Flavonoids from matteuccia struthiopteris and their anti-influenza virus (H1N1) activity

Article abstract of DOI:10.1021/np500879t

Seven new flavonoid glycosides (1-7), matteflavosides A-G, together with 12 known flavonoids (8-19) were isolated from the rhizomes of Matteuccia struthiopteris (L.) Todar. Their structures were established via the analyses of extensive spectroscopic data. All compounds were evaluated for their anti-influenza virus (H1N1) activity using the neuraminidase inhibition assay. The results showed that compound 7 exhibited significant inhibitory activity against the H1N1 influenza virus neuraminidase with an EC₅₀ value of 6.8 ± 1.1 μM and an SI value of 34.4, and compounds 8 and 17 showed moderate inhibitory activity.

Full text of DOI:10.1021/np500879t

Article
pubs.acs.org/jnp
Flavonoids from Matteuccia struthiopteris and Their Anti-influenza
Virus (H1N1) Activity
Bo Li, Yang Ni, Ling-Juan Zhu, Feng-Bo Wu, Fei Yan, Xue Zhang,* and Xin-Sheng Yao*,†,∥
†
,§
†
†
⊥
†
,†,‡
†College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
‡State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
§
Department of Natural Products Chemistry, College of Pharmacy, China Medical University, Shenyang 110001, People’s Republic of
China
⊥
Department of Pharmacy, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China
∥
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s
Republic of China
*
S Supporting Information
ABSTRACT: Seven new flavonoid glycosides (1−7), matte-
flavosides A−G, together with 12 known flavonoids (8−19)
were isolated from the rhizomes of Matteuccia struthiopteris
(
L.) Todar. Their structures were established via the analyses
of extensive spectroscopic data. All compounds were evaluated
for their anti-influenza virus (H1N1) activity using the
neuraminidase inhibition assay. The results showed that
compound 7 exhibited significant inhibitory activity against
the H1N1 influenza virus neuraminidase with an EC value of
50
6
1
.8 ± 1.1 μM and an SI value of 34.4, and compounds 8 and
7 showed moderate inhibitory activity.
nfluenza is an acute respiratory infectious disease caused by
the influenza virus. It occurs in annual epidemics, with a
Matteuccia struthiopteris (L.) Todar (Onocleaceae) is widely
distributed in the temperate regions of the northern hemi-
sphere and has been used as both a health food in many
I
peak occurrence during the winter in temperate regions and
during rainy seasons in some tropical countries, resulting in
countries and a traditional medicine in China since ancient
1
9−12
significant morbidity and mortality. The influenza virus is a
times.
The rhizomes of M. struthiopteris, possessing
1
1,13−16
member of the Orthomyxoviridae family, which is divided into
types A, B, and C based on the antigenicity of the
nucleoprotein (NP) and matrix protein (M1). Type A is
primarily responsible for annual epidemics or pandemic
flavonoids, phenolics, stilbenes, and steroids,
are used
as traditional Chinese and folk remedies for the treatment of
pinworm, enterozoic abdominalgia, dysentery hematochezia
and metrorrhagia, and prevention of influenza, Japanese
2
10
outbreaks. It is widely accepted that vaccination remains the
encephalitis, and viral parotitis. These traditional uses suggest
most effective approach for the prevention of the viral
infections, but the development of vaccines is a relatively
hysteretic process because of the high mutation rate of the
virus. Therefore, anti-influenza drugs play a critical role in the
prevention and management of influenza infections. Currently,
there are two classes of drugs licensed for influenza infections in
humans: the M2 ion channel blockers amantadine and
rimantadine, and the neuraminidase (NA) inhibitors oseltami-
that this plant may contain compounds with anti-influenza virus
activities. Therefore, systematic studies of the chemical
constituents of the rhizomes of M. struthiopteris and their
anti-influenza virus (H1N1) activity have been carried out.
Seven new flavonoid glycosides (1−7), matteflavosides A−G,
together with 12 known flavonoids (8−19) were isolated from
the 60% EtOH extract of the rhizomes of M. struthiopteris. In
the neuraminidase inhibition assay, compound 7 exhibited
potent antiviral activity against the influenza A (H1N1) virus,
and compounds 8 and 17 showed moderate inhibitory activity.
No previous investigations regarding the anti-influenza virus
activity of the constituents isolated from the genus Matteuccia
have been reported. In this paper, the isolation, structural
3
,4
vir, zanamivir, and peramivir. However, problems regarding
these drugs have been reported due to adverse effects, risk of
emergence of resistant viruses, loss of efficacy due to serotype
variation, and, for the adamantanes, lack of activity against
5
−8
influenza B.
As a result, the discovery process for new anti-
influenza drugs has resulted in much attention being paid to
natural products as sources of new antiviral compounds with
high efficiency and low toxicity.
Received: November 6, 2014
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np500879t
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Journal of Natural Products
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1H NMR spectrum of 1 exhibited resonances for meta-coupled
characterization, and anti-influenza virus (H1N1) activity of the
isolated flavonoids are described.
aromatic protons at δ 6.46 and 6.78 (each 1H, d, J = 2.0 Hz)
H
in ring A, an AA′XX′ coupling system at δ 7.83 and 6.95 (each
H
2
H, d, J = 8.8 Hz) in ring B, and two phenolic hydroxy groups
at δH 12.67 and 10.27 (each 1H, s). Based on the above
evidence, the aglycone of 1 was identified as kaempferol. The
proton signals at δ 5.50 (1H, br s)/0.89 (3H, d, J = 6.4 Hz),
H
4
1
.12 (1H, m)/0.98 (3H, d, J = 6.4 Hz), and 5.55 (1H, br s)/
.12 (3H, d, J = 6.0 Hz) were attributed to three rhamnosyl
1
moieties in the H NMR spectrum of 1. Two of these
rhamnosyl moieties were linked to the aglycone at C-3 and C-7,
as indicated by the HMBC correlations (Figure 1) of H-1″/C-3
and H-1⁗/C-7, respectively. The HMBC correlations of H-1‴/
C-2″ and H-2″/C-1‴ established that the remaining rhamnosyl
unit was attached to C-2″ of the C-3 rhamnosyl moiety. On the
basis of the above evidence, the structure of compound 1 was
determined to be kaempferol-3-O-[α-L-rhamnopyranosyl-(1→
2
)-α-L-rhamnopyranosyl]-7-O-α-L-rhamnopyranoside.
Matteflavoside B (2) was isolated as a yellowish, amorphous
powder, [α]2 −30.4 (c 0.5, MeOH). The molecular formula
5
D
RESULTS AND DISCUSSION
■
13
C H O was determined by+ C NMR data and HRESIMS
33
40 19
Matteflavoside A (1) was obtained from the 60% EtOH extract
of the rhizomes of M. struthiopteris as a yellowish, amorphous
ion at m/z 741.2242 [M + H] (calcd 741.2242). In addition,
the HRESIMS spectra of compound 2 showed fragment ions at
powder, [α]2 −13.4 (c 0.5, MeOH). The UV absorption
5
D
+
m/z 579.1714 [M + H − C H O ] , 433.1136 [M + H −
6
10
5
maxima of 1 were at 266 and 346 nm, characteristic of a 3-
substituted flavonol. The IR spectrum showed absorption
+
C H O − C H O ] , and 287.0558 [M + H − C H O − 2
6
10
5
6
10
4
6
10
5
+
−
1
× C H O ] , suggesting that there were two deoxyhexose and
6 10 4
17
bands for the hydroxy group(s) (3427 cm ), carbonyl
−1
one hexose unit in 2. The acid hydrolysis and HPLC analysis,
group(s) (1655 cm ), and aromatic ring(s) (1602, 1492,
in combination with the 13C NMR data and the coupling
constant of the anomeric proton (H-1‴, 6.8 Hz), confirmed the
presence of α-L-rhamnose and β-D-galactose. Compound 2
displayed UV maximal absorptions and IR absorption bands
−1
and 1450 cm ). Its molecular formula was deduced as
1
3
C H O by C NMR data and HRESIMS, giving a
3
3
40 18
+
quasimolecular ion peak [M + H] at m/z 725.2296 (calcd
25.2293). In addition, the HRESIMS spectra of compound 1
7
1
13
+
similar to those of 1. The H and C NMR spectra of 2
(Tables 1 and 2) were also similar to those of 1. The differences
involved the substitution of a galactosyl unit at C-2″ of the C-3
rhamnosyl moiety instead of a rhamnosyl unit, according to the
HMBC correlation between the anomeric proton of galactose
showed fragment ions at m/z 579.1721 [M + H − C H O ] ,
6
10
4
+
4
×
33.1134 [M + H − 2 × C H O ] , and 287.0554 [M + H − 3
6
10
4
+
C H O ] , suggesting that there were three deoxyhexose
6 10 4
units in 1. The acid hydrolysis and high-performance liquid
17
chromatography (HPLC) analysis, in combination with the
1
3
C NMR data, confirmed the presence of α-L-rhamnose. The
at δ 4.19 (H-1‴) and C-2″. Thus, the structure of compound
H
1
1
Figure 1. Key HMBC (arrows) and H− H COSY (bold lines) correlations of compounds 1 and 5−7.
B
DOI: 10.1021/np500879t
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1
Table 1. H NMR Spectroscopic Data (DMSO-d ) for Compounds 1−7 (J in Hz)
6
a
a
a
a
b
a
a
position
1
2
3
4
5
6
7
2
3
6
8
2
3
6
8
4
4
5
3
5.40 dd (12.0, 2.8)
3.14 m
6.46 d (2.0)
6.78 d (2.0)
7.83 d (8.8)
6.95 d (8.8)
6.46 d (2.0)
6.78 d (2.0)
7.80 d (8.4)
6.94 d (8.4)
6.46 d (2.0)
6.78 d (2.0)
7.78 d (8.4)
6.97 d (8.4)
6.46 d (2.0)
6.79 d (2.0)
7.81 d (8.8)
6.96 d (8.8)
6.46 d (2.4)
6.78 d (2.4)
7.79 d (9.0)
6.92 d (9.0)
6.47 d (2.0)
6.80 d (2.0)
7.79 d (8.8)
6.93 d (8.8)
′/6′
′/5′
-CH3
6.46 s
2.08 s
2.09 s
3.68 s
-CH3
′-OCH3
′-OH
-OH
10.27 s
12.67 s
10.26 s
12.57 s
10.31 s
12.52 s
10.29 s
12.49 s
10.30 s
12.65 s
10.27 s
12.50 s
12.08 s
′/5′-OH
9.20 s
(
3-O-Rha)1″
″
″
″
″
″
″-COCH3
‴
‴
‴
‴
‴
‴
‴-OCH3
‴-COOCH3
‴-OH
‴-CH3
⁗
⁗
⁗
⁗
5.50 br s
5.61 br s
4.02 m
5.65 br s
5.60 br s
5.38 br s
4.10 m
5.39 d (1.2)
4.32 m
(7-O-Glc) 4.59 d (7.2)
3.30 m
2
3
4
5
6
4
1
2
3
4
5
6
1
3
3
5
1
2
3
4
5
6
1
1
2
3
4
5
6
4.01 d (2.4)
4.06 d (2.8)
3.75 br s
4.15 d (2.8)
3.78 br s
c
c
c
c
3.54−3.58
3.13 br s
3.52−3.54
3.13 m
3.56−3.60
2.96 m
3.58−3.62
3.17 m
3.24 m
c
4.64 t (10.0)
4.65 d (10.0)
3.58 dd (6.0, 10.0)
0.78 d (6.0)
2.04 s
3.10−3.14
c
c
c
3.42−3.48
0.89 d (6.4)
3.22 m
3.38−3.42
0.76 d (6.4)
3.12−3.16
0.81 d (6.0)
3.23 m
3.07 m
c
0.86 d (6.0)
0.85 d (6.0)
3.40−3.44, 3.62 m
2.02 s
4.12 m
4.19 d (6.8)
4.14 d (7.6)
4.21 d (8.0)
3.02 m
c
c
c
3.26−3.30
3.26−3.30
3.35−3.40
3.35−3.40
3.30−3.33
3.30−3.33
3.64 m
3.30−3.33
3.30−3.33
3.64 m
5.13 s
4.02 t (6.0)
4.88 d (6.0)
c
c
c
c
3.12−3.16
3.12−3.16
2.97 br s
c
c
1.98 m
4.26 m
c
c
3.30−3.33
3.39 m, 3.47 m
3.26 br s
0.98 d (6.4)
3.37 m, 3.47 m
3.41 m
3.64 s
3.61 s
5.99 s
6.08 d (6.0)
1.12 d (6.0)
5.55 br s
3.83 br s
3.63 m
5.55 br s
3.84 s
5.55 br s
3.83 m
3.64 m
5.55 br s
3.84 br s
3.64 br s
5.55 br s
3.85 br s
4.53 q (6.8)
1.26 d (6.8)
3.62 m
3.64 dd (3.2, 9.2)
c
c
c
c
c
3.26−3.30
3.40−3.45
1.12 d (6.0)
3.27−3.31
3.40−3.45
1.13 d (6.4)
3.28−3.33
3.40−3.45
1.13 d (6.0)
3.28−3.32
3.40−3.45
1.13 d (6.0)
3.28−3.32
3.40−3.45
1.12 d (6.0)
c
c
c
c
c
⁗
⁗
⁗-OCH3
⁗′
⁗′
⁗′
⁗′
3.64 s
5.56 br s
3.84 m
3.61 m
c
3.26−3.30
⁗′
⁗′
3.43 m
1.13 d (6.0)
a
b
c
Measured at 400 MHz. Measured at 600 MHz. Signals overlapped.
1
13
2
was established as kaempferol-3-O-[β-D-galactosyl-(1→2)-α-
with that of 3. The H and C NMR data (Tables 1 and 2) for
4 were similar to those of 3, except for a D-glucosyl unit
replacing the D-galactosyl unit, which was supported by the
L-rhamnopyranosyl]-7-O-α-L-rhamnopyranoside.
Matteflavoside C (3), a yellowish, amorphous powder, [α] D2 5
17
−
70.4 (c 0.5, MeOH), gave a molecular formula of C H O
result of the acid hydrolysis and HPLC analysis. The HMBC
correlation between the anomeric proton of glucose at δ 4.21
3
5
42 20
13
+
by C NMR data and HRESIMS (783.2366, [M + H] ). The
H
1
13
H and C NMR data (Tables 1 and 2) for 3 were similar to
(H-1‴) and C-2″ further confirmed the above conclusion. The
β-configuration of the glucosyl moiety was assigned from the
large coupling constant (8.0 Hz) of the anomeric proton. Based
on the above evidence, the structure of compound 4 was
deduced as kaempferol-3-O-[β-D-glucopyranosyl-(1→2)-4-O-
acetyl-α-L-rhamnopyranosyl]-7-O-α-L-rhamnopyranoside.
Matteflavoside E (5) was a yellowish, amorphous powder,
those for 2, except for the presence of an acetyl group. The
HMBC correlation between H-4″ and the carbonyl carbon of
the acetyl group at δ 169.8 indicated the acetylation of 4″-OH
of the C-3 rhamnosyl moiety. Therefore, the structure of 3 was
elucidated as kaempferol-3-O-[β-D-galactopyranosyl-(1→2)-4-
O-acetyl-α-L-rhamnopyranosyl]-7-O-α-L-rhamnopyranoside.
Matteflavoside D (4) was obtained as a yellowish, amorphous
powder, [α] −25.2 (c 0.5, MeOH). Its molecular formula was
established as C H O by C NMR data and HRESIMS ion
at m/z 783.2345 [M + H] (calcd 783.2348), in agreement
C
[α]2 −79.2 (c 0.5, MeOH). The HRESIMS showed a
quasimolecular ion peak at m/z 737.2292 [M + H] (calcd
737.2293), indicating a molecular formula of C H O , which
was consistent with the C NMR data. The H and C NMR
5
D
2
5
+
D
1
3
35
42 20
34 40 18
+
13
1
13
C
DOI: 10.1021/np500879t
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Journal of Natural Products
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Table 2. 13C NMR Spectroscopic Data (DMSO-d ) for Compounds 1−7
6
a
a
a
a
b
a
a
position
1
2
3
4
5
6
7
2
3
4
5
6
7
8
9
1
1
2
3
4
6
8
4
157.5 C
134.8 C
177.9 C
160.9 C
99.4 CH
161.7 C
94.6 CH
156.0 C
105.6 C
120.2 C
130.6 CH
115.5 CH
160.3 C
157.6 C
134.5 C
177.8 C
160.9 C
99.4 CH
161.7 C
94.6 CH
156.0 C
105.7 C
120.2 C
130.6 CH
115.4 CH
160.2 C
157.9 C
134.3 C
177.6 C
160.9 C
99.4 CH
161.7 C
94.6 CH
156.1 C
105.7 C
120.0 C
130.6 CH
115.4 CH
160.3 C
157.7 C
134.6 C
177.7 C
160.8 C
99.4 CH
161.7 C
94.6 CH
156.0 C
105.7 C
120.0 C
130.6 CH
115.4 CH
160.3 C
157.8 C
134.4 C
177.9 C
161.0 C
99.5 CH
161.7 C
94.6 CH
156.1 C
105.7 C
120.2 C
130.7 CH
115.5 CH
160.3 C
157.7 C
134.8 C
177.8 C
160.8 C
99.4 CH
161.7 C
94.6 CH
156.1 C
105.7 C
120.1 C
130.6 CH
115.3 CH
160.2 C
77.7 CH
42.4 CH2
198.1 C
157.8 C
111.2 C
161.4 C
110.0 C
157.1 C
104.8 C
134.0 C
105.4 CH
150.7 C
135.4 C
8.6 CH3
9.2 CH3
59.6 CH3
0
′
′, 6′
′, 5′
′
-CH3
-CH3
′-OCH3
(
3-O-Rha)1″
″
″
″
″
″
″-CO
″-COCH3
‴
‴
‴
‴
‴
‴
‴-OCH3
‴-CO
‴-COOCH3
‴-CH3
⁗
⁗
⁗
⁗
101.1 CH
81.3 CH
70.1 CH
71.7 CH
70.2 CH
17.3 CH3
100.7 CH
80.9 CH
70.1 CH
71.7 CH
70.4 CH
17.3 CH3
100.2 CH
80.7 CH
67.5 CH
73.4 CH
67.4 CH
16.9 CH3
169.8 C
100.5 CH
80.9 CH
67.5 CH
73.3 CH
67.4 CH
16.9 CH3
169.8 C
100.5 CH
74.9 CH
69.9 CH
71.3 CH
70.6 CH
17.7 CH3
100.7 CH
78.5 CH
70.0 CH
71.1 CH
70.6 CH
17.2 CH3
(7-O-Glc) 104.1 CH
74.0 CH
2
3
4
5
6
4
4
1
2
3
4
5
6
1
3
3
5
1
2
3
4
5
6
1
1
2
3
4
5
6
76.3 CH
69.9 CH
77.0 CH
61.0 CH2
20.7 CH3
106.6 CH
70.8 CH
72.9 CH
67.5 CH
74.9 CH
59.6 CH2
20.7 CH3
106.1 CH
73.6 CH
76.1 CH
69.1 CH
76.6 CH
60.3 CH2
106.6 CH
70.9 CH
73.1 CH
70.4 CH
70.7 CH
16.2 CH3
106.5 CH
71.1 CH
73.0 CH
67.6 CH
74.9 CH
59.7 CH2
170.7 C
107.2 C
83.7 C
72.9 CH
103.4 CH
39.9 CH2
75.2 CH
51.6 CH3
171.0 C
51.6 CH3
22.6 CH3
98.4 CH
69.8 CH
70.2 CH
71.5 CH
70.1 CH
17.9 CH3
98.5 CH
69.8 CH
70.2 CH
71.6 CH
70.0 CH
17.8 CH3
98.4 CH
69.8 CH
70.2 CH
71.6 CH
70.0 CH
17.8 CH3
98.4 CH
69.7 CH
70.2 CH
71.5 CH
70.0 CH
17.8 CH3
98.4 CH
69.7 CH
70.2 CH
71.5 CH
70.0 CH
17.8 CH3
172.0 C
71.1 CH
18.4 CH3
⁗
⁗
⁗-OCH3
⁗′
⁗′
⁗′
⁗′
51.3 CH3
98.4 CH
69.7 CH
70.2 CH
71.5 CH
70.0 CH
17.8 CH3
⁗′
⁗′
a
b
Measured at 100 MHz. Measured at 150 MHz.
data (Tables 1 and 2) for 5 were similar to those for 1, except
group at C-3‴ and a methyl group located at C-5‴.
Additionally, the correlation between −OCH (δ 3.61) and
1
for the absence of the rhamnosyl moiety. In addition, the H
3
H
and 13C NMR spectra of 5 exhibited one carbonyl, one acetal
group, one oxygenated tertiary carbon, one oxygenated
methine, one methylene, one methyl, one methoxy group,
the carbon at δ 171.0 in the HMBC spectrum of 5 indicated
C
the presence of a −COOCH group attached to C-3‴. The
3
above tetrahydrofuran ring fragment was linked to C-2″ of the
C-3 rhamnosyl moiety through an oxygen atom, as indicated by
the correlations of H-2‴/C-2″ and H-2″/C-2‴ in the HMBC
spectrum. The NOESY spectrum of 5 showed the correlations
for 3‴-OH/H-2‴, H-5‴, and 3‴-COOCH /5‴-CH (Figure
1
1
and one hydroxy group. The H− H COSY correlations
between H-5‴ and H-4‴, 5‴-CH and HMBC correlations for
3
3
2
‴-OH/C-2‴, C-3‴, C-4‴, H-2‴/C-4‴, C-5‴, and H-4‴/C-
‴, C-3‴ (Figure 1), in combination with the molecular
3
3
formula and index of hydrogen deficiency, revealed the
2), suggesting that the relative configuration of the tetrahy-
drofuran ring was 2‴S*3‴R*5‴R* or 2‴R*3‴S*5‴S*. On the
presence of a tetrahydrofuran ring fragment with a hydroxy
D
DOI: 10.1021/np500879t
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Journal of Natural Products
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Matteflavoside G (7), a yellowish amorphous powder, [α]2
5
D
−
4.0 (c 0.5, MeOH), had a molecular formula of C H O on
24 28 12
13
the basis of C NMR data and HRESIMS at m/z 509.1661 [M
+
+
H] (calcd 509.1659). The IR spectrum showed absorption
−
1
−1
bands for hydroxy (3442 cm ) and carbonyl (1633 cm )
−1
groups and aromatic ring(s) (1601, 1515, and 1442 cm ). The
UV absorption maxima of 7 were at 283 and 357 nm,
suggesting the presence of a flavanone skeleton in the structure.
The H and C NMR spectra of 7 were similar to those of the
known flavanone ophiofolius A (17), except for the presence
1
13
18
of a sugar moiety. Acid hydrolysis followed by HPLC analysis
1
7
using an authentic sample as reference confirmed the
presence of D-glucose. Additionally, the configuration of the
anomeric carbon was deduced to be β based on the coupling
constant of the anomeric proton (7.2 Hz). The glucosidic
linkage was established by the HMBC correlation (Figure 1)
Figure 2. Key NOESY (arrows) correlations of compound 5.
basis of the above evidence, the structure of compound 5 was
determined to be kaempferol-3-O-[(2,3-dihydroxy-3-methox-
ycarbonyl-5-methyltetrahydrofuran-2-yl)-(2→2)-α-L-rhamno-
pyranosyl]-7-O-α-L-rhamnopyranoside.
between H-1″ (δ 4.59) and C-7, indicating that the glucosyl
H
moiety was attached to C-7. The electronic circular dichroism
(ECD) spectrum of 7 showed a negative Cotton effect at 283
nm and a positive Cotton effect at 354 nm, suggesting that the
Matteflavoside F (6) was obtained as a yellowish, amorphous
powder, [α]2 −8.4 (c 0.5, MeOH). The C NMR data and
5
13
D
+
19
HRESIMS (783.2351 [M + H] ) revealed the molecular
absolute configuration at C-2 was S. Therefore, the structure
of 7 was identified as (2S)-6,8-dimethyl-4′-methoxy-5,3′,5′-
trihydroxyflavanone-7-O-β-D-glucopyranoside.
Additionally, 12 known compounds were isolated and
identified as kaempferol-3-O-β-D-glucopyranoside (8),
kaempferol-3-O-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside
9), kaempferol-3,7-di-O-α-L-rhamnopyranoside (10),
kaempferol-3-O-(α-L-3-O-acetylrhamnopyranosyl)-7-O-α-L-
rhamnopyranoside (11), kaempferol-3-O-(α-L-2-O-acetyl-
rhamnopyranosyl)-7-O-α-L-rhamnopyranoside (12), kaemp-
ferol-3-O-(α-L-4-O-acetylrhamnopyranosyl)-7-O-α-L-rhamno-
pyranoside (13), kaempferol-3-O-[β-D-glucopyranosyl-(1→
)-α-L-rhamnopyranosyl]-7-O-α-L-rhamnopyranoside (14),
1
13
formula as C H O . In the H and C NMR spectra, the
3
5
42 20
differences between 6 and 1 were the absence of a rhamnosyl
moiety and the presence of two carbonyls, one acetal group,
two oxygenated methines, one methyl, two methoxy groups,
20
1
1
and one hydroxy group. The H− H COSY correlations at H-
2
‴/H-3‴, 2‴-OH, and H-2⁗/H-3⁗ and the HMBC
21
22
(
correlations for H-3‴/C-2⁗ (Figure 1) indicated the presence
of a fragment CH CHOCH(O)CH(OH)−. Additionally, the
23
3
correlations between −OCH (δ 3.64) and the carbons at δ
24
3
H
C
1
70.7 and 172.0 in the HMBC spectrum of 6 showed the
presence of two −COOCH fragments, one of which was
3
25
linked to C-2⁗, in accordance with the HMBC correlations of
2
6
2
H-2⁗ and H-3⁗/C-1⁗. The second −COOCH fragment was
3
kaempferol-3-O-[1,2,4-trihdroxy-3-oxo-5-methyltetra-
hydropyran-(1→2)-α-L-rhamnopyranosyl]-7-O-α-L-rhamno-
attached to C-2‴, in accordance with the correlations of 2‴-
OH, H-2‴, and H-3‴/C-1‴. The above six-carbon chain was
attached to C-2″ of the C-3 rhamnosyl moiety, according to the
correlation between H-3‴ and C-2″ in the HMBC spectrum.
Based on the above evidence, the structure of compound 6 was
established as kaempferol-3-O-{methyl 2,3-dihydroxy-3-[(1-
methoxy-1-oxopropan-2-yl)oxy]propanoate-(3→2)-α-L-
rhamnopyranosyl}-7-O-α-L-rhamnopyranoside.
27
28
pyranoside (15), protoapigenone (16), ophiofolius A
18
29
(
17), apigenin-4′-O-β-D-glucopyranoside (18), and mat-
30
teuorien (19) by comparison of observed and published data.
Compounds 8−18 are reported from the Matteuccia genus for
the first time, and compound 19 has not been previously
reported to come from this plant.
Figure 3. (A) Docked conformation of compound 7 with neuraminidase. (B) Binding modes of compound 7 with the key residues of neuraminidase
that are essential for the binding.
E
DOI: 10.1021/np500879t
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Journal of Natural Products
Article
Figure 4. (A) Docked conformation of compound 8 with neuraminidase. (B) Binding modes of compound 8 with the key residues of neuraminidase
that are essential for the binding.
Figure 5. (A) Docked conformation of compound 17 with neuraminidase. (B) Binding modes of compound 17 with the key residues of
neuraminidase that are essential for the binding.
All of the isolated compounds were evaluated for their anti-
influenza virus (H1N1) activity using the neuraminidase
inhibition assay. Cell viability was measured in parallel with
the AlamarBlue assay to exclude the bioactivity resulting from
the cytotoxicity of tested compounds. Ribavirin, an approved
antiviral drug, was used as a positive control and showed an
EC50 value of 19.7 ± 1.0 μM and a selective index (SI, CC50/
EC ) value greater than 10.2. Compounds 7, 8, and 17
visualized: these fits extended deep into the active site pocket,
forming several hydrogen bonds and hydrophobic interactions
with the key residues of the active site (Figures 3−5). The
docking results showed that the hydrogen bond interactions
played important roles in the ligand−protein interactions.
Compound 7 could form six hydrogen bonds with six residues
(Trp178, Glu277, Arg292, Asn294, Arg371, and Tyr406);
compound 8 could form five hydrogen bonds with four
residues (Ser179, Glu277, Arg292, and Tyr406); and
compound 17 could form only three hydrogen bonds with
three residues (Ser179, Glu277, and Tyr406). The above
molecular docking results suggested that compound 7 could
combine tightly with neuraminidase, consistent with the results
of the neuraminidase inhibition assay. The specific molecular
mechanism needs to be clarified by further investigations.
50
exhibited inhibitory activity against neuraminidase from H1N1
influenza virus with EC50 values of 6.8 ± 1.1, 30.5 ± 1.0, and
72.8 ± 1.1 μM, and SI values of 34.4, 12.4, and 3.1, respectively,
while significant cytotoxicity was found for compound 16. The
other tested compounds showed low cytotoxicity but were
inactive in the neuraminidase inhibition assay. Therefore,
matteflavoside G (7) may be a potential antiviral agent against
influenza A (H1N1) on the basis of the SI value.
To further investigate the binding conformations of the
active compounds to neuraminidase, molecular docking
modeling was carried out using the Libdock method to dock
compounds 7, 8, and 17 into the active sites of neuraminidase.
The docked conformations of the best-fit ligands were
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO P-1020 digital polarimeter (l = 1 cm)
(JASCO, Kyoto, Japan). IR spectra (KBr disks) were obtained with a
Bruker IFS-55 spectrometer (Bruker, Rheinstetten, Germany). UV
F
DOI: 10.1021/np500879t
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
spectra were recorded on a Shimadzu UV-2201 UV−vis recording
spectrophotometer (Shimadzu, Kyoto, Japan). ECD spectra were
performed on a Biologic MOS-450 spectrometer (BioLogic Science
Instruments, Grenoble, France). The 1D and 2D NMR spectra were
obtained using Bruker AVANCE-400 or AVANCE-600 NMR
spectrometers (Bruker). HRESIMS data were acquired using a Waters
Synapt G2 QTOF mass spectrometer (Waters, Milford, MA, USA).
The analytical HPLC was performed using an Agilent 1200 (Agilent
Technologies, Santa Clara, CA, USA) equipped with a DAD detector
using a reversed-phase C18 column (5 μm, 4.60 × 250 mm;
Phenomenex Gemini, Phenomenex Inc., Torrance, CA, USA).
Semipreparative HPLC was carried out on a Shimadzu LC-6AD
(eluted with CH Cl /MeOH, 8:2) was subjected to an ODS column
2
2
and eluted with a gradient of MeOH/H O. Fr. C6-2 (eluted with
2
MeOH/H O, 30:70) was further purified by pHPLC (MeOH−0.05%
2
TFA, 30:70, monitored at 260 nm) to afford 18 (12.5 mg). Fr. C6-3
(eluted with MeOH/H O, 40:60) was passed over a Sephadex LH-20
2
column, eluted with CH Cl /MeOH, 1:1, and purified by pHPLC
2
2
(MeOH−0.05% TFA, 37:63, monitored at 260 nm) to yield 1 (8.0
mg). Fr. C7 (eluted with CH Cl /MeOH, 7:3 and 1:1) was subjected
2
2
to an ODS column and eluted with a gradient of MeOH/H O. The
2
40% MeOH eluate was passed over a Sephadex LH-20 column, eluted
with CH Cl /MeOH, 1:1, and purified by pHPLC (MeOH−0.05%
2
2
TFA, 37:63, monitored at 260 nm) to afford 2 (11.4 mg) and 14 (20.8
(
Shimadzu) with a UV SPD-20A detector using a reversed-phase C18
mg).
Matteflavoside A (1): yellowish, amorphous powder; [α]2 −13.4 (c
5
column (5 μm, 10 × 250 mm; Phenomenex Gemini, Phenomenex).
Column chromatography was performed using macroporous adsorp-
tive resins (Diaion HP20, Mitsubishi Chemical Corporation, Tokyo,
Japan), silica gel (200−300 mesh, Qingdao Haiyang Chemical Co.,
Ltd., Qingdao, China), ODS (60−80 μm, YMC, Tokyo, Japan), and
Sephadex LH-20 (GE Healthcare Biosciences AB, Uppsala, Sweden).
Silica gel G plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao,
China) were used for TLC analysis.
Plant Material. The rhizomes of M. struthiopteris were collected
from Liaoning province, China, in September 2011, and authenticated
by Professor Jin-Cai Lu, Shenyang Pharmaceutical University. A
voucher specimen (YLBMS-2011) was deposited at the College of
Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, China.
D
0.5, MeOH); UV (MeOH) λmax (log ε) 241 (4.34), 266 (4.50), 286
(4.14), 346 (4.37) nm; IR (KBr) νmax 3427, 2923, 1655, 1602, 1492,
−
1 1
1450, 1383, 1205, 1176, 1114, 961, 820, 621 cm ; H NMR (DMSO-
d6, 400 MHz), see Table 1; 13C NMR (DMSO-d , 100 MHz), see
6
+
Table 2; HRESIMS m/z 725.2296 [M + H] (calcd for C H O ,
3
3
41 18
725.2293).
Matteflavoside B (2): yellowish, amorphous powder; [α]2 −30.4 (c
5
D
0.5, MeOH); UV (MeOH) λmax (log ε) 241 (4.14), 266 (4.30), 286
(3.94), 346 (4.16) nm; IR (KBr) νmax 3418, 2923, 1656, 1602, 1492,
−
1 1
1450, 1384, 1208, 1177, 1114, 962, 821, 620 cm ; H NMR (DMSO-
d6, 400 MHz), see Table 1; 13C NMR (DMSO-d , 100 MHz), see
6
+
Table 2; HRESIMS m/z 741.2242 [M + H] (calcd for C33
H
41
O
19
,
741.2242).
Matteflavoside C (3): yellowish, amorphous powder; [α]2
5
−70.4 (c
Extraction and Isolation. The air-dried rhizomes (9 kg) of M.
struthiopteris were refluxed with 60% EtOH (v/v, 2 × 70 L, 2 h each).
The combined extracts were concentrated in vacuo to afford a yellow
D
0.5, MeOH); UV (MeOH) λmax (log ε) 264 (4.12), 343 (3.96) nm; IR
(KBr) νmax 3442, 2922, 1654, 1600, 1448, 1384, 1205, 1175, 1116, 620
−
1
1
13
residue (670 g), which was dissolved in H O (approximately 2.7 L)
cm ; H NMR (DMSO-d
(DMSO-d , 100 MHz), see Table 2; HRESIMS m/z 783.2366 [M +
H] (calcd for C35 20, 783.2348).
Matteflavoside D (4): yellowish, amorphous powder; [α]
6
, 400 MHz), see Table 1; C NMR
2
and subjected to column chromatography (CC) over Diaion HP20
6
+
macroporous adsorptive resins and eluted with EtOH/H O (0:100 to
H O
43
2
2
5
95:5). The 95% EtOH (v/v) eluate (27 g) was subjected to column
D
−25.2
chromatography on silica gel and eluted with CH Cl /MeOH (100:0
(c 0.5, MeOH); UV (MeOH) λmax (log ε) 227 (3.98), 264 (4.07), 342
(3.87) nm; IR (KBr) νmax 3421, 2923, 1726, 1655, 1600, 1492, 1450,
2
2
to 0:100) to afford eight fractions (Fr. D1−D8). Compound 19 (11.0
−1 1
mg) was crystallized in MeOH from Fr. D2 (eluted with CH Cl /
1384, 1206, 1175, 1127, 970, 840, 620 cm ; H NMR (DMSO-d ,
6
2
2
400 MHz), see Table 1; 13C NMR (DMSO-d
, 100 MHz), see Table
MeOH, 99:1). Fr. D3 (eluted with CH Cl /MeOH, 98:2) was passed
6
2
2
+
over a Sephadex LH-20 column with CH Cl /MeOH (1:1) as the
2; HRESIMS m/z 783.2345 [M + H] (calcd for C35
H
43
O
20
,
2
2
eluent and a silica gel column and a cyclohexane/acetone stepwise
gradient; it was then applied to an ODS column and eluted with
783.2348).
Matteflavoside E (5): yellowish, amorphous powder; [α]2
5
−79.2 (c
D
MeOH/H O. The 40% MeOH eluate yielded 17 (76.2 mg), and the
0.5, MeOH); UV (MeOH) λmax (log ε) 265 (4.17), 342 (4.02) nm; IR
(KBr) νmax 3428, 2920, 1665, 1662, 1597, 1443, 1384, 1206, 1176,
2
3
(
0% MeOH eluate was further purified by preparative HPLC
pHPLC) using 40% MeOH (monitored at 254 nm) as the eluent
to afford 16 (12.4 mg). Fr. D5 (eluted with CH Cl /MeOH, 90:10)
−1 1
1138, 840, 723, 620 cm ; H NMR (DMSO-d
, 600 MHz), see Table
, 150 MHz), see Table 2; HRESIMS m/z
737.2292 [M + H] (calcd for C34 18, 737.2293).
Matteflavoside F (6): yellowish, amorphous powder; [α]
6
13
1; C NMR (DMSO-d
2
2
6
+
was subjected to an ODS column and eluted with a gradient of
H O
41
2
D
5
MeOH/H O. 7 (25.8 mg), 8 (67.2 mg), 11 (57.0 mg), 12 (13.7 mg),
−8.4 (c
2
and 13 (24.1 mg) were obtained from the 40% MeOH eluate by
purification with pHPLC (MeOH−0.05% TFA, 45:55, monitored at
0.5, MeOH); UV (MeOH) λmax (log ε) 265 (4.13), 342 (3.95) nm; IR
(KBr) νmax 3428, 2925, 1741, 1660, 1600, 1449, 1384, 1206, 1176,
−1 1
2
60 nm). Fr. D5-5 (eluted with MeOH/H O, 60:40) was further
1137, 841, 620 cm ; H NMR (DMSO-d
6
, 400 MHz), see Table 1;
, 100 MHz), see Table 2; HRESIMS m/z
783.2351 [M + H] (calcd for C H O , 783.2348).
2
13
purified by pHPLC (MeOH−0.05% TFA, 55:45, monitored at 260
nm) to afford 5 (8.5 mg) and 6 (10.3 mg). Fr. D6 (eluted with
CH Cl /MeOH, 85:15) was applied to an ODS column eluted with a
C NMR (DMSO-d
6
+
35
43 20
2
5
Matteflavoside G (7): yellowish, amorphous powder; [α]D −4.0 (c
0.5, MeOH); UV (MeOH) λ (log ε) 283 (4.08), 357 (3.41) nm;
ECD (c = 0.5 × 10 , MeOH) Δε (nm): −16.3 (282.6), 6.38 (353.6);
IR (KBr) ν 3442, 2928, 1633, 1601, 1515, 1442, 1384, 1286, 1188,
2
2
gradient of MeOH/H O. The eluents of 40% MeOH and 50% MeOH
2
max
−3
were purified by pHPLC (45% MeOH and 50% MeOH, respectively,
monitored at 260 nm) to yield 15 (39.1 mg) and 10 (20.1 mg),
respectively. Fr. D7 (eluted with CH Cl /MeOH, 8:2/7:3) was
max
−1 1
1124, 993, 891, 616 cm ; H NMR (DMSO-d , 400 MHz), see Table
2
2
6
13
separated by an ODS column and eluted with a gradient of MeOH/
1; C NMR (DMSO-d , 100 MHz), see Table 2; HRESIMS m/z
6
+
H O. Fr. D7-3 (eluted with MeOH/H O, 40:60) was passed over a
509.1661 [M + H] (calcd for C H O , 509.1659).
2
2
24 29 12
Sephadex LH-20 column, eluted with 50% MeOH−H O, and then
Acid Hydrolysis and HPLC Analysis. The absolute config-
2
further purified by pHPLC (MeOH−0.05% TFA, 40:60, monitored at
urations of the sugar moieties in the structures were determined by the
1
7
2
5
60 nm) to afford 9 (12.3 mg). Fr. D7-5 (eluted with MeOH/H O,
0:50) was further subjected to a silica gel column and eluted with
method of Tanaka et al. Compound 1 (3 mg) was hydrolyzed with 2
2
M HCl for 2 h at 90 °C. The mixture was evaporated to dryness in
CH Cl /MeOH (9:1 to 7:3). Purification of Fr. D7-5-3 (eluted with
vacuo, and the residue was dissolved in H O and extracted with
2
2
2
CH Cl /MeOH, 8:2) by pHPLC (MeOH−0.05% TFA, 45:55,
CHCl . After the aqueous layer was dried in vacuo, the residue was
2
2
3
monitored at 260 nm) gave 3 (8.1 mg) and 4 (5.8 mg).
dissolved in pyridine (1 mL) containing L-cysteine methyl ester (1 mg)
(Sigma, USA) and heated at 60 °C for 1 h. o-Tolyl isothiocyanate (5
mL) (Alfa Aesar, U.K.) was added, and the mixture was heated at 60
°C for 1 h and directly analyzed by HPLC. Analytical HPLC was
performed on a reversed-phase C18 column (5 μm, 4.60 × 250 mm;
The 50% EtOH (v/v) eluate (20 g) from the Diaion HP20
macroporous adsorptive resin CC was subjected to column
chromatography on silica gel and eluted with CH Cl /MeOH
2
2
(
100:0 to 0:100) to afford eight fractions (Fr. C1−C8). Fr. C6
G
DOI: 10.1021/np500879t
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Journal of Natural Products
Article
Phenomenex Gemini) at 35 °C with isocratic elution using 25%
Molecular Docking. The structure building of all the compounds
was performed by molecular modeling software package Discovery
Studio 3.5 (Accelrys Inc., San Diego, CA, USA, 2012) and then
minimized using the CharMM27 force field and the MMFF94 charge
with a distance-dependent dielectric and conjugate gradient method.
The optimized structures were used for all subsequent calculations.
The X-ray crystal structure of the neuraminidase in complex with
zanamivir was obtained from Brookhaven Protein Data Bank (entry
CH CN containing 0.1% formic acid for 40 min at a flow rate 0.8 mL/
3
min. The peaks were detected with a UV detector at 250 nm. The
standard monosaccharides, L-rhamnose, D-glucose, L-glucose, and D-
galactose (Sigma, USA), were subjected to the same process. The
peaks of the standard monosaccharide derivatives were recorded at tR
1
7.1 (D-Gal), 17.9 (L-Glc), 19.5 (D-Glc), and 32.8 (L-Rha) min.
Following the above procedure, the derivatives of 1, 5, and 6 afforded
3
B7E). The Libdock module embedded in Discovery Studio was used
one peak at t = 32.8 min (L-Rha), and the derivatives of 2 and 3 both
R
to dock the compounds to the binding site of neuraminidase. The
active site was defined as including all atoms within a 6.5 Å radius of
the cocrystallized ligand, and the default parameters were used.
gave two peaks at tR = 17.1 (D-Gal) and 32.8 (L-Rha) min; the
derivative of 4 gave two peaks at t = 19.5 (D-Glc) and 32.8 (L-Rha)
R
min, and one peak of the derivative of 7 was observed at t = 19.5 (D-
R
Glc) min.
ASSOCIATED CONTENT
Supporting Information
Structures of compounds 8−19 and HRESIMS, UV, IR, and
NMR spectra of compounds 1−7. The Supporting Information
is available free of charge on the ACS Publications website at
DOI: 10.1021/np500879t.
Neuraminidase Inhibition Assay. The influenza virus strain A/
PR/8/34 H1N1 was maintained and cultured in Wuhan Institute of
Virology, Chinese Academy of Science. Madin-Darby canine kidney
■
*
S
(
(
MDCK) cells were grown in Dulbecco’s modified Eagle medium
DMEM) containing 10% fetal bovine serum at 37 °C with 5% CO2.
Neuraminidase plays a key role in the release of virions from the
infected host cells and in their movement through the respiratory
31
tract, which makes it an important target for the development of
AUTHOR INFORMATION
Corresponding Authors
*(X. Zhang) Tel: +86-24-23993994. Fax: +86-24-23993994. E-
mail: syzxalice@163.com.
(X.-S.Yao) Tel: +86-24-23993994. Fax: +86-24-23993994. E-
mail: tyaoxs@jnu.edu.cn.
Notes
anti-influenza drugs. A fluorimetric assay was used to determine the
■
32
influenza virus NA activity. In the assay, the fluorescent product
resulting from the NA-specific substrate 4-methylumbelliferyl-α-D-N-
acetylneuraminate (MUNANA) catalyzed by NA was quantified,
which sensitively reflects the NA activity. The reported method was
*
3
2,33
adopted with some modifications.
MDCK cells in a 96-well
4
microplate (2 × 10 cells/well) were infected with influenza virus (A/
PR/8/34 H1N1, MOI = 0.01), and various concentrations (0.7 to 166
μM) of the tested samples in DMEM containing 1% DMSO
The authors declare no competing financial interest.
(
dimethylsulfoxide) were added to the wells simultaneously. After
ACKNOWLEDGMENTS
being incubated at 37 °C for 1 h, the culture supernatant was removed
and the virus-infected cells were washed with phosphate buffered
saline. The fresh DMEM containing the tested samples at various
concentrations was added to the corresponding wells. The plate was
incubated at 37 °C for 48 h. The infection medium containing the
tested samples (40 μL) was transferred to a 96-well black microplate,
and the substrate (20 μL) was added to start the reaction. After
incubation for 45 min at 37 °C, the reaction was terminated. The
fluorescence intensity was measured with a multilabel plate reader
■
The authors are grateful to Professor Jin-Cai Lu, Shenyang
Pharmaceutical University for authentication of the plant
material. Thanks are also given to the Institute of Traditional
Chinese Medicine and Natural Products, Jinan University, for
measuring the HRESIMS and optical rotation data. This work
was supported by grants from State Key Laboratory of
Bioactive Substance and Function of Natural Medicines (No.
GTZK201304) and the Key Projects of the National Science
and Technology Pillar Program (No. 2012BAI30B02).
(Wallac Envision 2102, PerkinElmer, MA, USA) (excitation 355 nm,
emission 485 nm). Ribavirin at different concentrations (0.8 to 200
μM) was used as a positive control. All tests were performed in
triplicate. The inhibition ratio was determined as follows: inhibition
activity (%) = [1− (F
1
concentration, Fvirus is the fluorescence of the influenza virus control,
and Fcellular control is the fluorescence of normal cells. The 50% effective
concentration (EC ) was determined by extrapolation of the results
REFERENCES
■
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is the fluorescence of the tested sample at a certain
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H
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