Chemical constituents from the roots and rhizomes of Asarum heterotropoides var. mandshuricum and the in vitro anti-inflammatory activity

Article abstract of DOI:10.3390/molecules22010125

Anti-inflammatory compounds were investigated from the ethanol extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum, a traditional Chinese medicine called Xixin and used for pain and inflammatory. Nine new compounds were isolated, including six new lignans, neoasarinin A-C (1-3), neoasarininoside A and B (4 and 5), and asarinin B (7), and one new monoterpene, asarincin A (8), two new amides, asaramid II and III (10 and 11), and one new natural monoterpene, asaricin B (9), along with 37 known compounds (6, 12-47). Their structures and absolute configurations were elucidated on the basis of spectroscopic methods and chemical analyses. This is the first report of the absolute configuration of asarinin A (6). The 8-O-40 neolignans (1-5) were reported in the genus Asarum for the first time. The 15 compounds 17, 19, 22-25, 28, 31, 36, 40, 42, 43, 45-47 were isolated from the genus Asarum, and compounds 16, 32, 33, 37 and 39 were isolated from A. heterotropoides var. mandshuricum for the first time. Thirty-seven of the isolates were evaluated for anti-inflammatory activity against the release of β-glucuronidase in polymorphonuclear leukocytes (PMNs) induced by the platelet-activating factor (PAF), and compounds 1, 4, 7, 8, 14, 17-19, 22, 24, 25, 29, 30, 32, 33, 40-43, 45, and 46 showed potent anti-inflammatory activities in vitro, with 27.9%-72.6% inhibitions at 10^-5 mol/L. The results of anti-inflammatory assay suggested that lignans obtained from the CHCl₃ extract might be the main active components of Xixin.

Full text of DOI:10.3390/molecules22010125

molecules
Article
Chemical Constituents from the Roots and Rhizomes
of Asarum heterotropoides var. mandshuricum and the
In Vitro Anti-Inflammatory Activity
Yu Jing 1,2, Yi-Fan Zhang 1, Ming-Ying Shang 1,*, Guang-Xue Liu 1, Yao-Li Li 1, Xuan Wang 1 and
Shao-Qing Cai 1,
*
1
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University Health Science Center, No. 38, Xueyuan Road, Beijing 100191, China;
jing_jy@126.com (Y.J.); zhangyifan@bjmu.edu.cn (Y.-F.Z.); guangxl@bjmu.edu.cn (G.-X.L.);
13466759800@163.com (Y.-L.L.); xuanwang6818@bjmu.edu.cn (X.W.)
Tonghua Gold-Horse Pharmaceutical Group Co., Ltd., Beijing 100025, China
Correspondence: myshang@bjmu.edu.cn (M.-Y.S.); sqcai@bjmu.edu.cn (S.-Q.C.);
Tel./Fax: +86-10-8280-2534 (M.-Y.S.); +86-10-8280-1693 (S.-Q.C.)
2
*
Academic Editor: Derek J. McPhee
Received: 14 December 2016; Accepted: 6 January 2017; Published: 13 January 2017
Abstract: Anti-inflammatory compounds were investigated from the ethanol extract of the roots
and rhizomes of Asarum heterotropoides var. mandshuricum, a traditional Chinese medicine called
Xixin and used for pain and inflammatory. Nine new compounds were isolated, including six new
lignans, neoasarinin A–C (
new monoterpene, asarincin A (
natural monoterpene, asaricin B (
1
–
3
), neoasarininoside A and B (
), two new amides, asaramid II and III (10 and 11), and one new
), along with 37 known compounds ( 12 47). Their structures and
4 and 5), and asarinin B (7), and one
8
9
6,
–
absolute configurations were elucidated on the basis of spectroscopic methods and chemical analyses.
0
This is the first report of the absolute configuration of asarinin A (
reported in the genus Asarum for the first time. The 15 compounds 17
45 47 were isolated from the genus Asarum, and compounds 16 32 33
A. heterotropoides var. mandshuricum for the first time. Thirty-seven of the isolates were evaluated for
anti-inflammatory activity against the release of -glucuronidase in polymorphonuclear leukocytes
(PMNs) induced by the platelet-activating factor (PAF), and compounds 14 17 19 22 24 25
6
). The 8-O-4 neolignans (
1
40
–
5
,
) were
42 43
37 and 39 were isolated from
,
19
,
22
–
25
,
28
,
31
,
36
,
,
,
–
,
,
,
β
1,
4,
7
,
8
,
,
–
,
,
,
,
29, 30, 32, 33, 40–43, 45, and 46 showed potent anti-inflammatory activities in vitro, with 27.9%–72.6%
inhibitions at 10−5 mol/L. The results of anti-inflammatory assay suggested that lignans obtained
from the CHCl3 extract might be the main active components of Xixin.
Keywords: Asarum heterotropoides var. mandshuricum; Aristolochiaceae; lignans; 8-O-40 neolignan;
amide; monoterpene; anti-inflammatory
1. Introduction
The roots and rhizomes of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag,
which is described as Xixin in Chinese or Asari Radix et Rhizoma (AR) in the Pharmacopoeia of the
People’s Republic of China (2010 edition), is one of the most important traditional Chinese medicines
of the genus Asarum (Aristolochiaceae). It was widely used in traditional medicine as an anodyne and
anti-allergic remedy to treat various diseases such as aphthous stomatitis, headache, toothache, and
inflammation in some countries of Asia [
var. mandshuricum resulted in the identification of chemical constituents with diverse structures
including volatile oil [ ], lignans [ ], amides [ ], terpenes [ ], flavones [ 12], and benzene
derivatives [ 11 14]. Additionally, the volatile oil of AR was considered the main effective component
1]. Previous phytochemical investigations of A. heterotropoides
2
3–
6
7
8
6,9–
6,
–
Molecules 2017, 22, 125; doi:10.3390/molecules22010125
www.mdpi.com/journal/molecules

Molecules 2017, 22, 125
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contributing to analgesia and anti-inflammation [15
,16]. However, when a clinical application of a
water decoction of AR was used to treat pain or inflammation diseases, the decocting process lasted
for a long time [17 18]. Moreover, several studies proved that the volatile oil content was very low
in the water decoction boiling for a long time of AR [19 20]. Meanwhile, in the course of our studies
,
,
on anti-inflammatory and anti-nociceptive effects in mice of water and ethanol extracts of AR [21],
it was also found that both 95% and 50% ethanol extracts showed significantly anti-inflammatory
effects in the xylene-induced mice ear edema test with inhibitory rates of 61.86% and 52.56% at
0.2 g/kg, respectively. According to the previous results, it could be concluded that non-volatile
constituents existed in AR also had the anti-inflammatory and analgesic activities. However, although
AR worked to control pain and inflammation in traditional Chinese medicine prescriptions, there was
not enough research to explain its non-volatile active components of analgesia and anti-inflammation,
in spite of a few previous papers reporting its potential anti-tussive, anti-tumor, and anti-inflammatory
effects [5,6,22,23], along with anti-allergic and anti-inflammatory components of Asiasarum sieboldi
F. Maekawa belonging to the same genus [24–26].
In order to investigate the anti-inflammatory constituents in the roots and rhizomes of
A. heterotropoides var. mandshuricum, the present study was therefore designed to describe the extraction,
isolation, structure characterization, and anti-inflammatory activity screening of non-volatile
compounds isolated from ethanol extract of this plant. As a result, 47 compounds were isolated and
identified, including six new lignans (1–5, 7), one new monoterpene (8), two new amides (10 and 11),
one new natural product (
elucidated on the basis of IR, UV, MS, CD, and 1D and 2D NMR spectra. Since
plays an important role in the inflammatory process, inhibitors of -glucuronidase release may be
9), and 37 known compounds. The structures of new compounds were
β-glucuronidase
β
accordingly considered therapeutic agents in inflammatory diseases [27]. Consequently, in order to
evaluate the anti-inflammatory activity of non-volatile constituents, 37 compounds were screened
in vitro by assessing the inhibitory rate of the release of
β-glucuronidase in polymorphonuclear
leukocytes (PMNs) induced by the platelet-activating factor (PAF) in this paper.
2. Results and Discussion
The EtOH extract of the roots and rhizomes of A. heterotropoides var. mandshuricum was
suspended in H2O and then partitioned with petroleum ether, CHCl3, EtOAc, and n-BuOH,
successively. The CHCl3, EtOAc, and n-BuOH extracts were separated and purified by various column
chromatographic methods to yield six new lignans: neoasarinin A (
), neoasarininoside A ( ), neoasarininoside B ( ), and asarinin B (
A ( ); two new amides: asaramid II (10) and III (11); and one new natural monoterpene: asaricin B (
together with 37 known compounds ( 12 47) (Figures 1 and 2). Compounds 12 19 21 25
27–33, 37, and 40–47 were tested for anti-inflammatory activity.
1
7
), neoasarinin B (
); one new monoterpene: asarincin
),
2), neoasarinin C
(3
4
5
8
9
6,
–
1
,
2,
4–9,
–
,
– ,
Compound 1, an amorphous powder, was shown to have a molecular formula of C19H24O7 by
HRMALDIMS (m/z 387.1403 [M + Na]+, calcd. 387.1414). The IR spectrum showed absorptions for
hydroxyl (3445 cm−1). The 1H-NMR spectrum showed an ABX and an AX system aromatic proton
signals at
H-5), and 6.41 (1H, d, J = 3.0 Hz, H-20) and 6.29 (1H, d, J = 3.0 Hz, H-60), respectively. The 1H-NMR
signals at 3.81 (3H, s), 3.80 (3H, s), 3.71 (3H, s), and 2.12 (3H, s) supported the existence of three
methoxyl groups and a methyl group. Through the analysis of HMBC spectrum, the correlations
between H-2 and C-4, C-7, H-6 and C-4, C-7, H-5 and C-3, C-1, the methoxyl protons ( 3.71) and C-3
indicated the presence of a 3-methoxyl-4-hydroxyphenyl unit. And the HMBC cross peaks between
H-20 and C-40, C-60, H-60 and C-40, C-20 and methyl carbon ( 2.12) and C-40,
15.7), methyl protons (
C-6 could be attributed to a 5-methyl-1,3-dimethoxylphenyl unit (Figure 3). The other 1H-NMR signals,
including two oxygenated methine protons ( 4.87 (1H, d, J = 4.2 Hz, H-7) and 4.18 (1H, m, H-8)),
δ 6.96 (1H, d, J = 2.4 Hz, H-2), 6.77 (1H, dd, J = 8.4, 2.4 Hz, H-6), and 6.73 (1H, d, J = 8.4 Hz,
δ
δ
δ
δ
0
δ
an oxygenated methylene group (
δ
3.83 (1H, m, H-9a) and 3.62 (1H, dd, J = 12.6, 3.6 Hz, H0-9b)), in
combination with the HMBC and 1H-1H COSY experiments, suggested that
1 could be a 8-O-4 type of
neolignan [28].

Molecules 2017, 22, 125
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OCH3
OH
OCH3
R1
OH
H C
O
S1:
S2:
H3C
HO
2
HOH2C
O
O
HOH2C
C
O
O
O
R2
R3
OH
O
7'
OH
8
H
HO
7
8'
H3CO
HO
R5
R6
7
H3CO
HO
R4
OH
OH
OH
O
C
C
C
O
O
OH
OH
H
H
HO
R1
R2
H
H
R3
R4
R5
R6
1
4
5
CH3
OCH3
H
OCH3 OCH3 OCH3 OH
2
3
7 R
7 S
6
S1
OCH3
H
H
OCH2O
OCH3 OH
S2
OCH3
HO
R1
7'
CH2OH
HOH2C
OH
HO
R2
R3
8
H
H3CO
HO
OCH3
OH
O
7
H3CO
HO
O
7
O
8'
H
H
8
7'
R4
R5
O
19
22 7 S, 8 R, 7' R
23 7 R, 8 S, 7' S
24 7 R, 8 S, 7' R
R1
R2
R3
R4
R5
O
O
7
OCH3 OH
OCH3 OH
OCH3 OCH3 OH
7 R, 8 S, 7' S, 8' S
7 S, 8 R, 7' R, 8' R
7 S, 8 R, 7' R, 8' R
7 S, 8 R, 7' S, 8' R
7 R, 8 S, 7' S, 8' S
O
HOH2C
OCH3
OCH3
13
14
15
16
17
18
20
OCH3
OCH2O
OCH2O
OCH2O
OCH2O
HO
H
H
H
H
H
H
OCH2O
OCH2O
H3CO
H3CO
H
OH
O
O
O
OH
OCH3 OH
OH
OCH3
OH OCH3 7 S, 8 R, 7' S, 8' R
7 S, 8 R, 7' R, 8' R
OCH3 OCH3 OH
21
25
OCH3 OH
H
OCH2O
7 R, 8 S, 7' R, 8' S
Figure 1. Chemical structures of compounds 1–7 and 13–25.
Figure 2. Chemical structures of compounds 8–12 and 26–47.
The coupling constant between H-7 and H-8 was measured to be 4.2 Hz, which resulted in
an erythro configuration at C-7 and C-8 [29 30]. Also, the NOE cross peaks observed between H-7
and H-8 indicated the relative configuration to be cis. A negative Cotton effect at 219 nm suggested
the absolute configuration of 7S, 8R in by analysis of its CD spectrum [31 32]. From these data,
the structure of , therefore, was established as erythro-(7S,8R)-7-(3-methoxyl-4-hydroxyphenyl)-8-
(50-methyl-10,30-dimethoxylphenoxyl) propane-7,9-diol, and it was named neoasarinin A.
,
1
,
1

Molecules 2017, 22, 125
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Figure 3. Key HMBC correlations (H → C) of compounds 1, 2, and 4–5.
The spectroscopic data of compound
1
are as follows: amorphous powder; [α]2D0 +10.0
) 231 (4.05), 280 (3.70), 308 (3.01) nm; CD (MeOH)
(c 0.60, MeOH); UV (MeOH)
λ
(log
ε
λ
max
max
(
c 0.56 × 10−4 mol/L, ∆ε) 219 (–0.48), 293 (–0.24); IR (KBr)
νmax 3445, 2939, 2873, 1602, 1516, 1493, 1464,
1332, 1274, 1209, 1152, 1123, 1090, 1056, 1033, 825 cm−1; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR
(CD3OD, 150 MHz) data, see Table 1; EIMS m/z 364 [M]+ (4), 316 (11), 288 (4), 194 (32), 181 (8), 168
(100), 153 (63), 125 (25), 109(11), 93 (19), 65 (17), 53 (10); HRMALDIMS m/z 387.1403 [M + Na]+ (calcd.
for C19H24O7Na, 387.1414).
Table 1. NMR spectroscopic data (CD3OD) for compounds 1–3.
1
2
3
Position
δC
δ
(J in Hz)
δC
δ
(J in Hz)
δC
δ (J in Hz)
H
H
H
1
2
3
4
5
6
7
8
133.1
110.1
147.4
145.5
114.4
119.2
73.0
—
132.8
110.5
147.4
145.7
114.3 a
119.6
72.8
—
132.2
110.4
147.5
145.9
114.5 b
119.4
72.8
—
6.96, d (2.4)
—
7.00, d (2.4)
—
7.01, d (1.8)
—
—
—
—
6.73, d (8.4)
6.77, dd (8.4, 2.4)
4.87, d (4.2)
4.18, m
6.73, d (8.4)
6.82, dd (8.4, 2.4)
4.82, d (5.4)
4.28, m
6.75, d (8.4)
6.85, dd (8.4, 1.8)
4.88, d (6.0)
4.20, m
85.1
85.3
86.3
3.83, m, H-9a
3.62, dd (12.6, 3.6), H-9b
3.70, dd (12.0, 3.6), H-9a
3.41, m, H-9b
3.75–3.83, m
9
60.2
60.8
60.5
0
1
2
3
4
5
6
7
8
152.9
97.4
156.0
138.2
132.6
106.6
—
—
134.6
112.8
150.6
146.1
118.3
120.7
39.4
—
6.75, d (1.8)
—
134.9
112.7
150.4
146.5
118.2
120.8
39.5
—
6.81, d (1.8)
—
0
6.41, d (3.0)
0
—
—
—
0
—
—
0
6.83, d (7.2)
6.64, dd (7.2, 1.8)
3.28, d (5.4)
5.92, m
6.98, d (7.8)
6.69, dd (7.8, 1.8)
3.33, m
0
6.29, d (3.0)
0
—
—
—
0
—
—
137.7
137.7
5.93, m
5.03, m
0
114.4 a
5.03, m
114.5 b
9
3-OCH3
54.9
54.5
55.0
15.7
3.71, s
3.80, s
3.81, s
2.12, s
55.4
55.3
3.79, s
55.0
55.2
3.82, s
0
1 -OCH3
0
3 -OCH3
3.76, s
3.84, s
0
5 -CH3
a−b
Assignments may be interchanged in the same column.
Compound
2
was isolated as an amorphous powder. The molecular formula, C20H24O6, was
were
indicative of the presence of a hydroxyl group (3423 cm−1) and a C=C double bond (1639 cm−1).
The 1H-NMR signals of two oxygenated methine protons (
4.82 (1H, d, J = 5.4 Hz, H-7) and 4.28 (1H,
m, H-8)), an oxygenated methylene group ( 3.75–3.83 (2H, m, H-9)), and HMBC correlations from
established by HRMALDIMS (m/z 383.1457 [M + Na]+, calcd. 383.1465). The IR absorptions of
2
δ
δ

Molecules 2017, 22, 125
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H-2 and H-6 to C-7, and H-8 to C-40 (Figure 3), supported the idea that
2
could be a 8-O-40 type of
neolignan [28]. The 1H-NMR spectrum showed the presence of a 3-methoxyl-4-hydroxyphenyl unit
(δ
7.00 (1H, d, J = 2.4 Hz, H-2), 6.73 (1H, d, J = 8.4 Hz, H-5), 6.82 (1H, dd, J = 8.4, 2.4 Hz, H-6) and
a methoxyl group at 3.79 (3H, s)), which was supported by the HMBC spectrum. In addition, the
1H-NMR spectrum of
δ
2
also showed an additional trisubstituted aromatic ring with an ABX system
protons at
δ
6.75 (1H, d, J = 1.8 Hz, H-20), 6.83 (1H, d, J = 7.2 Hz, H-50), 6.64 (1H, dd, J = 7.2, 1.8 Hz,
H-60) and a methoxyl group at
δ
3.76 (3H, s). Furthermore, by the analysis of H-, 13C-NMR and
1
1H-1H COSY spectra, the signals at
δ
3.28 (2H, d, J = 5.4 Hz, H-70) and 39.4 (C-70), 5.92 (1H, m,
δ
H-80) and 137.7 (C-80), and 5.03 (2H, m, H-90) and 114.4 (C-90) showed the existence of an allyl group.
The long-range correlations between H-20 and C-40, C-70, H-60 and C-40, C-70, H-50 and C-10, C-30, and
methoxyl protons (
trisubstituted aromatic ring being determined as a 1-allyl-3-methoxylphenyl unit.
The relative configuration of was established from the NOESY spectrum and the coupling
δ
3.76) and C-30 were observed in the HMBC spectrum (Figure 3), which led to the
2
constants between H-7 and H-8. The NOE cross peak of H-7 and H-8 was observed in the NOESY
spectrum, suggested the cis-configuration between H-7 and H-8. Moreover, the coupling constant
J
7,8 = 5.4 Hz demonstrated the relative configuration to be erythro [29
appearing at 241 nm in the CD spectrum indicated the absolute configuration of
Thus the structure of was determined to be erythro-(7R,8S)-7-(3-methoxyl-4-hydroxylphenyl)-8-
(10-allyl-30-methoxylphenoxyl) propane-7,9-diol, which named as neoasarinin B.
The spectroscopic data of compound
are as follows: amorphous powder; [α]2D0 –3.6
) 211 (4.03), 229 (3.95), 280 (3.55) nm; CD (MeOH)
,
30]. The positive cotton effect
2
was 7R, 8S [31 32].
,
2
2
(c 0.55, MeOH); UV (MeOH)
λ
max
(log
ε
λ
max
(c 0.58 × 10−4 mol/L, ∆ε) 241 (+0.54), 274 (+0.04), 300 (–0.15); IR (KBr)
ν
3423, 2936, 1639, 1605,
max
1511, 1463, 1429, 1268, 1223, 1153, 1126, 1031, 916, 817 cm−1; H-NMR (CD3OD, 600 MHz) and
13C-NMR (CD3OD, 150 MHz) data, see Table 1; HRMALDIMS m/z 383.1457 [M + Na]+ (calcd. for
C20H24O6Na, 383.1465).
1
Compound
3 was isolated as an amorphous powder. The molecular formula of it was determined
as C20H24O6 on the basis of HRESIMS (m/z 383.1455 [M + Na]+, calcd. 383.1465). Compound
3
had
1
the same molecular formula as
2
. And IR, UV, H- and 13C-NMR spectra of the two compounds
were very similar to each other, which revealed the identical plane structure of them with a
3-methoxyl-4-hydroxyphenyl unit and a 1-allyl-3-methoxylphenyl unit. The major differences between
2
and
1-allyl-3-methoxylphenyl unit. The chemical shift values of
the 1-allyl-3-methoxylphenyl unit of were all more downfield than those of
and 6.64 (H-60)). Furthermore, the more downfield chemical shift of C-8 (
and the chemical shifts of H-9 ( 3.70 (1H, dd, J = 12.0, 3.6 Hz, H-9a) and
were more highfield than those of 3.75–3.83 (2H, m)). Through the analysis of the above data, it
could be concluded that and were stereoisomers with different configurations at the chiral centers
of C-7 and C-8, respectively.
The coupling constant between H-7 and H-8 was J7,8 = 6.0 Hz, suggesting the relative configuration
to be threo [29 30], which confirmed no correlation between H-7 and H-8 in the NOESY spectrum.
The CD spectrum of showed the positive cotton effect at 238 nm, which established that the
absolute configurations at C-7 and C-8 were 7S and 8S, respectively [31 32]. On the basis of the
above data, the structure of was concluded to be threo-(7S,8S)-7-(3-methoxyl-4-hydroxyphenyl)-8-
(10-allyl-30-methoxylphenoxyl) propane-7,9-diol, and it was named neoasarinin C.
The spectroscopic data of compound
are as follows: amorphous powder; [α]2D0 –5.3
) 205 (3.33), 230 (2.77), 279 (2.38) nm; CD (MeOH)
3 were the chemical shift values of H-8, 9, and C-8, 9, and three aromatic protons for the
0 0 0
δ
6.81 (H-2 ), 6.98 (H-5 ) and 6.69 (H-6 ) for
0
0
3
2
(
δ
6.75 (H-2 ), 6.83 (H-5 )
86.3) of was observed,
3.41 (1H, m, H-9b)) of
δ
3
δ
δ
3
2 (δ
3
2
of
3
,
3
,
3
3
(c 0.38, MeOH); UV (MeOH)
λ
max
(log
ε
λ
max
(c 0.61 × 10−4 mol/L, ∆ε) 238 (+0.18), 288 (–0.18); IR (KBr)
νmax 3421, 2921, 1700, 1606, 1511, 1462, 1429,
1266, 1222, 1132, 1030, 874 cm−1; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz) data,
see Table 1; HRESIMS m/z 383.1455 [M + Na]+ ( calcd. for C20H24O6Na, 383.1465).
Compound
4 was obtained as an amorphous powder. Its molecular formula was determined to be
C24H18O13 by HRESIMS (m/z 525.1605 [M + H]+, calcd. 525.1602). The IR spectrum showed absorptions

Molecules 2017, 22, 125
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for hydroxyl (3133 cm−1) and conjugated carbonyl groups (1630 cm−1). The 1H- and 13C-NMR data
were similar to those of the known threo-guaiacylglycerol-
-O-40-vanillic acid ether, which revealed
the plane structure of
to be a breakdown product of 8-O-40 type of neolignan [33]. However, the
major difference between and threo-guaiacylglycerol-
-O-40-vanillic acid ether was the presence
of a methylenedioxy at 5.77 (2H, s) in . The HMBC correlations between H-2 and C-4, H-5 and
C-3, C-1, H-6 and C-4, and the methylenedioxy protons ( 5.77) and C-3, C-4 suggested the existence
β
4
4
β
δ
4
δ
of a 3,4-methylenedioxyphenyl group instead of the 3-methoxyl-4-hydroxyphenyl unit (Figure 3).
1
In addition, the H- and 13C-NMR spectra of
4
exhibited the signal of a set of
5.58 (1H, d, J = 7.8 Hz, H-1”) and the six characteristic aliphatic
94.8, 77.5, 76.7, 72.7, 69.7, and 61.0. The glycosidic linkage was determined to be at
165.1) by the correlation observed between H-1” and C-70 in HMBC experiments
β-glucopyranose,
including the anomeric proton at
carbon signals at
the formyl group (
(Figure 3). Acid hydrolysis of
δ
δ
δ
4
afforded a D-glucose. The HMBC spectrum also showed the correlations
between H-2, H-6 and C-7, H-8 and C-40, and H-20, H-60 and C-70, which further supported the
above conclusions.
The coupling constant value of 4.8 Hz between H-7 and H-8 revealed the erythro configuration [29,30].
Furthermore, a negative Cotton effect at 240 nm indicated that the absolute configuration of
8R [31,32]. From these data, the structure of 4
4
was 7S,
was, therefore, established as erythro-(7S,8R)-7-(3,4-
β
-D-glucopyranosyl)-30-methoxylphenoxyl) propane-7,9-diol,
methylenedioxyphenyl)-8-[10-(formyl-
and named as neoasarininoside A.
The spectroscopic data of compound
4
are as follows: amorphous powder; [α]2D0 +30.6
) 220 (4.01), 263 (3.81), 289 (3.71) nm; CD (MeOH)
(c 0.52, MeOH); UV (MeOH)
λ
(log
ε
λ
max
max
(
c 0.36 × 10−4 mol/L, ∆ε) 240 (–0.13), 264 (+0.30), 298 (+0.14); IR (KBr)
νmax 3133, 1630, 1605, 1400, 1349,
760 cm−1; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz) data, see Table 2. ESIMS
m/z 542 [M + NH4]+, 547 [M + Na]+; HRESIMS m/z 525.1605 [M + H]+ (calcd. for C24H29O13, 525.1602).
Table 2. NMR spectroscopic data (CD3OD) for compounds 4 and 5.
4
5
Position
δC
δ
(J in Hz)
δC
δ
(J in Hz)
H
H
1
2
3
4
5
6
7
8
135.1
107.4 a
147.5
147.1
107.1 a
120.4
72.5
—
136.2
111.0
149.1
146.1
116.21 d
119.8
72.4
—
6.87, d (1.8)
—
7.09, d (1.2)
—
—
7.07, m
—
6.59, d (7.8)
6.78, m
4.71, d (4.8)
4.44, m
6.94, dd (7.8, 1.2)
4.84, d (4.8)
83.8
84.1
4.47, m
3.85–3.76 e, m, H-9a
3.65, dd (12.0, 4.8), H-9b
9
3.77–3.74 c, m
60.8
61.0 b
0
1
2
3
4
5
6
7
8
122.1
113.0
149.7
152.9
114.7
123.8
165.1
—
—
94.8
72.7
76.7
—
150.4
150.2
116.15 d
122.0
128.4
110.8
144.8
116.11 d
169.5
101.6
73.6
—
—
0
7.46, d (1.2)
0
—
—
6.93, d (7.8)
7.04, dd (7.8, 1.8)
—
0
0
6.92, d (8.0)
7.52, dd (7.8, 1.2)
0
7.12, m
0
—
—
—
7.54, d (15.6)
6.31, d (15.6)
—
0
0
9
1”
2”
3”
4”
5”
6”
5.58, d (7.8)
4.80, d (7.2)
76.5
69.9
76.8
61.2
3.62–3.32, m
3.45–3.36 , m
3.85–3.76 e, m
69.7
77.5
3.77–3.74 c, m
61.0 b
-OCH2O-
3-OCH3
100.9
—
—
5.77, s
—
—
—
55.3
55.3
—
—
3.79, s
3.79, s
—
0
2 -OCH3
0
3 -OCH3
55.2
3.75, s
a−e
Assignments may be interchanged in the same column.

Molecules 2017, 22, 125
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Compound
5
was isolated as a needle crystal. The molecular formula of it was determined as
C26H32O13 on the basis of HRESIMS (m/z 551.1776 [M
showed absorptions for hydroxyl (3422 cm−1) and conjugated carbonyl groups (1688 cm−1). The 1H-
and 13C-NMR spectrum of -O-40-ferulic
, similarity with the known compound guaiacylglycerol-
acid ether [34], were attributed to two 1,3,4-trisubstituted aromatic rings ( 7.09 (1H, d, J = 1.2 Hz,
−
H]−, calcd. 551.1770). The IR spectrum
5
β
δ
H-2) and 111.0 (C-2), 7.07 (1H, overlapping, H-5) and 116.21 (C-5), and 6.94 (1H, dd, J = 7.8, 1.2 Hz,
H-6) and 119.8 (C-6), 7.12 (1H, overlapping, H-60) and 110.8 (C-60), 6.93 (1H, d, J = 7.8 Hz, H-30)
and 116.15 (C-30), and 7.04 (1H, dd, J = 7.8, 1.8 Hz, H-40) and 122.0 (C-40)), a (E)-formylvinyl unit
(δ
7.54 (1H, d, J = 15.6 Hz, H-70) and 144.8 (C-70), 6.31 (1H, d, J = 15.6 Hz, H-80) and 116.11 (C-80), and
carboxyl carbon at
and 72.4 (C-7), and 4.47 (1H, m, H-8) and 84.1 (C-8)), an oxygenated methylene group (
(1H, overlapping, H-9a,), 3.65 (1H, dd, J = 12.0, 4.8 Hz, H-9b) and 60.8 (C-9)), and two methoxyl
groups ( 3.79 (6H, s) and 55.3), indicating
was an 8-O-40 type of neolignan derivative. Comparing
the NMR data of with those of guaiacylglycerol-
-O-40-ferulic acid ether, there was not a ferulic
δ
169.5 (C-90)), two oxygenated methine protons (
δ
4.84 (1H, d, J = 4.8 Hz, H-7)
δ
3.85–3.76
δ
5
5
β
acid unit in t0he structure of0 5 but an isoferulic acid unit, whic0h further supported by the correla0tions
0
0
0
0
0
0
0
0
0
between H-6 and C-7 , C-4 , H-3 and C-1 , C-5 , H-4 and C-2 , H-7 and C-6 , C-4 and C-9 , H-8 and
C-50, the methoxyl protons (
correlations between H-8 and C-10, H-7 and C-2, C-6, H-2 and C-7 further confirmed the above
conclusions (Figure 3). The presence of a -glucopyranosyl moiety in the molecule was suggested by
the NMR spectrum, which showed six carbon signals at 101.6, 76.8, 76.5, 73.6, 69.9, and 61.2, and an
anomeric proton signal at 4.80 (1H, d, J = 7.2 Hz, H-1”). Acid hydrolysis and GC analysis proved
the absolute configuration of the sugar moiety to be D-glucose. The HMBC correlation between H-1”
δ
3.79) and C-20 in HMBC spectrum (Figure 3). Moreover, the HMBC
β
δ
δ
and carboxyl carbon (δ 169.5) was observed, confirming the β
-D-glucopyranosyl group linked to C-90.
The assignments of the 1H and 13C-NMR data were established by the analysis of the 1H-1H COSY,
HMQC and HMBC spectra. The coupling constant J7,8 was 4.8 Hz, which was indicative of the erythro
configuration between C-7 and C-8 [29,30]. The absolute configurations, 7S, 8R, were assigned on
the basis of a negative Cotton effect at 224 nm [31,32]. As a result, 5 was determined to be erythro-(7S,8R)-
7-(3-methoxyl-4-hydroxyphenyl)-8-{50-[(E)-formylvinyl- -D-glucopyranosyl]-20-methoxylphenoxyl}
β
propane-7,9-diol, and named neoasarininoside B.
The spectroscopic data of compound
5
are as follows: needle crystal (MeOH); m.p. 178–179 ◦C;
max (log ) 205 (4.10), 284 (3.89), 319 (3.91) nm; CD (MeOH)
3422, 2935, 1688, 1632, 1598, 1510,
[α]2D0 –23.3 (c 0.60, MeOH); UV (MeOH)
λ
ε
−4
λ
max
(c 0.39
×
10 mol/L, ∆ε) 224 (–1.17), 279 (–0.45); IR (KBr)
ν
max
1464, 1421, 1265, 1136, 1074, 1030, 755 cm−1; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD,
150 MHz) data, see Table 2; ESIMS m/z 570 [M + NH4]+, 575 [M + Na]+; HRESIMS m/z 551.1776
[M − H]− (calcd. for C26H31O13, 551.1770).
Compound 6 was obtained as an amorphous powder, with the molecular formula of C22H28O7
derived from the ion peak at m/z 427.17261 [M + Na]+ (calcd 427.1727) in HRESIMS. The IR spectrum
indicated the presence of a hydroxyl group (3412 cm−1). All the 1H-NMR, 13C-NMR, 1H-1H COSY, and
HMBC data are in good agreement with those of 4-[1-ethoxy-1-(4-hydroxy-3-methoxy) phenyl]
methyl-2-(4-hydroxy-3-methoxy)phenyl-3-hydroxymethyl-tetrahydrofuran [35]. The NOESY correlation
between H-7 and H-9 indicated that the two protons, H-70and H-8, were oriented as trans with each
0
0
0
other. The NOE cross peaks were observed at H-7 and H-9 a, H-8 and H-9 b, which indicated that the
relative configuration between C-70 and C-80 was trans. On the other hand, the trans configuration
between C-8 and C-80 was determined by the NOESY correlations between H-80 and H-9, H-70 and
H-8 (Figure 4). The absolute configuration of
with those of tanegool and analogues [36 37]. The CD spectrum of
at 235 and 278 nm, which were identical with the known compound tanegool. Thus, the structure
of
was determined to be (7S,8R,70R,80S)-70-[70-ethoxyl-70-(40-hydroxyl-30-methoxylphenyl)]methyl-
7-(4-hydroxyl-3-methoxylphenyl)-8-hydroxymethyl- tetrahydrofuran and named asarinin A.
6
was determined by comparing the CD spectrum of
6
,
6
showed positive absorption peaks
6

Molecules 2017, 22, 125
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Figure 4. Key NOESY correlations (H ↔ H) of compound 6.
Compound
determined as C21H24O7 on the basis of HRESIMS (m/z 411.1409 [M + Na]+, calcd. 411.1414). The IR
absorption of
was indicative of the presence of hydroxyl (3446 cm−1). The UV spectrum showed
7 was obtained as a white amorphous powder. The molecular formula of it was
7
maximum absorption at 241 and 280 nm, which corresponded to the furofuran type of lignan [38].
The 1H- and 13C-NMR spectra of
lignan. The H-NMR signals of an ABX system at
7
also indicated that it was an unsymmetrically substituted furofuran
6.91 (1H, d, J = 1.2 Hz, H-20), 6.89 (1H, d,
1
δ
J = 7.8 Hz, H-50) and 6.83 (1H, dd, J = 7.8, 1.2 Hz, H-60), the other two aromatic protons at 6.60
(2H, s, H-2, 6), together with three methoxyl groups at 3.91 (3H, s) and 3.90 (6H, s) showed
the existence of a 4-hydroxyl-3-methoxylphenyl unit and a 4-hydroxyl-3,5-dimethoxylphenly unit.
Comparing NMR data of
with those of (+)-de-4,40-O-dimethylepimagnolin [39], which was a
microbial transformation product of (+)-epimagnolin by a specific type of Aspergillus, compound
presented identical spectroscopic data with the known compound. Although exhibited the same
δ
δ
7
7
7
1H-NMR data as (+)-de-4, 40-O-dimethylepimagnolin A, which has the absolute configuration 7S, 8R,
7 R, 8 R with optical rotation value of [α]1D9 +107.7 (c 0.1, CHCl3), its optical rotation, [α]2D0 –126.7 (c 0.60,
CHCl3), was opposite. Accordingly, the absolute configuration was assigned as 7R, 8S, 70S, 80S in
0
0
7
[
40]. Finally, compound
70-(40-hydroxyl-30-methoxylphenyl)-10,100-dioxabicyclo[3.3.0]octane, and was named asarinin B.
The spectroscopic data of compound
are as follows: amorphous powder; [α]2D0 –126.7 (c 0.60,
CHCl3); UV (CHCl3) (log ) 241 (3.86), 280 (3.58) nm; IR (KBr) 3446, 1612, 1517, 1457,
7
was determined as (7R, 8S,70S, 80S)-7-(4-hydroxyl-3,5-dimethoxylphenyl)-
7
λ
max
ε
ν
max
1
1426, 1359, 1277, 1242, 1204, 1161, 1111, 1069, 1031, 998, 731 cm−1; H-NMR (CDCl3, 600 MHz)
and 13C-NMR (CDCl3, 150 MHz) data, see Table 3. HRESIMS m/z 411.1409 [M + Na]+ (calcd. for
C21H24O7Na, 411.1414).

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Table 3. NMR spectroscopic data for 7 (in CDCl3).
7
Position
δC
δ
(J in Hz)
H
1
2
3
4
5
6
7
8
133.1
102.4
147.1
133.7
147.1
102.4
87.8
—
6.60, s
—
—
—
6.60, s
4.44, d (6.6)
2.90, dd (7.2, 13.8)
4.13, d (9.6)
54.5
9
71.1
3.87–3.83, m, H-9b
0
1
2
3
4
5
6
7
8
129.6
108.6
146.8
145.4
114.3
119.2
82.3
—
6.91, d (1.2)
—
0
0
0
—
0
6.89, d (7.8)
6.83, dd (7.8, 1.2)
4.85, d ( 6.0)
3.34–3.31, m, H-8
3.87–3.83, m, H-9 a
0
0
0
0
50.2
0
0
0
9
69.7
3.34–3.31, m, H-9 b
0
7 -OCH2-
-CH3
—
—
—
—
3-OCH3
5-OCH3
3 -OCH3
56.4
56.4
56.0
—
3.90, s
3.90, s
3.91, s
5.60 a, brs
5.48 a, brs
0
4-OH
0
4 -OH
—
a
Assignments may be interchanged in the same column.
Compound
by HRESIMS (m/z 153.1273 [M + H]+, calcd. 153.1273). The IR absorption spectrum of
presence of a double bond (1608 cm−1). The 1H-NMR and HMQC spectra contained the signals for
an olefinic proton at 5.54 (1H, t, J = 4.2, 1.2 Hz, H-2), an oxygenated methine at 3.94 (1H, t, J = 3.0
2.4 Hz, H-4), two methylenes ( 2.09 (1H, m, H-6a), 1.77 (1H, m, overlapping, H-6b), 1.94 (1H, dt,
J = 13.8, 2.4 Hz, H-5a), 1.35 (1H, ddd, J = 13.2, 3.5 Hz, H-5b)), a methylene at 1.77 (1H, m, overlapping,
H-7) and three methyl groups (
1.75 (3H, s, CH3-10), 1.15 (6H, s, CH3-8, 9)). The 13C-NMR data of
showed the existence of two olefinic carbons at 134.0 (C-3) and 124.8 (C-2), an oxygenated quaternary
carbon at 71.4 (C-1), an oxygenated methine carbon at 67.9 (C-4), a methine carbon at 38.4 (C-7),
two methylene carbons at 32.8 (C-5) and 26.7 (C-6), and three methyl carbons ( 25.7 (C-8), 25.6
(C-9), and 19.8 (C-10)). Combined analysis of the spectroscopic data of suggested that it was very
8
was isolated as a white powder. Its molecular formula was found to be C10H16O
8
showed the
δ
δ
,
δ
δ
δ
8
δ
δ
δ
δ
δ
δ
8
similar to 2-methyl-5-isopropyl-7-oxabicyclo[2.2.1]hept-2-ene [41]. The principal differences between
these two compounds were the substitution sites of the isopropyl group and the methyl group, linked
to the 7-oxabicyclo[2.2.1]hept-2-ene moiety, respectively. In the HMBC spectrum, the correlations
between H-2 and C-10, the methyl protons at
δ 1.75 and C-4, indicated that the methyl at δ 1.75 was
attached to C-3. Moreover, the isopropyl group was determined to connect with C-1, which was
supported by HMBC correlations from H-2 to C-7, from H-6 to C-7, and the two methyl protons at
1.15 to C-1 (Figure 5). All other HMBC correlations observed were consistent with the structure of
δ
8.
The relative configuration of
8 was determined by analysis of its NOESY spectrum, which showed
no NOE correlation between H-4 and CH3-8, 9, suggesting that H-4 was on the opposite face of the
molecule relative to the isopropyl group. Finally, the assignments of all protons and carbons were

Molecules 2017, 22, 125
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made by HMQC, HMBC, 1H-1H COSY spectra (Figure 5). Thus, the structure of
1-isopropyl-3-methyl-7-oxabicyclo[2.2.1]hept-2-ene and named asaricin A.
8
was elucidated as
Figure 5. Key HMBC (H → C) and 1H-1H COSY correlations (H ↔ H) of compounds 8 and 9.
The spectroscopic data of compound
UV (MeOH) (log ) 202 (3.07) nm; IR (KBr) ν
max
8
are as follows: white powder; [α]2D0 –13.3 (c 0.60, MeOH);
3340, 2975, 2931, 2891, 1608, 1471, 1435, 1378,
λ
max
ε
1313, 1293, 1253, 1159, 1054, 1033, 918, 666, 617 cm−1; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR
(CD3OD, 150 MHz) data, see Table 4. EIMS m/z 152 (15), 137 (29), 109 (100), 95 (27), 79 (66), 59 (90), 55
(22), 43 (71); HRESIMS m/z 153.1273 [M + H]+ (calcd. for C10H17O, 153.1273).
Table 4. NMR spectroscopic data (CD3OD) for compounds 8 and 9.
8
9
Position
δC
δ
(J in Hz)
δC
δ (J in Hz)
H
H
1
2
3
4
71.4
124.8
134.0
67.9
—
67.0
136.0
129.6
68.6
3.90, d (3.0)
—
5.46, d (1.2)
5.54, t (4.2, 1.2)
—
3.94, t (3.0, 2.4)
3.85, dd (3.0, 1.2)
1.94, dt (13.8, 2.4), H-5a
1.35, ddd (13.8, 3.5), H-5b
2.09, m, H-6a
1.77, m, H-6b
1.77, m,
5
32.8
41.5
1.58, m
1.71, dt (13.2, 3.0), H-6a
1.38, ddd (13.2, 4.2), H-6b
2.10, m
6
26.7
29.5
7
8
9
38.4
25.7
25.6
19.8
25.6
20.0
15.7
19.3
1.15, brs
1.15, brs
1.75, s
0.96, d (6.6)
0.81, d (7.2)
10
1.76, s
Compound
9 was obtained as a colorless prismatic crystal. The molecular formula of it was
determined as C10H16O by HRESIMS (m/z 153.1271 [M + H]+, calcd. 153.1273). The IR spectrum
displayed the absorption of a double bond (1632 cm−1). Comparing the 1H- and 13C-NMR data of
9
with those of asiasarinol [42], the existence of a 7-oxabicyclo[2.2.1]hept-2-ene moiety, a isopropyl
group, and a methyl group in was inferred. Furthermore, the locations of the isopropyl group and the
methyl, connected with , were determined by an HMBC experiment. The HMBC correlations of the
methyl protons at 1.76 with C-3 and C-1, H-3 and C-10 supported the idea that the methyl at 1.76
was linked to C-2. In addition, the HMBC correlations from H-8, H-9 to C-5 revealed that the isopropyl
was attached to C-5. The 1H-1H COSY spectrum of
further reinforced the above conclusions by the
9
9
δ
δ
9

Molecules 2017, 22, 125
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correlation of H-3 with H-4, H-4 with H-5, H-5 with H-6, H-6 with H-1 (Figure 5). On the basis of the
above evidence, the structure of was identified as 2-methyl-5-isopropyl-7-oxabicyclo[2.2.1]hept-2-ene,
9
named asaricin B. Although reported as an intermediate product during the synthesis process of
α-Phellandrene previously [41], asaricin B was first isolated from a plant.
T◦he spectroscopic data of compound
9
are as follows: colorless prismatic crystal (EtOAc); mp
(log ) 205 (4.64) nm; IR (KBr) 3259, 2963,
98–99 C; [α]2D0 +13.6 (c 0.66, MeOH); UV (MeOH)
λ
max
ε
ν
max
2948, 2883, 1632, 1465, 1446, 1384, 1365, 1255, 1212, 1165, 1128, 1072, 1027, 989, 868, 707 cm−1; 1H-NMR
(CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz) data, see Table 4. EIMS m/z 152 (2), 137 (9), 127
(16), 111 (74), 109 (40), 100 (83), 71 (100), 55 (29), 43 (67); HRESIMS m/z 153.1271 [M + H]+ (calcd. for
C10H17O, 153.1273).
Compound 10 was obtained as a white amorphous powder. Its molecular formula was established
as C16H27NO3 by HRESIMS (m/z 282.2069 [M + H]+, calcd. 282.2063). ESI-MS gave the ion peak at m/z
282 [M + H]+, 264 [M + H
−
H2O]+, 246[M + H
−
2H2O]+, indicating the presence of two hydroxyl
groups in the structure of 10. The IR bands at 3417 cm−1 and 1676 cm−1 were attributed to hydroxyl
and carbonyl groups, respectively. The UV maximal absorption near 256 nm and the 1H-NMR signals
of 10 at
δ
0.91 (6H, m, H-30, 40), 1.78 (1H, m, H-20) and 3.05 (2H, d, J = 7.0 Hz, H-10) suggested
the presence of an N-isobutylamide group [43]. Comparing the 1H- and 13C-NMR data of 10 with
those of asaramid [42], it showed the presence of the (2E,4E)-2,4-dienamide moiety and an allylic
1
methylene (
δ
2.35 (2H, m, H-6) and 28.9 (C-6)). In addition, the H-, 13C-NMR and DEPT spectra
of 10 indicated the existence of one methyl, two oxygenated methines, two olefinic methines, and a
methylene. Furthermore, the geometry of the additional double bond between C-10 and C-11 was
determined to be the E-form, which was supported by the coupling constant of J10,11 = 15.0 Hz. The
1H-NMR signals of two downfield shifted oxygenated methine protons at
δ 3.38 (1H, m, H-8) and 3.81
(1H, m, H-9) implied that C-8 and C-9 bear one hydroxyl group. Moreover, the locations of double
bonds between C-10 and C-11, and two hydroxyl groups at C-8 and C-9, respectively, were further
confirmed by the 1H-1H COSY experiment, in which the methyl protons at
δ
1.70 (3H, d, J = 6.0 Hz)
coupled with the olefinic proton at
δ
5.71 (1H, m, H-11), and the other olefinic proton at 5.48 (1H,
δ
dd, J = 15.0, 7.2 Hz, H-10) coupled with H-9, and H-9 coupled with H-8 (Figure 6). According to the
IR, UV, 1H- and 13C-NMR, DEPT, 1H-1H COSY, and MS data, the structure of 10 was elucidated as
(2E,4E,10E)-8,9-dihydroxy-N-isobutyl-2,4,10-dodecatrienamide, and named asaramid II.
Figure 6. Key 1H-1H COSY correlations (H ↔ H) of compounds 10 and 11.
The spectroscopic data of compound 10 are as follows: white amorphous powder; [α]2D0 –1.7
(c 0.60, MeOH); UV (MeOH) λmax (log ε) 256 (3.66) nm; IR (KBr) νmax 3417, 2962, 1676, 1550, 1437, 1388,
1271, 1203, 1135, 1027, 719 cm−1; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz), see
Table 5; ESIMS m/z 282 [M + H]+; HRESIMS m/z 282.2069 [M + H]+ (calcd. for C16H28NO3, 282.2063).

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Table 5. NMR spectroscopic data (CD3OD) for compounds 10 and 11.
10 11
Position
δC, Mult.
δ
(J in Hz)
δC, Mult.
δ (J in Hz)
H
H
1
2
3
4
5
6
7
8
9
167.7, C
121.8, CH
140.7, CH
128.0, CH
142.2, CH
28.9, CH2
31.6, CH2
73.6, CH
75.8, CH
130.6, CH
128.6, CH
16.7, CH3
—
167.6, C
121.8, CH
140.7, CH
127.1, CH
142.2, CH
28.9, CH2
31.3, CH2
73.6, CH
69.9, CH
129.7, CH
128.7, CH
12.3, CH3
—
5.94, d (15.0)
7.10, dd (15.0, 10.8)
6.21, m
5.92, d (15.0)
7.10, dd (15.0, 10.8)
6.22, m
6.10, m
2.35, m
2.23, m
3.38, m
6.11, m
2.38, m
2.22, m
3.49, m
3.81, m
4.29, m
10
11
12
5.48, dd (15.0, 7.2)
5.71, m
5.46, dd (9.0, 7.2)
5.65, m
1.70, d (6.0)
1.67, d (7.2)
0
1
46.5, CH2
28.3, CH
19.1, CH3
3.05, d (7.2)
1.78, m
0.91, m
46.7, CH2
28.4, CH
19.1, CH3
3.05, d (7.2)
1.78, m
0.91, d (6.6)
0
2
0
0
3 , 4
Compound 11 was obtained as a white amorphous powder, and had the same molecular formula
as 10, which was determined to be C16H27NO3 by HRESIMS (m/z 282.2063 [M + H]+, calcd. 282.2063).
The IR, UV, 1H-, and 13C-NMR data of 11 were very close to those of 10 except for the different chemical
shift values of C-9 and C-12. The chemical shift values of 11 at
δ 69.9 (C-9) and 12.3 (C-12) were at
higher fields than 75.8 (C-9) and 16.7 (C-12) of 10, respectively, which suggested that the geometry of
δ
the double bond between C-10 and C-11 was Z-form [44]. Moreover, the Z-configuration of the double
bond was further confirmed by the coupling constant J10,11 = 9.0 Hz instead of 15.0 Hz. On the basis
of IR, UV, 1H- and 13C-NMR, DEPT, 1H-1H COSY (Figure 6), and MS data, the structure of 11 was
identified as (2E,4E,10Z)-8,9-dihydroxy-N-isobutyl-2,4,10-dodecatrienamide, and named aramid III.
The spectroscopic data of compound 11 are as follows: white amorphous powder; [α]2D0 –3.3
(c 0.60, MeOH); UV (MeOH) λmax (log ε) 257 (3.64) nm; IR (KBr) νmax 3408, 2962, 1678, 1551, 1437, 1386,
1270, 1204, 1136, 1026, 720 cm−1; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz), see
Table 5; ESIMS m/z 282 [M + H]+; HRESIMS m/z 282.2063 [M + H]+ (calcd. for C16H28NO3, 282.2063).
The 37 known compounds (6, 12–47), which comprise six structural classes, were fully identified
by comparison of their physical and spectroscopic data with those reported in the corresponding literature.
They were 14 lignans: asarinin A (6) [35], (1R,2S,5R,6R)-50-O-methylpluviatilol (13) [26], (–)-asarinin (14) [3]
,
(–)-sesamin (15) [
(19) [48], (–)-piperitol
3
], xanthoxylol (16) [45], clemaphenol A (17)0 [46], epipinoresinol (18) [47], neo-olivil
0
(
20) [49], episesaminone (21) [50], (7 R)-7 -hydroxylariciresinol (22) [51], tanegool
(23) [35], (–)-tanegol (24) [52], morinols G (25) [53]; six flavanones: (2S)-naringenin (26) [54], (2S)-naringenin-
5-O-β-D-glucopyranoside (27) [55], (2S)-liquiritigenin-7-O-β-D-glucopyranoside (28) [56], (2S)-naringenin-
7-O-β-D-glucopyranoside (29) [57], (2S)-naringenin-5,7-di-O-
β
-D-glucopyranoside (30) [58], naringenin-
-D-glucosyl-5-hydroxy-borneol
], car-3-ene-2,5-dione (35) [ ]; four straight chain amide
derivatives: N-trans-coumaroyltyramine (36) [60], asaramid (37) [42], pellitorine (38) [ ], N-isobutyl-3,4-
methylenedioxy cinnamide (39) [61]; seven benzene derivatives: 3-methoxyl-5-methylbenzene-1-O-β-D-
0
5,4 -di-O-
β-D-glucopyranoside (31) [59]; four monoterpenes: 2-exo-O-
β
(
32) [42], aisasarinol (33) [42], asiarinol A (34) [
8
8
7
0
0
glucopyranoside (12) [62],1-(3 ,4 -dihydroxy) phenyl-6,7-dihydroxy-isochroman (40) [63], trans-ferulic
acid (41) [64], 2-(3,4-dihydroxy)-phenyl-ethyl-O- -D-glucopyranoside (42) [65], vanillic acid 1-O- -D-
β
β
glucopyranoside (43) [66], 3-(3,4-methylenedioxyphenyl)-1,2-propanediol (44) [67], 2,3-dihydro-2-methyl-6-
hydroxybenzofuran-5-carboxylic acid (45) [68]; and two fatty glycerides: glyceryl-1-stearate (46) [69],
2,3-dihydroxypropyl-16-hydroxyhexadecanoate (47) [70]. The 15 known compounds 17
31 36 40 42 43, and 45 47 were isolated for the first time from the genus Asarum, and five known
compounds 16, 32, 33, 37, and 39 were first reported from A. heterotropoides var. mandshuricum.
, 19, 22–25, 28,
,
,
,
,
–

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Although previous studies had reported the isolation of bisepoxylignans and monoepoxylignans
from the genus Asarum [ , were reported in this genus for the first
], five 8-O-40 type neolignans,
time. In addition, 1-O-feruloyl- -D-glucopyranoside was found in Asiasarum sieboldi [58], but it was
interesting that contained the 1-O-isoferuloyl- -D-glucopyranosyl unit in its structure, which was
3
,5
1–5
β
5
β
not reported in the compounds isolated from the genus Asarum.
As mentioned in the introduction, a few papers have reported some anti-inflammatory compounds
isolated from A. heterotropoides var. mandshuricum and A. sieboldi, which showed the potent inhibition of
(–)-sesamin (15) in both LPS-induced NO production in BV-2 microglial cells and TNF
α
-induced NF-
κ
B
0
transcriptional activity in HepG2 cell assays [25
,
26], along with (1R, 2S, 5R, 6R)-5 -O-methylpluviatilol
(
13), (–)-asarinin (14) and epipinoresinol (18) in LPS-activated macrophages RAW264.7 assay [
there has been no prior report on the evaluation of the anti-inflammatory effects of non-volatile
compounds obtained from A. heterotropoides var. mandshuricum in PAF-induced -glucuronidase
release in a PMNs cell assay. Thus, 37 of the isolates, including 18 lignans ( 13 19 and 21 25),
five flavanones (27 31), four monoterpenes ( 32 and 33), one amide derivative (37), seven benzene
derivatives (12 and 40
45), and two fatty glycerides (46 and 47), at 10−5 mol/L, were screened for
anti-inflammatory activity in vitro by assessing the inhibitory rate of the release of -glucuronidase in
5], but
β
1,
2,
4–
7
,
–
–
–
8, 9,
–
β
polymorphonuclear leukocytes (PMNs) induced by the platelet-activating factor (PAF). Ginkgolide B
was used as the positive control and gave inhibitory rates of 64.1%–80.9% for four different 96-well
plates (1#–4#) at 10−5 mol/L. The Student’s t-test for unpaired observations between the model
(stimulated by PAF alone) and the control (cultured in medium alone) or tested samples was carried
out to identify statistical differences; * p < 0.05, ** p < 0.01, *** p < 0.001; p < 0.05 were considered as
significantly different. The results showed that 10 of the lignans (
of the flavonones (29 and 30), three of the monoterpenes ( 32 and 33), five of the benzene derivatives
40 43 and 45), and one fatty glyceride (46) possessed varying degrees of anti-inflammatory activity, as
shown in Figure 7 and Table S1 in Supplementary Materials. However, the I.R. values of compounds
12 13 16 21 28 31 37, and 47 were all less than 20%. Compound 19, a monoepoxylignan, exhibited
1, 4, 7, 14, 17–19, 22, 24 and 25), two
8
,
(
–
6
,
9
,
,
,
,
,
,
,
the highest inhibitory activity with a rate of 135.6% (p < 0.001), much more than that of the positive
control of the 3# 96-well plate (75.7%). Two other monoepoxylignans, compounds 22 and 24, displayed
anti-inflammatory activity, with inhibitory rates of 49.7% (p < 0.05) and 34.7% (p < 0.05), respectively.
Four bisepoxylignans, 7, 14, 17, and 18, also gave significant inhibition with a rate of 27.9%–57.9%.
These lignans showing anti-inflammatory activities were observed to commonly bear a tetrahydrofuran
0
ring in their structure. In addition, two 8-O-4 neolignans, compounds
1 and 4, also produced a strong
inhibitory effect with a rate of 40.5% (p < 0.05) and 69.9% (p < 0.01), respectively. Moreover, one
monoterpene, , three benzene derivatives, 40 43, and 45, and one fatty glyceride compound, 46,
8
,
exhibited high inhibitory activity with rates of 55.8%, 72.6%, 66.9%, 56.9%, and 69.4% (p < 0.01, for
each), respectively. For the other active compounds, monoterpenes 32 and 33, flavonone glucosides
29 and 30, and benzene derivatives 41 and 42, the inhibitory rates of release of β-glucuronidase were
31.0%–44.4% (p < 0.05, for each). The other compounds showed weak or no inhibition activity in
this study.
In this anti-inflammatory activity assay in vitro, we also tested the inhibitory activity of
β-glucuronidase release of the different polarity extracts, including Pet. extract, CHCl3 extract, EtOAc
extract, and n-BuOH extract obtained from the ethanol extract of roots and rhizomes of A. heterotropoides
var. mandshuricum. The results showed that the CHCl3 extract displayed strong inhibitory activity with
a rate of 76.8% (p < 0.01) at 10
activity (Figure 7). Moreover, by detailed analysis of the 21 anti-inflammatory active compounds,
it was found that 13 of them were isolated from the CHCl3 extract, comprising nine lignans (
14 17 19 22 24, and 25), two benzene derivatives (41 and 45), one monoterpene ( ), and one fatty
µg/mL, while the other three extracts exhibited no anti-inflammatory
1, 7,
,
–
,
,
8
glyceride (46). According to the results of the anti-inflammatory study, it was inferred that the
lowly polar components that existed in the CHCl3 extract, especially lignans, as well as benzene
derivatives, were the main active anti-inflammatory components of this traditional Chinese medicine.

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In addition, the anti-inflammatory active compound 14 was found to be the most abundant among all
isolates—the amount obtained from 231 g CHCl3 extract was about 10 g, likely responsible for most
of the anti-inflammatory effect of the CHCl3 extract. Based on these results, it is possible that one
of the mechanisms for the anti-inflammatory effect of A. heterotropoides var. mandshuricum is due to
the inhibition of the release of β-glucuronidase in PMN cells. However, a number of mediators and
mechanisms are also involved in inflammatory reaction, so additional investigations are required to
find the anti-inflammatory mechanisms and active components of A. heterotropoides var. mandshuricum.
160.0%
***
140.0%
120.0%
100.0%
*
**
**
***
**
80.0%
60.0%
40.0%
20.0%
0.0%
**
**
**
**
**
**
**
*
*
*
*
***
*
*
**
*
*
*
*
**
Figure 7. Isolated compounds and extracts from roots and rhizomes of Asarum heterotropoides var.
mandshuricum inhibit the release of -glucuronidase in PMN cells induced by the PAF. Asterisks
β
indicate significant differences comparing with control, * p < 0.05, ** p < 0.01, *** p < 0.001.
3. Experimental Section
3.1. General
Melting points were obtained on an XT-4A micromelting point apparatus (Shanghai Hui Tong
Optical Instrument Co., Ltd., Shanghai, China) without correction. Optical rotations were determined
on a Perkin-Elmer 243B digital polarimeter (Boston, MA, USA). UV spectra were carried out on a
Cary 300 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). CD spectra were
measured on a JASCO J-810 spectropolarimeter (Jasco, Tokoyo, Japan). A Nicolet NEXUS-470 FTIR
spectrophotometer (Madison, WI, USA) was used for scanning IR spectroscopy. NMR spectra were
recorded on Varian INOVA-500 (Walnut Creek, CA, USA) and JOEL JNM-ECA600 spectrometers
(JEOL, Tokyo, Japan). The chemical shifts are expressed as
δ (ppm) values using solvent as an internal
standard, and coupling constant, J, are in Hz. Mass spectra were detected with Bruker APEX IV
FT (Bruker, Billerica, MA, USA) and ABI Q-STAR mass spectrometers (Navarre, FL, USA). GC was
carried out on a Shimadzu GC-2010 series system fitted with a FID detector (Shimadzu Corporation,
Kyoto, Japan) and performed with a DB-1701 column (30 m
Agilent Technologies, Santa Clara, CA, USA). Semi-preparative HPLC was conducted on an Alltima
C18 column (10 mm i.d. 250 mm, 10 m, Alltech, Nicholasville, KY, USA) equipped with an
×
0.25 mm i.d., 0.25 µm film thickness,
×
µ
Alltech 426 HPLC pump and an Alltech UVIS 2000 detector (Alltech, Nicholasville, KY, USA). Column
chromatography was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Co., Ltd.,
Qingdao, China) or Sephadex LH-20 gel (Pharmacia Co., Ltd., Shanghai, China). TLC analysis was
performed on silica gel (400 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and precoated
polyamide plates (0.2 mm, Zhejiang Siqing Biochem Co., Ltd., Taizhou, China) plates. D-glucose was

Molecules 2017, 22, 125
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obtained from Beijing Chemical Reagent Company. All other chemical solvents used for isolation were
of analytical grade (Beijing Beihua Fine Chemicals Co., Ltd., Beijing, China). Fractions were monitored
by TLC and spots were detected by UV illumination or visualized by heating silica gel plates sprayed
with 10% H2SO4 in 95% EtOH.
3.2. Plant Material
The roots and rhizomes of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag
were purchased from Benxi City, Liaoning province, China, in May 2005, and authenticated by Professor
Shao-Qing Cai of Peking University Health Science Center. The voucher specimen (No. 050526) was
deposited in the Herbarium of Pharmacognosy, School of Pharmaceutical Sciences, Peking University,
Beijing, China.
3.3. Extraction and Isolation
The air-dried and powdered roots and rhizomes of A. heterotropoides var. mandshuricum (36 kg)
were extracted three times (2 h, 1.5 h, 1.5 h for each) under reflux with 95% ethanol and then three times
(2 h, 1.5 h, 1.5 h for each) with 50% ethanol successively. The combined extracts were concentrated
under reduced pressure to give a dark brown residue (9.2 kg), then 7.8 kg of it was suspended in H2O
◦
(16.5 L) and partitioned sequentially with petroleum ether (Pet.) (60–90 C) (4
× 5 L), CHCl3 (4 × 5 L),
EtOAc (4
×
5 L), and n-BuOH (4
×
5 L), respectively. The CHCl3 layer (231 g) was fractionated on
silica gel CC eluting with a gradient of petroleum ether–EtOAc (10:1 to 0:1) to obtain 16 fractions,
C1–C16. Fraction C11 was subjected to silica gel CC eluting with a gradient of CHCl3–MeOH (1:30
to 1:35) to yield four subfractions C11-1–C11-4. C11-1 was then subjected to chromatography on
silica gel CC eluting with petroleum ether–Me2CO to produce seven subfractions C11-1-1–C11-1-7.
C11-1-5 was chromatographed over silica gel with petroleum ether–Me2CO (2.5:1) as eluent, and
subfraction was then separated by Sephadex LH-20 (CHCl3–MeOH, 6:4) and semi-preparative HPLC
eluted with a gradient of MeOH–H2O to yield
with petroleum ether–Me2CO (4.5:1) to yield
7
(18 mg). C11-3 was subjected to silica gel CC eluting
9
(4 mg). Fraction C12 was chromatographed on silica
gel with a gradient of CHCl3–MeOH to give four fractions C12-1–C12-4. C12-3 was further divided
into five subfractions C12-3-1–C12-3-5 with repeated silica gel CC eluting with CHCl3–MeOH (80:1 to
15:1). Chromatography of C12-3-1 on silica gel with CHCl3–EtOAc (5:1, 4:1) and CH2Cl2–Me2CO (7:1)
as eluent successively and purified by semi-preparative HPLC eluting with a gradient of MeOH-H2O
to yield
with CHCl3–MeOH (70:1 to 15:1) and further purified by C18 reversed-phase silica gel CC eluting
with 70% MeOH in H2O to afford (43 mg). Fraction C13 was chromatographed with a gradient
1 (23 mg), 2 (11.9 mg), 3 (2.1 mg) and 6 (5.1 mg). C12-4 was separated on silica gel CC eluting
8
of CHCl3–MeOH (35:1, 20:1, 5:1) as eluent to give six subfractions C13-1–C13-6. C13-6 was then
subjected to silica gel CC with petroleum ether–Me2CO (30:1), and subfraction was further separated
by semi-preparative HPLC with a gradient of MeOH–H2O as the mobile phase to yield 10 (12 mg) and
11 (6.5 mg).
The EtOAc layer (50 g) was fractionated on silica gel CC eluting with a gradient of CHCl3–MeOH
(10:1 to 0:1) to yield 10 fractions, E1–E10. Fraction E6 was chromatographed on silica gel CC eluting
with CHCl3–MeOH–H2O (15:1:0.1, 10:1:0.1), and purified by Sephadex LH-20 with MeOH to afford 12
(8.1 mg). Fraction E8 was separated on silica gel CC eluting with CHCl3–MeOH–H2O (8:1:0.1, 5:1:0.1)
and then purified by semi-preparative HPLC with MeOH–H2O as eluent to yield 4 (3.9 mg).
The n-BuOH layer (400 g) was fractionated on silica gel CC eluting with a gradient of
CHCl3–MeOH (7:1 to 0:1) to yield 14 fractions, B1–B14. Fraction B6 was further separated on silica gel
CC eluting with CHCl3–MeOH–H2O (5:1:0.1 to 2:1:0.1) to give nine subfractions B6-1–B6-9. B6-7 was
further divided by silica gel CC using CHCl3–MeOH–H2O (5:1:0.1), and purified by Sephadex LH-20
eluting with MeOH to afford
5 (20 mg). The extraction and isolation of known compounds 13–47 is
described in the Supplementary Materials.

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3.4. Acid Hydrolysis of Compounds 4 and 5, and GC Analysis
◦
4 (1 mg) and 5 (2 mg) in 2 N HCl (3 mL) were refluxed at 80 C for 1 h. After
Compounds
cooling, the reaction mixture was neutralized with NaHCO3 and successively extracted with CHCl3
(4 3 mL). The aqueous layer was then evaporated to dryness and the residue was dissolved
in anhydrous pyridine (200
×
µ
L), and L-cysteine methyl ester hydrochloride (0.06 mol/L, 200
µ
L)
◦
was added. The mixture was stirred at 60 C for 1 h. Then 150
µL trimethylsilylation reagent
hexamethyldisilazane–trimethyl chlorosilane (HMDS–TMCS, 3:1) was added, and the mixture was
◦
stirred at 60 C for an additional 30 min. After centrifugation, the supernatant was concentrated under
a stream of N2. The residue was portioned between n-hexane and H2O (0.2 mL each), and the n-hexane
layer (2
µ
L) was analyzed by GC for sugar identification (detector, FID; injection temperature, 260 ◦C;
detector temperature, 280 ◦C; temperature gradient system for the oven, 160 ◦C for 1 min and then
◦
◦
raised to 230 C at a rate of 5 C/min; carrier gas, N2; flow rate, 1 mL/min). D-Glucose was identified
for compounds and by comparison with retention time of authentic D-glucose (tR = 20.76 min) after
4
5
treatment in the same manner.
3.5. Anti-Inflammatory Activity Assay
Compounds and extracts used to test were dissolved in DMSO at the concentration of 10−5 mol/L
and 10
250
L) was incubated at 37 ◦C for 15 min with the presence of the test sample (2.5
Then cytochalasin B (1 mmol/L, 2.5
5 min, followed by activating with platelet-activating factor (PAF) (1
µ
g/mL. A suspension of rat polymorphonuclear leukocytes (PMNs) (2.5
×
106 cells/mL,
µL) (n = 3) [71].
µ
µL, Sigma-Aldrich, Shanghai, China) was added and incubated for
µ
mol/L, 2.5 L, Sigma-Aldrich,
µ
Shanghai, China). After 10 min, the reaction was terminated in an ice bath. The supernatant was
◦
obtained by centrifugation at 4000 rpm for 5 min (4 C), and then the supernatants (25
µL) and
phenolphthalein glucuronic acid (2.5 mmol/L, 25
with acetic acid buffer (0.1 mol/L, 100 L, pH 4.6) at 37 C, 5% CO2, for 18 h. Finally, NaOH (0.2 mol/L,
150 L) was added to terminate the reaction. The results were quantified by reading the absorbance at
µL, Sigma-Aldrich, Shanghai, China) were incubated
◦
µ
µ
550 nm by BIO-RAD model 450 enzyme-labeling instrument. The inhibitory rate (I.R.) was obtained
by the following formula:
I.R. (%) = (ODmodel − ODsample)/(ODmodel − ODcontrol) × 100%.
ODmodel, ODsample, and ODcontrol refer to the average absorbance of the four wells of PAF, the
three wells of the test sample, and the control group, respectively.
4. Conclusions
The roots and rhizomes of A. heterotropoides var. mandshuricum are used in traditional Chinese
medicine for the treatment of pain and inflammatory diseases [
some compositions of the volatile oil, monoterpenes, lignans, and amides isolated from the genus
Asarum showed potential anti-inflammatory activity [ 15 16 24 26]. In this paper, 47 compounds,
1]. Previous research proved that
5
, , , –
including nine new compounds, one new natural compound, and 37 known compounds, were
isolated and identified from the ethanol extract of this plant. Meanwhile, 8-O-40 neolignans were
reported in this genus for the first time. In order to evaluate the potential anti-inflammatory effects of
non-volatile constituents separated from the roots and rhizomes of A. heterotropoides var. mandshuricum,
37 compounds were tested to assess the inhibitory rate of the release of
induced by PAF. The results showed that 10 of the lignans ( 14
the flavonones (29 and 30), three of the monoterpenes ( 32 and 33), five of the benzene derivatives
40 43 and 45) and one fatty glyceride (46) possessed varying degrees of anti-inflammatory activity.
The lignans, especially the tetrahydrofuran ring in their structures ( 14 17 19 22, and 24), displayed
significant anti-inflammatory activity. From an integrated analysis of previous research [ 25 26], it
could be inferred that lignans are the main non-volatile constituents with anti-inflammatory activity.
β-glucuronidase in PMNs cells
1,
4,
7,
,
17 19 22 24, and 25), two of
–
,
,
8
,
(
–
7,
,
–
,
5, ,

Molecules 2017, 22, 125
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Additionally, the flavonones are commonly found in the genus Asarum [
9
,55
,57
,72
,
73], but there
is no study reporting whether the flavonones obtained from this genus possess anti-inflammatory
activity since this type of compound had been proved to be potentially useful for inhibiting the
inflammatory response [74
anti-inflammatory activity against the release of
,
75]. This study demonstrated that two flavonones (29 and 30) showed
-glucuronidase in PMNs cells, which is the first
β
report of the anti-inflammatory effects of flavonones from this genus. Interestingly, (–)-asarinin (14),
the effects of which had been reported for LPS-activated NO production in macrophages RAW264.7
cells and PAF-induced β-glucuronidase release in PMN cells in the literature [5] and in this paper,
respectively, was the most abundant in this plant (Xixin) among all isolates. This primary finding
provides support for further study of this compound for the development of a novel anti-inflammatory
agent. The results provide a scientific explanation for the use of this plant as an herbal medicine in the
treatment of inflammatory diseases.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/22/
1/125/s1. The NMR, HR-MS and HPLC-MS/MS spectra of Compounds 1–11, and the extraction and isolation of
known compounds 13–47, and anti-inflammatory activity assay in vitro are included in the Supplementary Data.
Acknowledgments: This research was financially supported by the National Natural Science Foundation of China
(grant No. 81274073), the 985 Project of Peking University, and the National Eleventh Five-year Key Technologies
R&D Program of China (No. 2006BAI14B01).
Author Contributions: Ming-Ying Shang and Shao-Qing Cai conceived and designed the experiments; Yu Jing
performed the experiments; Yu Jing and Ming-Ying Shang analyzed the data; Yi-Fan Zhang, Guang-Xue Liu,
Yao-Li Li and Xuan Wang contributed reagents/materials/analysis tools; Yu Jing, Yi-Fan Zhang and Ming-Ying
Shang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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