Anti-hepatitis B virus effects of the traditional Chinese herb Artemisia capillaris and its active enynes

Article abstract of DOI:10.1016/j.jep.2018.06.005

Ethnopharmacological relevance: Artemisia capillaris (Yin-Chen) is a famous traditional Chinese medicine (TCM) for treating acute and chronic hepatitis in China. Enynes are one type of characteristic constituents in this herb, while their anti-hepatitis B virus (anti-HBV) properties have not been systemically investigated. Aim of the study: This study is to reveal the active part of A. capillaris, and systemically investigate the enynes and their anti-HBV activity. Materials and methods: The total extract and each fraction of A. capillaris were assayed for the anti-HBV activity to reveal the active part. Bioassay-guided fractionation using various chromatographic techniques yielded the enynes, whose structures were elucidated by spectroscopic analyses and ECD calculations. The anti-HBV properties inhibiting HBsAg and HBeAg secretions and HBV DNA replication were evaluated on HepG 2.2.15 cell line in vitro. Results: ACT-2 and ACT-3 was revealed to be the respective active and toxic part of A. capillaris. Twelve enynes (1–12) involving four new ones (1–4) and two unusual enyne analogs (13–14) were isolated from the active part (ACT-2). All the isolates were assayed for their anti-HBV activity, and the preliminary structure-activity relationships were summarized based on the structural features. In particular, compound 4 could significantly inhibit the secretions of HBsAg and HBeAg, and HBV DNA replication with IC₅₀ values of 197.2 (SI > 5.1), 48.7 (SI > 20.5) and 9.8 (SI > 102) μM. Conclusions: Enynes are responsible for the anti-HBV effects of A. capillaris. Hydroxyl and glycosyl groups are preferable for maintaining activity. This is the first time to systematically investigate the anti-HBV activity of enynes in A. capillaris, which provides valuable information for understanding the ethnopharmacological application of Yin-Chen.

Full text of DOI:10.1016/j.jep.2018.06.005

Author’s Accepted Manuscript
Anti-hepatitis B virus effects of the traditional
Chinese herb Artemisia capillaris and its active
enynes
Chang-An Geng, Tong-Hua Yang, Xiao-Yan
Huang, Jing Yang, Yun-Bao Ma, Tian-Ze Li, Xue-
Mei Zhang, Ji-Jun Chen
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https://doi.org/10.1016/j.jep.2018.06.005
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To appear in: Journal of Ethnopharmacology
Cite this article as: Chang-An Geng, Tong-Hua Yang, Xiao-Yan Huang, Jing
Yang, Yun-Bao Ma, Tian-Ze Li, Xue-Mei Zhang and Ji-Jun Chen, Anti-hepatitis
B virus effects of the traditional Chinese herb Artemisia capillaris and its active
e n y n e s , Journal
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Anti-hepatitis B virus effects of the traditional Chinese herb Artemisia capillaris and its active
enynes
a
a
a
a
a
a
Chang-An Geng , Tong-Hua Yang , Xiao-Yan Huang , Jing Yang , Yun-Bao Ma , Tian-Ze Li , Xue-
a
a,b,*
Mei Zhang , Ji-Jun Chen
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming
6
50201, PR China
b
University of Chinese Academy of Sciences, Beijing 100049, PR China
chenjj@mail.kib.ac.cn
*
Corresponding Author: Ji-Jun Chen, State Key Laboratory of Phytochemistry and Plant Resources in
#
West China, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming
6
50201, Yunnan, PR China. Tel: +86-871-65223265. Fax: +86-871-65227197.
Abbreviations:
CC, column chromatography; ECD, electronic circular dichroism; HBeAg, HBV e antigen; HBsAg,
HBV surface antigen; HBV, hepatitis B virus; HPLC, high performance liquid chromatography; MPLC,
medium pressure liquid chromatography; MS, mass spectrum; NMR, nuclear magnetic resonance; SI,
selection index; TCM, traditional Chinese medicine
List of compounds
8
S-deca-9-en-4,6-diyne-1,8-diol (1)
(S)-deca-4,6,8-triyne-1,3-diol (2)
(S)-3-hydroxyundeca-5,7,9-triynoic acid (3)
3
S-Hydroxyundeca-5,7,9-triynoic acid 3-O-β-D-glucopyranoside (4)
atractylodin (5)
dendroarboreol B (6)
dehydrofalcarinol (7)
dehydrofalcarindiol (8)

(
E)-deca-2-en-4,6-diyne-1,10-diol (9)
Z)-deca-2-en-4,6-diyne-1,10-diol (10)
(
8
3
5
2
S-deca-9-en-4,6-diyne-1,8-diol 1-O-β-D-glucopyranoside (11)
S,8S-dihydroxydec-9-ene-4,6-diyne 1-O-β-D-glucopyranoside (12)
-benzylthiophencarboxylic acid (13)
-methyl-6-phenyl-4H-pyran-4-one (14)
ABSTRACT
Ethnopharmacological relevance: Artemisia capillaris (Yin-Chen) is a famous traditional Chinese
medicine (TCM) for treating acute and chronic hepatitis in China. Enynes are one type of characteristic
constituents in this herb, while their anti-hepatitis B virus (anti-HBV) properties have not been
systemically investigated.
Aim of the study: This study is to reveal the active part of A. capillaris, and systemically investigate the
enynes and their anti-HBV activity.
Materials and methods: The total extract and each fraction of A. capillaris were assayed for the anti-
HBV activity to reveal the active part. Bioassay-guided fractionation using various chromatographic
techniques yielded the enynes, whose structures were elucidated by spectroscopic analyses and ECD
calculations. The anti-HBV properties inhibiting HBsAg and HBeAg secretions and HBV DNA
replication were evaluated on HepG 2.2.15 cell line in vitro.
Results: ACT-2 and ACT-3 was revealed to be the respective active and toxic part of A. capillaris.
Twelve enynes (1–12) involving four new ones (1–4) and two unusual enyne analogs (13–14) were
isolated from the active part (ACT-2). All the isolates were assayed for their anti-HBV activity, and the
preliminary structure-activity relationships were summarized based on the structural features. In
particular, compound 4 could significantly inhibit the secretions of HBsAg and HBeAg, and HBV DNA
replication with IC50 values of 197.2 (SI>5.1), 48.7 (SI>20.5) and 9.8 (SI>102) μM.
Conclusions: Enynes are responsible for the anti-HBV effects of A. capillaris. Hydroxyl and glycosyl
groups are preferable for maintaining activity. This is the first time to systematically investigate the anti-
HBV activity of enynes in A. capillaris, which provides valuable information for understanding the
ethnopharmacological application of Yin-Chen.
Graphical abstract:

Keywords: Artemisia capillaris, Enynes, Anti-HBV activity, Structure-activity relationships
1
. Introduction
Hepatitis B caused by hepatitis B virus (HBV) infection is a serious health problem, which affects
57 million people all over the world. As one of the top ten causes of death, hepatitis B leads to 0.89
2
million deaths per year. In China, there are about 90 million people have been infected with HBV,
occupied one third of the world's infections (World Health Organization, 2017). Vaccinations are
effective in preventing hepatitis B, whereas they are invalid to people already suffering from HBV
infection. Interferons and nucleoside analogs are the main treatment strategies, but their application are
limited by the drug-resistance and adverse side effects (Bhattacharya and Thio, 2010; Mirandola et al.,
2
011). Traditional Chinese medicines (TCMs) that have been used for medicinal purposes in China for
thousands of years are fascinating sources for searching new anti-HBV candidates (Geng et al., 2013).
Artemisia capillaris (Yinchenhao in Chinese) belonging to the Artemisia genus of the family
Compositae is documented as the authentic resource of Yin-Chen in Chinese Pharmacopoeia (Chinese
Pharmacopoeia Commission, 2015). As a famous TCM, Yin-Chen is widely used for choleretic,
antiinflammatory and diuretic purposes in the treatment of epidemic hepatitis in China (Chen et al., 2008;
Wu et al., 2001; Yan et al., 2001; Zhao et al., 2007). The folk application of Yin-Chen could be traced
back to the first Chinese dispensatory “Shennong's Herbal Classic of Materia Medica (Shennong Bencao
Jing)”. Afterwards, many TCM classics involving “Treatise on Febrile Diseases (Shanghan Lun)”,
“
Synopsis of Golden Chamber (Jinkui Yaolue)” and “Compendium of Materia Medica (Bencao
Gangmu)” restate its effectiveness in treating hepatitis. Furthermore, many formulae with Yin-Chen as
the main composition are used for curing viral hepatitis and hepatoprotective purpose in clinic. Enynes,
coumarins, flavonoids, lignans, terpenes and phenolic acids are the main constituents of Yin-Chen
(
Abad et al., 2012; Wu et al., 2001). In the previous investigation, a series of active chlorogenic acids,

sesquiterpenes and phenolics were isolated from A. capillaris which was collected in Shaanxi Province,
China (Zhao et al., 2014; Zhao et al., 2014; zhao et al., 2015). However, the anti-HBV activity of enynes,
the characteristic constituents in Artemisia plants, has not been systemically studied (Geng et al., 2015;
Yano, K., 1983). In the TCM theory, the producing area always plays important roles in the efficacy of
medical herbs, based on which the concept of “Genuine Medicinal Materials” is proposed. As a
comparison, A. capillaris collected from Hunan Province (China) was systematically investigated in this
study to characterize the typical enynes and their anti-HBV properties.
2
. Materials and methods
2
.1. General experimental procedures
Optical rotations were performed on a Jasco model 1020 digital polarimeter (Horiba, Tokyo,
Japan). UV spectra were measured by a Shimadzu UV2401PC spectrophotometer (Shimadzu, Kyoto,
Japan). Electronic circular dichroism (ECD) spectra were recorded on an Applied Photophysics
Chirascan instrument (Agilent, Santa Clara, United States). IR (KBr) spectra were performed on a Bio-
Rad FTS-135 spectrometer (Hercules, California, USA). 1D and 2D NMR were measured on an
AVANCE III-600 spectrometer (Bruker, Bremerhaven, Germany). High-resolution mass spectra were
determined by a LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). MPLC separation was
performed on a Dr-Flash II apparatus (Lisui, Suzhou, China) using a CHP20P MCI gel column (75~200
μm). HPLC purification was achieved on Chuangxin Tongheng LC3000 apparatus (Beijing Chuangxin
Tongheng Science and Technology, Beijing, China) with an Agilent Eclipse XDB-C18 column (9.4×250
mm, i.d., 5 μm).
2
.2. Plant material
The plant materials of Artemisia capillaris Thunb. were collected from Huaihua, Hunan Province
China) in May 2012, and authenticated by Dr. Li-Gong Lei (Kunming Institute of Botany, CAS). A
(
voucher specimen (No. 201205001) was deposited in the Laboratory of Anti-virus and Natural
Medicinal Chemistry, Kunming Institute of Botany, CAS.
2
.3. Extraction and isolation

The dried aerial parts of A. capillaris (1 kg) were powdered and extracted with 90% EtOH (8 L)
under reflux for two times. The combined extract was condensed in vacuo to give the total extraction
(
ACT). ACT was dissolved in H
CC) which was eluted with EtOH-H
~ACT-3. According to the bioassay in vitro, the active part ACT-2 was further separated by MPLC on
a CHP20P MCI gel column using MeOH-H O gradient (1:9, 3:7, 5:5 and 10:0, v/v) as the mobile phase
to yield four fractions, ACT-2-1~ACT-2-4. ACT-2-3 was subjected to silica gel CC and eluted with
CHCl -MeOH system (9:1) to give two fractions, ACT-2-3-1 and ACT-2-3-2. ACT-2-3-1 was purified
by repeated silica gel CC (acetone-petroleum ether, 3:7) and Sephadex LH-20 CC (MeOH-CHCl , 5:5)
to give 5 (10 mg), 7 (8 mg) and 14 (10 mg). ACT-2-3-2 was separated by silica gel CC (CH Cl -MeOH,
:1), then followed by semi-preparative HPLC (MeOH-H O, 35:65) to provide 1 (3 mg), 2 (1.5 mg), 9
4 mg) and 10 (3 mg). ACT-2-2 was loaded on silica gel CC and eluted with CHCl -MeOH gradient
from 9:1 to 8:2) to give three fractions, ACT-2-2-1~ACT-2-2-3. ACT-2-2-1 was purified by semi-
O (25:75) as the elution to yield 6 (7 mg), 8 (5 mg) and 13 (11 mg).
ACT-2-2-2 was isolated by Sephadex LH-20 CC (MeOH) and then with semi-preparative HPLC
MeOH-H O, 30:70) to give 3 (4 mg) and 11 (6 mg). ACT-2-2-3 was purified by repeated silica gel CC
using CHCl -MeOH-H O gradient and semi-preparative HPLC (MeCN-H O, 30:70) to provide 4 (8 mg)
and 12 (6 mg).
2
O and fractioned by D101 macroporous resin column chromatography
(
2
O gradient (0:10, 5:5 and 9:1, v/v) to provide three fractions, ACT-
1
2
3
3
2
2
9
2
(
(
3
preparative HPLC using MeCN-H
2
(
2
3
2
2
2
2
.4. Spectroscopic data of the isolates
.4.1. 8S-Deca-9-en-4,6-diyne-1,8-diol (1)
1
8
Colorless oil; [α]
D
: – 37 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 255 (2.38), 241 (2.55), 201
(
3.38) nm; CD (0.0008 M, MeOH) λmax (∆ε) 197 (− 11.8) nm; IR (KBr) νmax 3417, 3398, 2926, 2254,
–
1
1
13
–
1
632, 1384, 1029, 987, 933 cm ; H and C NMR data see Table 1; HRESIMS m/z 163.0759 [M–H]
, 163.0765, ‒ 0.6 mDa).
(calcd for C10
H
11
O
2
2
.4.2. (S)-deca-4,6,8-triyne-1,3-diol (2)
2
0
1
13
Colorless oil; [α]
D
: + 22 (c 0.19, MeOH); H and C NMR data see Table 1; HRESIMS m/z
+
1
63.0768 [M+H] (calcd for C10
H
10
O
2
, 163.0754, + 1.4 mDa).
2
.4.3. 3S-hydroxyundeca-5,7,9-triynoic acid (3)

2
2
Colorless oil; [α]
D
: +8.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (5.02) nm; IR (KBr) νmax
–
1
1
13
3
427, 2926, 2220, 1741, 1722, 1637, 1422, 1386, 1172, 1049, 1022 cm ; H and C NMR data see
+
Table 2; HRESIMS m/z 191.0660 [M+H] (calcd for C11
H
10
O
3
, 191.0730, − 4.3 mDa).
2
.4.4. 3S-Hydroxyundeca-5,7,9-triynoic acid 3-O-β-D-glucopyranoside (4)
1
8
Colorless oil; [α]
D
: –48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (5.10) nm; CD (0.0003 M,
MeOH) λmax (∆ε) 196 (− 8.5), 214 (+ 5.2) nm; IR (KBr) νmax 3421, 2924, 2222, 1715, 1631, 1573, 1414,
–
1
1
13
+
1
162, 1075, 1033 cm ; H and C NMR data see Table 2; HRESIMS m/z 375.1049 [M + Na] (calcd
Na, 375.1050, − 0.1 mDa).
20 8
for C17H O
2
.5. Acidic hydrolysis
Compound 4 (3.0 mg) was hydrolyzed by 1M HCl (2 mL) under reflux for 5 h. After
neutralization with NaHCO , the reaction was extracted with CH Cl . The aqueous layer was purified by
repeated Si CC to give the D-glucose which was certified by comparing with the authentic sample on
silica gel TLC (CHCl -MeOH-H O, 6:4:0.4) and the [α] value of + 29 (c 0.05, MeOH).
3
2
2
3
2
D
2
.5. Anti-HBV assay on HepG 2.2.15 cell line in vitro
HepG 2.2.15 cells were cultured in RPMI-1640 medium (Gibco) which was supplemented with 10%
fetal calf serum (Gibco), 100 μg/mL G148 (Gibco), 100 IU/mL penicillin (Gibco) and 100 IU/mL
streptomycin (Gibco). The anti-HBV assay was performed according to the previous report (Geng et al.,
2
011), with tenofovir (Jiangxi Chenyang Pharmaceutial, China, purity > 97.6%) as the positive control.
In brief, the inhibition of the secretions of HBsAg and HBeAg was assayed by the ELISA method; the
inhibition of HBV DNA replication was tested by the real-time quantitative PCR method; and the
cytotoxicity was evaluated by the modified MTT method. All the samples were dissolved in appropriate
DMSO to make sure the final concentration of DMSO not higher than 2.5% (v/v) in culture.
3
. Results and discussion
3
.1. Characterization of the isolates
The aerial parts of A. capillaris were extracted with 90% EtOH to give the total extraction (ACT),
which was fractionated by D101 macroporous resin CC to yield three fractions, ACT-1~ACT-3. Based
on the bioassay on HepG 2.2.15 cells, the active ACT-2 was separated by repeated silica gel CC,

Sephadex LH-20 CC, MPLC and HPLC to afford four new enynes (1–4), eight known enynes (5–12)
and two enyne analogs (13–14) (Fig. 1).
Fig. 1. Structures of compounds 1–14.
12 2
Compound 1 was characterized with the molecular formula of C10H O from the negative mode
–
HRESIMS at m/z 163.0759 ([M–H] , calcd. for 163.0765, −0.6 mDa) with five degrees of unsaturation.
The existence of hydroxyls and acetylenic bonds was confirmed from the characteristic IR absorptions at
–
1
13
3
417, 3398 and 2254 cm . In accordance with the molecular formula, the C NMR (DEPT) spectrum
displayed ten carbon resonances involving four methylenes, two methines (one oxygenated) and four
quaternary carbons, of which two alkynyl [δ 82.1 (s), 65.2 (s), 70.5 (s) and 76.6 (s)] and one alkenyl [δ
38.6 (s) and 115.8 (t)] groups were obviously recognized. The presence of a terminal double bond was
C
C
1
H
verified from the proton signals at δ 5.91 (1H, ddd, J = 17.0, 10.2, 5.4 Hz), 5.38 (1H, dt, J = 17.0, 1.4
Hz) and 5.14 (1H, dt, J = 10.2, 1.4 Hz). All the five unsaturated degrees were occupied by the alkynyl
and alkenyl functionalities, which required compound 1 to possess an acyclic skeleton. The NMR data
of 1 were similar to these of deca-9-en-4,6-diyne-1,8-diol 8-O-β-D-glucopyranoside (Kurimoto et al.,
2
010), except for the absence of the glucosyl group and the obviously shielded C-8 from δ
C
70.1 to 63.4
(Table 1). From the above analysis, compound 1 was deduced to be the aglycone of deca-9-en-4,6-
1
1
diyne-1,8-diol 8-O-β-D-glucopyranoside, which was totally validated by the H H COSY correlations of
OH-1/H-1/H-2/H-3 and OH-8/H-8/H-9/H-10, and HMBC correlations from H-2 to C-4, from H-3 to C-4
and C-5, from H-8 to C-6 and C-7, and from H-9 to C-7. The absolute configuration of C-8 was deduced
D
to be S by the negative [α] value of –37.1 (Pan et al., 2006), and the good match between the

experimental and calculated ECD spectra. Hence, the structure of 1 was determined as 8S-deca-9-en-4,6-
diyne-1,8-diol.
1
13
a
6
Table 1. H and C NMR data of compounds 1–2 in acetone-d .
1
2
No.
1
13
1
13
H NMR
C NMR
H NMR
C NMR
3
3
.74, m
.68, m
1
3.61, td, 6.2, 5.4
60.8, t
58.8, t
2
3
4
5
6
7
8
9
1.71, m
2.41, t, 7.0
−
−
−
−
32.2, t
16.1, t
82.1, s
65.2, s
70.5, s
76.6, s
63.4, d
138.6, d
1.87, m
4.63, td, 6.1, 5.8
41.1, t
60.2, d
80.5, s
68.9, s
64.1, s
59.2, s
64.7, s
78.4, s
–
–
–
–
–
–
4.91, dd, 6.3, 5.4
5.91, ddd, 17.0, 10.2, 5.4
5
5
.38, dt, 17.0, 1.4
.14, dt, 10.2, 1.4
1
0
115.8, t
2.00, s
4.0, q
OH-1
OH-8(3)
3.71, t, 5.4
4.80, d, 6.3
−
−
3.79, t, 5.1
4.77, d, 5.8
–
–
a 1
13
H NMR recorded in 600 MHz, C NMR recorded in 150 MHz.
Compound 2 was assigned with the molecular formula of C10
H
10
O
2
from the protonated ion
+
1
(
[M+H] ) at m/z 163.0768 (calcd. for 163.0754, +1.4 mDa) in the positive HRESIMS. Its H NMR
spectrum showed the presence of one oxygenated methine (δ
and 3.68), one methylene (δ 1.87) and one tertiary methyl (δ
.77 and 3.79 which showed no correlation with any carbon in HSQC spectrum. In the C NMR
spectrum, all ten carbons were well resolved involving six sp-hybridized carbons (three alkynyls), one
H
4.63), one oxygenated methylene (δ
H
3.74
H
H
2.00), as well as two active protons at δ
H
1
3
4
1
1
methine, two methylenes and one methyl. The coherent H H COSY correlations of OH-1/H-1/H-2/H-
/OH-3 constructed the 1,3-dihydroxypropyl moiety [HOCH CH CH(OH)-] which was deduced to be
3
2
2
linked with the alkynyl system according to the HMBC experiment: from H-2, H-3 and OH-3 to C-4,
and from H-3 to C-5. Similarly, the HMBC correlations from H-10 to C-9 and C-8 verified the
connection of methyl with the alkynyl bond to fulfill the structure (Fig. 2). The only stereocenter of C-3
D
was determined to be S-configuration based on the positive optical rotation in [α] experiment (Bentley
et al., 1969). Thus, the structure of 2 was elucidated to be (S)-deca-4,6,8-triyne-1,3-diol.

Fig. 2. Selected 2D NMR correlations of compounds 1–4.
10 3
Compound 3 had the molecular formula of C11H O which was proposed from the positive
+
HRESIMS at m/z 191.0660 ([M+H] , calcd. for 191.0730, −4.3 mDa). In accordance with its molecular
formula, all 11 carbons including one carboxy, six acetylenic carbons, one methine, two methylenes and
13
1
1
one methyl were recognized in the C NMR spectrum (Table 2). The H H COSY correlations of H-
/H-3/H-4, and HMBC correlations from H-3 to C-1 constructed the 3-carboxy-2-hydroxypropyl moiety.
Three pairs of triple bonds (δ 76.3, 68.0, 61.5, 60.0, 65.1 and 76.4) were proposed to connect the 3-
2
C
carboxy-2-hydroxypropyl moiety and the residual methyl based on the HMBC correlations from H-3 to
C-5, from H-4 to C-6, and from H-11 to C-10 and C-9. Similar to 2, the positive optical rotation
indicated an S configuration of C-3 (Yan et al., 1994). Therefore, the structure of 3 was characterized as
3
S-hydroxyundeca-5,7,9-triynoic acid.
1
13
a
3
Table 2 H and C NMR data of compounds 3–4 in CD OD .
3
4
No.
1
1
13
1
13
H NMR
C NMR
H NMR
C NMR
−
175.0, s
−
173.2, s
2
2
.45, dd, 15.6, 8.1
.56, dd, 15.6,4.7
2.74 (1H, dd, 16.0, 6.8)
2.67 (1H, dd, 16.0, 5.5)
2
42.0, t
38.9, t
3
4
5
6
7
8
9
4.14, m
2.55, d, 5.2
−
−
−
−
−
−
67.6, d
28.3, t
76.3, s
68.0, s
61.5, s
60.0, s
65.1, s
76.4, s
3.7, q
−
4.32 (1H, m)
2.75 (2H, d, 5.4)
−
−
−
−
−
−
72.8, d
23.3, t
73.7, s
65.8, s
59.4, s
57.7, s
62.8, s
74.2, s
1.4, q
1
0
1
1
1.94, s
−
−
1.97 (3H, s)
4.43 (1H, d, 7.8)
3.18 (1H, dd, 8.5, 7.8)
1
2
'
'
101.1, d
72.6, d
−

3
4
5
'
'
'
−
−
−
−
−
−
3.37 (1H,dd, 8.5, 8.5)
3.30 (1H, m)
3.27 (1H, m)
75.6, d
69.1, d
75.6, d
3
3
.86 (1H, dd, 12.0, 2.0)
.68 (1H, dd, 12.0, 5.6)
6
'
−
−
60.4, t
a 1
13
H NMR recorded in 600 MHz, C NMR recorded in 150 MHz.
20 8
Compound 4 with the molecular formula of C17H O was deduced from the positive HRESIMS
+
experiment that displayed [M+Na] ion at m/z 375.1049 (calcd. for 375.1050, −0.1 mDa), indicating
1
3
eight unsaturated degrees. In the C NMR (DEPT) spectrum, the characteristic carbons at δ
2.6 (d), 75.6 (d), 75.6 (d), 69.1 (d) and 60.4 (t) indicated a glucosyl moiety which was determined to be
β-D-configuration based on the large coupling constant (J = 7.8 Hz) of the anomeric proton (H-1') and
the positive [α] value of the glucose after acidic hydrolysis. In addition to the glucosyl moiety, the
C
101.1 (d),
7
D
leaving 11 carbons corresponding to one tertiary methyl, two methylenes, one methine and seven
quaternary carbons (involving three alkynyl bonds and one carboxy) suggested a C11 polyacetylene. The
NMR data of the aglycone part showed high similarity with those of 3 except for the obviously
C
deshielded shift of C-3 from δ 67.6 in 3 to 72.8 in 4, as well as the slight drift of the adjacent carbons.
Thus, compound 4 should be the 3-O-glucoside of 3, which was verified from the HMBC correlations
from H-1' to C-3 and from H-3 to C-1'. The above deduction was totally consistent with the 2D NMR
1
1
experiment: the H H COSY correlations of H-2/H-3/H-4 and HMBC correlations from H-3 and C-1
and C-5, and from H-11 to C-10 and C-9. The absolute configuration of C-3 was determined to be S by
the experimental ECD spectrum that showed a positive Cotton effect at λmax 216 nm (Berova et al., 2007)
and well matched with the calculated one (Fig. 3). Eventually, the structure of 4 was elucidated as 3S-
hydroxyundeca-5,7,9-triynoic acid 3-O-β-D-glucopyranoside.
Fig. 3. Experimental and calculated ECD spectra of compounds 1 and 4.
The known compounds were identified as atractylodin (5) (Chen et al., 2012), dendroarboreol B
(
6), dehydrofalcarinol (7), dehydrofalcarindiol (8) (Bernart et al., 1996), (E)-deca-2-en-4,6-diyne-1,10-
diol (9), (Z)-deca-2-en-4,6-diyne-1,10-diol (10) (Jente et al., 1979), 8S-deca-9-en-4,6-diyne-1,8-diol 1-

O-β-D-glucopyranoside (11) (Kurimoto et al., 2010), 3S,8S-dihydroxydec-9-ene-4,6-diyne 1-O-β-D-
glucopyranoside (12) (Zhao et al., 2014), 5-benzylthiophencarboxylic acid (13) (Knght and Nott, 1980)
and 2-methyl-6-phenyl-4H-pyran-4-one (14) (Kishida et al., 1969) by comparing their spectra data with
those reported in literatures.
3
.2. Anti-HBV activity of extracts
The total extract (ACT) and each fraction (ACT-1~ACT-3) of A. capillaris were assayed for their
anti-HBV activity on HepG 2.2.15 cell line in vitro. As shown in Table 3 and Fig. 4, ACT showed
moderate inhibition on the secretions of HBsAg and HBeAg with IC50 values of 689.6±153.3 μg/mL
(
SI>1.4) and 216.3±61.7 μg/mL (SI>4.6), and on HBV DNA replication with an IC50 value of 52.8±13.8
μg/mL (SI>18.9). A relative low cytotoxicity for ACT was revealed from the cell viability being around
4.0% at the highest tested concentration (1000 μg/mL).
6
a
Table 3 Anti-HBV activity of extracts from A. capillaris .
HBsAg
HBeAg
IC50 (μg/mL)
>1.4 216.3±61.7
>1000.0
>6.0 78.0±21.3
HBV DNA
IC50 (μg/mL)
>4.6 52.8±13.8
>250.0
>12.8 15.4±5.8
Fractions
CC50 (μg/mL)
IC50 (μg/mL)
689.6±153.3
>1000.0
166.8±49.3
461.0±64.1
>1000.0
SI
SI
SI
>18.9
–
>64.9
14.9
>192.3
ACT
>1000.0
>1000.0
>1000.0
556.1±72.5
>1000.0
ACT-1
ACT-2
ACT-3
–
–
1.2
–
318.8±40.1
>1000.0
1.7
–
37.4±9.5
5.2
b
Tenofovir
a
b
Data were expressed as mean ± S.D. (n = 3). Tenofovir was used as the positive control.
After separated by macroporous resin D101 CC, the obtained three fractions (ACT-1~ACT-3) were
further assayed for their anti-HBV effects to certify the active parts. ACT-1 was inactive on both the
secretions of HBsAg and HBeAg and HBV DNA replication, as well as low cytotoxicity. ACT-2
displayed obviously increased activity on both HBsAg and HBeAg secretions and HBV DNA
replication, with IC50 values of 166.8 (SI>6.0), 78.0 (SI>12.8) and 15.4 (>64.9), respectively. Although
ACT-3 showed slightly enhanced anti-HBV activity, the obvious cytotoxicity (CC50=556 μg/mL) gave
rise to low selective indices. From the above analyses, ACT-2 and ACT-3 should be corresponding to
the active and toxic parts of A. capillaris. Thus, ACT-2 was selected for the subsequent investigation,
from which 12 enynes (1–12) and two enyne analogs (13–14) were obtained.

Fig. 4. Anti-HBV activity of fractions from A. capillaris. Results were expressed as mean ± S.D. (n = 3).
3
.3. Anti-HBV activity of the isolates
The anti-HBV activity of compounds 1–14 was evaluated on HepG 2.2.15 cell line in vitro, which
was expressed as IC50 values of HBsAg, HBeAg, and HBV DNA replication (Table 4). Based on their
structures, the isolates can be classified into enediynes (1, 5–10), enediyne glucosides (11, 12),
enetriynes (2, 3), enetriyne glucoside (4) and enyne analogs (13, 14).
a
Table 4. Anti-HBV activity of compounds 1-14 .
HBsAg
IC50 (μM)
HBeAg
IC50 (μM)
HBV DNA
Compounds
CC50 (μM)
SI
SI
IC50 (μM)
SI
1
2
3
4
5
6
7
8
9
463.4±23.5
365.3±18.7
889.4±56.9
>1000.0
563.3±24.1
347.2±10.3
217.8±21.6
197.2±17.8
368.1±30.4
225.4±35.6
201.2±18.8
325.4±32.1
247.3±32.4
200.6±11.4
174.2±23.5
150.3±21.5
147.1±18.8
117.3±26.1
>1000
<1
<1
245.2±14.7
301.6±23.6
98.4±14.7
48.7±13.6
157.9±22.4
145.7±26.5
88.4±19.5
41.3±11.8
165.1±22.7
114.5±14.8
67.4±7.6
1.9
1.2
9.0
>20.5
6.0
5.8
4.2
9.7
4.0
4.8
12.9
13.0
2.1
3.3
56.9±10.6
66.4±14.3
16.7±13.2
9.8±4.4
8.1
5.5
4.1
>5.1
2.6
3.7
1.8
1.2
2.6
2.7
5.0
5.1
1.5
2.8
53.3
>102.0
27.7
15.4
8.1
5.6
7.5
7.1
79.3
42.2
3.5
4.8
>294.1
945.3±54.1
842.4±10.6
367.4±14.7
401.5±22.3
652.4±34.8
550.4±27.6
872.4±30.1
771.6±44.3
224.6±17.9
324.1±11.6
>1000
34.1±9.8
54.6±12.6
45.6±9.4
71.4±12.3
87.3±10.9
77.8±14.0
11.0±4.9
18.3±8.7
65.1±12.4
67.1±22.1
3.4±1.1
1
0
1
1
1
1
1
2
3
4
59.2±13.4
108.9±20.3
98.2±17.1
>1000
b
Tenofovir

a
b
Data were expressed as mean ± S.D. (n = 3). Tenofovir was used as the positive control.
The enediynes showed moderate anti-HBV potency compared to their glucosides, with IC50 values
(HBV DNA) in the range of 34.1~87.3 μM. The furan derivative (5) could obviously inhibited HBV
DNA replication with an IC50 value of 34.1 uM (SI=27.7), indicating the importance of furan ring.
Compounds 6 and 8 are a pair of isomers with different position and configuration of the double bond.
Compound 8 showed higher activity on HBeAg secretion (IC50=41.3 μg/mL, SI=9.7) than 6 (IC50=145.7
μg/mL, SI=5.8), indicating that the E-form double bond and conjugated system are unfavorable.
Compound 7 with the absence of OH-7 exhibited decreased inhibition on HBeAg, suggesting the
importance of hydroxyl group. Compounds 9 and 10 are a pair of E/Z-isomeric enediynes showed
similar activity inhibiting HBsAg and HBeAg secretions and HBV DNA replication, suggested the
conformation of double bond plays little role in maintaining activity. Compared to 9 and 10, compound
1
showed similar inhibition on HBeAg secretion and HBV DNA replication, but slightly decreased
activity on HBsAg. Three glucosides (4, 11, 12) showed higher activity than the aglycones: compound 4
as the most active compound could inhibit the secretions of HBsAg and HBeAg, and HBV DNA with
IC50 values of 197.2 (SI>5.1), 48.7 (SI>20.5) and 9.8 (SI>102) μg/mL, respectively (Fig. 5); compounds
1
1 and 12 also showed potency inhibiting the secretions of HBsAg (IC50=174.2 and 150.3 μg/mL,
SI=5.0 and 5.1, respectively) and HBeAg (IC50=67.4 and 59.2 μg/mL, SI=12.9 and 13.0, respectively),
and HBV DNA replication (IC50=11.0 and 18.3 μg/mL, SI=79.3 and 42.2, respectively). When carboxyl
group was involved in the structure, the anti-HBV activity was obviously increased (3 vs 2), suggesting
the polar group is preferable. Two enyne analogs (13 and 14) showed moderate activity inhibiting the
secretions of HBsAg and HBeAg, and HBV DNA replication.

Fig. 5. Cytotoxicity and anti-HBV effects of 4 inhibiting HBsAg, HBeAg and HBV DNA replication.
Results were expressed as mean ± S.D. (n = 3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs
control group.
4
. Conclusion
Yin-Chen as a famous TCM has been used for treating hepatitis B in China for thousands of years.
Enynes are the characteristic constituents, while their anti-HBV potency has not been systemically
investigated. In this investigation, 12 enynes (1–12) involving four new (1–4) and two unusual enyne
analogs (13–14) were isolated from the active parts of A. capillaris. Their inhibition on HBsAg and
HBeAg secretions and HBV DNA replication was systematically investigated in vitro. The enediyne
glucosides exhibited higher anti-HBV potency than the aglycones. Particularly, compound 4 could
inhibit the secretions of HBsAg and HBeAg, and HBV DNA with IC50 values of 197.2 (SI>5.1), 48.7
(SI>20.5) and 9.8 (SI>102) μM. Although compound 13 has been synthesized in 1980 (Knght and Nott,
1
980), this is the first time to report it as a natural product and its biological activity. From a
biosynthetic point of view, compounds 13 and 14 might be derived from the phenylalkyne-like precursor
that was also present in Artemisia plants. Based on the bioassay in vitro, the structure-activity
relationships were summarized as follows: 1) hydroxyl group is important for maintaining activity; 2)

glucosylation is preferable for increasing activity and decreasing cytotoxicity; 3) the configuration of the
double bond affects the anti-HBV activity. Tenofovir alafenamide (TAF), a novel prodrug of tenofovir,
was approved for the treatment of chronic HBV infection in 2016. Compared to tenofovir disoproxil
fumarate (TDF), TAF has the advantages of improved renal and bone safety and reduced plasma
tenofovir concentrations. Similar to other nucleoside analogs, TAF also takes effect by targeting HBV
reverse transcriptase. In addition, longer term follow-up data are still required to verify the difference in
viral/biochemical response and renal/bone safety between TAF and TDF (Chan et al., 2016; Ogawa et
al., 2017). This is the first time to systematically investigate the anti-HBV activity of enynes in A.
capillaris, which will provide valuable information for understanding the ethnopharmacological
application of Yin-Chen and searching new anti-HBV candidates from natural sources.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the CAS "Light of West China" Program (Western Youth Scholars
"A"), the Youth Innovation Promotion Association CAS, the National Natural Science Foundation of
China (No. 81202436), the Program of Yunling Scholarship, the Applied Basic Research Programs of
Yunnan Province (2017FB137).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at .
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