31 μg/mL (SI = 2.5)
and 49.13 μg/mL (SI = 13.4), respectively. In order to clarify its
anti-HBV compounds, our current investigation on the active
⁎
Corresponding author. Tel.: +86 871 65223265; fax: +86 871 65227197.
E-mail address: chenjj@mail.kib.ac.cn (J.-J. Chen).
http://dx.doi.org/10.1016/j.fitote.2014.03.017
0367-326X/© 2014 Elsevier B.V. All rights reserved.

188
Y. Zhao et al. / Fitoterapia 95 (2014) 187–193
parts led to the isolation of 38 compounds, including three new
polyacetylenes, 8-(Z)-decene-4, 6-diyne-1, 3, 10-triol (1), 1, 3S,
8S-trihydroxydec-9-en-4, 6-yne (2), and 3S, 8S-dihydroxydec-9-
en-4, 6-yne 1-O-β -D-glucopyranoside (3), and one new glucosyl
caffeoate, 1-O-ethyl-6-O-caffeoyl-β -D-glucopyranose (4), to-
gether with 34 known compounds (4–38). Among them, 19
compounds showed activity inhibiting HBsAg secretion, 20
compounds showed activity inhibiting HBeAg secretion and 25
compounds possessed inhibitory activity against HBV DNA
replication according to our anti-HBV assay on HepG 2.2.15 cell
line in vitro. The most active compound 12 could inhibit not
only the secretions of HBsAg and HBeAg but also HBV DNA
replication with IC50 values of 15.02 μM (SI = 111.3), 9.00 μM
(SI = 185.9) and 12.01 μM (SI = 139.2). In this paper, we
described the isolation, structural elucidation and anti-HBV
activities of the isolates.
17.5 × 35 cm) eluted with MeOH–CHCl3 (0:100, 10:90,
20:80, 30:70, 40:60, 50:50 v/v) to give 9 fractions (A–G).
Fraction A was chromatographed on a silica gel column,
eluted with EtOAc–petroleum ether (PE) (5:95) to yield
compounds
fraction
compounds
8
(470 mg) and 10 (2.6 g). Purification of
on Si CC with acetone–PE (5:95) afforded
(8 mg) and 37 (44 mg). Fraction was
B
7
C
subjected to Si CC and further purified with Sephadex
LH-20 column (MeOH–CHCl3, 50:50) to give compounds 17
(3 mg), 25 (6 mg) and 30 (5 mg). Fraction D was separated
by repeated Si CC [EtOAc–PE, 20:80, 30:70, 40:60] and
Sephadex LH-20 columns (MeOH) to yield compounds 9
(18 mg), 11 (12 mg), 14 (20 mg), 15 (36 mg), 16 (21 mg),
19 (18 mg), 23 (10 mg), 32 (28 mg) and 33 (360 mg).
Compounds 1 (10 mg), 2 (9 mg), 22 (7 mg) and 34 (10 mg)
were isolated from fraction E by Si CC (acetone–PE, 30:70).
Fraction F (30 g) was performed on a MCI CHP-20P gel CC
(490 g, 5.04 × 50 cm) and eluted with MeOH–H2O (30:70,
50:50, 70:30) to yield 3 sub-fractions (F-1–F-3). Fraction F-1
was loaded on a Si CC (MeOH–CHCl3, 20:80) to yield
compounds 26 (14 mg), 27 (19 mg) and 28 (43 mg).
Fraction F-2 was chromatographed on a reversed phase
C-18 column (320 g, 5.04 × 50 cm) and eluted with MeOH–
H2O to give compounds 6 (5 mg), 12 (43 mg), 18 (38 mg),
29 (13 mg) and 31 (19 mg). Fraction F-3 was purified by
Sephadex LH-20 chromatography (MeOH) affording com-
pounds 4 (45 mg), 20 (145 mg), 21 (6 mg) and 24 (43 mg).
Fraction G was loaded on a silica gel column eluted with
MeOH–CHCl3 (10:90), then further purified with Sephadex
LH-20 column (MeOH) to give compounds 13 (10 mg) and
35 (9 mg). Fraction H was purified by Sephadex LH-20
column (MeOH) to give two sub-fractions. Compound 36
(3 mg) was isolated from fraction H-1 by Sephadex LH-20
column (MeOH). Fraction H-2 was further separated by HPLC
(Agilent ZORBAX SB-C18, 5 μm, 9.4 × 250 mm) with MeOH–
H2O (33:67) as the eluent to afford compounds 5 (3 mg,
tR = 10 min) and 38 (39 mg, tR = 12 min). Fraction I was
performed on a silica gel column with the elution of MeOH–
H2O (30:70) to yield compound 3 (12 mg).
2. Experimental
2.1. General experimental procedures
Mass spectra were determined by a LCMS-IT-TOF (Shimadzu,
Kyoto, Japan) or a Waters AutoSpec Premier P776 (Waters, USA)
mass spectrometer. Optical rotations were measured through a
Jasco model 1020 digital polarimeter (Horiba, Tokyo, Japan). UV
and IR (KBr) spectra were recorded on a Shimadzu UV2401PC
spectrophotometer (Shimadzu, Kyoto, Japan) and a Bio-Rad
FTS-135 spectrometer (Hercules, California, USA), respectively.
Electronic circular dichroism (ECD) spectra were performed on
an Applied Photophysics Chirascan instrument (Agilent, Amer-
ica). 1D and 2D NMR were recorded on Bruker AM-400, Bruker
DRX-500 or AVANCE III-600 spectrometers (Bruker, Bremerha-
ven, Germany). Silica gel (200–300 mesh) for column chroma-
tography and TLC plates (GF254) were purchased from Qingdao
Makall Chemical Company (Makall, Qingdao, China) and
Qingdao Haiyang Chemical Company (Haiyang, Qingdao,
China), respectively. Sephadex LH-20 (20–50 μm) for chroma-
tography was obtained from Pharmacia Fine Chemical Co., Ltd.
(Pharmacia, Uppsala, Sweden). Waters Alliance 2695 appara-
tus with an Agilent ZORBAX SB-C18 (5 μm, 9.4 × 250 mm)
column (Agilent, USA) was used for HPLC separation. Fractions
were visualized by heating silica gel plates sprayed with 10%
H2SO4 in ethanol or colored by sulfuric acid-vanillin reagent.
Compound 1: pale yellow oil; [α]21 D: −20.6 (c 0.10,
MeOH); UV (MeOH) λmax (log ε): 284 (3.69), 268 (3.80), 254
(3.65), 241 (3.37) nm; IR (KBr) νmax: 3381, 2954, 2928, 2888,
2232, 1743, 1630, 1416, 1385, 1334, 1050, 1024 cm−1; 1H NMR
(400 MHz) and 13C NMR (100 MHz) data, see Tables 1–2;
HREIMS m/z 180.0784 (C10H12O3, calcd for 180.0786).
2.2. Plant material
Compound 2: pale yellow oil; [α]23 D: −94.9 (c 0.10,
MeOH); UV (MeOH) λmax (log ε): 283 (2.80), 267 (2.89), 253
(2.88), 241 (2.90) nm; IR (KBr) νmax: 3395, 2959, 2930, 2890,
2152, 1641, 1410, 1312, 1050, 1021 cm−1; 1H NMR (400 MHz)
and 13C NMR (100 MHz) data, see Tables 1–2; HREIMS m/z
180.0781 (C10H12O3, calcd for 180.0786).
The whole plants of A. capillaris Thunb. were collected
from wide sources in Chengcheng County, Shaanxi Province,
China, in April 2012 and identified by Dr. Prof. Li-Gong Lei
(Kunming Institute of Botany, CAS). A voucher specimen (No.
20120428) was deposited in the Laboratory of Anti-virus and
Natural Medicinal Chemistry, Kunming Institute of Botany,
CAS.
Compound 3: pale yellow oil; [α]18 D: −51.4 (c 0.20,
MeOH); UV (MeOH) λmax (log ε): 284 (3.05), 268 (3.12), 254
(3.12), 242 (3.13) nm; IR (KBr) νmax: 3419, 2925, 2894, 1641,
2.3. Extraction and isolation
1419, 1384, 1078, 1032 cm−1 1H NMR (500 MHz) and 13C
;
NMR (100 MHz) data, see Tables 1–2; HRESIMS m/z 365.1266
The aerial part of A. capillaris (10 kg) was extracted with
90% ethanol under reflux for two times, 1 h each time. The
extract was concentrated in vacuo and partitioned between
ethyl acetate (EtOAc) and water. The EtOAc part (400 g) was
subjected on silica gel column chromatography (Si CC, 3 kg,
([M + Na]+, C16H22O8Na+, calcd for 365.1207).
Compound 4: brown gum; [α]27 D:-25.8 (c 0.12, MeOH);
UV (MeOH) λmax (log ε): 329 (4.26), 216 (4.27) nm; IR (KBr)
ν
max: 3423, 2977, 2920, 1692, 1632, 1605, 1521, 1446, 1383,
1284, 1181, 1053 cm−1 1H NMR (400 MHz) and 13C NMR
;

Y. Zhao et al. / Fitoterapia 95 (2014) 187–193
189
Table 1
1H NMR data of compounds 1–4 (1–3 in CD3OD, 4 in acetone-d6, δ in ppm, J in Hz).
Position
1
2
3
4
1
2
3.72 (m)
1.89 (m)
3.70 (m)
1.89 (m)
3.72 (m) 3.99 (m)
1.97 (m)
−
6.31 (d, 15.9)
7.56 (d, 15.9)
−
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
4.59 (t, 6.8)
4.55 (t, 6.7)
4.63 (t, 6.5)
−
−
−
−
−
−
7.17 (d, 1.8)
−
−
−
−
−
−
−
−
5.64 (d, 11.0)
4.88 (overlap)
4.87 (overlap)
5.91 (ddd, 16.6, 9.8, 5.4)
5.20 (d, 9.8) 5.39 (d, 16.6)
4.27 (d, 7.8)
3.17 (m)
6.86 (d, 8.1)
6.23 (dt, 11.0, 6.4)
4.30 (d, 6.4)
5.91 (ddd, 17.0, 10.2, 5.5)
5.20 (d, 10.2) 5.40 (dd, 17.0, 1.2)
7.05 (dd, 8.1, 1.8)
−
4.30 (d, 7.5)
3.54 (m)
3.39 (m)
3.41 (m)
3.19 (m)
4.30 (m)
4.47 (dd, 11.8, 1.9)
3.84 (m)
3.55 (m)
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
3.30 (m)
3.27 (m)
3.36 (m)
3.68 (dd, 11.9, 4.5)
3.87 (dd, 11.7, 1.0)
−
1''
2''
−
−
−
1.14 (t, 7.1)
(100Hz) data, see Tables 1–2; HRESIMS m/z 393.1147
2.5. Anti-HBV assay on HepG 2.2.15 cell line in vitro
([M + Na]+, C17H22O9Na+, calcd for 393.1156).
The anti-HBV assay was performed according to our previous
report, with tenofovir (Jiangxi Chenyang Pharmaceutial Co., Ltd,
China, purity N97.6%) as the positive control [6].
2.4. Acid hydrolysis of compound 3
Compound 3 (4 mg) was refluxed with 2 M HCl (5 mL) at
80 °C for 5 h. After neutralization with NaHCO3 and extrac-
tion with CHCl3, the aqueous layer was concentrated and
detected by TLC over silica gel (H2O–MeOH–CHCl3, 4:40:60).
The presence of glucose was confirmed by comparison with
authentic samples (Rf 0.4). The aqueous part was further
purified by Si CC and identified to be D-glucose based on its
[α]D value ([α]13 D + 46.6, c 0.089, MeOH).
3. Results and discussion
Compound 1 had a molecular formula of C10H12O3 by
HREIMS ([M]+ m/z 180.0784, calcd for 180.0786). The
absorption bands at 3381, 2232 and 1630 cm−1 in the IR
spectrum indicated the presence of hydroxyl, alkynyl and
alkenyl groups. The UV absorptions at 284, 268, 254 and
241 nm were typical for polyacetylenes [7]. The 13C NMR
(DEPT) spectrum of 1 displayed 10 carbons, including three
methylenes, three methines and four quaternary carbons, of
which two alkynyls [δC 85.9 (s, C-4), 69.1 (s, C-5), 74.9 (s, C-6)
and 78.6 (s, C-7)] and one alkenyl [δC 109.2 (d, C-8) and 147.7
(d, C-9)] were obviously recognized. The above analyses
suggested that compound 1 should be a C10 polyacetylene.
The NMR data of compound 1 were similar to these of the
known compound 8-(E)-decene-4, 6-diyne-1, 3, 10-triol (1a)
[7], except for the different coupling constants between H-8
and H-9 (JH-8/H-9 = 11.0 Hz in 1, JH-8/H-9 = 15.9 Hz in 1a).
From the above analysis, the double bond was deduced as Z
configuration in 1 instead of E configuration in 1a. Since there
was only one chiral center (C-3) in both of the two compounds,
the absolute configuration of compound 1 was determined to
be R identical to 1a from the similar [α]D value. Thus,
compound 1 was determined as 8-(Z)-decene-4, 6-diyne-1, 3,
10-triol as shown in Fig. 1.
Table 2
13C NMR dataa,b of compounds 1–4 (1–3 in CD3OD, 4 in acetone-d6, δ in
ppm).
Position
1
2
3
4
1
2
3
4
5
6
7
8
59.0, t
41.3, t
60.3, d
85.9, s
69.1, s
74.9, s
78.6, s
109.2, d
147.7, d
61.1, t
−
57.6, t
39.8, t
58.8, d
80.5, s
67.6, s
68.7, s
78.1, s
62.5, d
136.5, d
115.5, t
−
66.7, t
38.8, t
60.1, d
82.0, s
68.9, s
70.1, s
79.5, s
63.9, d
138.0, d
116.8, t
104.5, d
75.0, d
77.9, d
71.5, d
78.0, d
62.6, t
−
167.5, s
115.2, d
145.9, d
127.5, s
115.4, d
146.2, s
148.8, s
116.3, d
122.5, d
−
103.9, d
74.8, d
71.3, d
77.8, d
74.8, d
64.3, t
9
10
1'
2'
3′
4′
5′
6′
1″
2″
Compound 2, had the same molecular formula of
C10H12O3 as compound 1 by HREIMS ([M]+ m/z 180.0781,
calcd for 180.0786). The NMR spectra of compound 2 were
almost identical to those of the known compound 1, 3R,
8R-trihydroxydec-9-en-4, 6-yne (2a) from the congener plant
Artemisia monosperma [8], indicating the same relative config-
uration. However, the [α]D value of compound 2 (−94.9) was
−
−
−
−
−
−
−
−
−
−
−
−
65.3, t
15.5, q
−
−
−

190
Y. Zhao et al. / Fitoterapia 95 (2014) 187–193
Fig. 1. Structures of compounds 1–4.
opposite to 2a (+125), suggesting that compound 2 should be
the enantiomer of 2a. Based on the above analyses, compound
2 was assigned as 1, 3S, 8S-trihydroxydec-9-en-4, 6-yne.
Compound 3 had a molecular formula of C16H22O8 by
HRESIMS ([M + Na]+ m/z 365.1266, calcd for 365.1207). The
13C NMR data of compound 3 was similar to compound 2
except for an additional glucosyl group and the obvious
down-field shift of C-1 from δC 57.6 in 2 to 66.7 in 3. Therefore,
compound 3 was proposed as a glycoside of 2 with the
glycosidation at C-1, which was confirmed by the correlations
of δH 4.26 (H-1′, d, J = 7.8 Hz) to δC 66.7 (C-1, t) in the HMBC
spectrum (Fig. 2). The β-glucose was deduced from the
coupling constant (J = 7.8 Hz) of the anomeric proton. Acid
hydrolysis of 3 provided D-glucose, which was confirmed by
comparison with an authentic sample on TLC and [α]D
experiment ([α]13 D: +46.6, c 0.089, MeOH). The identical
ECD spectra shown in Fig. 3 suggested that compounds 2 and 3
had the same absolute configuration. Therefore, compound 3
was assigned as 3S, 8S-dihydroxydec-9-en-4, 6-yne 1-O-β-D-
glucopyranoside.
Fig. 2. Selected HMBC and COSY correlations of compounds 1–4.

Y. Zhao et al. / Fitoterapia 95 (2014) 187–193
191
10
5
compound 2
compound 3
0
200
250
300
350
400
-5
-10
-15
wavelength λ/nm
Fig. 3. Experimental ECD spectra of compounds 2 and 3.
Compound 4 had a molecular formula of C17H22O9 by
positive HRESIMS (m/z 393.1147 [M + Na]+
calcd for
393.1156). The presence of hydroxyl (3423 cm−1), carbonyl
(1692 cm−1), and aromatic ring (1605, 1521, 1446 cm−1
acid (36) [35], stigmast-4-en-3-one (37) [36] and tuberonic
acid (38) [37]] by comparing the data with literatures.
,
According to our anti-HBV assay on HepG 2.2.15 cells in
vitro, 26 compounds showed activity inhibiting the secretions
of HBsAg, HBeAg and HBV replication (Table 3). Three caffeic
acid derivatives (4, 31, 33) possessed potent anti-HBV
activity against HBV DNA replication with IC50 values of
6.84, 16.83 and 25.77 μM, indicating that caffeoyl moiety
might be the active group [38]. Of the 5 coumarins (14–18),
compound 18 with hydroxyl groups in both C-6 and C-7
exhibited the most promising activity against HBV DNA
replication with an IC50 value of 33.76 μM. Other aromatic
compounds such as furofurans lignans (22 and 23), flavo-
noids (19, 20 and 21), and benzenoids (24–30, 32 and 34)
showed moderate anti-HBV activity, of which compounds 22,
23, 24, 26 and 29 could inhibit HBV DNA replication with IC50
values in the range of 9.38 to 32.60 μM. Importantly, two
eudesmane sesquiterpenoids (11 and 12) exhibited promis-
ing activity against HBV DNA replication with IC50 values of
19.70 and 12.01 μM, with high SI values of 105.5 and 139.2. In
addition to the inhibition on HBV DNA, compound 12 could
also suppress the secretions of HBsAg and HBeAg with the
IC50 values of 15.02 μM (SI = 111.3) and 9.00 μM (SI =
185.9). Compound 35 as a heterozygote of purine and phenol
possessed potent activity against HBV DNA replication with
an IC50 value of 13.06 μM (SI = 148.6), as well as activity
against the secretions of HBsAg and HBeAg. This is the first
investigation on the anti-HBV constituents of A. capillaris,
which will provide valuable information for understanding
its active principles.
)
was deduced from the IR spectrum. The NMR spectra of
compound 4 were similar to those of 1-O-methyl-6-O-caffeoyl-β -
D-glucopyranose (4a) [9], and the only difference was that the
methoxyl group in 4a was changed to be ethoxyl group in 4.
The above deduction was supported by the HMBC correlations
from H-1″ to C-1′ and from H-6′ to C-1. The coupling constant
of 7.5 Hz for H-1′ indicated the β configuration of the glucosyl
group. Its absolute configuration was proposed as D-glucosyl
due to the similar [α]D values of compounds 4 (−25.8) with 4a
(−18.1).
The other 34 compounds were determined as [8-(E)-decene-
4, 6-diyne-1, 10-diol (5) [10], deca-9-en-4, 6-diyne-1, 8-diol
1-O-β -D-glucopyranoside (6) [11], dendroarboreol B (7)
[12], (3S)-16, 17-didehydrofalcarinol (8) [12], (3S, 8S)-16,
17-dehydrofalcarindiol (9) [12]]; 4 terpenoids [phytol (10)
[13], 7-eudesm-4 (15)-ene-1β, 6α-diol (11) [14], pumilaside A
[15] (12), (6R, 9R)-3-oxo-α -ionol-9-O-β -D-glucopyranoside
(13) [16]]; 5 coumarins [umbelliferone (14) [17], scopoletin
(15) [18], isoscopoletin (16) [18], scoparone (17) [19], esculetin
(18) [18]]; 3 flavonoids [rhamnocitrin (19) [20], isorhamnetin
3-O-β -D-glucopyranoside (20) [21], naringenin 7-O-β -D-
glucopyranoside (21) [22]], 2 lignans [(+)-epipinoresinol (22)
[23], (+)-medioresinol (23) [24]]; 11 benzenoids [salicylic acid
(24) [25], 4-hydroxy-3, 5-dimethoxybenzaldehyde (25) [26], 3,
4-dihydroxybenzaldehyde (26) [27], 4-hydroxybenzoic acid
(27) [28], ethyl 4-hydroxybenzoic acid (28) [29], protocatechuic
acid (29) [30], 1-(4-hydroxy-3-methoxyphenyl) propan-
1-one (30) [31], caffeic acid (31) [30], ethyl p-hydroxy-trans-
cinnamate (32) [32], ethyl caffeoate (33) [32], sonchifolinin
B (34) [33]]; 4 other compounds [6-amino-9-[1-(3, 4-
dihydroxyphenyl) ethyl]-9H-purine (35) [34], 4-methoxynicotinic
Conflict of interest statement
There are no conflicts of interests among all authors in
this manuscript.

192
Y. Zhao et al. / Fitoterapia 95 (2014) 187–193
Table 3
Anti-HBV activities of compounds 4, 11, 12, 14–35 and 38.a
Compounds
CC50 [μM]
HBsAgb
HBeAgc
DNAd
IC50 [μM]
SIe
IC50 [μM]
SI
IC50 [μM]
SI
N155.0
105.5
139.2
1.1
2.7
2.1
11.7
161.5
N8.3
4
N1059.89
2079.54
1672.27
488.76
1819.11
1625.68
2892.33
N5452.75
N4084.97
N2584.29
N2552.63
480.08
1086.20
4331.38
1504.70
1392.46
N7002.79
4617.47
N8339.68
2759.50
N4302.83
3258.54
5049.38
3067.44
1902.25
N4945.35
N8710.80
28.08
155.29
15.02
855.74
885.36
451.09
516.26
43.60
b32.67
446.74
370.69
56.11
N37.7
13.4
111.3
b1.0
2.1
126.70
107.52
9.00
N8.4
19.3
185.9
b1.0
b1.0
1.4
21.8
N6.7
N125.1
N7.8
N6.5
9.4
6.84
19.70
12.01
448.15
682.14
761.67
206.31
33.76
11
12
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
38
Tenofovirf
717.22
2911.25
1206.56
110.24
811.29
b32.67
331.92
389.72
50.98
3.6
4.7
N125.1
N125.1
N5.8
N6.9
8.6
490.87
16.42
41.59
N157.4
N61.4
51.2
9.38
836.73
3620.51
2343.88
b55.72
3531.43
b44.88
308.57
543.48
2166.50
802.74
b52.79
1643.93
1423.21
N4945.35
5531.26
1.3
1.2
381.82
4652.54
3717.16
b55.72
159.87
b44.88
66.69
2.8
10.92
25.80
763.22
27.17
274.94
59.52
32.60
481.66
16.83
1046.46
25.77
198.69
13.06
99.5
167.9
2.0
51.2
N25.5
77.6
N255.8
5.7
N255.6
3.1
195.9
15.4
148.6
N29.8
N2604.2
b1.0
b1.0
N25.0
N43.8
N101.6
N125.1
22.9
N64.1
7.2
b1.0
N25.0
N2.0
N101.6
N27.0
5.1
N2.0
4.1
N95.7
1.9
120.66
67.17
454.15
b52.79
446.31
527.34
N4945.35
N8710.80
N95.7
6.9
3.6
1.3
166.06
3.34
N1.6
a
All values are the mean of two independent experiments.
HBsAg: HBV surface antigen.
HBeAg: HBV e antigen.
DNA: HBV DNA replication.
CC50 = 50% cytotoxic concentration, IC50 = 50% inhibitory concentration, SI (selectivity index) = CC50/IC50
Tenofovir, an antiviral agent used as a positive control.
b
c
d
e
f
.
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 81202436), the National Science
Foundation of China for Distinguished Young Scholars (No.
81025023), and the Youth Innovation Promotion Association,
CAS. We are grateful to the staff of the analytical group of the
State Key Laboratory of Phytochemistry and Plant Resources
in West China, Kunming Institute of Botany, CAS.
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Appendix A. Supplementary data
1D and 2D NMR, HREIMS, HRESIMS, IR, UV and [α]D spectra
of compounds 1–4, ECD spectra for 2 and 3, and structures of
known compounds (5–38) are available as Supporting Infor-
mation. Supplementary data to this article can be found online
at http://dx.doi.org/10.1016/j.fitote.2014.03.017.
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