C-Methylated Flavonoid Glycosides from Pentarhizidium orientale Rhizomes and Their Inhibitory Effects on the H1N1 Influenza Virus

Article abstract of DOI:10.1021/acs.jnatprod.7b00677

Thirteen C-methylated flavonoid glycosides (1-13), along with 15 previously known flavonoids (14-28), were isolated from rhizomes of Pentarhizidium orientale. Among these compounds, matteuorienates D-K (1-8) were obtained as analogues of matteuorienates A-C (14-16), which contain a characteristic 3-hydroxy-3-methylglutaryl (HMG) moiety. The structures of 1-13 were characterized by spectroscopic analysis and chemical derivatization. The isolates were evaluated for their antiviral activities against influenza virus (H1N1), with compounds 21, 22, 23, 25, and 26 showing inhibitory effects (IC₅₀ of 23.9-30.3 μM) against neuraminidases.

Full text of DOI:10.1021/acs.jnatprod.7b00677

Article
pubs.acs.org/jnp
C‑Methylated Flavonoid Glycosides from Pentarhizidium orientale
Rhizomes and Their Inhibitory Effects on the H1N1 Influenza Virus
Jungmoo Huh,† Thi Kim Quy Ha,† Kyo Bin Kang,† Ki Hyun Kim,‡ Won Keun Oh,† Jinwoong Kim,†
and Sang Hyun Sung*,†
†College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Gwanak-gu, Seoul 08826,
Republic of Korea
‡School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea
S
* Supporting Information
ABSTRACT: Thirteen C-methylated flavonoid glycosides (1−13), along with 15 previously known flavonoids (14−28), were
isolated from rhizomes of Pentarhizidium orientale. Among these compounds, matteuorienates D−K (1−8) were obtained as
analogues of matteuorienates A−C (14−16), which contain a characteristic 3-hydroxy-3-methylglutaryl (HMG) moiety.
The structures of 1−13 were characterized by spectroscopic analysis and chemical derivatization. The isolates were evaluated
for their antiviral activities against influenza virus (H1N1), with compounds 21, 22, 23, 25, and 26 showing inhibitory effects
(IC50 of 23.9−30.3 μM) against neuraminidases.
Pentarhizidium orientale (Hook.) Hayata (Onocleaceae) (syn.
Matteuccia orientalis) is a perennial pteridophyte that is distri-
buted mainly in East Asia. It has been used traditionally as a
diuretic and helminthic in Korean folk medicine. Flavonoids,
isocoumarins, phthalides, and stilbenes have been reported from
P. orientale, among which the flavonoids are characterized by
C-methylation at C-6 and C-8.1−3 These C-methylated flavonoids
were reported to exhibit various pharmacological activities; for
example, hypoglycemic activity in streptozotocin (STZ)-induced
diabetic rats4 and anti-influenza virus (H1N1) inhibitory effects5
have been shown. As part of the present investigation on the
bioactive compounds from a Korean medicinal plant, the
phytochemicals in the 80% aqueous MeOH extract of P. orientale
rhizome were purified. As a result, flavonoids 1−27 and a
chromone (28) were isolated. Compounds 14−28 were iden-
tified as matteuorienates A−C (14−16),1 matteuorienin (17),1
demethoxymatteucinol 7-O-β-D-glucoside (18),6 matteucinol
7-O-β-D-glucoside (19),6 myrciacitrin II (20),7 demethoxymat-
teucinol (21),4 matteucinol (22),4 matteucin (23),8 farrerol
(24),9 methoxymatteucin (25),8 3′-hydroxy-5′-methoxy-6,8-
dimethylhuazhongilexone (26),10 naringenin (27),11 and
leptorumol (28),12 by comparison of spectroscopic data with
previous literature data. The structural elucidation of the new
compounds 1−13 and the antiviral activity evaluation of all
isolates obtained are described herein.
RESULTS AND DISCUSSION
■
Compound 1 was isolated as a yellowish powder. Its molecular
formula was determined to be C30H36O14 from its HRESIMS
deprotonated ion peak at m/z 619.2025 [M − H]− (calcd for
619.2027). UV absorption bands at 214, 282, and 349 nm
suggested that 1 is a flavanone derivative. Its 1H and 13C NMR
data (Table 1) were similar to those of 14, which suggested
that 1 is an analogue of a 5,6,7,8,4′-pentasubstituted flavanone
glycoside containing a 3-hydroxy-3-methylglutaryl (HMG)
moiety. The HMBC correlation between the methoxy protons
(δH 3.77) and a carbon at δC 159.4 (C-4′) confirmed the location
of the methoxy group at C-4′. The glycosidic linkage was located
at C-7 and determined by an HMBC correlation between signals
of the anomeric proton [δH 4.68 (d, J = 7.7 Hz, H-1″)] and C-7
(δC 161.2). The HMBC correlation between H-4″ (δH 4.60) and
C-1‴ (δC 169.9) confirmed the HMG group substitution at C-4″
of the sugar moiety. The sugar was confirmed as D-glucose by
acid hydrolysis followed by HPLC analysis after arylthiocarba-
moyl-thiazolidine derivation.13 The absolute configuration at
C-2 in 1 was identified as (2S) due to the negative Cotton effect at
282 nm in the electronic circular dichroism (ECD) spectrum.14
Received: August 8, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00677
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Chart 1
Table 1. 1H and 13C NMR Data of Compounds 1−4 in DMSO-d6 (1H 500 MHz, 13C 125 MHz)
matteuorienate D (1)
matteuorienate E (2)
matteuorienate F (3)
δH, (J in Hz)
matteuorienate G (4)
position
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
δC, type
δC, type
δH, (J in Hz)
2
77.9, CH
42.7, CH2
5.57 dd (12.5, 2.9)
2.86 dd (17.1, 3.0)
3.33 m
78.0, CH
42.3, CH2
5.65 dd (12.5, 3.0)
2.92 dd (17.1, 3.1)
3.34 m
77.9, CH
5.57 dd (12.5, 3.0)
78.0, CH
5.65 dd (12.5, 3.0)
3
42.2, CH2 2.86 dd (17.1, 3.1)
3.34 m
42.3, CH2 2.93 dd (17.1, 3.1)
3.33 m
4
198.5, C
157.9, C
111.7, C
161.2, C
110.6, C
157.2, C
104.9, C
130.8, C
198.2, C
157.9, C
111.3, C
161.2, C
110.2, C
157.1, C
104.9, C
138.9, C
198.5, C
198.3, C
5
157.8, C
157.8, C
6
111.3, C
111.4, C
7
161.1, C
161.1, C
8
110.2, C
110.2, C
9
157.2, C
157.1, C
10
104.9, C
104.9, C
1′
130.8, C
138.9, C
2′
3′
4′
5′
128.0, CH 7.46 d (8.7)
114.0, CH 6.99 d (8.8)
159.4, C
126.3, CH 7.53 d (7.3)
128.6, CH 7.44 dd (7.3, 7.3)
128.4, CH 7.38 t (7.3)
128.6, CH 7.44 dd (7.3, 7.3)
126.3, CH 7.53 d (7.3)
103.9, CH 4.69 d (7.7)
128.6, CH 7.46 d (8.7)
114.0, CH 6.99 d (8.8)
159.4, C
126.3, CH 7.54 d (7.3)
128.6, CH 7.44 dd (7.3, 7.3)
128.4, CH 7.38 t (7.3)
128.6, CH 7.44 dd (7.3, 7.3)
126.3, CH 7.54 d (7.3)
103.7, CH 4.74 d (7.7)
114.0, CH 6.99 d (8.8)
128.0, CH 7.46 d (8.7)
103.9, CH 4.68 d (7.7)
114.0, CH 6.99 d (8.8)
128.6, CH 7.46 d (8.7)
103.7, CH 4.73 d (7.7)
6′
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
6‴
CH3-6
CH3-8
74.1, CH
73.5, CH
71.3, CH
74.4, CH
60.8, CH2
169.9, C
45.9, CH2
69.2, C
3.44 m
74.1, CH
73.5, CH
71.3, CH
74.4, CH
60.8, CH2
169.9, C
3.45 m
72.0, CH
77.4. CH
67.6, CH
76.6, CH
3.46 m
72.0, CH
77.4, CH
67.6, CH
76.6, CH
3.47 m
3.52 t (9.2)
4.60 t (9.5)
3.37 m
3.52 m
4.84 t (9.4)
3.33 m
4.84 t (9.4)
3.34 m
4.60 t (9.5)
3.38 m
3.20 m
3.21 m
3.33 m 3.46 m
3.33 m 3.46 m
60.7, CH2 3.43 m; 3.62 m
170.1, C
60.7, CH2 3.44 m; 3.62 m
170.1, C
2.62 d (13.8) overlap 45.9, CH2
69.2, C
2.62 d (13.8) overlap 46.6, CH2 2.65 d (13.3) overlap
69.3, C
46.7, CH2 2.65 d (13.3) overlap
69.3, C
45.9, CH2
173.0, C
27.3, CH3
8.7, CH3
9.3, CH3
2.42 s
45.9, CH2
173.0, C
27.3, CH3
8.7, CH3
9.3, CH3
2.42 s
45.6, CH2 2.38 d (14.8) 2.31 d (14.8) 45.6, CH2 2.36 d (15.3) 2.29 d (14.8)
173.8, C
173.9, C
1.27 s
2.09 s
2.07 s
3.77 s
1.27 s
2.10 s
2.10 s
27.8, CH3 1.27 s
27.9, CH3 1.27 s
8.7, CH3
9.3, CH3
2.08 s
2.06 s
8.7, CH3
9.3, CH3
2.09 s
2.09 s
OCH3-4′ 55.2, CH3
55.2, CH3 3.77 s
In turn, absolute configuration at the HMG moiety C-3‴ was
determined as (S) by a concerted method including amidation,
reduction, and acetylation15 as well as by comparison of the
1H NMR data with a reference value.16 Thus, compound 1
(matteuorienate D) was characterized as (2S)-matteucinol-7-O-
[4″-O-((S)-3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside.
Compound 2 gave a molecular formula of C29H34O13, as
1
indicated by HRESIMS. The UV, IR, H NMR, and 13C NMR
data of 2 were similar to those of 1, but they differed in their
B-ring proton couplings. The 1H NMR spectrum of 2 suggested
a monosubstituted B-ring substructure [δH 7.53 (d, J =
7.3 Hz, H-2′, 6′), 7.44 (dd, J = 7.3, 7.3 Hz, H-3′, 5′), and 7.38
B
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Table 2. 1H and 13C NMR Data of Compounds 5−8 in DMSO-d6 (1H 600 MHz, 13C 150 MHz)
matteuorienate H (5)
matteuorienate I (6)
δH, (J in Hz)
matteuorienate J (7)
matteuorienate K (8)
position
δC, type
δH, (J in Hz)
δC, type
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
2
73.6, CH
41.5, CH2
5.63 dd (13.3, 2.8)
2.73 dd (17.0, 2.8)
3.28 m
73.4, CH
41.0, CH2
5.69 dd (13.8, 2.4)
74.0, CH
41.3, CH2
5.69 dd (13.1, 2.8)
2.81 dd (17.0, 2.8)
3.25 m
74.2, CH
41.3, CH2
5.74 dd (13.1, 2.7)
2.80 dd (17.1, 2.8)
3.29 m
3
2.67 dd (17.2, 2.4) 3.39 overlap
4
198.9, C
157.9, C
111.0, C
161.1, C
110.1, C
157.8, C
104.8, C
117.4, C
155.7, C
101.3, CH
160.3, C
104.8, CH
127.9, CH
104.0, CH
73.9, CH
76.0, CH
69.8, CH
73.6, CH
63.1, CH2
199.4, C
158.0, C
110.5, C
161.1, C
110.7, C
158.0, C
104.8, C
117.3, C
157.2, C
198.6, C
198.8, C
157.8, C
111.0, C
161.1, C
110.0, C
157.8, C
104.8, C
124.9, C
155.0, C
115.8, CH
129.3, CH
118.8, CH
126.9, CH
104.0, CH
74.0, CH
76.1, CH
70.0, CH
73.7, CH
63.0, CH2
a
5
157.9,
110.0,
C
C
b
6
7
161.1, C
b
8
111.2,
157.6,
C
a
9
C
10
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
104.8, C
125.7, C
148.1, C
6.46 br s
101.7, CH 6.50 d (2.5)
160.5, C
116.3, CH
114.3, CH
152.1, C
6.82 d (8.8)
6.91 d (8.1)
7.18 m
6.79 dd (9.0, 2.8)
6.47 m
104.1, CH 6.41 dd (8.5, 2.5)
127.9, CH 7.32 d (8.6)
104.0, CH 4.62 d (7.7)
6.85 t (7.5)
7.44 d (8.6)
4.60 d (7.7)
3.28 m
7.36 d (8.2)
4.62 d (7.6)
3.29 m
112.2, CH
104.0, CH
74.0, CH
76.1, CH
69.8, CH
73.6, CH
63.0, CH2
7.03 d (2.9)
4.63 d (7.7)
3.32 m
73.9, CH
75.9, CH
70.2, CH
73.6, CH
63.1, CH2
3.29 m
3.26 m
3.24 m
3.25 m
3.24 m
3.19 m
3.15 m
3.20 m
3.18 m
3.30 m
3.30 m
3.31 m
3.31 m
4.04 dd (11.8, 6.0)
4.20 d (11.5)
3.96 dd (11.8, 7.0)
4.21 d (11.6)
4.02 dd (11.8, 6.0)
4.22 d (10.2)
3.96 dd (11.7, 6.5)
4.23 m
1‴
2‴
170.3, C
170.4, C
170.4, C
170.5, C
45.4, CH2
2.61 d (14.1)
2.47 d (14.1)
46.3, CH2
2.28 d (13.5)
2.23 d (13.6)
45.9, CH2
overlap 2.41 d (13.8)
46.5, CH2
2.32 s
3‴
4‴
68.9, C
68.9, C
68.9, C
68.9, C
45.5, CH2
2.45 d (2.4)
47.5, CH2
2.12 d (14.7)
2.00 m
48.6, CH2
2.35 d (14.8)
2.29 d (14.8)
47.0, CH2
2.16 d (15.0)
2.03 m
5‴
6‴
CH3-6
CH3-8
OCH3-4′
172.4, C
27.2, CH3
8.7, CH3
9.2, CH3
55.1, CH3
175.5, C
27.9, CH3
8.5, CH3
9.2, CH3
55.0, CH3
173.6, C
27.3, CH3
8.7, CH3
9.1, CH3
55.4, CH3
175.4, C
27.7, CH3
8.6, CH3
9.1, CH3
1.18 s
2.05 s
2.00 s
3.71 s
1.04 s
2.12 s
2.00 s
3.71 s
1.14 s
2.05 s
2.05 s
3.69 s
1.05 s
2.05 s
2.03 s
a,b
Exchangeable signals.
(t, J = 7.3 Hz, 4′)]. The absolute configuration of the HMG
group was also determined to be (S) using the same method
mentioned above. Consequently, compound 2 (matteuorienate
E) was identified as (2S)-demethoxymatteucinol-7-O-[4″-O-
((S)-3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside.
(Table 2) were similar to those of 14 (Table S1, Supporting
Information), except for the proton coupling pattern of the
flavonoid B-ring [δH 6.46 (br s, H-3′), 6.47 (m, H-5′), and 7.36
(d, J = 8.2 Hz, H-6′)]. From these data, either a 1,3,4-, 1,2,5-, or
1,2,4-trisubstituted aromatic ring system could be implied for
the B-ring, and it turned out to be a 1,2,4-trisubstituted ring by
analyzing the 2D NMR data. The methoxy moiety was located at
C-4′, which was determined by the HMBC correlation between
the signals at δH 3.71 (OCH3) and δC 160.3 (C-4′). The mole-
cular formula of compound 6 was the same as 5, C30H36O15,
which was determined by HRESIMS. The 1H and 13C NMR data
were similar, and the planar structure was identical to 5; however,
several differences were observed in the chemical shifts of
H-2/H-3 and the aromatic protons on the B-rings of 5 and 6.
This suggested that 6 is a 2-epimer of 5; this was confirmed from
the ECD spectra of these two substances. Compounds 5 and 6
showed negative and positive Cotton effects at 289 nm, respec-
tively, which indicated a (2S)-configuration for 5 and a (2R)-
configuration for 6. Compound 7 was isolated as a yellowish
powder, and its molecular formula was determined to be
C30H36O15 by HRESIMS. The 1H and 13C NMR data of 7 were
similar to those of 5, with the only difference being that 7 could
be assigned a 1,2,5-trisubstituted aromatic ring system [δH 6.82
(d, J = 8.8 Hz, H-3′), 6.79 (dd, J = 9.0, 2.8 Hz, H-4′), and 7.03
Compound 3 showed the molecular formula C30H36O14, the
1
same as that of 1. The UV, IR, H NMR, and 13C NMR data
were similar to those of 1. The location of the HMG moiety was
different between 1 and 3, and it was revealed that compound 3
has an HMG substituent at C-3″ instead of C-4″ due to the
HMBC correlation between H-3″ (δH 4.84) and C-1‴ (δC 170.1).
Like compounds 1 and 3, the spectroscopic data of compound 4
(C29H34O13) resembled those of 2, although the HMBC
correlation between H-3″ and C-1‴ indicated a difference in
location of the HMG unit. The (S)-configurations of 3 and 4
at C-2 and C-3‴ were also identified from the ECD spectra
and the same series of reactions mentioned above. Thus, com-
pounds 3 and 4 (matteuorienates F and G) were identified as
(2S)-matteucinol-7-O-[3″-O-((S)-3-hydroxy-3-methylglutar-
yl)]-β-D-glucopyranoside and (2S)-demethoxymatteucinol-7-O-
[3″-O-((S)-3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside,
respectively.
The molecular formula of 5 was determined to be C30H36O15
1
by HRESIMS. The UV, IR, H NMR, and 13C NMR data
C
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(d, J = 2.9 Hz, H-6′)]. Compared with 7, the molecular formula
of 8, C29H34O14, suggested the absence of a methoxy group.
and 284 (m/z 293.0807) [M − C6H10O4 − H2O − 120 − H]−,
which indicated the compound to be a C-glycosylflavonoid with
an additional deoxyhexose moiety (Figure S67, Supporting
1
Based on the H and 13C NMR, the modified pattern of the
1
B-ring and the absence of a methoxy group at C-5′ were
observed. The ECD spectra of compounds 7 and 8 showed a
negative Cotton effect, indicating that the absolute configuration
at C-2 is (S).
Information). The H NMR resonance of an anomeric proton
[δH 4.70 (d, J = 9.8 Hz, H-1′)] and the HMBC correlation
between H-1′ and C-8 (δC 103.3) suggested the presence of an
8-C-β-glucosyl group (Figure S68 and Table S3, Supporting
Information).18 The position of the interglycosidic linkage was
determined as being located on C-2″ by the HMBC cross-peak
between δH 5.13 (H-1‴) and δC 75.5 (C-2″). The deoxyhexose
unit was confirmed as L-rhamnose using the HPLC analysis
method mentioned above.13 Compared with 11, compound 12
was identified as a 4′-hydroxylated analogue, which was suggested
by its molecular formula of C28H34O14 and the A2B2 coupling
Experiments for determining the absolute configuration of the
HMG moiety in 5 and 6 were not conducted due to their scarce
amounts obtained. However, the absolute configuration of the
HMG moiety at C-3 was deduced to be (S) because the naturally
occurring HMG group is known to be in an (S)-configuration,17
and other HMG-containing flavonoid glycosides from this plant
(1−4, 7, and 8) were verified as having a (3S)-HMG moiety.
Accordingly, based on the natural biogenetic pathway of the
HMG group, compounds 5 and 6 were identified as (2S)-2′-
hydroxymatteucinol-7-O-[6″-O-((S)-3-hydroxy-3-methylglutar-
yl)]-β-D-glucopyranoside and (2R)-2′-hydroxymatteucinol-7-O-
[6″-O-((S)-3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside
and were named matteuorienate H and matteuorienate I, respec-
tively. Additionally, compound 7 (matteuorienate J) was charac-
terized as (2S)-methoxymatteucinol-7-O-[6″-O-((S)-3-hydroxy-
3-methylglutaryl)]-β-D-glucopyranoside, while compound 8
(matteuorienate K) was confirmed as (2S)-matteucin-7-O-[6″-
O-((S)-3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside.
1
pattern in the H NMR spectrum. The molecular formula of
1
compound 13, C29H36O14, was obtained by HRESIMS. Its H
and 13C NMR data were similar to those of 12. The only dif-
ference between the two compounds was the presence of a
methoxy group [δH 3.79; δC 55.1] at C-4′. The absolute configu-
ration of compounds 11−13 at C-2 was determined as (S) via
their ECD spectra. Therefore, based on the above evidence,
compounds 11−13 were identified as (2S)-6-methylpino-
cembrin-8-C-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside,
(2S)-6-methylnaringenin-8-C-[α-L-rhamnopyranosyl-(1→2)]-
β-D-glucopyranoside, and (2S)-6-methylisosakuranetin-8-C-[α-
L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside and named
matteuorienins B−D, respectively.
1
The H and 13C NMR spectra of 9 (Table S3, Supporting
Information) suggested this compound to be an analogue of 8
without the HMG moiety, which was also supported by the
molecular formula of 9, C23H26O10. Comparison of its ECD
spectrum with 8 showed a positive Cotton effect at 283 nm in the
ECD spectrum of 9, indicating an (R)-configuration at C-2.
Therefore, compound 9 was identified as (2R)-matteucin-7-O-β-
D-glucopyranoside. The molecular formula of compound 10,
C23H24O9, was obtained by HRESIMS. Its 1H and 13C NMR data
were similar to those of matteuorien.1 The presence of an
anomeric proton at δH 4.66 (d, J = 7.7 Hz, H-1′) suggested 10 as
having a glucose unit, and the position of the glycosidic linkage was
identified from the HMBC correlation between δH 4.66 (H-1″)
and δC 159.2 (C-7). The sugar was determined to be D-glucose
using the same method mentioned above.13 Thus, compound 10
was characterized as matteuorien-7-O-β-D-glucopyranoside.
Compound 11 was obtained as a yellow powder, and its mole-
cular formula was determined to be C28H34O13 by HRESIMS.
The spectroscopic data of 11 were similar to those of matteuorienin
(17),1 but they differed in their B-ring proton coupling. The 1H
NMR spectrum of 11 suggested a monosubstituted B-ring
substructure [δH 7.56 (d, J = 7.4 Hz, H-2′, 6′), 7.45 (dd, J = 7.5,
7.5 Hz, H-3′, 5′), and 7.39 (t, J = 7.3 Hz, H-4′)]. In the tandem
mass spectrum, compound 11 showed a series of neutral losses of
120 (m/z 457.1488), 164 (m/z 413.1231) [M − C6H10O4 −
H2O − H]−, 266 (m/z 311.0912) [M − 120 − C6H10O4 − H]−,
1
During the structural elucidation of the isolates, the H and
13C NMR resonances of the flavonoid glycosides (14−20) were
also fully assigned (Tables S1 and S2, Supporting Information).
The structures of 14−16 were previously reported, but the
absolute configurations of their HMG moieties were not verified.
These were investigated using the method described above, and
it was confirmed that the HMG moieties in compounds 14−16
also possess the (S)-configuration.
Based on a consideration about previously reported anti-
influenza activity of C-methylated flavonoids,5,19,20 several
isolated compounds (1−4, 7−9, 11−23, 25, 26, and 28) from
P. orientale were screened for their neuraminidase (NA)
inhibitory activity of H1N1 influenza virus. Compounds 5, 6,
and 10 were not investigated due to their scarcity. Compounds
24 and 27 were not examined, because other researchers
reported that these compounds have no inhibitory activity on
influenza viruses.21,22 As shown in Figure S70 (Supporting
Information), the flavonoid aglycones, such as compounds 21−
23, 25, and 26, showed more potent inhibitory activities than the
glycosylated aglycones. Thus, these aglycones were further
investigated to determine their half-maximal inhibitory concen-
trations (IC50) on the neuraminidase activities of the influenza
H1N1 A/PR/8/34 and H9N2A/chicken/Korea/01210/2001
viruses. As indicated in Table 3, compounds 21−23, 25, and 26
a
Table 3. Inhibitory Effects of Selected Compounds on Neuraminidase Activities in the H1N1-Induced CPE Assay
compound
IC50 H1N1 (μM)
IC50 H9N2 (μM)
EC50 (μM)
CC50 (μM)
21
22
23
25
26
30.3 3.0
31.3 5.7
30.7 2.0
26.9 1.3
22.9 2.0
23.0 3.4
21.4 2.0
0.6 0.2
77.6 2.6
>100
25.2 2.4
27.2 3.2
23.9 3.0
24.1 1.3
>100
24.5 1.5
24.6 0.8
>100
24.4 2.0
23.1 1.7
>100
b
c
oseltamivir
77.6 9.5 (nM)
19.1 1.3 (nM)
N.D.
a
Compounds 21−23, 25, and 26 were selected based on the screening data for H1N1 neuraminidase inhibitory activity (Figure S70, Supporting
b
c
Information). Positive control. Not detected.
D
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afford five (BA31−BA35), seven (BA51−BA57), and three (BA61−BA63)
subfractions, respectively. Compounds 1 (10.0 mg) and 2 (11.5 mg)
were isolated from subfraction BA33 by semipreparative HPLC eluted
with a 50% EtOH aqueous solution. Using the same conditions,
subfractions BA31, BA32, and BA34 were subjected to semipreparative
HPLC to yield compounds 18 (3.1 mg), 19 (3.7 mg), 14 (13.0 mg), 15
(24.0 mg), 3 (6.1 mg), and 4 (6.4 mg). Compound 12 (10.2 mg) was
isolated from subfraction BA51 via semipreparative HPLC using 30%
aqueous acetonitrile. Subfraction BA53 was purified with semi-
preparative HPLC (acetonitrile/H2O, 3:7) to afford compounds 7
(12.6 mg) and 8 (4.7 mg). Compounds 5 (3.6 mg) and 6 (1.1 mg) were
isolated from subfraction BA54 by semipreparative HPLC eluted with
29% aqueous acetonitrile. Subfraction BA57 was further purified with
Sephadex LH-20 eluted with MeOH to give compound 16 (15.0 mg).
Compounds 17 (4.1 mg), 11 (8.2 mg), and 13 (10.2 mg) were isolated
from subfraction BA62 by preparative HPLC with 27% aqueous
acetonitrile. Fraction B70 was further separated into eight subfractions
(B70_1−B70_8) using silica gel CC eluted with CHCl3/MeOH/H2O
(ranging from 25:4:1 to 5:4:1). Subfraction B70_2 was subjected to
preparative HPLC (acetonitrile/H2O, 3:7), yielding five subfractions
(B70_21−25). Compounds 9 (4.6 mg) and 20 (10.3 mg) were isolated
from subfraction B70_24 by semipreparative HPLC eluted with 20%
aqueous acetonitrile.
exhibited NA inhibitory activities with IC50 values ranging from
23.1 1.7 to 31.3 5.7 μM and were compared with oseltamivir
(Hoffman-La Roche Ltd., Basel, Switzerland) as a positive
control. Based on the NA results, these compounds were further
investigated with cytotoxicity and H1N1-induced cytopathic
effect (CPE) assays. The data in Table 3 showed that compound
21 was cytotoxic to MDCK cells (CC50, 77.6 2.6 μM), while
the other compounds (22, 23, 25, and 26) did not have strong
antiproliferative effects at a concentration of 100 μM. Com-
pounds 22, 23, 25, and 26 showed EC50 values in the range from
21.4 to 30.7 μM, while the positive control exhibited an EC50 of
0.6 μM.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO P-2000 polarimeter (JASCO, Easton, MD,
USA). All UV and CD spectra were recorded with a Chirascan CD
spectrometer (Applied Photophysics, Leatherhead, UK). IR spectra
were acquired on a JASCO FT/IR-4200 spectrometer. The NMR
spectra were obtained with AVANCE-500, 600, and 850 NMR
spectrometers (Bruker, Billerica, MA, USA), equipped with a cryogenic
probe (600 and 850 MHz). All HRESIMS data were recorded on a
Waters Xevo G2 QTOF mass spectrometer (Waters Co., Milford, MA,
USA). Column chromatography was performed with Kieselgel 60 silica
gel (40−60 μm, 230−400 mesh, Merck, Darmstadt, Germany),
Sephadex LH-20 (25−100 μm, Pharmacia, Piscataway, NJ, USA), and
Diaion HP20 (200−300 mesh, Mitsubishi Chemical Co., Tokyo, Japan).
TLC was performed using Kieselgel 50 F254 coated normal-phase silica
gel TLC plates and TLC silica gel 60 RP-18 F254s (Merck). Preparative
HPLC was performed with a G-321 pump (Gilson, Middleton, WI,
USA) and a G-151 UV detector (Gilson). All solvents were purchased
from Daejung Chemicals & Metals Co. Ltd. (Siheung, Korea).
The reagents for aldose discrimination (L-cysteine methyl ester hydro-
chloride and o-tolyl isothiocyanate) were obtained from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan). Authentic sugars were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
The aqueous residue (25.4 g) was separated with a Diaion HP20 resin
using a MeOH/H2O gradient (0, 50, and 100% MeOH) to yield three
fractions (W0, W50, and W100). Fraction W50 was subjected to passage
over a Diaion HP20 resin with a MeOH/H2O gradient system (0, 25,
and 100% MeOH) to afford three subfractions (W50_0, W50_25, and
W50_100). Further purification of subfraction W50_100 by RP-MPLC
(MeOH/H2O, ranging from 10 to 100%) afforded 10 subfractions
(W50_100_1-W50_100_10). Compound 10 (1.2 mg) was isolated
from subfraction W50_100_8 using preparative HPLC and eluted with
25% aqueous acetonitrile.
Matteuorienate D (1): yellow amorphous powder; [α]2D0 −20.1
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (3.31), 282 (3.62), 349
(3.18); ECD (MeOH) λmax 214 (9.05), 287 (−5.55), 349 (0.98); IR νmax
3393, 2897, 1730 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data,
see Table 1; HRESIMS m/z 619.2025 [M − H]− (calcd for C30H35O14,
619.2027).
Plant Material. Pentarhizidium orientale was collected in September
2013 at Ulleung Island, Gyeongsangbuk-do, Korea, and authenticated
by S. I. Han (Medicinal Plant Garden of the College of Pharmacy, Seoul
National University). A voucher specimen (SNUPT01) of the plant has
been deposited at the Herbarium at the Medicinal Plant Garden of the
College of Pharmacy, Seoul National University.
Matteuorienate E (2): yellow amorphous powder; [α]2D0 −29.0 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 250 (3.26), 282 (3.64), 350 (3.14);
ECD (MeOH) λmax 216 (5.38), 285 (−4.23), 351 (0.79); IR νmax 3336,
2853, 1730, 1715 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data,
see Table 1; HRESIMS m/z 589.1929 [M − H]− (calcd for C29H33O13,
589.1921).
Extraction and Isolation. Dried P. orientale rhizomes (900 g) were
extracted three times with 80% MeOH (4 L × 3, 90 min) at room
temperature. After being concentrated in vacuo, the extract (119.9 g)
was suspended in H2O and successively partitioned with CH2Cl2 and
n-BuOH. The CH2Cl2 fraction (8.1 g) was fractionated by silica gel
column chromatography (CC) and eluted with mixtures of n-hexane/
EtOAc (ranging from 50:1 to 1:1) followed by CHCl3/MeOH (ranging
from 20:1 to 1:1), to afford 14 subfractions (D1−D14). Compounds 21
(5.0 mg) and 22 (4.3 mg) were isolated from subfraction D3 using
preparative HPLC (acetonitrile/H2O, 1:1). Subfraction D5 was
subjected to Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to afford four
subfractions (D51−D54). Compounds 23 (16.2 mg), 27 (3.2 mg), and
28 (2.6 mg) were isolated from subfraction D54 by semipreparative
HPLC (45% aqueous acetonitrile). Subfraction D6 was separated into
four subfractions (D61−D64) using Sephadex LH-20 and eluted with
CH2Cl2/MeOH (1:1). Subfraction D64 was further purified using
semipreparative HPLC and eluted with 42% aqueous acetonitrile under
isocratic conditions to yield compounds 24 (1.0 mg), 25 (2.3 mg), and
26 (24.8 mg).
Matteuorienate F (3): yellow amorphous powder; [α]2D0 −5.1 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 252 (3.26), 282 (3.54), 351 (3.10);
ECD (MeOH) λmax 214 (7.21), 288 (−3.51), 349 (0.98); IR νmax 3335,
2928, 1722, 1715 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data,
see Table 1; HRESIMS m/z 619.2032 [M − H]− (calcd for C30H35O14,
619.2027).
Matteuorienate G (4): yellow amorphous powder; [α]2D0 −16.6 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 250 (3.26), 282 (3.64), 350 (3.14);
ECD (MeOH) λmax 216 (6.08), 285 (−3.40), 350 (0.79); IR νmax 3384,
2882, 1730, 1716 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data,
see Table 1; HRESIMS m/z 589.1923 [M − H]− (calcd for C29H33O13,
589.1921).
Matteuorienate H (5): yellow amorphous powder; [α]2D0 −26.5 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 252 (3.05), 283 (339), 362 (2.91);
ECD (MeOH) λmax 217 (5.84), 289 (−5.18), 352 (1.28); IR νmax 3420,
2853, 1730, 1715 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data,
see Table 2; HRESIMS m/z 635.1974 [M − H]− (calcd for C30H35O15,
635.1976).
The n-BuOH fraction (67.9 g) was subjected to passage over Diaion
HP20 resin using a MeOH/H2O gradient (0, 30, 70, and 100% MeOH
and 100% acetone) to yield five fractions (B0, B30, B70, B100, and BA).
Fraction BA was separated using silica gel CC and eluted with CH2Cl2/
MeOH (ranging from 15:1 to 1:1) to yield seven subfractions (BA1−BA7).
Further purification of BA3, BA5, and BA6 was conducted by preparative
HPLC with 30% aqueous acetonitrile under isocratic conditions to
Matteuorienate I (6): yellow amorphous powder; [α]2D0 −10.4 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 250 (3.34), 284 (3.77), 350 (3.26);
ECD (MeOH) λmax 229 (−1.74), 288 (0.91), 361 (−0.06); IR νmax
3446, 2853, 1730, 1715 cm−1; 1H (600 MHz) and 13C NMR (150 MHz)
data, see Table 2; HRESIMS m/z 635.1974 [M − H]− (calcd for
C30H35O15, 635.1976).
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Matteuorienate J (7): yellow amorphous powder; [α]2D0 −42.0 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 252 (3.20), 286 (3.55), 350 (3.07);
ECD (MeOH) λmax 219 (2.55), 288 (−5.32), 351 (0.38); IR νmax 3446,
2853, 1730, 1715 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data,
see Table 2; HRESIMS m/z 635.1985 [M − H]− (calcd for C30H35O15,
635.1976).
aqueous HCl. A yellowish residue was obtained after drying under N2
gas. The residue was separated using a silica gel Waters Sep-Pak Plus
Long column (CH2Cl2/MeOH, 30:1, 20:1, 15:1, and 10:1) to obtain
amide A and was identified by LC-MS analysis.15 LiBH4 (15 equiv of A)
was added to the solution containing A and THF (0.3 mL) under ice-
cooling. The solution was stirred for 24 h at 25 °C, and then the reaction
was quenched with dilute aqueous HCl. The resultant mixture was
extracted with EtOAc. The resulting extract was separated using a silica
gel Waters Sep-Pak Plus Long column (CHCl3/MeOH, 20:1, 15:1,
10:1, and 5:1) to furnish B. The product was confirmed by LC-MS
analysis and then acetylated with Ac2O (5 equiv of A) in pyridine
(30 μL). The reaction mixture was stirred for 24 h at 25 °C, diluted with
H2O, and extracted with EtOAc; a colorless oil C was obtained after
Matteuorienate K (8): yellow amorphous powder; [α]2D0 4.0 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 254 (2.94), 285 (3.23), 350 (2.75);
ECD (MeOH) λmax 226 (2.48), 279 (−2.13), 350 (0.31); IR νmax 3446,
2853, 1731, 1715 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data,
see Table 2; HRESIMS m/z 605.1862 [M − H]− (calcd for C29H33O14,
605.1870).
Matteucin 7-O-β-D-glucoside (9): yellow amorphous powder; [α]2D0
4.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 248 (3.78), 283 (4.34),
361 (3.76); ECD (MeOH) λmax 230 (−0.93), 283 (0.62), 305 (0.33); IR
1
refining by HPLC. The H NMR spectrum (Figure S69, Supporting
Information) of C was found to be consistent with that of (3R)-5-O-
acetyl-1-[(S)-phenylethyl]mevalonamide upon comparison, rather than
the (3S) isomer that was previously reported.16 (1: 5.1 mg, 8.22 μmol; 2:
5.3 mg, 8.97 μmol; 3: 5.1 mg, 8.22 μmol; 4: 5.1 mg, 8.64 μmol; 7: 5.6 mg,
8.8 μmol; 8: 5.3 mg, 8.74 μmol, 14: 4.2 mg, 6.77 μmol; 15: 5.8 mg,
9.82 μmol; and 16: 5.5 mg, 9.34 μmol.)
1
νmax 3274, 2603, 1730, 1715 cm−1; H (850 MHz) and 13C NMR
(213 MHz) data, see Table S3, Supporting Information; HRESIMS m/z
461.1446 [M − H]− (calcd for C23H25O10, 461.1448).
Matteuorien 7-O-β-D-glucoside (10): yellow amorphous powder;
[α]2D0 27.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 247 (3.97), 280
(4.41), 349 (3.74); IR νmax 3337, 2927, 1758, 1670, 1640, 1607,
1186 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data, see
Table S3, Supporting Information; HRESIMS m/z 443.1337 [M − H]−
(calcd for C23H23O9, 443.1342).
Neuraminidase Inhibition Assay. This enzyme-inhibition assay
was performed as previously reported, with slight modifications.20
In brief, for the preparation of neuraminidase enzymes, MDCK cells
were infected with influenza viruses (H1N1 A/PR/8/34 virus or H9N2
A/chicken/Korea/01210/2001 virus) on a large scale. To inactivate the
viral infectivity, the suspension was supplemented with formaldehyde
solution at a final concentration of 0.1% at 37 °C and incubated for
30 min. The neuraminidase inhibition assay was performed using a
4-methylumbelliferyl-α-D-N-acetylneuraminic acid sodium salt hydrate
(4-MU-NANA) (Sigma-Aldrich) as the fluorescent substrate in 32.5 mM
2-(N-morpholino)ethanesulfonic acid and 4 mM CaCl2 (pH 6.5) as the
enzyme buffer. In general, the assay was performed with the following
steps: (1) 10 μL of virus suspension and 10 μL of diluted test compound
at various concentrations in the enzyme buffer were added to 96-well
plates; (2) after 30 min of incubation at 37 °C, 30 μL of diluted 4-MU-
NANA substrate was added to each well; (3) the reaction was maintained
at 37 °C for 2 h and quenched by treatment with 150 μL of stop solution
(25% EtOH and 0.1 M glycine, pH 10.7); and (4) the fluorescence
intensity of the reaction was measured at an excitation wavelength of
360 nm and an emission wavelength of 440 nm with a fluorescence
microplate reader (SpectraMax GEMNI XPS, Molecular Devices,
Sunnyvale, CA, USA).
Matteuorienin B (11): yellow amorphous powder; [α]2D0 24.6 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 257 (3.84), 291 (4.25), 332 (3.83);
ECD (MeOH) λmax 222 (9.3), 288 (−5.06), 331 (1.08); IR νmax 3392,
2927, 1729, 1516 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data,
see Table S3, Supporting Information; HRESIMS m/z 577.1920
[M − H]− (calcd for C28H33O13, 577.1921).
Matteuorienin C (12): yellow amorphous powder; [α]2D0 24.5 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 258 (3.91), 291 (4.17), 331 (3.83);
ECD (MeOH) λmax 218 (6.7), 290 (−2.95), 335 (0.99); IR νmax 3397,
2930, 1729, 1519 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data,
see Table S3, Supporting Information; HRESIMS m/z 593.1874
[M − H]− (calcd for C28H33O14, 593.1870).
Matteuorienin D (13): yellow amorphous powder; [α]2D0 41.8 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 256 (3.83), 291 (4.25), 331 (3.79);
ECD (MeOH) λmax 218 (10.6), 289 (−5.03), 335 (1.69); IR νmax 3397,
2930, 1730, 1518 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data,
see Table S3, Supporting Information; HRESIMS m/z 607.2030
[M − H]− (calcd for C29H35O14, 607.2027).
Acid Hydrolysis of Compounds 1−13 and Determination of
the Resulting Sugars. The absolute configurations of the sugar
moieties in 1−13 were confirmed using the method of Tanaka et al.13
Compounds (0.5 mg) were hydrolyzed with 0.5 N HCl (0.1 mL) for 2 h
at 90 °C and neutralized with NH4OH. The mixture was dried in vacuo,
and the residue was dissolved in pyridine (0.1 mL) containing L-cysteine
methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. Then, a
0.1 mL solution of o-tolyl isothiocyanate in pyridine was added to the
mixture. The mixture was heated at 60 °C for 1 h, and then the reaction
mixture was directly analyzed by HPLC. Analytical HPLC was acquired
on an YMC-Triart C18 column (4.60 × 250 mm, 5 μm) at 35 °C with
a 25% acetonitrile isocratic solvent system for 40 min (0.8 mL/min).
The authentic sugars, D-glucose, L-glucose, and L-rhamnose, were
subjected to the same derivatization procedure, and all the peaks were
detected with a UV detector at 250 nm. The derivatives of D-glucose in
1−10 and L-rhamnose in 11−13 were confirmed by comparison of the
retention times with those of the standard sugar. The authentic sugar
derivative peaks were recorded at 19.7 (L-glucose), 21.7 (D-glucose), and
37.1 (L-rhamnose) min.
Determination of the Absolute Configuration of HMG in
Compounds 1−4, 7, 8, and 14−16. (S)-1-Phenylethylamine
(2 equiv), triethylamine (Et3N, Sigma-Aldrich) (3 equiv), (benzo-
triazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP, Sigma-Aldrich) (1.5 equiv), and 1-hydroxybenzotriazole
(HOBt, Sigma-Aldrich) (2 equiv) were added to a solution containing
a compound and 0.3 mL of N,N-dimethylformamide (DMF, Sigma-
Aldrich) under ice-cooling. The mixture was stirred at room
temperature for 9 h, and the reaction was quenched with diluted
Cytopathic Effect Inhibition Assay. Madin-Darby canine kidney
(MDCK) cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) (HyClone, Logan, UT, USA) containing 10% fetal bovine
serum (HyClone) at 37 °C and 5% CO2. The cells were seeded onto
96-well plates at 1 × 105 cells per well and incubated for 24 h. Then,
the influenza H1N1 A/PR/8/34 virus was incubated with the cell
monolayers for 2 h via infection using DMEM containing 0.15 μg/mL
trypsin and 5 μg/mL bovine serum albumin. The medium was then
replaced with fresh DMEM containing 10 μg/mL trypsin with different
concentrations of the test compounds. Virus inhibition was examined
with each test compound concentration in triplicate. After 3 days of
incubation, the cells were replaced with fresh DMEM and 20 μL of
2 mg/mL MTT solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide] (Sigma-Aldrich) was added to each well and
incubated for 4 h at 37 °C. The formazan crystals were dissolved with
100 μL of DMSO, and the absorbance was measured at 550 nm using a
microplate reader (VersaMax, Randor, PA, USA).
Cytotoxicity Assay. The cell viability was calculated using an MTT
method. Briefly, the MDCK cells were grown on 96-well plates at
1 × 105 cells per well. After 24 h of incubation, the cells were washed with
phosphate-buffered saline and replaced with fresh DMEM containing
the test compounds at various concentrations. The compounds were
tested at each concentration in triplicate. The cultures were incubated
for 2 days, and 2 mg/mL MTT solution was added to each well, followed
by further incubation for 4 h. The supernatant was removed, and
formazan crystals were dissolved in 100 μL of DMSO and measured at a
wavelength of 550 nm.
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Statistical Analysis. The data are expressed as the means SD of
three independent experiments. Statistical analysis of the half-maximal
inhibitory concentration (IC50), effective concentration (EC50), and
cytotoxic concentration (CC50) was performed using the Sigma Plot
Statistical Analysis software (SPCC Inc., Chicago, IL, USA).
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Kamo, T. Tetrahedron: Asymmetry 2007, 18, 1183−1186.
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Phytochemistry 2004, 65, 2517−2520.
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(18) Markham, K. R.; Geiger, H. In The Flavonoids: Advances in
Research since 1986; Harborne, J. B., Ed.; Chapman & Hall: London,
1994; pp 441−497.
(19) Dao, T. T.; Tung, B. T.; Nguyen, P. H.; Thoung, P. T.; Yoo, S. S.;
Kim, E. H.; Kim, S. K.; Oh, W. K. J. Nat. Prod. 2010, 73, 1636−1642.
(20) Ha, T. K. Q.; Dao, T. T.; Nguyen, N. H.; Kim, J.; Kim, E.; Cho, T.
O.; Oh, W. K. Fitoterapia 2016, 110, 135−141.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.7b00677.
■
S
Chemical structures of compounds 14−28; fully assigned
1H and 13C NMR data of compounds 14−20; raw 1D and
1
2D NMR data of 1−13; scheme and H NMR data for
determination of absolute configurations of HMG
moieties; screening data of neuraminidase inhibitory
assay for isolated compounds (PDF)
(22) Kaihatsu, K.; Kawakami, C.; Kato, N. Nat. Prod. Chem. Res. 2014,
2, 2−9.
AUTHOR INFORMATION
Corresponding Author
*Tel (S. H. Sung): (+82)-2-880-7859. E-mail: shsung@snu.ac.kr.
■
ORCID
Kyo Bin Kang: 0000-0003-3290-1017
Ki Hyun Kim: 0000-0002-5285-9138
Jinwoong Kim: 0000-0001-9579-738X
Sang Hyun Sung: 0000-0002-0527-4815
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF), which was funded by the Ministry of Science, ICT and
Future Planning (NRF-2015M3A9A5030733). We would like to
thank Y.-J. Ko of the National Centre for Interuniversity
Research Facilities for her assistance with the NMR experiments.
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DOI: 10.1021/acs.jnatprod.7b00677
J. Nat. Prod. XXXX, XXX, XXX−XXX